Environmental and Molecular Mutagenesis - Legacy Tobacco ...

Environmental and 

Molecular 

Mutagenesis 

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An international journal speciplipng jp :~ 

• Molecular mechanisms of mutaganesis 

• DNA dama e and repair '? 

• Genetic an d cytogenetic methodology 

• Screening for mutagens and antimutagens 

• Mutagenic hazards for humans ; 

• Genetic toxicology and'risk assessment 

ISSN 0893-6692

Environmental and Molecular Mutagenesis 

JOURNAL OF THE ENVIRONMENTAL MUTAGEN SOCIETY 

OFFICERS, ENVIRONMENTAL MUTAGEN SOCIETY 

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EDITOR-IN-CHIEF 

Richard J. Albertini 

Vermont Regional Cancer Center 

Burlington, Vermont 

REVIEWS EDITORS 

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NCI-Frederick Cancer Research Facility 

Frederick, Maryland 

Barry W . Glickman 

York University, Toronto, Ontario, Canada 

Michael D . Shelby 

National Institute of Environmental Health Sciences, 

Research Triangle Pork, North Carolina 

Raymond R . Tice 

Brookhaven National Laboratory, Upton, New York 

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Vermont Regional Cancer Center 

Burlington, Vermont 

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University of North Carolina, Chapel Hill, North Carolina 

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Hope College, Holland, Michigan 

REFERENCE EDITORS 

Elizabeth S . Von Halle 

Oak Ridge National Laboratory, Oak Ridge, Tennessee 

Kathleen Mavournin 

Oak Ridge Notional Laboratory, Oak Ridge, Tennessee 

Seymour Abrahamson Sheila M . Galloway James T. MacGregor Herbert S . Rosenkranz 

University of Wisconsin Merck Institute for Therapeutic U .S . Department of Agriculture Case Western Reserve University 

Madison, Wisconsin Research Berkeley, California Cleveland, Ohio 

John Ashby Imperial 

Chemical Industries PLC 

Alderley Pork, Cheshire, 

United Kingdom 

Herman E . Brackman 

Illinois State University 

Normal, Illinois 

Anthony Carrano 

Lawrence Livermore Laboratory 

Livermore, California 

June H . Carver 

West Point, Pennsylvania 

Mary Esther Goulden 

University of Texas 

Dallas, Texas 

Philip E . Hartman 

The Johns Hopkins University 

Baltimore , Maryland 

George R . Hoffmann 

Centre Nationole de la Recherche 

Scientifique 

Strasbourg, France 

Donald G. MacPhee 

La Trobe University 

Bundooro, Australia 

Toijiro Matsushima 

University of Tokyo 

Tokyo,Japon 

J . Justin McCormick 

Michigan State University East Lansing, Michigan 

Robin W. Morgan 

Gary A . Sega 

Oak Ridge Notional Laboratory 

Oak Ridge, Tennessee 

Richard B . Setlow 

Brookhaven Notionol Laborotory 

Upton, New York 

Thomas R. Skopek 

Chemical Industry Institute of 

Toxicology 

Research Triangle Park, 

North Carolina 

Chevron Environmental Health Center 

University of Delaware 

Richmond, California 

Vicki L. Dellorco 

Environmental Protection Agency 

Washington, DC 

David M . DeMorini 

Environmental Protection Agency 

Research Triangle Park, 

North Carolina 

Henry E . Holden 

Pfizer Central Research 

Groton, Connecticut 

Joseph R . Landolph 

University of Southern California 

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Los Angeles, California 

Newark, Delaware 

Kristien E . Mortelmons 

SRI International 

Menlo Park, California 

Earle R . Nestmonn 

CanTox Inc ., Oakville 

Ontario, 

Canada 

Sheldon Wolff 

University of California 

Son Francisco, California 

Friedrich E . Wurgler 

University 

of Zurich 

Schwerzenboch, Switzerlond 

Errol Zeiger 

Notionol Institute of Environmental 

Eric Eisenstadt L . Gayle Littlefield Miahael J . Prival Health Sciences 

Harvard School of Public Health Oak Ridge Associated Universities Food and Drug Administration Research Triangle Pork, 

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http://legacy.library.ucsf.edu/tid/clb93d00/pdf

Environmental and Molecular Mutagenesis Volume 14, Supplement 15 : 1-252 (1989) 

Abstracts of the 

Fifth International Conference 

on 

Environmental Mutagens 

Case Western Reserve University 

Cleveland, Ohio 

J u ly 10-15, 1989 

National Organizing and Program Committee Members 


Frederick J . deSerres, Chairman 

Richard J. Albertini John Mirsalis 

David Brusick R . Julian Preston 

Eugene L. Elmore Sheila Galloway 

Mortimer L. Mendelsohn Herbert Rosenkranz

ABSTRACTS 

I THE KINETICS OF SODIUM FLUORIDE-INDUCED ENDOREDUPLICATION IN CHINESE NAMSTER OVARY 

CELLS . M .J . Aardema,'D .P . Gibson, R .A . LeBoeuf . The Procter & Gamble Company, 

Cincinnati, OH 45239 (USA) . 

We previously reported endoreduplication in Chinese hamster ovary (CHO) cells 

exposed to high doses of NaF (Mut . Rea ., 1989) . To inveatigate further the induction 

and the mechanism of NaF-induced endoreduplication, we conducted kinetic studies after 

NaF exposure . With a 4 h exposure to 1 .276 mg/ml NaF, the highest percentage of 

endoreduplicated cells in the culture was observed 20 h after exposure . When cells 

were exposed to 20 yM BrdU for 1 h or 2 h before NaF treatment, the endoreduplicated 

cells harvested 20 h after exposure had 2-6 or 2-7 differentially stained chromosomes, 

respectively, or had no differential staining . The differential staining pattern corresponded 

to late replicating chromosomes . This indicates that cells which were sensitive 

to the induction of endoreduplication were in late S or G2 of the cell cycle . 

To examine the timing of the DNA synthesis phase leading to andoreduplication, cells 

were pulse labeled with tritiated thymidine for 15 min at 2 h intervals following NaF 

exposure and harvested 20 h after NaF treatment . The results indicated that DNA 

synthesis leading ~o endoreduplication lasts at least 18 h and is followed by a normal 

2 h G2 phase . The delayed appearance of endoreduplicated cells, the sensitivity of 

S/G2 cells, and the long endoreduplication DNA synthesis phase observed in our studies 

are very similar to that reported for other agents (e .g . cytosine arabinoside) which 

are proposed to induce endoreduplication by inhibition of DNA synthesis . These results 

are consistent with a mechanism of NaF-induced endoreduplication by an inhibition of 

DNA synthesis as we previously suggested for NaF-induced chromosome aberrations . 

L 

COMPARATIVE MUTAGENICITY TESTING OF A DRUG CANDIDATE : BROPIRIMINE, A 

POTENTIALLY POWERFUL ANTIVIRAL COMPOUND . C.S . Aaron, A . Thilagar, V . Kumaroo, 

T. Petry, J . Mayo, D . Branstetter, J . Mazurek, The Upjohn company, Kalamazoo, MI ; Sitek 

Research Laboratories, Rockville, MD . 

Comprehensive genetic risk assessment of potential drugs involves more detailed analysis than the 

assignment of positive or nagative values to the outcome of a battery of tests . Bropirimina was subjected 

to a variety of qenotoxicity ssssYs and presents a complex risk assessment problam. Bropirimine was 

negative in the Ames sssay, 

. UDS, rst micronudeus assay and the LS178Y TKt mouse 

lymphoma assa Ho, wever slka induced the line elution a compound stronyIy non-Iinear incraase In chromosome 

aberrations (CA)y(as high as 42 % of the cells had aberrations at S00 pg/ml) in CHO ceils . The CA were only 

observed in the presence of inetabolic activation (S 9) . Extensive experiments were carried out in order to 

evaluate this observation . For exampie, chromosome aberration induction in LS17gY cells was 

demonstrated, despite the absence of mutagenesis in this assay . Furthermore, gene mutations (HPRT) 

were demonstrated in CHO cells over the same dose range which yielded CA . Several studies were carried 

out to characterize the influence of metabolism . Heat inactivation of the S-9 failed to eliminate 

chromosome aberration induction . Furthermore, no significant quantities of metabolites were formed !n 

vitro by S-9 acting on bropirimine. No binding to DNA or protein could be detected following metabolism 

of bropirimine by S•9 in vitro . These latter experiments suggest that the positive findings In in vitro 

systems may be due to enhanced transport (caused by interadion with protein) coupled to deleterious 

effects on chromatin structure caused by the unchanged drug . This drug did not cause rat bons marrow 

CA or micronucleus formation, but did induce significant frequencies of mouse micronucleus formation . In 

addition, bropirimine induced transofrmation of Balb 3T3 cells . This array of genetic toxicology findings is 

similar to an alternative therapy acylrovir . The risk assessment process for drugs such as bropirimine must 

incorporate a balancing of relative risks . 

3 

COMPARATIVE MUTAGENICITY TESTING OF A DRUG CANDIDATE : U-68,553B . 

EVALUATION OF U-68,553B REVEALS AN UNUSUAL ABILITY TO BREAK SPECIFIC CHO 

CHROMOSOMES AND LACK OF IN VIVO GENOTOXICITY . C .S . Aaron, A. Thilapar, V . 

Kumaroo, J . Mazurek, J . Mirsalis, K. Steinmetz, L . Stankowski, R . Sorp The Upjohn 

Company, Kalamazoo, MI ; SITEK Research Laboratories, Rockviile, MD ; SRI, Menlo Park, 

CA; Pharmakon Research International, Inc . Waverly, PA . 


1989 EMS Abstracts 3 

Notes

4 1989 EMS Abstracts 


Notes U•68,5538 is a novel drug under development by The Upjohn Company . This compound was marginally 

positive in the Ames assay In the presence of S•9 and in In vitro cytopenetics in CHO cells In the absence of S- 

9 . The result of testing in the In vitro UDS assay In rat hepatocytes was a clear positive . The compound was 

not mutagenic in the mouse micronucleus test and was negative in both the CHO/HPRT and ASS2/xvRT 

mammalian cell mutation assays . A variety of follow-up studies has revealed that the In vitro findings 

probably do not reflect a 9enotoxic hazard . For exampie, no evidence of Induction of chromosome 

aberrations in vivo was observed In rat bone marrow . Furthermore, the kinds of aberrations Induced In vitro 

were primarily chromosome breaks and appeared to occur selectively in a few chromosomes . We followed 

up this observation by G-bsndinq the chromosomes and determining the distribution of breaks in the 

9enome . In fact, approximately 90% [207/234) of all the chromosomal aberrations were breaks in the three 

longest chromosomes in the complement, with a larye majority in Chromosome 3. These breaks appear to 

occur at discrete locations In the chromosomes that are broken . Finally, the In vitro UDS findings were 

pursued by treatment of rats In vivo (the so-called "in vivolin vitro UDS") and there was no UDS observed In 

vivo . Thus, the genetic toxicology profile of U-68,S63t yields several positive In vitro findings which are not 

relevant to In vivo hazard . The assessment of risk due to this compound is clearly an example of the need for 

rational risk assessment when in vitro tests give positive results . The full data and evaluation of the results 

will be presented in the context of the risk assessment . 

IN VIVO GENOTOXICITY OF CHEMICALS WHEN ADMINISTERED IN DIPPERENT SOLVENT/CARRIERS . 

N .G . Abbott and A .F tlcFee, Oak Ridge Associated Oniversities, Oak Ridge, TN (USA) 

Soae ehemieal coapounds to be tested for mutagen/eareinogen potency are insoluble in 

both the aqueous and oil solvents eomaonly used for in vivo adainistration . Diaethyl 

sulfoxide (DMSO) has often been used for injecting such compounds, but its in vivo 

behavior and possible interaetion with the solute have besn questioned . We quantified 

sister ehroaatid exchanges (SCE) in marrow leukoeytes and aieronuelei (MN) in 

polychromatic erythrocytes (pCE) of mice after injections of compounds having various 

degrees of solubility in phosphate-buffered saline (PBS), corn oil (00), and DHSO . 

Equal quantities of chemicals were adainistered as solutions or mechanical suspensions 

in each of the carriers, and aice vsre killed 24 hr later . SCEs were scored in 25 

metaphases from each of 4 mice psr treatment, and fluorescence microscopy was used to 

enuoerate MN among 2,000 FCE in each of 5 aice per treatment . SCEs induced by 10mg/kg 

of 7,12-diaethylbensanthracene (DqBA) were significantly higher when administration was 

in CO solution than in PBS suspension, and even higher when in DNSO solution . MN 

induction was significantly higher following administration of 50 ag/kg of DMBA in OD 

or DMSO solutions than with PBS suspension . The number of SCE induced by 

diglycidylresorcinol ether showed a relationship to ita solubility in the various 

carriers, whereas MN induetion was increased most sharply when injeetions were in DMSO . 

Doses of 1,2-dibroao-3-chloropropane in DNS0 solution repeatedly produced much higher 

nuabers of SCE than the same amounts given in CO solution or PBS suspension . The data 

identify some compounds which express greater genotoxicity when administered in DpSO 

solution and some with a possible chemical interaction with DdSO . Supported by NIEBS 

Interagency Agreement Y01-ES-20100 and DOE/0RA0 Contract DE-ACOS-760R00033 . 

5 

METABOLIC ACTIVATION OF Me1Q TO A MUTAGEN BY HEPATIC PREPARATIONS FROM DIFFERENT RAT 

STRAINS . Amal Abu-Shakra, Cellular and Genetic Toxicology Branch, National Institute 

of Environmental Health Sciences, Research Triangle Park, MC 27709 . 

MeIQ (2-amino-3,4-dimethylimidazo[4,5-f]Quinoline) was activated to a mutagen in 

the Ames test by hepatic microsomal preparations from Aroclor- pretreated Mistar . 

Sprague-Dawley and Fischer rats . NeIQ-Rwtapenicity was catalysed by cytochromes P448, 

which are induced by Aroclor . Microsomes from the uninduced rats were deficient In 

the cytochromes P448 and could not activate MeIQ . Purified microsomes from induced 

rats, regardless of the strain, activated MeIQ to give 860-920 rev/ng . This strong 

mutagenic response was enhanced when the microsomes were supplemented with their 

respective cytosolic fractions, to generate the •resuspended• $9 . Strain differences 

became apparent with this procedure . Resuspension caused Fischer-S9 to be noticeably 

weaker than Wistar-S9 and Sprague-Dawley-S9 . Because the strains were similar in their 

microsome-mediated responses, it seemed that differences in S9-activation could be 

attributable to the cytosol . However, in a cross-over study, where the cytosol of one 

strain was mixed with the microsomes of the other, Fischer-cytosol mixed with Wistaror 

Sprague-Dawley-microsomes provided stronger MeIQ-activation than did Fischercytosol 

with Fischer-microsomes . Also, Wistar-and Sprague-Dawley-cytosol, which were 

excellent enhancers of their respective mlcrosane-mediated responses, failed to 

enhance that of the Fischer-microsomes . Therefore, strain differences are not only due 

to different levels of cytosolic activity, but are affected by tnteractions between 

the cytosol and the microsome-generated metabolites . 

4

6 1989 EMS Abstracts 5 

COhiPA:jArIVi; N:LTAGT13S13 O? VITAVAC . N . A3hikari an3 LS, 3rover, Notes 

school of Life Sciences, Guru Nanak Dev University, Amritsar, In31a . 

Vitavax (carboxin), an important systomic fungicide, has an annual 

consumption in In3ia of about 80 M .T, Reports on its differential 

mutaienic response in in vitrq, (both with animal an3 plant microEomal 

activation) an3 Ip, vivo bioassays are scarce . Iience a comparison of 

its genotoxicity in Jn y~~ (Ames test) an3 in_ vivo chromosomal 

aberrations an3 MN in3uction in bone marrow cells of rats was done . 

Neqative control (DMSO) an3 appropriate positive controls (ao3itm azi3e- 

TA100r NPD-TA97al FMS-rats) were usej concurrently. In Ames test, the 

pestici3e was teste3 both in the presence an3 absence of S9 mix an3 two 

S14s (see3ling homogenaW of Zea mavs an3 Btasg1a over a range of 7 

log concentrations . However, it faile3 to elicit any positive 

response + S3/S14s. C ytogene tic 3amage in rats was screene3 for thre e 

9oses ( 1/40 LDS , 1/20 LD an3 1/10 LD ) with the abarrant cells 

ranging from 4,90 to 16 .90Oper cent . Fa"q the MN test, three 3ose 

levels, 1/4 MTD (maximum tolerate3 9ose) , 1/2 MTD an3 bITO were testa3 . 

Micronucleate3 pCEs range3 from 3. 60-11 .60/1000 PCSS . Significant 

cytog=netic 3amage in both the assays was observe3 at the two highest 

3oses teste3. Though vitavax 3i3 not elicit any significant response 

in Anas assay, yet its ability to in3uce an appreciable clastogenic 

3ama :3e warrants further stu3ies . 

7 

SURVEILLANCE OF TOBACCO-ARECA NUT CHEWERS WITH CYTOGENETIC MARKERS . 

Adhvaryu S .G ., Dave B .J ., Trivedi A .H ., Cell Biology Div ., Gujarat Cancer Research 

Institute, Ahmedabad, 380016, INDIA . 

Role of tobacco-areca nut chewing in causation of oral cancer and oral submucous 

fibrosis (SMF) is well documented, however, thorough human studies are rare . In 

the present study; (i) controls not consuming tobacco or areca nut in any form, (ii) 

chewers with no appreciable change in buccal mucosa (nor .chw .), (iii) chewers with 

oral SMF and (iv) chewers with oral cancer, have been studied for frequencies of 

sister chromatid exchanges (SCE) & chromosome aberrations (CA) in lymphocytes and 

micronuclei (MN) in exfoliated buccal mucosa cells . The study design was expected to 

provide information about genotoxic effects of the habit on target as well as nontarget 

tissue . The observations were : 

Frequency Of :- SCE/cell C .A ./cell % MN cells . 

Controls 6 .185 0 .050 0 .193 

Nor .Chw . 7 .219 0 .097 0 .740 

Or . SMF Pts . 7 .617 0 .127 0 .753 

Oral Ca . Pts . 8 .500 0 .144 - 

Values obtained for 

all three groups of 

chewers were significantly 

higher with p


Notes GENETIC TOXICOLOGY OF FOOD PRODUCTS 

H .U . Aeschbacher, Nestld Research Centre, Nestec Ltd . 

Lausanne 26 (Switzerland) . 


9 

Vers-chez-les-Blanc CH-1000 

There is evidence from epidemiological studies that food is involved in cancer 

etiology . Such a relationship has not generally been established for non 

carcinogenic .genotoxic food components . This might be due in part to the difficulty to 

provide evidence for genetic defects in man but might also be due to the fact that a 

variety of food components which are positive In vitro are not harmful to man . 

For instance, a number of compounds such as some vitamins, plant phenolics, etc are 

mutagenic in vitro due to their antioxidant activity but might as a matter of fact 

protect man from cancer . Food components also affect other mechanisms e .g . adsorption 

to fiber, alteration of gut flora, etc, which can hardly be taken into account by In 

vitro test systems . 

It is therefore essential to permit for a correct risk assessment to thoroughly 

investigate food-borne compounds with possible mutagenic or carcinogenic potential . 

SYNEYISTIC GENOTOXF EFFECT OF.3 SMOKING AtiD LEAD. YR Ahulal, Talitha T 

RaJah , B DineshKumar , NV Prasad , K Kashyap , K Krishnaswamy . 1)Dept . of 

Genetics, Osmania University,2)National Institute of Nutrition 3)Govt Printing Press 

4)Bureau of Polic . R6D, Hyderabad, INDIA . 

Both smoking (S) and lead (Pb) have been shown to be genotoxic when tested 

alone . Since the results of the studies on the Interacting effects between S and 

Pb exposure are controversial, the present stud y was undertaken . Eighteen adult 

male workers (8 of whom were smokers) exposed to Pb In a printing press and 

25 controls were included in our study . Blood Pb levels were determined in 15 

of the exposV subjects . Atmospheric Pb levels were also determined . Atmospheric 

Pb (3 .1qjig/m ) was significantly higher as compared to the unpolluted environment 

(0 .5ug/m ) . Blood Pb level was also significantly elevated (41 .55i5 .91,ug/dl) as 

compared to controls (23 .6:3 .29,ug/dl) . Chromosome aberrations (CA) (structural+ 

numerical) were increased significantly in those who were exposed to Pb (15 .78%) 

as compared to controls (6 .7%) . Amongst the Pb exposed workers, there was no 

difference in the frequency of CA between smokers and nonsmokers (14 .16 vs 

14 .70%)i however smokers had a significantly higher frequency of micronuclei 

than nonsmokers (0 .85 vs 0 .44%) suggesting that 8 In combination with Pb is 

clastogenic leading to cell death . The mitotic Index was also depressed in Pb 

exposed smokers indicating that Pb In combination with smoking is cytotoxic . 

Our results suggest that smoking interacts synergistically with Pb in causing genetic 

damage . 

Financial Assistance from ICMR is acknowledged . 

NUCLEOID SEDIMENTATION ANALYSIS OF DNA DAMAGE PRODUCED BY NUTAGENS IN RAT LYMPHOCYTES . 

Anane Aidoo, Ning Gao and Robert H . Heflich, National Center for Toxicological Research, 

Jefferson, AR (USA), and Drug Inspection Institute of Inner Mongolia, Huhhot (PRC) 

Assays based on detecting DNA damage in lymphocytes are used to monitor exposure to 

potential genotoxic chemicals in vivo . In this study, we have examined the ability of 

the model genotoxic agents, 2-acetylaminofluorene (2-AAF), benzo(a)pyrene (BP) and 

several of their metabolites to produce DNA damage in rat lymphocytes . Lymphocytea, 

isolated from the spleens of male Fischer 344 rats, were exposed to the agents for 

one hour In serum-free medium . DNA damage was determined by the sedimentation rate of 

nucleoids from the treated cells as compared with the appropriate controls . The 

relative efficiency of producing DNA damage in rat lymphocytes by BP and its metabolites 

was : BP anti-7,8-diol-9,10-epoxide > BP 4,5-oxide > BP trans-7,8-dihydrodiol a 

BP, with the latter two compounds eliciting essentially no damage . For 2-AAF and its 

derivatives, the order of potency was : N-acetoxy-2-AAF > N-hydroxy-2-aminofluorene 

(N-hydroxy-2AF) a N-hydroxy-2-AAF > fluorene 3- 2-AF a 2-MF . The latter three compounds 

produced very little damage . Paraoxon eliminated the DNA damage produced by Nacetoxy-2-AAF, 

but both paraoxon and salicylamide had no affect on N-hydroxy-2-AAFinduced 

damage . Rat lymphocytes also mediated a mutagenic response with N-hydroxy- 

2-AAF in Salmonella ~t himuriu~m TA98 . These results indicate that rat lymphocyte 

nucleoid seation detecS DNA damage produced by reactive metabolites of 2-AAF 

and BP . While rat lymphocytes were capable of metabolizing N-acetoxy and N-hydroxy- 

2-AAF to DNA-damaging species, they displayed little ability for activating the parent 

compounds or BP trans-7,8-dihydrodiol . 

10 

11


THEORETICAL BASIS OF THE TRANSPLACENTAL CARCINOGENICITY AND TERATOGENICITY OF DIMETHY- 

LNITROSAMINE AND N-ETHYL-N-NITROSOUREA . Dunstan A .A . Akintonwa, Department of Biochemistry, 

College of Medical Sciences, University of Calabar, Calabar - Nigeria . 

Dimethylnitrosamine and N-ethyl-N-nitrosourea are mutagens in the Ames test and they 

are carcinogens whilst thalidomide is a teratogen and neither a mutagen nor a carcinogen 

. The evidence that nitrosamines are not active in transplacental carcinogenesie 

except when administered late in pregnancy has been challenged . Mechanism of transplacental 

carcinogenesis of dimethylnitrosamine (DMNA) and N-ethyl-N-nitrosourea (ENU) 

has been presented based on the presence of monooxygenases (EC1 .14 .14 and EC1 .14 :15) 

and smooth endoplasmic reticulum and rough endoplasmic reticulum in 13 week and 16 

week old human foetal livers . Plausible mechanism for the teratogenicity of ENU based 

on chelating propensity of its metabolites has been presented . Probable mechanism for 

the basis of the2#eratogenicity 21 a metabolite of thalidomide based on chelation 

with calcium (Ca ) and zinc (Zn ) comparable to similare chelation of ethylene 

diaminetetraacetate (EDTA) has also been presented . From the molecular basis of carcinogenesis 

and teratogenesis DMNA was found to be negative (-) as a theoretical (T) 

and experimental (E) traneplacental carcinogen in rodent and (-) and positive (+) for 

T if the maternal metabolite or the intact DMNA passes through the placenta respectively 

and not known (?) for (E) in human . For ENU, (+) for (T) and (E) in rodent 

and (+) for (T) and (?) for (E) for human . In terstogenesis for DMNA, (-) for (T) and 

(E) in rodent and (-) for (T) and (?) for (E) in human were obtained . Por ENU, (+) 

for (T) and (E) in rodent and (+) and (?) for (T) and (E) respectively were obtained . 

13 

COMPARISON OF CTTOGBNETIC, SPRT uD GLLCOP80RIN A 8?UDIL9 IN A-B01D $URYIYORS 

Mitoshi Akiyama, M .D .,?, Seishi Hyoisumi, Ph .~ .,1, Yuko Hirai, Ph .D .?, 

Masayuki Hakoda, M .D .,1, Nori Nak~aura, Ph .D . and Akio A . Awa, Ph .D . 

Department of Radiobiology and Department of Genetics, 

Radiation Effects Research Foundation, Hiroshima 732, Japan 

It is well known that somatic mutation are induced by various environmental 

genotoxic substances including ionizing radiation . At present, only a few methods 

are available for measuring the frequency of in vivo mutants in human somatic 

cells . One such method is to detect the mutant lymphocytes lacking HPRT, and 

second method is to detect the variant erythrocytes lacking surface protein, 

glycoprotein A (GPA), third is the HLA somatic mutation method recently developed 

by Morley et al . The frequencies of somatic mutation were measured by using the 

former two methods in atomic bomb survivors in Hiroshima, and were found to be 

significantly correlated with the DS 86 estimated dose and, as well, to the 

chromosome aberration frequency of lymphocytes, respectively . However, the 

variant frequency from HPRT and GPA study are not correlated significantly with 

each other . One possible reason for this lack of correlation may be in the fact 

that the lymphocytes bearing sex-linked HPRT mutations, presumably large 

deletions (based on other studies) ∎ay have been at greater selective 

disadvantage than erythrocytes containing glycophorin mutations . 

14 

hurt MUTATIONS IN VIVO IN HUMAN T-LYMP110CYTES : FREQUBNCIES, SPECTRA AND CLONALITY, R .J . 

Albertini, J .P . O'Neill, J .A . Nicklas, M .J . McGinniss, L . Recio, and T .R . gkopek, VRCC 

Genetics Lab, Univ . of Vermont . Burlington, VT and CIIT, Research Triangle Park, NC . 

hort mutations in vivo in human T-lymphocytes reveal (i) mutant frequency values, (!i) 

v v mutational spectra, (iii) pra- versus post-thymic origin of the !n vivo mutations 

and (iv) clonality of )= mutants . Mean mutant frequency values differ by ten-fold 

between newborns and young adults, being 0 .64 t 0 .41 x 10-6 for 45 placental blood samples 

and 6 .5 ± 4 .8 x 10-6 for a large cohort of adults . h= mutational spectra also differ 

between newborns and adults ; more than 85% of the newborn mutations show deletions of h= 

exons 2 and 3 while only 151 of 326 b= mutants from normal adults show b= structural 

alterations detectable by Southern blot analyses . In adults, no type of alteration 

predominates . Deletions are common and deletion breakpoints are distributed randomly 

along the length of the gene . Direct sequencing studies of adult b= mutants are 

determining the actual mutations tn mutants with normal Southern blots . bQ=j, mutations 

in newborns and adults differ also in their cells of origin with those recovered from 

newborns tending to arise in pre-thymie cells while those recovered from adults tending 

to arise in post-thymic T-cells . Iterative analyses of 1)= alterations and TCR gene 

rearrangement patterns in wild type and b= mutant isolates from adults reveal clonality 

among the mutant but not the wild type isolates . •Doublets•, 'triplets' and higher orders 

of clonality are routinely observed among an individual's recovered mutants . This has 


Notes


Notes led to the hypothesis that proliferating rather then "resting" T-lyaphocytes are 

preferentially susceptible to gsne mutations in vivo . This may have significant 

implications for interpreting human in vivo mutagenicity studies . Research supported by 

NCI CA30688 and DOE FG0287ER60502 . 


INFLUENCE OF DIFFERENT FACTORS ON THE INDUCTION or KALSEGREGATION IN 

SACCHAROMYCES CEREVISIAE D61 .M BY BAVISTAN . 

Silvio Albertini, Pharmaceutical Research, Department of Toxicology, 

F .Hoffmann-La Roche o Co ., Ltd, CH-4002 Basel, Switzerland . 

Bavistan is known as a potent inducer of malsegregation in Sacharom ces 

cerevisiae . with both standard protocols used with S . cerev s ae D .M 

overnight incubation and cold treatment protocol, respectively) 

bavistan induced malsegregants in the same range . The frequencies obtained 

were not influenced by the plating volume used on selective medium 

. The higher the YEP content the more distinct the toxicity and the 

induction of malsegregants was . The physiological temperature for 

yeasts of 28°C led to a more pronounced induction than 33°C and 37°C . A 

clear observable induction by bavistan was detectable after 8 hours 

incubation (overnight protocol) and 1 .5 hours of the second incubation 

period (cold treatment protocol), respectively . 

Comparison of the growth of mixed cultures of red, cycloheximide 

sensitive cells and white, cycloheximide resistant, leucine auxotrophic 

cells prepared at different ratios revealed, that there is a strong 

selection towards red cells and against the malsegregants . 

PLASKID COPY NUMBER AND MUTANT FREQUENCIES IN S .TYPHIMURIUM TA102 

Silvio Albertini and Elmar Gocke, Pharmaceutical Research, Department 

o Tox co ogy, c .Hoffmann-La Roche i Co . Ltd, CH-4002 Basel,Switserland . 

Tetracycline and chloramphenicol increase the number of mutant colonies 

of strain TA102, which carries the reverting gene on the plasmid pAQl . 

Determination of the plasmid content by agarose gel analysis shows that 

the increase of the mutant colony number is paralleled closely by an 

increase of the number of pAQl plasmids per cell, indicating that the 

two compounds do not increase the frequency of mutants "per gene", but 

only enhance the number of genes at which mutations can occur . Thus, 

not considering the molecular processes could result in mistakenly 

attributing the increase in the number of mutants per plate (respective 

to the number of mutants per cell) to a mutagenic activity of the antibiotics 

. An other antibiotic, kanamycin, acts by reducing the translation 

fidelity which leads to read through of the ochre stop codon and 

thereby to an accumulation of enough functional enzyme so that enhanced 

cellular growth is possible . 

These different mechanisms all mimic mutagenic responses with some 

analogies to the effect of growth enhancing compounds (e .g . complex 

mixtures containing histidine) . It is discussed whether such false 

positive results are likely to occur in routine testing with TA102 and 

how to avoid possible misinterpretation . 

17 

INDIRECT EFFECT OF SOME DNA DAMAGING AGENTS ON LAMBDA RECOMBINATION . D . Alcintara-Dfaz 

and N . Brefia-Valle, Instituto Nacional de lnvsstigaciones Nucleares, Kixico, D .F . (NE- 

IQCO) . 

Recombinant progeny of undamaged lambda phage, increases up to four times when infecting 

E . coli hosts previously irradiated with UV light. A similar effect occurs in 

the mutant recA-441 (tif-1) SOS induced by hest, but so far none could be detected in 

lexA (ind-) bacteria (Alcfintara and BreBa, data submitted for publicatioa) . All these 

results suggeat an SOS-dependent stimulation of undamaged lambda recombination . The 

present work shows more data supporting the former view . Phage crosses were carried 

out according to standard procedures in hosts with distinct repair capabilities treated 

with either UV or three other genotoxicants . An increase of 7 times the normal recombinant 

progeny was found in umuC cells exposed to 50 J/m2 of W light, a fact which 

to our view proves that these ara true recombinants and not mutants due to the well 

known W effect . On the other hand the experiments using FRlC, l04S and MJNG as recombina 

tion stimulants show that whereas no affect could be found in treated E . coli lexA 

(ind-) hosts, a significant increase in phage recombinaits occurred whan Tnfecting 

15 

16

1989 EMS Abstracts 

wild type cells exposed to either chemical . We conclude that this is due to some recom Notes 

binational event(s) associated in some way to SOS induction . Nevertheless, in the case 

of the chemicals we cannot exclude the possibility that at least part of the effect 

could be adscribed to phage DNA damage by the agent itself . For these reason further 

experiments on the subject are being carried on presentely . 

Acknow)edgements : We wish to thank Mrs . Rufina Gbmez and Mrs . Guadalupe Martinez 

for technical assistance . 

18 

METABOLISM OF HETEROCYCLIC AMINES IN COOKED FOOD . 

J . Alexander, H . Wallin, G . Becher, J .A . Holme and A . Mikalsen . 

Dept . of Toxicology, Natl . Inst . Publ . Health, Geitmyrsvn .75, N-0462 Oslo 4, NORWAY 

Metabolic fate of ingested heterocyclic amines involves both metabolic activation to 

prnximate or ultimate mutagens/carcinogens and detoxication reactions . Metabolic pathways 

of aminoamidazoazaarenes (AIA) were studied in isolated rat hepatocytes as well 

as microsomes and a reconstituted P450 system . Isolated hepatocytes contain both oxidative- 

and conjugation enzymes for xenobiotics and modulation of inetatalism can easily 

be done with enzyme inducers and inhibitors . Radiolabelled parent ea :pouuxi and cofactor 

in conjugation reaction are useful .Metabolites are excreted into the incubation buffer 

which is far less carplex than bile or urine, making metabolite isolation for chemical 

characterization (e .g . MS and NhIIt) less cotnplicated . Formation of maonarplecular adduct 

(i .e . with DNA or proteins) and genotoxic events in the hepatocyte(e .g . UDB, DNA-strand 

breaks) or in coincubated bacteria or cells are good indicators ofinetabolic activation . 

P450 dependent activation with N-hydroxylation of the exocyclic amino group is moet 

inportant and further activation can also take place to more reactive products (e .g . 

O-actetyl or O-prolyl esters) . But estrification to semistabile products (e .g . 0-glucuronides) 

may also occur . Formation of glutathione conjugates have also been noted . 

Important detoxication pathways for the I¢coffpounds are, provided low P450 activity, 

direct conjugation of the amino group to sulphate or glucuronic acid, while N-acetylation 

seems much less important . With high P450 activity ring hydmxylations followed 

by conjugation to glucuronic acid and sulphate are major detoxication pathways both 

for the IQ-campounds and PhIP . P45OBNF performs both activation and detoxication reactions, 

while P450b is inactive . 

19 

REPAIR OF O6-METHYT.GUANINE AND MNNG INDUCED MUTAGENESIS IN LIVER 

EPITHELIAL CELLS . N .K . Alvi, P .G . Foiles and G .M . Williams . American 

Health Foundation, Valhalla, NY 10595 USA . 

Methylation of guanine at the 06 position is considered to be a promutagenic 

lesion in mammalian DNA . An assay measuring mutations at the 

hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in adult 

rat liver epithelial cells (ART~,) was employed to study the mutagenic 

effects of O-methylguanine (O MeG) resulting from N-methyl-N'-nitro-Nnitrosoguanidine 

(MNNG) egposure . ARL cells were pretreated witg a non 

toxic dose of exogenous O MeG (0 .6 mM for 4 hr .) to deplete of Omethylguanine 

methyltransferase (MT) . Subsequently, cells were exposed 

to the various doses of MNNG . A dose dependent induction of 6-thioguanine 

resistnt (TGr) mutations was evident at lower levels of MNNG 

treatment in o6MeG pretreated and MeG pretreated cultures . ARL cells 

partially depleted of MT activity still repaired about 60t of 06MeG 

present in DNA as compared to untreated c~ltures . An immunoslot study 

employing a mouse antibody specific for O feG confirmed that the ARL 

cells were hi hly proficient in removing O MeG from their DNA . The 

kinetics of OgMeG elimination indicated that the half life of O6MeG 

repair in ARL cells was 1 hour and 4 hours respectively at two different 

doses of MNNG treatment . 

20 

A PROSPECTIVE STUDY COMPARING 6-TNIOGUANINE-RESISTANT VARIANT FREQUENCIES WITH CBROMO- 

SOME ABERRATION FREQUENCIES IN LYMPHOCYTES FROM RADIOTHERAPY AND CBEMOTf1ERAPY PATIENTS . 

M .M . Ammenheuserl, J .B . Ward, Jr .l, W .W . Aul, and J .A . Bel1i2, 1Departeent of Preventive 

Medicine and Community Health . 2Department of Radiation Therapy, The University of 

Texas Medical Branch, Galveston, TX 77550 (USA) . 

We used the autoradiographic 6-thioguanine-resistant (TGr) somatic cell sLtation 

assay to determine the frequency (Vf) and timing of appearance of variant T-lymphocytes 

from 6 patients receiving pelvic x-irradiation for uterine or bladder cancer . The 

patients received 180cGy/day, 5 days/week for 5 weeks . Blood samples were obtained 

before treatment and at weekly intervals during treatment . An aliquot of each sample 

was also analyzed for chromosome aberrations . The mean TGr Vf increased from 3 .47 x 


9


Notes 10-6 pre-treatment to 9 .43 x 10-6 2 weeks after6beginning radiotherapy, b~ was significantly 

higher (p

0 .40, and 0 .60% EMS in phosphate buffer to maintain pH 8 for 4 hr at 30'C . The treated 

teeds were washed with running water for 4 hr, sown on blotter 'sandwiches', and placed 

n a growth chamber with a temperature of 30'C . Eight days after the treatment, the 

seedling height was measured, seedlings pere planted individually in soil contained in 

polyethylene bags, transplanted to the field after 6 weeks, and grown to maturity . 

Seeds from self-pollinated M plants were planted for the M2 progeny . The dose of 0 .30, 

0 .45, and 0 .60% reduced the lercentage of germination to 95 .6, 92 .2, and 66 .7X respectively 

. Seedling height of those treated with 0 .15% was 83 .2% while those treated with 

0 .30, 0 .45, and 0 .60% were 76 .2 . 58 .4, and 49 .2% of control respectively . The survival 

percentage of 6-week-old M plants treated with 0 .15, 0 .30, 0 .45, and 0 .60% were 98 .7, 

89 .1, 88 .3, and 57 .1% of cAntrol respectively . Nine M tricotyledonous seedlings were 

observed, two of which were grown successfully to maturity . Although the effects of 

the treatment on the percentage of germination, seedling height, and survival of M1 

plants were significant, except for the tricotyledonous plants with short stature and 

small leaves, genetic effects of the treatment which could have been manifested in the 

form of chlorophyll mutations and other types of mutations in the M2 plants, were not 

observed . Ricinus communis L . which is possibly a polyploid, has probably a certain 

mechanism of buffering some genetic changes . 

24 

4nt tFFECTS OF GnM4n KAD1AT10N AND >:THYL Mk:'1'NANESULFONATk UN NUSTO( : LINI:KIA 

A .'1' . Aranez, lnstitute of Biology, University of the 1'hilippines, Uiliman tYhilippinss) 

Nostoc is a blue-green alga which is more closely related to bacteria than other 

algal groups . The objectives ot this study were to determine the etfects of gamma 

radiation and ethyl metnaneeulfonate (l+MS) on general morphology of calls, the ttequency 

of heterocysts, and the growth rate . Finely divided suspension of colonies of 

Nostoc suspended in distilled water were treatad with 75, 150, 300, and 45U kr gamma 

radiation . Anotner set was treated with U .6, 1 .2, 1 .8, and 2 .41 e:MS in phosphate 

butter at pN 8 for 4 hr at 3U-l: . Morphological changes produced by both treatments 

were enlarged and abnormally shaped vegetative cells, enlarged heterocysts, and chalns 

ot heterocysts . tlranched filaments were observed in Noatoc treated with gamma radiation 

. An increase in the number of beterocytts was observed in both treatments . Seven 

weeks atter the treatments, except for the presence of some elongated cslls and cells 

that were not in perfect alignment in Nostoc subjected to high doses ot gamma radiation 

and tP1S, treated Noatoc appeared as normal . keduction in growth rates were observed 

in the first week after treatment for algae treated with gamma radiation and the first 

two weeks, for algae treated with EMS . Afterwards, growth rates of treated Nostoc 

ihcreased . Seven weeks after the treatment, the population densities or the treated 

and untreated Nostoc were more or less similar . Organisms treated with U .b and 1 .Yx 

r:MS solution lost the capacity to form large aggregates for 7 days and those subjected 

to 1 .8 and 2 .4s formed small aggregates 10 days attsr the treatment . Nostoe was 

observed as not very sensitive to gamma radiation and CMS . (This study was funded by 

the Natural Science Nesearch institute, University of the Yhitippines) 

25 

TRANSMISSIBLE GENETIC EFFECTS IN MICE FOLLOWING POST-MEIOTIC EXPOSURE 

TO METHYL METHANESULFONATE 

M. Aravindakshan and P.S. Chauhan, Molecular Biology and Agriculture Division, Bhabha 

Atonic Research Centre, Banbey 400085,India . 

Majority of chenical mutagens unlike ionizing radiation induce genetic danage specifically 

in post-meiotic phase of epehnatogenesis . It is therefore inportant to investigate heritable 

effects arising from mutagenic exposure of post-meiotic germ cells . In the present study 

trananissible genetic effects followirnl post{neiotic exposure of mice to methyl methanesulfonate 

(MMS) have been investigated. Adult Swiss male mice, injected Lp. with 50 mg/kg 

of MMS or the solvent were individually paired with virgin fenales fran 7-13 days posttreaen 

ent, the m ost sensitive period of MMS m utagenesis in m ice . The fern ales, random ised 

into two groups were either evaluated for the induction of dominant lethals at r~ ~d tertn 

pregnancy or allowed to deliver to obtain subsequent generation . Based on two tndepenGent 

experinants, the incidence of daninant lethal mutations was 51 .9% in MMS treated -nice 

against 5.0% in controls. A higher incidence of post-implantation lethal mutations (PLM) 

in the test group (23.3%) as compared with controls (7 .5%) was observed in the Flgeneration . 

The mutation frequency was 15.5% and 5.9% respectively in test and control groups In 

the F2generation. However, in the F3g eneration, the incidence of PLM was canp arable 

in the control (7 .1%) and test group (7 .2%). Daninant lethal mutations are self I'mitirg 

as these are el'minated fran the progeny . However, these observations clearly demonstrate 

that post{n eiotic exposure of m ale m ice to MMS induce heritable genetic alterations which 

express as lethals in the early ernbryonic development in the subsequent generations and 

that these effects are gradually elininated 



Notes


Notes Temporal control of AP endonuclease !n human flbroblasts and Blooms syndrome cells . P . 

Arenaz, S . Trevizo, P . Anaya, V . Pelaez and L . Winkfield, Dept . Biology, University of 

Texas El Paso, El Paso, TX (USA) 

Previous reports have indicated that DNA repair and replication are coordinately 

regulated within the defined context of the cell cycle and that this temporal 

regulation acts as a screen to ensure replicative fidelity . Certain putative DNA 

repair deficient syndromes exhibit an aberrant pattern of gene expression of several 

DNA repair enzymes . We have investigated the temporal regulation of AP endonuclease 

in human fibroblasts and in Bloom's syndrome cells . Celie were synchronized by either 

serum starvation or hydroxyurea and cells collected at various time points after 

release from the block . DNA repair and replicative parameters were assayed using cell 

extracts . AP endonuclease activity, measured in serum synchronized cells was maximal 

at 18 h, 3 h prior to DNA synthesis and one hour prior to the fnduction of uracil DNA 

g)ycosylase . This same pattern was observed for AP endonuclease activity after 

hydroxyurea synchronization . In Bloom's syndrome cells, AP endonuclease was again 

Induced prior to DNA synthesis . However, as reported before uracil DNA glycosylase 

was induced concomitant with DNA synthesis . These data suggest that AP endonuclease 

ia Induced within the defined context of the cell cycle in both normal human 

fibroblasts and Bloom's cells . Furthermore, 1t appears that AP endonucleaoe and 

uracil DNA glycosylase are not concomitantly controlled and suggests that there may be 

different control signals involved in the induction of these enzymes . 

Supported in part by NIH grant RR08012 and a grant from the University of Texas at E1 

Paso . 


CHARACTERIZATION OF A DIRECT-ACTING MUTAGEN FORMED FROM N-NITROSOPIPERIDINE AND 

PHOSPHATE WITH NEAR-ULTRAVIOLET LIGHT IRRADIATION 

Sakae Arimoto, Hiromi Shimada, Satoko Ukawal, Masataka Mochizukil and Hikoya Hayatsu 

Faculty of Pharmaceutical Sciences, Okayama University . Tsushima, Okayama 700, and 

1Kyoritsu College of Pharmacy, Shibakoen, Minato-ku, Tokyo 105, Japan 

Previously we found that direct-acting autagens can be formed from N-nitrosodialkylamines 

on exposure to UVA . We have now isolated the product formed from N-nitrosopiperidine 

(NPIP) and investigated its structure . NPIP (40 mM) in 20 mM sodium phosphate 

at pH 7 .4 was irradiated with UVA (313-400 nm, 10 yN/mm2) for 3 hr . Direct-acting 

mutagenicity of the solution on S . typhimurium TA 1535 was 44000 His+ revertants for 

1 mmole equivalent of NPIP . The reaction mixture was freeze-dried and the residue was 

extracted with methanol . The methanol extract was evaporated to dryness under reduced 

pressure, and the residue was fractionated by HPLC on an ODS column . Direct-acting 

mutagenicity was observed only in one UV-absorbing peak . The compound in this peak 

fraction was identical to an authentic sample of a-phosphonooxy-NPIP in terms of the 

retention time in HPLC, UV spectrum, sensitivity to phosphatase and the mutagenic 

potency as determined on the basis of A231 units . Moreover, the 1H-NMR spectrum of the 

isolated compound was identical to that of the authentic specimen . Thus, it waa 

established that N-nitrosodialkylamines can be transformed into their 0-hydroxy phosphate 

esters by the action of near UV light in the presence of inorganic phosphate . 

This reaction represents a new, non-enzymatic activation of promutagenic N-nitrosodialkylamines 

. 

28 

ANALYSIS OF MUTATIONS INDUCED BY NEAR-UV RADIATION . J .D . Armstrong, M . Glattke, L . 

Kohalmi and B .A . Kunz, Microbiology Department, The University of Manitoba, Winnipeg, 

Manitoba, Canada R3T 2N2 

Ultraviolet (UV) wavelengths found in solar radiation are autagenic and carcinogenic 

. Attenuation by atmospheric ozone limits the total amount of solar UV in our 

environment and restricts incident solar UV wavelengths to the naar-W (NUV : 300-400 

nm) region so that virtually no UVC (200-280 nm) wavelengths are present . To begin 

probing the mechanism(s) responsible for solar UV mutagenesis, we have used DNA sequencing 

to characterize 120 autetlons induced in the SUP4-o gene of yeast by NUV 

and have compared the resulting spectrum to that for 185 UVC-induced mutations . In 

both cases, single base-pair substitutions accounted for approx . 90% of the induced 

mutations but the fraction of double-pair changes was 3-fold greater for NW and all 

double mutations were tandem events compared to only 330 for UVC . For each agent, 

approx . 90% of the substitutions were transitions but NUV induced substantially more 

G•C -> A•T events than UVC (88t vs . 681, respectively) . Of the substitutions that 

could be assigned to the 5' or 3' base of particular dipyrimidines, 800 of those induced 

by NUV occurred at the 3' base of S'-TC-3' or 5'-CC-3' sites compared to 60% 

for UVC . In addition, S'-TT-3' sites that were hotapots for UVC were not targets for 

NUV mutagenesis although hotspots at 5'-TC-3' sites coincided . Wavelengths involved 

in photoreactivation of cyclobutane dimers or photolysis of [6-41 photoproducts, are 

present in NUV radiation . On this basis, our data suggest that either (6-41 photophotoproducts, 

or photoproducts resulting from photolysis of these lesions, are the 

major premutational NW lesions . (Supported by NSERC Canada) 

27


TRANSFECTION WITH HUMAN STOMACH DNA AND BiCNU RESISTANCE OF CHO CELLS Notes 

I .I . Arzimanoglou, C . Troungos, and S . Kyrtopoulos, N .H .R .F ., Athens 11635, Greece 

The prem,tagenic, precarcinogenic alkylation lesion 06-alkylguanine is repaired 

in repair-proficient cells by the enzyme O6-alkylguanine-DNA-alkyltranaferase (06-AGT), 

an enzyme which in E .coli can be induced under conditions known as "adaptation" . The 

phenomenon of adaptation has not been clearly demonstrated in eucaryotic cells . We 

have exposed CHO cells (with very low 06-AGT activity) to low concentrations of ?RiNG 

and then tested them for 06-AGT induction . No such induction was achieved . However, 

following such pretreatment, a reduction in SCEs induction by a challenge dose of NNU 

was observed, suggesting the induction of protective mechanism other than 06-AGT . In 

an attempt to examine the expression of an eucaryotic ACT gene, we co-transfected 

CHO cells with human DNA, and the bacterial neomycin gene, which is enclosed in 

Cosmid Homer-6 . Control cells were transfected only with the latter .G418-rasistant 

colonies were continuously cultured for a number of cycles of further selection with 

the cross-linking, cytostatic, alkylating agent BiCNU (Carmustine) . 06-AGT has been 

demonstrated to confer resistance, against the toxic effects of the cytostatic drug 

BiCNU . The main results of the study are the following : 1) Following selection with 

BiCNU, colonies with increased amounts of AGT were obtained from both transfected 

and control cells, 2) While the average increase in 06-AGT levels was roughly the same 

in colonies derived from transfected and control cells, larger number of BiCNU - 

resistant colonies were obtained from transfected cells, 3) Cells containing increased 

06-ACT, showed increased resistance against BiCNU and decreased mutability (to thioguanine-resistance) 

by HNNG . 

30 

FUTURE DEVELOPI[ENY OF SHORT TERM TESTS 

J . Ashby . ICI Central Toxicology Laboratory, Alderley Park, Cheshire . 

Two models of chemically-induced rodent carcinogenicity are currently competing for 

attention . The first requires that all carcinogens induce cancer by virtue of their 

assumed ability to modify directly DNA structure or functlon, a property that 

presumably can be determ3ned in vitro given appropriate assays . The second model 

recognizes the potential impoTr7Rf~'Zff biologlcal disturbances lnduced in rodents by 

the administration of chemicals . These disturbances may be due to the chemical's 

overt toxicity or to its ability to induce subtle changes in tissue hoaeostasis and 

gene expression . If the chemical administration is chronic the associated biological 

changes may assume pseudo- permanence and lead to modification of tumor lncidences in 

the treated animal at death, in particular to 'carcinogenic' increases . If the 

chemical is also genotoxic, then truly irreversible changes in gane expression .ay 

accompany the temporal changes, and this could lead to more overt carcinogenic 

consequences . This second model fits the available facts of rodent carcinogenicity 

better than does the first model, however, the first model is sufficlent to account 

for the large majority of trans-species and multiple site carcinogens, ie, those 

carcinogens generally considered most likely to pose a hazard to humans are genotoxie 

in vitro . Progress in this field will depend upon which of these two models is 

accep e for development . If the goal is to detect DNA modifying carcinogens, such as 

benzpyrene, then the only need !s to organize and refine current techniques and 

testinh strategies . In contrast, if the requirement is to predict all future rodent 

carcinogens, then other disciplines must be invoked to understand the nature and 

significance of the many non-specific toxicities elicited by chemicals in rodents . 

From such studies may emerge useful predictive assays for non-genotoxic rodent 

carcinokens such as saccharin and limonene . 

31 

THE SPONTANEOUS MUTATION SPECTRUM IN H13mp2 PHAGE 

Gen-ichi Atsumll, Keiko Matsumotol, Tadayoshi Besshol, Kazuo Negishi2 

and Hikoya Hayatsul 

1 Faculty of Pharmaceutical Sciences . Okayama University, Tsushima, Okayama 700 

2 Gene Research Center, Okayama University, Tsushima, Okayama 700, Japan 

Naturally occurring mutations include almost all conceivable changes in DNA 

sequences . These random genetic changes are likely to be harmful to organisms ; genetic 

defects and cancer are believed to be caused by mutations . We have analyzed spontaneous 

mutations in the phage M13mp2 lac promoter-lac Za region ; the forward mutations in the 

lac promoter-lac Za region that is necessary for a-complementation to restore the Bgalactosidase 

activity in E. coli NR9099 were analyzed . Mutant a-complementationdeficient 

phages were detected as colorless or pale blue plaques among the wild blue 

plaques in the plates containing X-gal and IPTG . Viral single stranded DNA was prepared 

from the isolated mutant plaques and sequenced by chain termination procedure of Sanger 

et al . using a sequencing kit with a-32P-dCTP . The frequency of spontaneous mutation 

was 4 .7 x 10-5 (55/1170000) . Of the 55 DNA samples, sequence changes were detected in 

30 samples . The spectrum of these changes comprised transitions, transverslons, 

multiple base-deletions, -additions, single base-deletions, and -additions . About a 

half of these mutations were single base additions and deletions in several hot spots . 

Analysis of the local DNA sequences suggests that the intensity of these hot spots 

depends on structural features of the DNA, i .e ., runs of more than four identical bases . 

The cause of these spontaneous mutations is, therefore, suggested to be slippage errors 

by DNA polymerase of the host bacterium E . coli . 


13 

0 

0


Notes 


IN UTERO AND MALE MEDIATED-EFFECTS OF 1,2-DIBROlIO-3-CHLOROPROPANE IN RATS . W.W. Au, 

D . Walker and M .S . Legator . Department of Preventive Medicine and Community Health, 

University of Texas Medical Branch, Galveston, Texas 77550, USA . 

In utero and male-mediated effects after oral exposure to Sprague Dawley rate to 1, 

2-dibromo-3-chloropropane (DBCP) were investigated . DBCP was administered to timedpregnant 

females on days 11 and 15 of gestation at single doses of 1, 10, 50 and 150 

mg/kg . Calls were harvested g hours after treatment . Chromatid breaks and exchange 

figures were significantly elevated in pooled whole-embryo cell suspensions and in 

pooled fetal liver cells from females treated on day 15 . A doae related response was 

observed, even at the lowest concentration . In the dominant lethal test, DBCP was 

administered in doses of 1, 10 and 50 mg/kg for 5 consecutive days, with 5 males in 

each group . The males were each mated to 2 untreated females 4 and 5 weeks later . In 

the combined 4 and S week results, a dose-responsive effect was seen in the dominant 

lethal index (number of live fetuses in treated compared to nontreated females) . In 

addition to the dominant lethal effect using the same protocol but allowing the pups 

to go to term, a post natal effect was found . A significant number of new born rats 

died within 24 hours after birth . This increase in post natal death was dose dependent, 

with a effect found at the lowest concentration used, 1 mg/kg/day for 5 days . 

AFLATOXIN ADDUCTS AS A MEASURE OF EXPOSURE . 

Autrup, H ., Fibiger Institute, Danish Cancer Society, DK-2100 Copenhagen 

Denmark . 

Aflatoxin 81, a potent human liver carcinogen, is metabolized by cytochrome 

P-450 oxidases to several metabolites, including the electrophilic 

8,9-epoxide . This metabolite will react with in nucleic acids and 

proteins . The major DNA adduct is formed in the reaction between the 

N-7 position of guanine and the epoxide . This adduct is unstable, and 

would either ringopen to the more stable AFB-FAPY adduct or depurinate . 

The latter product is excreted in urine . Human exposure to aflatoxin 

has been determined by measuring the amount of AFS-FAPY in human liver 

samples from China, and by detecting the depurinated product in urine 

collected in Kenya . The sensitivity of both assays is 5 adducts per 

1oE7 nucleotides . Aflatoxin 8,9-epoxide also reacts with serum albumin, 

and the major adduct is formed with lysine, This adduct is detected in 

blood collected in China, and a positive association between exposure 

level and binding to serum albumin has been established . Three different 

methological approached with equal sensitivity and specificity have 

been developed to assess the biological active dose of aflatoxin Bl in 

humans . 

jLl SITU DETECTION OF DNA DAMAGE IN SINGLE CELLS OR TISSUE SECTIONS BY QUANTITATIVE 

IMMUNOFLUORESCENCE MICROSCOPY 

RA Baan, L Roza, GP van der Schans, MWM van Loon and CJM van der Wulp 

TNO Medical Biological Laboratory, P0 Box 45, 2280 M, Rijswijk, The Netherlands 

The interaction of reactive chemical species with DNA - leading to the formation of DNA 

adducts - is considered to be an important step in mutation and cancer initiation . To 

study the correlation between the presence of DNA adducts and mutation induction, methods 

are required to detect and quantify DNA damage . Correlations between DNA lesions and 

biological effects may be used to develop rlsk-asses,ment procedures . Molecular dosimetry 

of DNA damage can be used to estimate the extent of genotoxic exposure . . 

The present work is aimed at the application of issiunoohemical methods to assess gsnotoxic 

damage in a limited number of cells or at the single-cell level . This approach involves 

the development of antibodies specific for a particular DNA lesion, and the use of such 

antibodies for detection of DNA damage in fixed calls or tissue sections by laser-scan 

immunofluorescence microscopy (IFM) . The fluorescence is quantified by analog-digital 

conversion and data-processing in a Microdutch 100 computer with the software package 

TCL-image . The IFN technique has been or will be used for various purposes : 

the study of induction and removal of thymine dimers, with dimsr-specific antibodies, 

in UV-irradiated cultured human fibroblasts or skin ∎ections . 

analysis of benzo(a)pyrene(BP)-DNA adducts in human white blood cells with antibodies 

specific for the BP-daoxyguanosine adduct, to monitor exposure to polycyclic aromatics . 

- detection of DNA damage induced in germ cells by ionising radiation, with antibodies 

specific for single-stranded regions in DNA, to study repair processes in such cells . 

50869 3526 

32 

33 

34

35 1989 EMS Abstracts 1 5 

A CYTOGBf7BTIC STUDYlIN CARBON PLACK SXPOSED JNDIVIDUALS OF TYRB INDUSTRY Notes 

P .P.Babu, V .S.Prasad , K .Ram Rao and Y .R .Ahuja . (1) Dept. of Gestetica .Osmania 

University . Hyderabad-500 007 .India. (2) Deputy Civil Surgeon . ESI Disptsttsary. 

Moulali, Secunderabad, India . 

Carbon black (CB) is of substantial industrial importance and is used in the 

manufacture of rubber and leather . Several studies have been done to investigate 

the mutagenic effect of CB. However, the evidence for the mutagenicity of CB is 

still considered to be controversial . A cytogenetic study was undertaken in the 

workers occuptaionally exposed to CB, over a period of 2 to 8 years. In the tyre 

Industry . For comparison age and sex matched controls were taken from the 

unexposed population . Peripheral blood samples were collected from 28 CB exposed 

workers and 15 controls, and cultured in RPMI 1640 medium for 48 hours . Chromosomal 

aberrations were scored in 100 metaphases from each subject . There was 

a significant increase in the frequency of chromosome aberrations in the workers 

exposed to industrial CB (5 .07%) as compared to the unexposed controls (2 .27%) . 

The present study indicates that CB is a genotoxic agent . 

36 

Financial Assistance from ICMR is acknowledged . 

STAGE-SPECIFIC SYNAPTONEMAL CQMPLEX AND CHROMQSOZtE DAMAGE INDUCED BY X-RAYS IN MALE 

MOUSE GERM CELLS . L .C . Backeri and J .W . AllenL . IEHRT, P .O. Box 12199, RTP, NC, 27709 ; 

2U . S . EPA, RTP, NC 27711 . (th1 . .b .DS.et do .s neo n .e .sssly e fl et U .S . stA oolsoy . ) 

The synaptonemal complex (SC) comprises the proteituceous axes of paired homologous 

chromosomes at meiotic prophase and is involved in pairing, crossing over, and 

segregation. SC analysis allows the observation of damage induced prior to and during 

meiosis. In the present study, male C57BL/6J nice were exposed to 0, 2, and 4 Gy of X 

rays. Harvest times were chosen to assess specific damage induced in different germ 

cell stages. 4 animals/dose were harvested at each time point and the results were 

based on 100 cells/animal . Within 2-4 hours of radiation exposure, there was a 4-fold 

increase (compared to controls) in SC breakage ; damage was observed in all prophase 

stages but was most prevalent in mid-pachytene cells . 4 days following exposure (at 

DNA synthesis [Sj), there were significant increases in SC breaks (3 .5-fold) and 

rearrangements (16/animal compared to 0 for controls) . Spermatocytes (Hls) showed 

significant increases in both chromosome and chromatid damage on days 9 and 11 

following exposure . Overall damage was most extensive on day 9(zygotene exposure) 

with >60 chromosome rearrangements (CRs) and 42 chromosome breaks (CBrs)/nouse 

compared to 0 .5 CR and 1 .0 CBr/mouse in controls . On day 11 (premeiotic S exposure), 

there were 9 CRs and 49 CBrs/mouse . Consistent with the literature, zygotene was more 

sensitive to the induction of damage that results in CRs than S phase was . on the 

basis of the above data and the trends observed in preliminary analyses of day 1 postexposure 

SCs, SC analysis is a sensitive method with which to detect damage that may 

be expressed as chromosome aberrations at metaphase . The types of damage induced by 

radiation, including rearrangements qualitatively different from those we have 

observed with chemical treatments, should prove useful for further studies of the 

relationships between specific types of SC damage and chromosome aberrations . 

37 

ANALYSIS OF DIESEL PARTICULATE MATTER AND VAPOR PHASE SAMPLES USING THE 

MICROSUSPENSION ASSAY. S. L. Stoltz and $. Z. Bag)liy, Michigan Technological University, 

Houghton, MI (USA) 

Organic extracts of diesel particulate matter and vapor phase (from XAD-2 resin) samples were assayed 

using the Salmonella microsuspension (MS) assay (Kado et at ., Mutat . Res . 121, 1983, 25) to determine if 

increased sensitivity could be obtained compared to the standard plate incorporation (STD) Ames assay, 

particularly with the vapor phase extracts which typically show little muts~enicity in the STD assay . The 

MS assay uses a 90 minute preincubation with 10-fold more cells (1X10 cells) and 20-fold leu sample 

volume (5 µl) than the STD assay; after the preincubation, the MS assay proceeds as for the STD assay . 

In this study, 100 pl and S pl aliquots of the same dilutions were used in the STD and MS assays, 

respectively . The MS assay with TA98 and the particulate and vapor phase extracts was 10 and 30-times, 

respectively, more sensitive than the STD assay. This increase In sensitivity was related to more 

proximate interaction bewteen cells and sample and not, for example, to sample interactions with Os 

during the preincubation period as MS assays +/- Os had no difference in responses . With the MS assay, 

tester strains TA98NR and TA98 l,8-DNPs had similar increases in sensitivity with the particulate 

extracts and mutagenicity due to nitroarenes was detected for the first time in the vapor phase extracts . 

Based on repeat analysis of the same diesel samples, the MS assay was as repeatable as the STD may . I.e., 

C.V.'s S 15% . The MS assay was also modified for use with smaller (60XI5-mm) petri dishes to save on 

assay costs, with no loss in assay sensitivity or repeatability; the small dish MS assay uses 10 mL bottom 

agar and 0 .7 mL top agar, containing 90 nmoles histidine and biotin. The increased sensitivity of the MS 

assay not only allows for detection of mutagenicity with use of 20-fold less diesel extract mass, but 

mutagenicity, including that due to nitroarenes, can also be detected in vapor phase samples having little 

activity in the STD assay. (Supported in part by contract No . 87-6 from the Health Effects Institute .) 



Notes 


ELECTROPHILICITY'OF NONGENOTOXIC CARCINOGENS AND GENOTOXIC NONCARCINOGENS AS MEASURED 

BY THE ke TEST. G . Bakale and R .D . McCreary, Case Western Reserve University, 

Cleveland, OH (USA) 

The results of applying a physico-chemical short-term carcinogen-screeaing test, 

the ke test, to probe the elctrophilic properties of nonganotoxic carcinogens and 

genotoxic noncarcinogens will be presented . The electrophilicity of a test chemical 

as measured by the ke test is based upon the reaction rate constant, ke, at which 

excess electrons attach to the chemical in a nonpolar liquid (e .g., cyclohexane) ; a 

diffusion-controlled ke is regarded as a positive indication that the test chemical 

is an electrophile, whereas the ke of a chemical that is less than diffusioncontrolled 

indicates an activation barrier to attachment and a non-electrophilic test 

chemical . The pulse-conductivity syste∎ used to measure ke's in the nanosecond time 

regime will be described as well as the results of screening with the ke test those 

chemicals that yield conflicting rodent carcinogeniclty and bacterial mutagenicity as 

determined, respectively, by National Toxicology Program (NTP) long-term animal 

studies and by the Ames Salmonella test . Of 47 chemicals that are NTP rodent 

carcinogens but which yield negative Ames test reponses, 26 are k-test 

electrophiles. Of 23 chemicals that are noncarcinogenic in the NTP animal teits but 

are mutagenic to the Ames Salmonella strains, 17 also yield positive electrophilicity 

responses in the ke test . The implications of the ke-electrophilicity/bacterial 

mutagenicity/rodent carcinogenicity rel}tionship to short-term screening of 

carcinogens will be discussed as well as the rationale for the k test yielding 

positive electrophilicity responses to procareinogens that 4have not been 

metabolically activated . 

PAN OBTAIIPRD BY HPLC FRACTIOd1ATION OF DIRSEL-SQGINg-EXU1UST-PARTICLE ERTRACTS ARE NOT 

ACTIVATED BY 59 TISSUE HOMOGHiATE . James C . Ball and Irving Salmeen, Research Staff, 

Ford Motor Company, Dearborn, MI 41821-2053 . 

Diesel-sngine-exhaust-particle extracts are active in the Ames assay without the 

addition of S9 . However, the interpretation of the indirect-acting mutagenicity (i .e . 

mutagenicity in the presence of S9 tissue hosogenate) of these samples is difficult 

because of the unknown effect of the S9 enzymes on the direct-acting mutagens . lie 

carried out Ames assays on an HPLC-fractionated methylene chloride extract of a 

diessl-engine-exhaust-particle aample using both TA98 and TA100 with and without the 

addition of exogenous tissue homogenate . These resulta (shown below) suggest that the 

"classic" PAN fraction (e .g . pyrene, chrysene, and benzo(a)pyrene) is not mutagenic 

even with the addition of exogenous metabolizing enzymes and cofactors . These results 

have implications for the interpretation of Ames assays of diesel-engine-exhaustparticle 

extracts . 

Bacterial Unfract . Fraction Number ; Rev/ug 

Strain Extract 1 2 3 4 5 6 7 8 

TA98 ; -S9 13 nm Q .8 5 180 66 20 39 6 

+S9 8 nm znl 27 120 56 15 60 4 

TA100 ;-59 1S mm Tm 7 130 70 21 38 8 

_ +S9 11 r~m nl SO 130 42 22 31 6 

1nm-not mutagenic ; nl~non-linear and less than 2x spont . rev . 

F.FFECT OF PROTEIN A ON DRUG META90LISIKG ENZYMES 

M .R .Bansal and Deepika Khanna 

Department of Biophysics, Panjab University, Chandigarh 160 014, India 

The phase I and phase II enzyme systems are responsible for conversion of the 

carcinogen into a reactive metabolite and for its detoxification . Protein A is 

known to regenerate cytochrome P-450 activity . To study the effect of protein A 

on drug metabolising enzymes, female Swiss Porten rata were fed Ja'3A (24 mg in 

olive oil) which caused 50% tumor incidence after five months . The palpable 

tumor-bearing rats and the DMBA-fed rata without any morphological sign of tumor 

were treated with 12 ug protein A in normal saline s .c . twice a week for 6 weeks . 

Cytochrame P-450 levels increased significantly after protein A treatment whereas 

there was no significant change in cytochrome b5 activity . DM3A-fed rate revealed 

increased glutathione and glutathione-S-transferase activities . Protein A 

edministration to non-tumor bearing rate showed that glutathione levels decreaseo 

and glutathione-S-transferase activity increased . However, no significant change 

in phase II system was observed in tumor-bearing rats treated with protein A . 

It is concluded that cytochrome P-450 activities are regenerated by protein A 

and hence metabolism of the carcinogen . 

38 

39 

40


THE MUTAGENIC IMPACT OF X-RAY TREATMENT ON ELECTROPHORETICALLY EXPRESSED 

LOCI IN THE MOUSE 

Lois B . Barnettt, Raymond A . Poppz . and Susan E . Lewist 

tResearch Triangle Institute . P .O . Box 12194 . Research Triangle Park, NC 27709 USA 

z0ak Ridge National Laboratory . P .O . Box Y . Oak Ridge, TN 37830 USA 

The induction of mutations by various doses of x-rays has been studied in the 

electrophoretic specific locus test in the mouse . Both female and male germ cells 

were studied . In order to study effects on specific germ cell stages, matings to 

produce (C57BL/6J x DBA/2J)F1 hybrid mice for electrophoretic analysis were made 

at appropriate times following x-ray treatment . When C576L/6J females are the 

treated parent . two visible loci (brown and dilute) can be studied in addition to 

the electrophoretic loci . The comparison of visible and electrophoretic loci in 

C57BL/6J females treated with 300R shows that the visible loci appear to be more 

mutable at this dose than are the electrophoretic loci . Hhile spermatogonia 

treated with a single dose of 300R gave higher frequencies of mutations on a per 

locus basis than did the 600R dose, the effects on electrophoretically expressed 

loci in all studies were less than in the visible specific locus test (supported 

in part by Contract No . Nol-ES-2-5012 . NIEHS) . 

42 THE SOS RESPONSE AND INDUCED MUTAGENESIS . ) .R . Battista, C.E . Donnelly, T. Ohta, V. 

lgras, and Graham C. Walker . Massachusetts Institute of Technology, Cambridge, MA 02139 

The products of the umuD and umuC genes are required for most UV and chemical mutagenesis in 

Escherichia colr. The genes are organized in an operon that is repressed by LexA and regulated as part of 

the SOS response. We have noted that UmuD shares amino acid similarities with the LexA protein of E . 

colr and also with gp45 of bacteriophage T4 and that UmuC shares amino acid similarities with gp44 and 

gp62 of T4 . We have carried out a series of genetic experiments that indicate that RecA-mediated 

cleavage of UmuD at its Cyss4-Glyss bond activates UmuD for its role in mutagenesis and that the 

COOH-terminal fragment of UmuD is both necessary and sufficient for that role . Other genetic 

experiments indicate that the primary role of SereO in UmuD function is to act as a nucleophile in the 

RecA-mediated cleavage reaction and that RecA has a third role in mutagenesis besides mediating the 

cleavage of LexA and UmuD. Their similarities to the T4 DNA polymerase accessory proteins, gp4S, 

gp44, and gp62, suggest possible roles for UmuD and UmuC in mutagenesis that are supported by 

preliminary evidence . Our observation that groEL and groES mutations suppress the cold-sensitivity of 

DNA replication caused by umuDC overexpression led to the discovery that groEL and groES mutants 

are largely nonmutable with UV. The fact that this nonmutability of groEL and groES mutants can be 

suppressed by overexpression of the umuDC operon suggests that these 'chaperone' proteins may affect 

the ability of UmuD and UmuC to exert their functions in mutagenesis . 

43 

ROLE OF RECOMBINANT CYTOCHROME P4501A2 IN THE METABOLISM AND GENOTOXICITY OF FOOD 

DERIVED MUTAGENS . N . Battula, H .A .J . Schut', E .G . Snyderwine and S .S . Thorgeirsson, 

Laboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, MD 

20892 ' Department of Pathology, Medical College of Ohio, Toledo, OH 43699 

To circumvent the inherent difficulties of purification and specificity in 

reconstituted cytochrome P450 systems, we have constructed several types of recombinant 

viruses containing the coding DNA sequences for individual P450s to efficiently 

transfer the P450s into mammalian cells including human cells . Analysis of cell lines 

containing the introduced sequences for cytochrome P4501A2 indicate that the enzyme 

is constitutively expressed in its natural microsomal environment . These cell lines 

have permitted us to carry out measurements of the biotransformation of substrates j„0 

jJyt under physiological conditions . To determine the role of P4501A2 in the 

metabolism of carcinogenic heterocyclic ar~lamines, the DNA of the exposed cells was 

analyzed for DNA-carcinogen adducts by P-postlabeling . DNA of IQ (2-amino-3methylimidazo[4,5-flquinoline) 

exposed cells showed five specific DNA-IQ adducts . The 

finger prints of the adducts formed in cells were chromatographically indistinguishable 

from those formed in rat and mouse liver after in vivo administration of IQ . Similar 

analysis with AF and AAF indicate specific adducts with the cell DNA but showed minor 

differences with the in vivo adducts . This novel approach of stably expressing 

specific P450s in situ and their catalytic measurements provide an excellent method 

to study the biotransformation of substrates and their role in toxicity, mutagenesis, 

and carcinogenesis . 


Notes


Notes 


DNA ADDUCT FORMATION IN RELATION TO TUMORIOENESIS IN MICE 

CONTINUOUSLY ADMINISTERED 2-ACETYLAMINOFLUORENE OR 4-AMINO- 

RIPHENYL Frederick A . Beland, Nancy F . Fuperton, M . Matilde Marques. William B . 

Melchior, Jr., Beverly A . Smith and eMiriam C . Poirier National Center for 

Toxicological Research, Jefferson, Arkansas 72079 and •National Cancer Institute, 

Bethesda, Maryland 20892 

Continuous administration of 2-acetylaminofluorene 12-AAFI or 4-aminobiphenyl (4• 

ABP/ to female BALB/c mice results in the formation of liver and bladder tumors . We 

have compared the relationship between the concentration of DNA adducts in these target 

tissues following one month of treatment and the reported tumor incidences after two 

years of dosing . One adduct, N-ldeoxyguanosin-8 y1F2•aminofluorene (dO-C8-AF1, was detected 

after 2-AAF feeding, and at each of the seven doses . the adduct concentration in 

the bladder was two- to four-fold higher than in the liver . Two major adducts, one of which 

was N-ldeoxyguanoain-8 y11-4-aminobiphenyl ldO-CB-ABPI, were found in mice treated with 

4-ABP, and In contrast to 2-AAF, the adduct levels were five- to eight-fold higher In the 

liver than in the bladder. At doses producing equivalent liver tumor incidences, hepatic 

4-ABP adduct levels were higher than those induced by 2-MF, which suggests that 

2-AAF adducts are more efficient at inducing tumors than 4-ABP adducts . In order to 

investigate differential processing of these adduct. . pBR322 DNA, modified with dO-C8- 

A13P and d0-C8-AF . was transformed Into 8OS-repair induced F. coH. Although both 

adducts induced a similar pattern of transversion and transition mutations, d0-C8-AF also 

caused deletions . Thus, thee turoorigenic and mutagenic potential of aromatic amine adducts 

appear to be compound specific . 

USE OF THE POLYMERASE CHAIN REACTION TO AMPLIFY A SEGMENT OF THE ~$ GENE OF 

Salmonella FOR DNA SEQUENCE ANALYSIS . Douglas A . gelll, Jaaes E . Lae2, tvnhi and David M 

. DeMarini . 1National Research Council, U .S . EPA, RTP, NC 27711 ; 2Duke 

University Medical Center, Durham, NC 27710 ; and 3U .S . EPA, RTP, NC 27711 (U .S .A .) . 

Previous DNA sequence analyses of revertants of the hisD303l gene of $ . t,yphimurium 

TA98 or TA1538 have employed either a colony hybridisation technique (Cebula et al . . 

Environ . Mol . Mutagen . 11, Suppl . 11 :21 ; 1988) or traditional cloning procedures 

(Fuscoe et al ., Mutat . Res . 201 :241 ; 1988) . pa have explored the polymerase chain 

reaction (PCR) to see if it might permit more rapid processing of revertants for DNA 

sequence analysis than do the other methods . Briefly, a revertant is streaked onto 

minimal medium supplemented with biotin . After grovth, a colony is boiled for 5 min 

in 200 yl of TE buffer and then centrifuged for 5 min . The supernatant contains the 

Salmonella genomic DNA used for the PCR . The two amplification primers (AP) flank a 

327-bp segment that contains the hi,gD3Q52 mutation approximately in the center . The 

sequence of AP1 is : 5' GTC TGA ACT ACT GGT CAT CG 3' ; AP2 is : CCC TOG TM TCG CAT 

CCA CC . The reaction is performed essentially as described for Taq polymerase in the 

Perkin Elmer Cetua GeneAmp KitTM using 10 µl of Salmonella genomic DNA/200-pl reaction 

volume . Amplification of ds-DNA is obtained using a 1 :1 ratio of the primers (50 

pmoles of each) and 30 cycles of an automatic thermocycler ; amplification of ss-DNA is 

obtained using a 1 :100 (0 .5 :50 pmoles) ratio of primers and 36 cycles . The amplified 

DNA is purified by ultrafiltration in dH20 in an Amicon CentriconTM 30 microconcentrator, 

followed by resn• .p,na3on in 9 pl of sequencing buffer . The feasibility 

of employing nested primers for sequencing the amplified DNA is being evaluated . 

(Abstract of proposed presentation that does not necessaril'y reflect U .S . EPA policy,) 

VIDEO MICROSCOPY AND DIGITAL IMAGE PROCESSING OF PRENATAL BRAIN CELLS EZPOSED 

TO LEAD NITRATE . Maxwell A . Bampong, Biology Department, Norfolk, VA 23504 . 

The effect of glutamic acid on lead-induced morphological changes io cultured 

brain cells derived from prenatal mouse, was examined . Fluorescence microscopy, 

video-enhanced/intensified microscopy and digital image processing were used to 

monitor dose-dependent, qualitative and quantitative changes in cells exposed to 

lead nitrate and incubated in the presence or absence of 102 glutamic acid . Leadtreated 

cells without concurrent glutamic acid exposure were characterized by 

cell rounding, spreading and extensive cytoplasmic vacuolation . Thirty ∎inute 

exposure to S ug/ml of rhodamine 123, resulted in fluoreicently stained cytoplasm 

as well as nuclei . Video-intensified microscopy confirmed lead-induced 

degenerated nuclear membrane . Other morphological changes observed to be associated 

with lead exposure included dendrite retraction, globular shaped cells 

with the attending increased shape factor (a fraction for estimating the amouni 

by which a cell varies from a circle), and increased total cell area . Theai 

stereological changes in lead-treated calls, were dose-dependent . Cells incubated 

concurrently in lead and glutamic acid medium, regardless of lead concentration, 

were mostly spindle in shape, bi- or multipolar, with very low shape factor 

values . These data from fluorescence and video-intensified microscopy suggest 

that intracellular membranes were the primary targets of lead action . Intracellular 

digestion leading to extensive vacuolation was effected by lead impact on 

lyaosome, surface membrane disruption was responsible for changes in cell shape 

and the appearance of rhodamine 123 in nuclear region resulted from lead induced 

nuclear membrane degeneration . 

44 

45 

46

47 

TUMORIGENESIS PROFILES IN THE NCI/NTP DATA BASE 

R . Benigni 

Istituto Superiore di Sanita'-Lab . Tossicologia Comparata ed Ecotossicologia 

Rome - Italy 

The tumor profiles of 139 chemicals,resulted to induce cancer in rodents in the NCI/NTP 

experimentation, were analysed by multivariate statistical methods . The study pointed 

to four main aggregations of the chemicals . Benzene had a profile non classifiable in 

any of the four classes . From the point of view of risk assessment, two classes of car- 

cinogens ( composed by only 7 and 9 chemicals with peculiar profiles ) were associated 

with Ames test mutagenicity . No apparent association with Ames test result was observed 

for the two other classes, that included the large majority of chemicals . Thus, the cl- 

assification of carcinogens based on the whole spectrum of hystopathological evidences 

did not parallel the repartition between Ames positive and Ames negative chemicals . 

This suggests that the case for "primary" carcinogens ( genotoxic, and assumed to pose 

a greater risk ), and "secondary" carcinogens ( presumably producing the carcinogenic 

effects via non genotoxic mechanisms ) is rather speculative, and at present cannot be 

taken as a basis for risk management . 

48 

MUTAGENICITY STUDIES ON SIX PESTICIDES IN lIICE . 

D .K .Benova, I . Roupova, A . Yagova, A . Vuglenov, M . Bineva' . Institute of Nuclear Medicine, 

Radiobiology and Radiation Hygiene, Sofia 1756, Bulgaria . *Cell . Biol . Lab ., Gen . Biol . 

Dept ., University of Plovdiv, Plovdiv, Bulgaria . 

Six pesticides widely used in agriculture were studied for their Sp vivo mutagenic 

activities . They were : (a) insecticides - Vaztak (Pyrethroid), Dursban (Organophosphate) ; 

(b) fungicide - Rubigan (Pyrimidine) ; and (c) herbicides - Cliphozat (N-Phosopho-methylglycine), 

Stomp (Nitroaniline) and Diquat (Dipyridilium) . The production of polychromatic 

erythrocytes with micronuclei at different times after oral administration of an 1/2 LD50 

dose was examined . All pesticides except Gliphozat are mutagens with Rubigan the weakest . 

Doses of 1/4 and 1/8 of the LDsa ware found to be ineffective . 

The frequency of chromosome aberrations in mouse bone marrow cells after administration 

of Stomp or Diquat in doses of 1/2 the LDyo were also examined . A tendency of increasing 

numbers of hyperdiploid calls and acentric fragments was observed only with Stomp in the 

late sampling hours (96 and 120h) . 

The mutagenic effect of selected pestides is discussed . The data suggest that these 

chemicals are (at least Stomp and Diquat) more aneugens than clastogens . 

49 

UNSCHEDULED DNA SYNTHESIS (UDS) AND DNA ADDUCTS IN HEPATOCYTES AND GERM CELLS OF 

2-ACETYLAMINOFLUORENE (2AAF)-EXPOSED F-344 RATS . K .S . Bentley, G .T . Arce, 

K . Randerath, P .K . Working, D .A . Agostinelli, T . Smith-Oliver, and B .E . Butterworth, 

Haskell Laboratory, E .I . du Pont de Nemours 3 Co ., Newark, DE (USA), Baylor College of 

Medicine, Houston . TX (USA), and CIIT, Research Triangle Park, NC (USA) . 

In vivo/in vitro hepatocyte and spermatocyte UDS assays are used to quantitate 

chemicalTyinduced DNA repair in liver and germ cells . The ability of these assays to 

detect DNA damage was compared to quantities of DNA adducts . Male F-344 rats were 

exposed by gavage to 0, 0 .5, 5, 50, or 250 mg/kg 2AAF . Twelve hours later, primary 

hepatocyte and speSmatocyte cultures were prepared and the cells were incubated in 

medium containing H-thymidine . UDS was measured by autoradiography as net nuclear 

grains (NG) . DNA was isolat~ from the remaining cell suspensions and 2AAF-DNA 

adducts were evaluated by the P-postlabeling method . A dose-related increase in NG 

was observed in hepatocytes ; but a positive UDS response was not detected until a dose 

of 5 mg/kg was reached . Both N-(deoxyguanosin-B yl)-2-aminofluorene (dG-C8-AF) and 

N-(deoxyguan%sin-8-yl)~AAF (dG-C8-AAF) were detected at doses as low as 5 mg/kg 

(approx . 10 and 10' adducts/nucl4otide, respectively) . Only dG-C8-AF could be 

quantitated at 0 .5 mg/kg (approx . 10- adducts/nucleotide) . A correlation between DNA 

adducts and the UDS response was observed . In spermatocytes, UDS was detected only at 

250 mg/kg . Unlike the liver, only dG-C8-AF was detected in the germ cells . It was 

present at doses as low as 5 mg/kg (


Notes URINARY BIOMARKERS 0F ENDOGENOUS DNA DAMAC+E 


D .S . Bergtold and M .G . Simic 

National Institute of Standards and Te chnoloNy, Gaithersburg, MD 20899 

The biological consequences of damage to DNA by exogenous agents have been focal points 

of enormous numbers of studies. Damage to DNA by andoRenous processes, in contrast, 

has been investigated to a much smaller extent due to intrinsic difficulties, e .q ., 

lack of controls, low levels of damage, lack of suitable biomarkera, etc. Using gas 

chromatography/mass spectroscopy, we have developed absolute measurements of oxidative 

urinary biomarkers based on the original work of Ames and coworkers. The urinary 

levels of products (thymine glycol, 8-hydroxyguanine, their nucleoside moieties, and 

others) have been correlated in different species to their metabolic rates, longevity, 

and size . Since many of the observed urinary biomarkers are identical to •OH radical 

reaction products with DNA bases, gamma irradiation has been used to elucidate the 

underlying mechanisms by which metabolic biomsrkers eriae . 

L .F .Bernini, A .T .Nstarajan . A .H .M .Schrauder-Rotteveel, P .C .Giordano, J .S .Ploem 

and A .Tates . 

Depart . of Human Genatics . Dept . of Radiation Genetics and Chemical Mutagenesis, 

Dept . of Histochemistry and Cytochemistry, University of Leiden, Sylvius 

Laboratoria, Wassenaarsavag 72, Leiden, The Netherlands . 

ASSAY FOR SOMATIC MUTATIONS OF HUMAN HB}IOGLOBINS . 

With the purpose of monitoring by cytoimmunocheaical methods somatic mutations 

of globin genes, we have raised in rabbits monospecific polyclonal antisera 

against a number of abnormal hemoglobins . Rare mutant calls are identified on 

peripheral blood smears by indirect issmmofluorescence and counted by an 

automatic scanning microscope especially constructed for the examination of Hb 

abeorbance and FITC fluorescence . With the aid of a suitable adapter, glass 

slides of 8x8 cm . containing about fifty million red cell acan be screened in 

three hours . All fluorescent objects found on the smear undergo a computer 

directed pattern recognition analysis . Only those objects having the size and the 

shape of a red cell and absorbing at 413 nm are eventually classified as mutants 

and checked through direct examination by an expariented observer . On the 

average, five Hb S cells/10 • erythrocytes have been found in the peripheral 

blood of healthy adult individuals . When, in the sam. person, three different 

mutations are monitored simultaneously with a mixture of the relative antisera, a 

correspondent increase of the frequency of mutant cells is observed . We report 

and comment the results obtained with this assay in people exposed to genotoxic 

agents or carriers of inherited abnormalities of DNA repair . 

READTHROUGH OF CHEMICALLY INDUCED DAMAGES IN DNA DURING KLENOW FRAGMENT-MEDIATED 

DNA SYNTHESIS . 

Tadayoshi Beeshol, Kazuo Negishi2 and Hikoya Hayatsul 

1Faculty of Pharmaceutical Sciences, 2Gene Research Center, Okayama University, 

Tsushima, Okayama 700, Japan 

In E . coli, DNA damages inducible by UV, X-ray irradiation and many chemical 

carcinogens block DNA replication and exert "SOS response" . A characteristic event 

accompanying this phenomenon is an enhanced mutation . A possible way to elevate the 

mutation rate is "translesion" DNA synthesis ; but its mechanism remains unclear . We 

have shown the direct role of RecA protein in such a translesion DNA synthesis, with 

N4-amino-5,6-dihydrocytosine-6-sulfonate residues as the DNA base damage . This 

modification can be formed on single stranded M13mp2 DNA by treatment with a bisulfitehydrazine 

mixture . From the analysis of in vitro DNA chain elongation products using 

sequencing gels, we were able to show that this damage can block DNA chain elongation 

at one nucleotide before the damage . In the presanse of RecA protein, Rlenow fragment 

was able to readthrough this damage ; but in the presense of dGMP, an inhibitor for 

exonuclease activity, the DNA chain elongation remained to be blocked . A high rate of 

dCMP/dCTP turnover during the translesion DNA synthesis was observed, as detected by 

chromatographic analysis of the nucleotides in the reaction mixture . Therefore, 

enhanced exonuclease activity and the binding of RecA protein to damaged DNA may affect 

the processivity of the polymerase and this effect may be important in the translesion 

DNA synthesis . 

50 

51 

52 

tn 

m 

CO 

Oh 

t0 

W 

tn W 

N


ESTIM4TION OF CYTOGENETIC Di4M4GE DUE TO ENVIRONMENTAL MUTAGENS BY Notes 

STUDYING CHROMOSOMAL ABERRATIONS AND MICRONUCLEI 

Bharati Bhatt and Sreedevi Balakrishnan, Molecular Biology and Agriculture Division, 

Bhabha Atonic Research Centre, Banbay 400085, India . 

Using lymphocyte culture method, da~e response curve was established in vitro for 

dicentrics by expoaing whole blood to Co gamma ray doses ranging fro .m 0L_'f-Gy to 

2.00 Gy, in our laboratory . It has enabled us to estinate equivalent whole body exposure 

dose in a number of cases of suspected over-exposure to radiation during the last 13 years, 

most of which were either trivial or negative . Recently sinilar dose response curve Is 

also established for m icronuclei in bi-nucleated cells, obtained by blocking cytokinesis 

u in cytochalasin B. This is a quick m ethod and can be of great asset in case of a m ishap 

w~e~ large nunber of cases have to be studied. Besides being quick and cheap It does 

not require an expert scorer to score m icronuclei, which are easy to identify . These two 

methods are evaluated in in vivo studies of exposures to other envirormental mutagens . 

54 

MODULATION OF MUTAGENICITY BY PLANT FLAVONOIDS 

R .R . Bhattacharya, Biochemistry Division, Bhabha Atomic Research Centre, Bombay 

400 085, INDIA 

The flavonoids are interesting natural compounds which are regularly consumed 

by humans through diet containing fruits and vegetables, and several of them 

possess anticarcinogenic property . Using structurally different flavonoids it 

was observed that polyhydroxylated flavonols were highly efficient in modulating 

mutagenicity of carcinogens, expressed in Salmonella tester strains . These 

compounds were, more effective towards aflatoxin B1~ (AFB ), a carcinogen requiring 

metabolic activation for its action, than towaYAs N-1ethyl-N'-nitro-N-nitrosoguanidine 

(MNNG), a direct acting carcinogen . Methylation of hydroxyl groups 

reduced the activity, although glycosylation did not . Isoflavone group was also 

effective for both carcinogens . Saturation introduced into the ry-pyrone ring of 

flavone nucleus (flavanone) rendered the flavonoid inactive for modulating AFB1 

mutagenicity, but increased the antimutagenic potency for MNNG . Dose-response 

data, calculated on molar basis, revealed exceptional activity for kaempferol and 

rutin towards AFBt , and for naringin towards MNNG . These flavonoids acted either 

by interfering with the enzymatic activation leading to DNA adduct formation, as 

in the case of AFBl, or by preventing passage of the carcinogen into bacterial 

cell and/or by altering some cellular process, as in the case of MNNG . 

55 

CARCINOGEN-INDUCED HOMOLOGOUS RECOMBINATION BETWEEN DUPLICATED GENES STABLY 

INTEGRATED WITHIN THE GENOME OF MAFPVILIAN CELLS, INCLUDING NORMALLY-REPAIRING AND 

REPAIR-DEFICIENT HUMAN CELLS . N .P . Bhattacharyya, T . Tsujimura, Y . Wang, J .J . 

McCormick and V .M . Maher, Carcinogenesis Laboratory, Michigan State UniversTfy, 

asnsing, MI 48824 (USA) 

We have been studying carcinogen-induced homologous recombination in a tk- mouse 

L cell line which contains a single integrated plasmid which duplicat-3 copies 

of the Herpes tk gene, each containing an 8bp Xhol site inserted in a different 

place and with tTie neo gene located in the interven~ng sequence . Only by undergoing 

a productive recomFFnational event between the two non-functional tk genes can 

a functional gene product be made and the recombinant be selected witT-HAT medium . 

With this system, we determined that the frequency of recombination induced by 

UV radiation or a series of mutagens that form multi-ringed DNA adducts is linearly 

related to the number of DNA adducts or photoproducts . The majority of the events 

are consistent with non-reciprocal transfer of genetic material, i .e ., gene 

conversion . To examine the mechanisms involved and the role of DNA repair, we 

have transfected human cells which have normal rates of 06-alkylguanine DNA repair 

and nucleotide excision repair or which are deficient in one or both processes 

with the plasmid carrying the Htk genes or a related plasmid carrying duplicated 

copies of the gene for hygromycin resistance . Using these 2 systems, we have found 

that there is no difference in the rate of spontaneous recombination among the 

human cell lines, but nucleotide repair=proficient cells have a lower frequency 

of recombination induced by UV and adducts than repair-deficient cells . (Supported 

by Grants from the NCI and by Contract 87-2 from the HEI .) 


21


Notes 


56 

STUDIES ON MUTAGENIC AND ANTIMUTAGENIC EFFECTS OF COBALT (1l) CHLORIDE 

IN SWISS MICE 

H .N . Bhilwade, R.C. Chaubey and P.S. Chauhan, Molecular Biology and Agriculture Division, 

Bhabha Atonic Research Centre, Bo-nbay 400085 . 

Cobalt (1l) chloride has been reported to inhibit the m utagenic effects of ionizing radiation 

aaj chan ical m utagens in prokeryotic systen s . Lack of a s'm ilar study in m amm als, which 

are more relevant for investigations on genotoxic prevention in man, prvnpted us to examine 

the ability of cobalt (11) chloride to modify the clastogenicityof a chemical mutagen methyl 

m ethanesulfonate (MMS) and ionizing radiation (gamm a rays) using bone m arrow m icronucleated 

cells in mice. Cobalt (Im) chloride at a single dose of 15.0 mg/kg provoked a mild increase 

in the frequency of micronucleated polychro~natic erythrocytes (MN-PCEs ` over controls 

which increased further at 30 and 60 mg/kg dose (p< 0.05). There was, however, $ lack 

of dose-response relationship . Pretreahn ent of m ice with a single dose of 45 m g/kg of 

cobalt (ll) chloride reduced the frequency of MMS induced MN-PCts, significantly . Exposure 

of mice to 15.0, 30.0, 45.0 and 60.0 mg/kg of cobalt (lI) chloride showed a dose dependent 

decrease in the levels of MN-PCEs in cvnparison with MMS alone. A moderate but significant 

(p e 0 .05) reduction in the frequency of MN-PCEs in gamm a Irradiated (1 .0 Gy) m ice was 

also observed at 30 .0 mg/kg of cobalt (1I) chloride. The multiple dosage reginen of cobalt 

(il) chloride were not of much consequence . 

NUTAGENICITY OF ORGANIDINo AND ITS MAJOR COMPONENT, 3-IODO-1,2-PROPANEDIOL 

J .B .Bisho ,K.L .Mitt,E .Zeiyer,J .Mason,N .D .Shelby, and J .E .French, NIEHS,RTP,N .C . 

ryan inm [5634-39-9], is an expectorant found in OTC cough preparations . In 

NTP 2-year rodent studies, it induced leukemias and thyroid tumors in male rats, 

and pituitary and harderian gland tumors in female mice . Organidin is mutagenic in 

bacterial and mamnalian cells . In contrast to the patented formulation, Jameson, et 

al .(1988) reported that Organidin consists of 33% 3-iodo-1,2-propanediol (IPO), 17% 

glycerol, 40% polymers of glycerol, iodo-plycerol and numerous 

other components, of which -10% were isomeric p-dioxanes . Jones (1975) purported 

metabolism of IPD to the mutagenic epoxide, glycidol ; therefore, we have examined 

the in vitro and in vivo genetic toxicity of Organidin, IPD and glycidol to better 

understand the bases of Organidin's mutagenic and carcinogenic activity . None of 

the 3 chemicals required exogenous metabolic activation in the bacterial strains or 

mammalian cells in which they were active . Organidin was muta9enic in base substitution 

strains of Salmonella (TA100 and TA1535) but not in frame shift strains 

(TA97 and TA98) ; glycidol was mutagenic in both base pair and frame shift strains 

(TA100, TA1535, TA1537, TA97 and TA98) (Cantor et al .1986) ; IPD, like Organidin, 

was mutagenic in TA100 but not TA98 . All 3 chemicals induced SLRL mutations In 

Drosophila (Glycidol>IPD>Orqanidin) . Glycidol and Organidin both induced ABS and 

SCE in CHO cells (Glyctdol>Or9anidin) . IPD is on test In CHO . Glycidol was weakly 

positive in a bone marrow micronucleus test using B6C3F1 mice, but IPD and 

Organidin were negative . The relevance of these responses to understanding the 

mutagenic and carcinogenic effects of Organidin will be discussed . 

NONACTIVATED MVfAGENICITY OF CHEMICALLY NITRATED OILS CORRELATES VITH S-9-DEfENDEPr[' 

ACTIVITY OF THE UNMODIFIED OIL IN THE AMES ASSAY . G .R . Blackburn, R .A . Deitch , S .E . 

IRVIN , C .A . Schreiner, and C .R . Mackerer , Mobil Environmental & Health Science 

Laboratory, P .O . Box 1029, Princeton, NJ 08540 . 

Previous studies In this laboratory have shown that modifications to the sampledelivery 

and activation procedures in the Ames Assay lead to a substantial increase 

in the correlation between mutagenic and dermal carcinogenic potency of petroleumderived 

materials . An adjunct method, providing equal predictability, but requiring 

no S-9 preparation, and hence no sacrifice of animals, relies upon preliminary 

chemical nitration of oil samples, followed by assay in atrain TA98 . One hundred ul 

aliquots of oil are nitrated in 70 X(v/v) nitric acid at 80°C, for 90 ∎in . The 

nitrated derivatives are extracted into dichloromethane, concentrated, and resolubilized 

In DMSO for testing . Doses equivalent to 2, 1, 0 .5, 0 .1, and 0 .05 ug 

oil/plate generate dose-response curves with slopes ranging from 0 to 500 revertants/ug 

. The correlation between slopes from the nitration method and the S-9 

dependent assay for 30 oils is 0 .85 (r), and between the former and the relative 

carcinogenic potency (reciprocal of the mean latent period to tumor formation) is 

0 .92 (27 oils) . Neither method provides reliable predictability for oils boiling 

under 500°F, apparently because many of these materials are not genotoxici the nitration 

method is further limited at the high end of the distillation range for 

petroleum, i .e . fractions boiling above about 900•F . The simplicity of the method 

makes possible the evaluation of as many as 20 oils in one assay, at very low rnet 

per sample . 

57 

58

59 

MODIFICATION OF 7,8-BENZOFLAVONE METABOLISM IN HAMSTER HEPATIC BUT NOT RENAL MICROSOMES 

BY LIVER TUMOR INDUCING AGENTS . G . Blaich, A .M . Tritscher, and M . Metsler . Institute of 

Toxicology, University of Wurzburg, Versbacher Str . 9, D-8700 Wurzburg, West Germany . 

Male Syrian golden hamsters chronically exposed to certain synthetic estrogens, e .g . 

diethylstilbestrol (DES) or 17u-ethinylestradiol (EE2), and fed a diet containing 7,8benzoflavone 

(BF) develop a high incidence (80-100%) of tumors in the liver but not in 

the kidney after 6-8 months . No hepatic tumors have been observed in animals treated 

with estrogen or BF alone . In order to clarify the role of BF metabolism in the mechanism 

of this hepatotumorigenesis, the effects of pretreatment of male Syrian golden hamsters 

with BF, with DES, with EE2, with BF plus DES, and with SF plus EE2 on the metabolism 

of 14C-BF were studied in hepatic and renal microsomes in vitro . Whereas hepatic 

microsomes from animals treated with DES or EE2 produced the same pattern of BF metabolites 

as control microsomes, a marked quantitative and qualitative alteration was observed 

after pretreatment with BF, with BF plus DES, and with BF plus EE2 : the metabolic 

rate was increased and several new metabolites were formed which were not observed with 

control hepatic microsomes . These metabolites, which were tentatively identified as BF 

dihydrodiol and di- and tri-hydroxy-BFa, were not formed in control or pretreated renal 

microsomes . Non-extractable binding of radioactivity to hepatic microsomal protein was 

observed but did not exhibit as pronounced a dependence on pretreatment as did the pattern 

of BF metabolites . It is concluded that BF induces its own oxidative metabolism to 

reactive intermediates in the hamster liver which may play an important role in hepatic 

tumor formation following prolonged treatment with BF plus DES or EE2 . It is proposed 

that BF acts as initiator and the estrogen as promotor in this animal tumor model . 

Support of this study by the Deutsche Forschungsgemeinschaft is acknowledged . 

60 

SALMONELLA MUTAGENICITY AND RODENT CARCINOGENICITY : QSAR 

B .W . Blake, K . Enslein, V .K . Combar, H .H . Borgstedt 

Health Designs, Inc ., 183 East Main Street, Rochester, NY 14604 

Discriminant analysis (DA) equations have been developed to achieve (1) a division of 

the carcinogens into genotoxic (Ames positive) and non-genotoxir groups, and (2) a 

division of the non-genotoxic compounds into carcinogenic and non-carcinogenic ones, 

solely on the basis of quantitative structure-activity relationships (QSAR) . An 

equation comprising eight sigma charge descriptors, two molecular connectivity indices, 

two kappa shape descriptors, and one substructure descriptor achieved discrimination 

between 56 genotoxic and 43 non-genotoxic carcinogens with an accuracy of 96 .7% . 

Another equation comprising eight sigma charge descriptors, three MCI's, one kappa 

shape desciptor, and twelve substructural descriptors achieved discrimination between 

39 non-genotoxic carcinogens and 48 non-genotoxic non-carcinogens with an accuracy of 

96 .4% . The QSAR models are suitable for the classification of genotoxic and nongenotoxic 

carcinogens in the absence of experimental data . 

61 

OSAR PREDICTION Of SALMONELLA MUTAGENICITY ASSAY RESULTS 

B .W . Blake, K . Enslein, V .K . Combar, H .H . Borgstedt 

Health Designs, Inc ., 183 East Main Street., Rochester, NY 14604 

An equation which discriminates Salmonella mutagens from non-mutagens based on 

quantitative structure-activity relationships (QSAR), including sigma charges, threedimensional 

shape descriptors, kappa shape indices, molecular connectivity indices, and 

substructural fragments was developed from : 

1 . The results of 795 Salmonella mutagenicity assays performed up to 1979, previously 

modeled with an accuracy of 94 .7% . 

2 . The results of 383 Salmonella/microsome mutagenicity assays recently reviewed by a 

Gene-Tox panel . 

3 . The results of Salmonella mutagenicity assays listed by Ashby and Tennant (1988), 

Mutation Research 204, 17-115 . 

The resulting equation permits predicition of Salmonella mutagenicity on unassayed 

compounds based on structure . 



Notes

24 1989 EMS Abstracts 6 2 


Notes THE AUGER ELEGTRON DOSIMETRY OF INDIUM IN THE NUCLEUS OF V79 CELLS . D.H . 

Blakey', J.R. McLean', G .R. Douglas', D . Wilkinson', and J.M . Bayley', 'Mutagenesis Section, Bureau of 

Chemical Hazards, and 'RadioPharmaceuticals Section, Bureau of Medical Devices, Department of National 

Health and Welfare, Ottawa, Canada, K1A 0I2. 

Many radiopharmaceuticals or their metabolites distribute to intracellular sites where the release of Auger 

electrons can damage radiosensitive structures such as DNA . The effect of intracellular "'udium eadne on 

chromosomal aberrations and cell killing was defined in terms of equivalent "Cobalt doses . About 30% of 

cell-associated "'In oxine was found in the nucleus and 7 .5% in the DNA. Based on the assumption that 

these effects result only from radioactivity in the cell nucleus, the radiation dose to a cell was estimated by 

chromosomal aberration analysis to be 9 .6 x 10' Gy/decay and, by the colony assay for cell survival to be 

10.1 x 10' Gy/decay. The dose based on chromosomal aberrations is probably underestimated, since data 

from cells with pulverized chromosomes were excluded from the calculations . Furthermore, the cellular 

radiation dose could be as high as 13 .0 x 10' Gy/decay if only the DNA-associated radioactivity was presumed 

to contribute to the biological endpoint . Using the method deaeribedby NCRP (Public . 63), the average 

radiation dose to the cell nucleus from introcxllular "'In was estimated to be about 3.3 x 10' Gy/decay. 

Accordingly, based on the biological dose estimates in this study, the relative biological effectiveness (RBE) 

for intracellular "'In was estimated to be as high as 3.1 (i.e. 10.1/3.3). Moreover, if the DNA-associated 

radioactivity is assumed to be the sole contributor to cellular damage, the RBE could be even higher . 

Virtually all of the energy absorbed in the nucleus would be from the nuclear-associated Auger electron, since 

thepe netrating emissions (x- and gamma rays, and internal conversion electrons) ~ntn'bute only 6 .1 x 1001 

Gy/decay. It would, therefore, appear that the conventional method of organ dosimetry, which presumes a 

uniform distribution of energy throughout the target volume, is inappropriate when applied to intracellular 

Auger electron emitting compounds such as "'In . 

NUTAGENICITY AND ANTIMDTACENICITY TESTING OF SIX CHEMICALS ASSOCIATED WITH 

THE PUNGENT PROPERTIES OF SPECIFIC SPICES. E. D. Elevins and Asliyati Asian, 

School of Public 6 Allied Health, Dept. of Health Sciencea, East Tennessee State 

Univeraity, Johnson City, Tli 37614. 

Six compounds, capsaiein, thymol, borneol . and allyl were screened for 

mutagenic activity using Salmonella t~hiauriom strains TA97, TA98 and TA100, 

with and without 89 metabolic activation. A11 six oompounds are associated with 

the pungent properties of some specific spices . It was observed that capsaicin 

was mutagenic using strain TA100 in the presence of 89. Capaaiein, found in the 

spice Cansicum annum, was detected and quantified using thin layer and gas 

chrosutographic techniques . The presence of an antimutagenic factor(s) in C. 

annum that could suppress the nutagenicity of capsaicin was detected. When the 

mutagens capsaicin and 2-aminoanthracene were assayed in the presence of C . 

annum acetone extract, using strain TA100 with 89 metabolic activation, the 

nutagenic response of both the mutagens were reduced by approxisutely 50%. 

Assaying capsaicin and 2-aminoanthracene in the presence of chlorophyll, the 

mutagenic response of the two smtagene were reduced by less than 401 . From this 

observation it was inferred that chloropbyll can successfully suppress the 

mutagenicity activities of capsaicin and 2-astinoanthracene, together with other 

antimutagenic factors that were present in the acetone extract of C. annum. 

THE DETECTION OF 1WTAGENECITY OF CKMfICAL FACTCRS IN INDUSTRY 

N. P.Boabko v 

Institute of Medical (ienetios ASS USSR, Mosco w 

The paper generalizes the results of the control of mutagenio effect 

of chemioals in real industry conditions (some plants) in the USSR . To 

detect possible mutageneoity danger of industry chemicals for a man oytogenetio 

analysis was made regarding the oooupational group of workers 

being in contact with chloroprene lead,dioetbyl phtalat dimethyl aoetamide,benao(a) 

pyrene,trichlorfon,d~methyl sulplute formaidelyde,ethylene 

ozide,vinyl chloride and also rubber industry wor>


investigation of Styrene Oxide-DNA Adducts and Their Detection in Notes 

Workers Exposed to Styrene . W . J . Bodell, K . Pongracz, S . Kaur, A . L . 

Burlingame, S . F . Liu and S . M. Rappaport Brain Tumor Research Center 

and Mass Spectrometry Facility, Univ . of California, San Francisco and 

School of Public Health, Univ . of California, Berkeley 

Styrene Oxide (SO), a metabolite of the industrial chemical styrsna 

is mutagenic and carcinogenic in test systems . The identification of 

DNA adducts formed by SO may lead to an understanding of the genotoxic 

mechanisms of SO and quantitation of the SO-DNA add~~ts may provide a 

molecular dosimeter for human exposure to styrene . P-postlabeling of 

DNA reacted with SO resulted in the detection of six adducts . 

Chromatography standards were synthesized to allow assignment of the 

structures . Adducts ; and Z, are diaddu5ts resulting from the reaction 

of two molecules of SO with guanne (N ,C-8) . Adducts g and A are 

isomers of SO reacted with2the 04 position of guanine . Adduct I re~}lts 

from aralkylation at the N-site of guanine . We have extended the Ppostlabeling 

measurements to mononuclear cells isolated from the blood 

of workers exposed to styrene . Styrene exposure varied from less then 1 

ppm (low) to 20 ppm (high) . Several SO-DNA adducts were detected in the 

high exposure group . These preliminary results suggest that there may 

be a correlation between styrene exposure and levels of SO-DNA adducts 

in human mononuclear cells . (Calif . Toxic Substances FUND, RR01614 and 

P42-ES04705 

66 

ANEUPLOIDY INDUCTION BY ALKYLATED BASES AND NUCLEOSIDES IN MAMMALIAN CELLS . 

S . Bonatti, G . Cercignanni, M . De Ferrari, P .L . Ipata, M . Rocco, M .G . Tozzi, S . Viaggi, 

and A . Abbondandolo, National Institute for Research on Cancer, Genova (Italy), University 

of Genova (Italy), LMD of CNR, Pisa (Italy), and University of Pisa, (Italy) 

The mechanism by which alkylating agents induce aneuploidy is not know . We discovered 

(Bonatti et al ., 1986) that 01-ethylguanine (O6etG) is a powerful inducer of aneuploidy 

and polyploidy when given to mammalian cells as a free base . To ascertain whether the in_ 

duction of aneuploidy is a peculiarity of 06etG or is shared by other alkylated bates, 

the effect of O'meG, 7meG, 7atG, 3meA, 3meC and of unmodified bases was tested in human 

lymphocytes . It resulted that : (i) hypodiploidy was induced at similar frequencies by a .il 

tested compounds ; (ii) alkylated bases were more active than unmodified bases in inducing 

hyperdiploidy ; (iii) a high polyploidy induction was observed with 06etG . Since knwledge 

of the metabolic fate of the alkylated bases into the mammalian cell seemed essential, a 

number of enzymatic reactions were studied . We found that O4meG (i) is not degraded by 

guanase, that readily deaminates guanine to xanthine ; (ii) is not demethylated by adenosine 

deaminase that demethylates 04 methylguano$ine (O6meGuo) ; (iii)_is not converted to 

O6MeGuo by purine nuclaoside phosphorylase ; (iv) is not converted to O6meGMP by HPRT . On 

the other hand, O'meG was efficiently converted to O'meGuo by bacterial adenosine phosphorylase 

. In conclusion, the induction of aneuploidy was a general feature of alkylatad 

bases and, to some extent, of normal bases . The mechanism of induction, whether by spaci_ 

fic alkylated products, by nucleotide pool imbalance, or other, remains to be clarified . 

67 

INHIBITION OF RADIOGENIC TRANSFORMATION BY a-LAPACHONE . David A . Boothman and Arthur 

B . Pardee, Division of Cell Growth & Regulation (D-810A), DAna-Farber Cancer 

Institute, 44 Binney Street, Boston, MA 02115 . 

p-lapachone is a potent inhibitor of DNA repair in mammalian cells . It activates 

topoisomerase I . We show that p-lapachone can prevent the radiogenic transformation 

of CHEF/18A cells . Potentially lethal DNA damage repair (PLDR) occurs while cells 

are held in medium containing low serum prior to replating . PLDR processes permitted 

survival recovery, but also drastically increased the number of foci/plate (i .e ., 

transformation) of CHEF/18A cells . By blocking PLDR with P-lapachone both survival 

recovery and enhanced transformation were prevented . At equivalent survival levels, 

exposure of X-irradiated cells to Q-lapachone resulted in an 8-fold decrease in the 

number of foci/dish as compared to the number of transformants produced in 

X-irradiated cells following PLDR . 

Early PLDR-derived increases in transformation may be the result of error-prone 

genetic rearrangements dependent upon topoisomerase I, which are thereby prevented 

by p-lapachone . a-Lapachone decreased the rejoining of DNA strand breaks and also 

appeared to produce additional double strand breaks in X-irradlated calls during 

PLDR . We hypothesize that the activation of topoisomerasa I by (i-lapachone may 

convert repairable single strand DNA breaks into the more repair-resistant double 

strand breaks, thereby preventing PLDR and radiogenic transformation . These results 

suggest a novel direction for the development of new anticarcinogenic agents . 

Supported by grant CA 22427 to Arthur B . Pardee from the National Cancer Inst . 



Notes MITOD8PRL88IVE El7ECT8 OF PJ1R11QUAT Ill 71LLIU![ CEPA . 

R . A . Boroffice . Department of Biological and Chemical Sciences, Lagos 

State University, Ojo . 

Cells of Allium ceoa root meristems were exposed to different 

concentrations of paraquat (5, 10, 25, and 50mg/i) . There was a doserelated 

reduction in mitotic index which was directly associated with 

length of exposure . There was a relative reduction in mitotic index in 

treatments allowed recovery periods . 


SCREFNING FOR BASE MUPATICNS IN THE HPRT AND PAH LflCUS USING THE POLYMFRASE CHAIN 

REACfION (PCR) IN COMIDINATICN WITH DENATURING GRADIENT GEL ELDCIROPHORESIS (DGGE) . 

A .-L . BOrresen,l E . Hbvig,l B . S . Smrensen,l H . Vrieling2 and A . Brogger .l 

Inst, Cancer Research, The Norwegian Radium Hospital, Oslo, Norway 

2) Dept . of Rai . Gen . and Chen . Mutagenesis, Univ . of Leiden, The Netherlands 

We have u9ej the polymerase chain reaction (pCR) and denaturing gradient gel 

electrophoresis (DGGE) to screen for base mutations in the PAH (phenylalamin 

hydroxylase) locus and the HPRT (Hypottanthine guanine phospho-ribosyl transferase) 

locus . For the PAH locus a 245 base pair fragnent containing exon 12 with flanking 

intronic sequences was amplifie8 . In this DNA sequence a single base substitution has 

beert found in 30% of the Norwegian PKU patients . This mutation gives a different 

melting profile of the 245 bp fragments resulting in a different migration on DGGE . 

The hu3e amplificaton of the genamic DNA fragments by PCR allows detection by direct 

EtBr staining of the gel making a rapid and easy method to screen for this PKU 

mutation in newborns . 

For the HPRT locus exon 3 was amplified fran a collection of 13 W and ENU induced 

mouse and hamster mutants, all shown to contain a mutation in the 3rd exon after 

sequencing . 8 of these mutants could be detected on DGGE after PCR . A collection of 

12 unknown HPRT mutants induced by porphyrins plus W were screened for mutations in 

exon 3 using these methods . No deletions of exon 3 were fourd, and no base mutations 

in the lower melting domain of exon 3 have been found so far . The use of PCR in 

combination with DG3E is a rapid and applicable method for screening both known and 

unknown mutations, inherited or induced in the lower melting domain of a given 

DNA-sequence . 

MEJ-41 AND OTHER REPAIR GENES OF DROSOPHILA . James B . Boyd and Satnam S . Bangs. Department of 

Genetics, University of California . Davis CA 95616 

Genetic studies of repair deficient mutants in Drosophila have revealed the existence of extensive overlap in the 

functions that participate in DNA repair, recombination and synthesis . Mutants in several genes are known, for 

example, to be deficient in both DNA repair and meiotk recombinadon . A forward genetie approach is currently 

being employed in this laboratory to clone the repeir related genea nui-9 and ntef-I1 . Mutants at these loci are 

highly pleiotropic in that they influettce both meiotic reeombbttdon and a broad tpectrttm of DNA repair responses . 

Yamamoto and Mason have succeeded in mutageniztng both ioci In crosaes that mobilize transposable P elements . 

A combination of in siru hybridization and tevusiott atulysis has established that the recovered mutants were indeed 

induced by transposon insertions. Chromosome walking has been employed to recover all of the etei-41 gene . 

Transcriptionally active sequences adjacent to the P insertioa sites have been used to isolate a 2 .2 kb embryonic 

cDNA clone whose homology extends over 14 kb of the ohrumosomal walk . That cDNA represents a mei-01 

transcript because It exhibits homology to genomio sequences on both sides of the P insertion sites . Norihern blots 

prepared with poly(A)+ RNA from both embryos and adult females have Identified a single 2.2 kb transcript with 

homology to the cDNA clone . 

EVALUATION OF GENOTOXICFIY OF N-NfTROSODIBENZYlA1vIINE IN CHINESE 

HAMSTER V79 CELIS AND IN SALMONEIdA . B.G. Boyes. C.O . Rogers, N .P. Sen 

and T.I . Matula, Toxicology Research and Food Research Divisions . Food Directorate 

and 

. Health and Welfare Canada . 

Ottawa~OntariolCANADAI• sion, Drugs Directorate 

Health concerns have arisen due to the formation of N-nitrasodibenzylamine 

(NDBzA) in pork processed in a new type of rubber netting . While NDBzA was 

reported to be non-carcinogenic in an early in vivo study (Druckrey ct 2 . 

Krebsforsch .69:103, 1967), the potent carclnogenicity of related nitrasamines eg . Nnitroso-n-dlbutylamine 

and N-nitrosodiethylamtne) prompted evahxation this 

compound for genotoxicity in y= in both Chinese hamster V79 cells and In 

Salmonella . Concentrations up to 25 Ng/ml were tested in V79 cells with and 

without activation by rat or hamster hepatocytes . Under any of these conditions 

there was no significant elevation of sister-chromatid exchange levels . Mutation to 

6-thioguanine resistance was also negative, except for a weakpoaitive in only one of 

three replicate experiments, at 25 pg/ml, and only In the absence of hepatocytes 

which was considered not biologically siArti9cant . At concentrations up to 1000 

69 

70 

71

N ml in the Salmonella assay using pre-incubation protocol . NDBzA was negative 

A 98 . and in TA 100 with rat S9 . but was positive at the highest dose in TA 100 

with hamster S9 . and more strongly with Aroclor 1254-induced hamster S9 . When 

activated bY rat or hamster hepatocytes . as opposed to S9. NDBzA was negative with 

all tester strains. Thus . NDBzA appeared to be weakly mutagenic to Salm Qp!~8 but 

was apparently non-genotoxic to V79 cells with or without activation by rat or 

hamster hepatocytes. 

72 

HUMAN SPERM CHROMOSOMES : ANALYSIS OF STRUCTURAL ABERRATIONS AND DNA 

REPLICATION . B . F. Brandriff, L . A . Gordon and A . V. Carrano . Lawrence Livermore 

National Laboratory, University of Callfornia, Livermore, CA . 

Structural aberrations similar to those Identified In human sperm chromosomes by 

cytogenetic analysis following fusion of sperm with hamster eggs may contribute to some 

rare human heritable genetic diseases, as well as to unexplained Infertility and early 

embryonic losses . We analyzed structural aberrations observed in 6000 sperm 

chromosomal complements from 24 men . The most frequentiy observed aberrations 

were chromosome breaks and fragments . In lonpltudinal studies, frequencies were 

stable over many months or even years for Individual men . Egg culture conditions, 

numbers of chromosome complements processed per egg, and different sperm 

pretreatments had no effect on frequencies and types of aberrations . We examined DNA 

replication following sperm-egg fusion by detectinp Incorporated bromodeoxyuridine 

with Immunocytochemical methods . Human sperm chromatin was available as template 

soon after entry Into eggs, before full pronuclear development was achieved . This early 

availability may be an expression of a more labile packaging In the human sperm head 

compared to rodent sperm chromatin, and may suggest one mechanism leadinp to the 

higher frequencies of structural aberrations In human compared to rodent sperm 

chromosomes . Work performed by the Lawrence Livermore National Laboratory under 

the auspices of the US Department of Energy under contract 6W-7405-ENG-48 . 

73 

BASIC MECHANISMS OF ENVIRONMENTAL MUTAGENESIS ~ 

Bryn A . Bridges, MRC Cell Mutation Unit, University of Sussex, Brighton BN1 9RR, 

Gt . Britain . 

Most types of DNA damage do not inevitably result,in the induction of mutations . 

The latter usually arise during the operation of cellular processes . These cellular 

processes are most easily studied in microorganisms and have led to the formulation of 

mechanistic models for error-prone DNA repair pathways, as well as for error-free DNA 

repair and damage tolerance pathways, and for fidelity mechanisms that eliminate polymerization 

errors even after they have occurred . Many of these pathways are induced 

by DNA damage and some function poorly or not at all in the absence of a significant 

level of mutagen . Current studies are seeking to discover the extent to which mechanisms 

established in bacteria and other microorganisms also operate in human and 

other mammalian cells . Sometimes mammalian processes show a similarity which reflects 

a mechanistic similarityp sometimes they mask differences that are far from trivial . 

In bacteria many if not most induced mutations arise by a process that requires 

proteins specified by the umuD and C genes . Partially homologous genes have been 

reported in bacteriophage T4, where they code for processivity proteins interacting 

with DNA polymerase, and in the eukaryote Schizosaccharomyces pomb . It is possible 

that such proteins operate throughout the evolutionary scale, giving further justification 

for mechanistic work with microorganisms . 

74 

THE CENTROMERE AND ANEUPLOIDYi DRUG-INDUCED FRAGMENTATION AND DETACHMENT OF KINETOCHORES 

OF MAMMALIAN CHROMOSOMES . B .R . Brinkley, R .P . Ziakowski, S .L. McCune and R .D . 

Balczon . Department of Cell Biology and Anatomy . University of Alabama at Birmingham, 

UAB Station, Birmingham, AL 35294 . 

The centromere, a specialized region of inetaphase chromosomes required for normal 

partitioning of the eukaryotic genome, has been implicated in aberrant chromosome 

distribution leading to aneuploidy . We have identified a unique type of drug-induced 

chromosome damage involving the kinetochore, a component of the centromere required 

for the attachment of spindle microtubules to chromosomes . Chinese hamster ovary 

(CHO) cells synchronized at the G1/S phase of the cell cycle with 2 mM bydroxyures 

for 20 h and subsequently treated with 5 mN caffeine for 2-6 h entered mitosis without 

completing DNA synthesis (Schlegel and Pardee, Science 232i1264, 1986) . A similar 

effect was induced when either Mitomycin C (1 .0 pg/ml) or Bleomycin (5 yg/ml) was 

used instead of caffeine in the above protocol . We termed these mitotic cells with 

unreplicated genomes (MUGs) and found that pre-S phase entry into ∎itosis was 

accompanied by the detachment of kinetochores from prematurely condensed, highly 

fragmented chromosomes . Using video microscopy, fluorescent image analysis, and 



Notes 

27


Notes electron microscopy, we determined that the detached elements were actually 

aub-fragments of the unreplicated kinetochore . These fragments are short repeated 

egments of 30 nm chromatin fibers which bind MTs and undergo the complete repertoire 

of mitotic movements . Based upon these studies, a model for kinetochore structure 

will be presented which explains new aspects of centromere structure, evolution and 

involvement in aneuploidy . 


MOLECULAR ANALYSIS OF IN VIVO, SOMATIC MUTATIONS OF THE HPRT 

GENE IN MOUSE AND MAN, Eleanor C . Brinson, Karolyn Burkhart- 

Schultz, I M• l "Io, and Cheryl L . Strout, Biomedical Sciences 

Division, Lawrence'.ivermore National Laboratory, Livermore, CA 94550 (USA) 

To analyze factors that affect the spectrum of mutations in mammalian cells in 

vivo, we are studying mutations of the hypoxanthine phosphoribosyltransferase 

gene (hprt) in lymphocytes of both mouse and man . Gbmparison of mutations 

detected in these systems with mutations detected in other in vivo and in vitro 

systems will lead to better understanding of the effect that species, tissue, locus, 

selection method, and sequence have on the spectra of both spontaneous and 

mutagen induced mutations . Particularly in the mouse we are able to manipulate 

factors that may affect the mutations recovered . in the mouse model, using 

restriction fragment length analyses, we have detected a high proportion of 

deletions among spontaneous mutations of the hprt gene . In nondeleted mutants 

we have detected base substitution and frameshift mutations of the coding regions 

by sequencing exon DNA, after amplification of genomic DNA using ahe 

polymerase chain reaction (PCR). Studies of mutants isolated from irradiated mice 

are in progress . Interspecies comparisons of spontaneous mutations are being 

initiated with human lymphocytes by sequencing PCR amplified hprt cDNA. This 

work performed under the auspicies of the U.S. Department of Energy by 

Lawrence Livermore National Laboratory under contract number W-7405-ENG~48, 

in part supported by an Interagenc A reement (Y01-ES-80171) between the 

National Institute of Environmental Healt~ Sciences and DOE . 

QICTOX .`C rlM BIOCMIICAL ACPIVITIES OF SCt'lE C3BACIMED ElHANES . 

Brorizetti G ., I•Iorichetti E ., Del Carratore R., Rosellini D ., Paolini M., Cantelli Forti 

G., Grilli S . and Vellosi R . 

Istituto de A9utagenesi e Differenziamento, CtJR, Pisa 

Istituto de Farmaoologia, Istituto di Canoerologia, Universita di Bologna, ITALY 

1,1,1,2,-4+etrachloroethane (TIM), Pentachlaroethane (PCE) and Hexmchloroethane (F1CE) 

have a wide range of applications in industrial processes as solvents, degreasers ard 

aooeLerators in rubber vulcanization . 7he increasirg production of these caipoucrls in 

the past few years led to major attention in un8erstanding their effects on huren health . 

In this work, TiCT:, PCE and HCE were tested in D7 strain of yeast-Sacctwr -Cerevisiae 

in suspension test with and withcut a mentnalian activation system S . TDCE, P(E~r Hf~ 

gave positive results on cells harvested fraa logarithmic


aberrations/cell, respectively . Alpha-particles and X-rays given alone resulted in a Notes 

linear increase in micronuclei/binucleated cell with respective slopes of 0 .77 1 0 .08 

and 0 .20 t 0 .05 micronuclei/binucleated cell/Gy . When 1 .0 Gy of a-dose was given 

simultaneously with 0 .0, 0 .75, 1 .5, or 3 .0 Gy of X-rays, the slope of the combined 

exposure dose-response curve for the X-ray induced nicronuclei had a slope similar to 

that observed after exposure to a-particles alone (0 .74 2 0 .05 micronuclei/binucleated 

cell/Gy) . These data sets both suggest a synergistic interaction between a- and Xray-induced 

damage . Regardless of exposure sequence, when alpha and X-ray exposures 

were separated in time by 1/2, 2, or 6 hrs, synergistic interactions between the 

damage induced by both exposures was no longer evident . These data demonstrate that 

X-rays and a-particles can interact and that repair of the chromosome damage involved 

in the interaction is very rapid . (Research sponsored by the U .S . Department of 

Energy's Office of Health and Environmental Research under Contract DE-AC04-76EV01013 .) 

78 

ORGAN-SPECIFIC GENOTOXIC EFFECTS OF CHEMICALS : THE USE OF ALKALINE ELUTION TO 

DETECT DNA DAMAGE IN VARIOUS ORGANS OF IN VIVO-EXPOSED ANIMALS 

G . Brunborg, ] .A . Holme, EJ Sederlund and E . Dybing, Department of Toxicology, National Institute of 

Public Health, Oslo, Norway 

The existence of genotoxic chemicals with marked organ specificity indicates that chemicals should be 

tested in more than one organ or cell type . We have developed techniques for detecting aad comparing 

DNA damage in different organs of experimental animals after in vivo administration of chemicals . 

Exposed anim als are anesthetized, organs are re moved, cooled rapidly in cold buffer, and processed as 

follows: The liver, kidney, testis, lung or spleen is minced with scissors aad forced through a stainless 

steel screen + one layer of cotton gauze . The stomach, small or large intestines or the urine bladder is 

rinsed, opened, the epithelial cells scraped off with a glass slide, and homogenized in a glass homogenizer . 

The brain is homogenized directly . The femur is opened, and bone marrow cells are rinsed out and 

homogenized . All samples are then centrifuged and counted . The procedure takes about 1 hour for 16 

samples. Two million cells or nuclei are loaded on an automated alkaline elution system (Brunborg et al ., 

Anal . Biochem . 1Z, 522-536, 1988) . 

Experiments with 1,2-dibromo-3-chloropropane (DBCP) (10 mg/kg i .p ., 1 br) demonstrate highly 

variable effects in the various organs : Maximum DNA damage was observed in the liver, kidney and 

duodenum, with intermediate damage in the lung, spleen, brain, testis and stomach . Higher doses (40 

mg/kg) were required to produce significant damage in bone marrow and colon . DBCP has been shown 

to induce tumors in rat liver, kidney and stomach . 

T he data indicate that an evaluation of the genotoxic properties of a chemical by means of short-term 

tests should involve several organs . We are currently utilizing the methods described to study the 

genotoxicity of various dietary mutagens . 

79 

THE EFFECTS OP MBTBAPYRILBNB oN IN VIVO SCE AND IN VITRO CBROMOSOME ABERRATION 

INDUCTION. J . Brunny, D . Kindig, an-d f.Garriott, LThy tesaarch Laboratories, 8li 

Lilly and Company, Greenfield, IN 46140 

The antihistamine methapyrilene hydrochloride (MP) has been shovn to be a rat 

liver carcinogen ; however, ∎ixed positive and negative results exist in short term 

genetic toxicology assays . To date, the only in vivo data available on MP are 

negative results from two unscheduled DNA synthe®Ts studies using rat (Mirsalis et 

al ., Environ . Mutagen . 7, Suppl . 3 :73, 1985 ; Steinmetz et al ., Carcinogenesis 9t95§, 

1988) and souse (Steinmetz et al ., op . cit .) hepatocytes and from an SCE assay in 

Fischer 344 rats (Iype et al ., Cancer Res . 42t4614, 1982) . Results are presented 

here from an in vivo SCS assay in male CD-1 Uce . Intravenous doses of 2 .5, 5, 10, 

and 20 mg/kg oT IffP vere administered and bone marrow harvested 21 hr later . The 

incidence of SCE was recorded and ranged from 3 .2 to 4 .3 SCE/metaphase, which was not 

significantly different from solvent controls . Because of positive findings in the 

L5178Y mouse lymphoma assay in which an increase in small colony mutants was observed 

and chromosome damage confirmed (Blazak et al ., Environ . Mutagen .• Bt229, 1986), MP 

vas also tested for its ability to induce cTiromosome aberrations in v1tro in cultured 

Chinese hamster ovary cells . Doses of 375, 450, and 550 yg/al of MP were tested both 

vith and without an S-9 activation system . The percent aberrations ranged from 0 to 

2 percent in the activated assay and from 0 to 3 percent in the nonactivated assay . 

These results were not significantly different from solvent controls . The negative 

results from these two assays lend further support to the theory that NP is carcinogenic 

through a nongenotoxic mechanism . 

80 

FffiFRE HAVE WE BFEN? 74fE IAST 20 YFJtRS OF FNVtRMlOWPAL 1'1t1DWMMIS 

~ 

Dnvid Brvsiak. Hazleton Laboratories, U .S .A . 

N 

It is wr#Miile to assess the peaks an! valleys of the road travelled by 

tp 

the discipline of envirormental autagenesis daa'ing the past 20 years . /' 


tr 

m 

00 

Q1 

29


Notes WitTbut question, this discipline has ocntributed significanGly to the 

protection of the genetic integrity of fuRazti+e ganesaticre of humans . In 

additicn, basic researth infcrmatim devuloped by members of this field 

during the past two decedes has lead to a clearer mechanistic 

tadezstarxiirg of the prrooesees of cancer, cellular diffezantiation and 

aging . mnowledge bases within the applied scienoes of inedicine and 

taxicology would be poorer today if it w+ .re not for research into mutation, 

aa repair, ssrleic acid biodwemistsy and da+omosmis funct.ions carried azt 

under the domain of genetic toocicity . 


qhile valleys have been enoaa*ared along the road travelled since 1969, 

their existence has generally resulted in renewed snthusiasm and scientific 

vigor . Any scientific discipline which tails to subject itself to critical 

assesseent for fear that it will reveal possible flaws or lead to &Ange, 

is docmed to be replaced. Ths field of erwiratnental mltagenesis is 

forWnate to hn+e had a lartre manber of creative and dedicated visionaries 

leading it Liuu4i i .i .ia uailwylny aua7 ew.iuig jwciery . 

A COMPUTER-ASSISTED PROCEDURE FOR THE ASSEMBLY AND ANALYSIS OF SHORT-TERM 

GENOTOXICITY TEST DATA 

D .J . Brusick, P . Lohman, M . Mendelsohn, M . Waters, S . Nesnow, D . Moore, F . de Serres, 

J . Ashby, B . Matte- r . T: Matsushima, ICPENC, Subcommittee 1 . 

Determining the genetic hazard of a chemical is generally approached by using an 

assortment of tests for measuring the DNA reactivity of a chemical or its resultant 

genotoxicity . Over 100 short-term tests employing a wide diversity of species and 

genetic mechanisms have been used to measure genetic hazard . To date, attempts to 

achieve a standard test battery for defining genetic hazard have not been successful . 

Consequently, testing for genetic hazard involves the use of test batteries with 

variable types and numbers of assays . This increases the difficulties of interpreting 

data sets since the data sets are often filled with inconsistent responses from 

diverse types of assays . 

Several years ago, the International Commission for Protection Against Environmental 

Mutagens and Carcinogens (ICPEMC) established a Committee to establish a method to 

compile and interpret diverse short-term test data . The Committee has produced a 

quantitative weight-of-evidence approach that combines test data using certain 

parameters such as dose, replication and metabolic capacity into a series of scores 

for test type, test class test family and an overall score that defines the total 

weight-of-evidence regard ;ng the genetic hazard of the agent . • 

SISTER CHROMATID EXCHANGE AND MICRONUCLEUS ANALYSIS IN RAT PERIPHERAL 

BLOOD LYMPHOCYTES AFTIR IN VIVO EIPOSURE TO BENZO(A)gYREVE . M .F . 

Bryanta, G .L. Eregson , P . Kwanyuen , and A .D. Kligerman , EHRT,Inc, 

RTP, NC 27709, and U .S . EPA, RTP, NC 27711 (USA) . 

In an effort to determine the persistence of sister chromatid 

exchange (SCE) and micronucleus (MN) induction following exposure to a 

polycyclic aromatic hydrocarbon known to exist at hazardous waste 

cleanup sites, experiments were conducted in peripheral blood 

lymphocytes (PBLs) of male Sprague-Dawley rats injected ip . with 

0, 100, or 250 mg benzo(a)pyrene/kg . Peripheral blood was removed by 

cardiac puncture from each rat at 1, 2, 5, 7, 14, and 21 days after 

injection. Isolated PBLs from three animals/dose/harvest time were 

cultured, according to previously published methods, for analysis of 

SCEs in second-division cells and MN in cytochalasin B-blocked 

binucleated lymphocytes. Both the SCE and MN frequencies remained 

elevated for 21 days post-injection . These results suggest that rat 

PBLs containing SCE and MN inducing lesions remain viable for at least 

three weeks . Due to variable SCE frequencies in isolated PBLs among 

replicate animals, a 56 day study was undertaken in which whole blood 

was used for SCE analysis . Results will be presented comparing SCE 

responses in whole blood and isolated PBLs . 

(This abstract does not necessarily reflect US EPA policy .) 

50869 3542 

81 

82

83 

FREQUENCIES OF CHROMOSSOMAL ABERRATIONS AND MICRONUCLEUS IN RODENTS COLECTED IN COAL- 

FIELD AND TOBACCO CULTURE REGION - CRICIOMA - SC - BRAZIL .Angela M . S . Bueno, Jeanete 

M . S . Agostini, Karina Gaidzinsk, Riroko Nitta, Josane Moreira, Ivan Brognoli - 

Departamento de Biologia - UFSC - Florianopolis - SC - Brazil - 88049 . 

Criciuma - SC - is a town situated in the coal-field of South Brazil . The present work 

has been done in the Basin of Sangao River, one of the local rivers that receiv the 

rejectes of the coal washing, like mercury, cadmium, lead, etc . in wich bank exist many 

proprieties of tobacco cultures where various organophosphorate and carbamate agrotoxics 

are used . Our purpose in this area is to try to detect and estimate the possible action 

of the environmental pollution in the organisms directly exposed to it - in this case 

rodents collected in the local - through the study of the frequency of chromossomal 

aberrations and micronucleus in bone marrow . Parallely the same methodology has been 

used for rodents collected in regions free from these polluter factors to set up a 

control group . With this finality we delimited two collect points in the Basin of Sangao 

River (A and B), one point out of this area (C) and some points in Florian6polis 040Km 

from Criciuma) free from the action of the above mentionate polluters . The points C and 

D are considered control points . Our parcial results have showed the following 

percentage for chromossomal aberration : A-3,62x, .B-1,30x, C-none, and D-1,46x, and the 

following results for micronucleus analisis : 20% . for Criciuma's points and 10x .for the 

control group .For the meantime we can say that these results suggest that this 

methodology can be applied to detect the action of the environmental pollution in the 

organisms . (FINEP) . 

84 

STUDIES ON A GENOTO%ICITY TEST USING P . PHOSPHOREUM 

Anthony A . Bulich, Mary Grace Schreibner, Ser-Eon , Charles C . Walbourn 

Microbics Corporation, 2232 Rutherford Roa , ars a, CA 92008 

Chemicals are essential for modern society, and proper application of them has 

greatly improved general living conditions . However, the chemical wastes, and the 

by-products of these wastes, are a major cause of environmental pollution . In order 

to monitor and control these pollutants, simple and fast tests are needed . Following 

the introduction of the Microtox (R) Acute Toxicity Test, we now propose a test for 

genotoxic compounds (Mutatox (TM)) . The test uses a dark mutant of luminous bacteria 

and determines the ability of the tested agent or sample to restore the luminescent 

state . Genotoxic agents which are mutagens, DNA damaging agents, DNA synthesis 

inhibitors, and DNA intercalating agents are detected with this test . This one-step 

test can be performed as an overnight assay, under non-sterile conditions . Fifty 

pure chemicals have been tested with the proposed method, and the results correlate 

vell with published data obtained with the Ames test . The predictive value of this 

method for carcinogenicity is also comparable to the Salmonella assay . Validation of 

the test with an additional 50 extensively studied chemicals from the National 

Toxicology Program is underway . The results from this study will be presented . 

85 

MOUSE MODEL FOR SOlATIC MUTATION AT TH$ HPRT GENE : MOLECULAR AND CELLULAR 

ANALYSES, Ic . Burkhart-Schults, C .L . Strout, and I_ H . Jenaa, Lawrence 

Livermore National Laboratory, Livesmore, CA 94550 (USA) 

We are using the mouse to study factors that affect the frequency and 

molecular nature of somatic mutations that occur in vivo . We have 

detected differential induction of thioquanine resistant mutants in thymus 

and spleen T lymphocytes ; tissue, mutaqen and age dependent patterns of 

mutant frequency with time ; and persistence of up to one year for both 

ethylnitrosourea (sNU) and radiation induced mutants . Whereas the mutant 

frequency increased linearly up to 72mg ZNO/kQ, and was not reduced by 

fractionation of ENt1 dose, it increased curvilinearly to 400 cOy 1,7Ce and 

was reduced by radiation dose fractionation . The molecular nature of 

spontaneous and induced mutations is being studied by Southern analysis 

and by polym.rase chain reaction based methods . Soon after acute exposure 

of young adult, male mice to radiation the mutation spectrum is enriched 

two-fold for total deletions of the hypoxanthine phosphoribosyltransferase 

(hprt) locus, whereas after ZNO no deletions were detected . The recovery 

of many deletions, of frameshifts, and of 3 different base substitutions 

affecting the protein synthesis initiation codon, indicate that selection 

of mutants with 2 .5 mq/ml thioguanine is highly stringent and may limit 

the spectrum of point mutations recovered . This work performed under the 

auspicies of the U .S . Department of inerqy by Lawrence Livermore National 

Laboratory under contract number N-7405-iNG-48 . 



Notes


Notes IN VIVO DETECTION OF UDS IN THE RAT GASTRIC NUCOSA 86 

B . Burlinson, D .G . .Gatehouse and D .J . Tweats, Dept . Genetic and Reproductive Tox ., 

Glaxo Group Res . Ltd ., Ware, Herts, England . 

An assay is needed to detect genotoxicity in vivo in tissues other than the liver 

or bone marrow . For the pharmaceutical industry the most obvious tissue to 

investigate is the stomach and although there are UDS assays already described for 

this organ they are relatively insensitive or require the use of hydroxyurea . An 

assay has been developed in which account is taken of the morphology of the gastric 

mucosa to enable the isolation of non-S-phase cells and the measurement of UDS by 

scintillation counting without the need of hydroxyurea . The assay is sensitive and 

has a stable control background, (209 * 83 dpa/ vg DNA (11 .34)) . The gastric 

carcinogen ICPNG was detectable at doses down to 12 .6mg/kg (316 t 87 dpm/ vg DNA 

(N :5)) . Indomethacin a non-genotoxic gastric lrritant, was inactive at doses known 

to induce moderate cellular damage indicating that false positive responses due to 

gastric irritancy should not occur . The value of this assay is clearly demonstrated 

by the results obtained with epichlorhydrin . This compound is a direct acting, 

forestomach carcinogen which is Ames positive but which is not detectable in the bone 

marrow micronucleus test or the in vivo liver UDS assay . It was however, easily 

detected in the stomach UDS assay at 50 mg/kg (LD50 .2S0 mg/kg) . From the data 

obtained so far this assay appears to be a rapid and reliable method of measuring UDS 

in gastric mucosa . It can be used in cases where, becacse of the pharmacokinetics or 

lability of the compound, negative results obtained using the marrow or liver as the 

indicator tissue offer little confidence . It is also of use for investigating drug 

nitrosation in vivo, this aspect of a test is currently being investigated . 


COHPLFMENTATION GBOUP ASSIGl0KM1TS FOR W-SENSITIVB CH0 CELIS . D .B . BUSCH, L.H. -" 

THCMON, AND G . ADAIR, Armed Forces Inatitute of Pathology, Washington, D .C. (USA), 

Lawrence Livermore National laboratory, Livermore, CA (USA), and University of Texas 

System Cancer Center, Smithville, TZ (USA) 

These studies were performed in order to determine the number of oomplementation 

groups of W-sensitive CH0 cells, and to investigate the effeot of mutagen treatment 

of the parental lines on the distribution of CH0 W mutant groups . Parental cells 

included E-rey sensitive (EM9), mitoaqoin-C (MM) sensitive (M05), and wild type 

(AA8) CH0 cells . Using a rapid screening procedure, 166 of approximatel,y 284 W 

mutant CH0 cells were assigned to a total of six groups . Over 90% of the assigned 

mutants were in the first two groups, with group 1 predominating after frameshift 

mutagen ICR-170 treatment, and class 2 predominating after use of miesense mutagena . 

The relative scarcity of group 2 mutants following ICR-170 is consistent with the 

hypothesis that the gene damaged in group 2 mutants, VX,2-, is an essential gene . The 

effect of mutagen treatment on the yield of mutant groups demonstrates the potential 

value of varying the mutagen treatment during mutant hunts . Our isolation of W 

mutants of X-ray sensitive, MAiC sensitive, and (in one case) W-sensitive CH0 oells 

shows that mammalian cells can yield viable double mutants in mutagen sensitivity . 

Work supported by the National Institutes of Health (grant numbers GM22021 and 

R1O0961) and performed under the auspices of the U.S . Department of Energy by 

Iewrence Berkeley laboratory grant number 7134700 and by the Lawrence Livermore 

National Isboratory under contract W-7405-11110-48 . 

RIiR 71gsEgsMENT OF s00o 1WTJ10sNgt 

by Lait Dusk 

Toxicology Laboratory, National tood Administration, box 622, 8-75126 Uppsala, Sweden . 

There is little doubt that pyrido-iaidasoles/indolss and imidasoasaarenes ferasd during 

normal cooking are eapable of producing cancer in man, sinoe they induoa malignant 

tumours at aultiple sites in both sexes of rats and mios . ginoa very few food autagens 

have been tested in anwlti-dose experiments, the possibility to perform hi9h-to-lov doss 

extrapolations is Ii .ited . Usinq the simieluantitativ. TDSO approach, sinql"ose 

exparisents can be used to provide rough estisutes of the potency in animals . Such 

calculations 4ive TDSO values in the 1-100 mg/kg dose range, indioatino a moderate carelnoqenio 

potency in high-dose animal experiments . It is not known whether this is true 

for low doses or if man and animals are equally sensitive . At present, the axposura of 

humans to food autagens cannot be adequately detar .ined, since Quantitativs data are 

∎canty . A very rough estimate from the available data suq0ests that humans might be 

exposed to 1 Y9 of oooked food autaq .ns/kq bw . Combining thes* very rough exposure data 

with the eQually rough cancer potency estiaates and assuming that aan and rodents are 

equally sensitive, that the dose-rasponse relationship is linear, it saams unlikely 

that the cooked food autaqens tssted so far represent a major threat to human health . 

However, the carcinogenicity of the muta9ens present at the highest levels in food have 

not yet been detersin.d and virtually nothing is known about aodifyiaq dietary factors 

and differences in sensitivity between animals and man . The need for furthar resaaroh 

in order to provide a data base for a swre meaningful human risk assessment will be 

disoussad . 

50869 3544 

88

89 

HUMAN LIVER CYTOCHROME P-450P IS PRIMARILY RESPONSIBLE FOR CAFFEINE 3-DEMETHYLATION 

AND CARCINOGENIC ARYLAMINE N-eXIDATION . Mary Ann Butler, Masahiko Iwasaki*, F . Peter 

Guengerich*, and Fred F . Kaglubar . National Center for Toxicological Research, 

Jefferson, AR 72079 aerbilt University School of Medicine, Nashville, TN 37232 

Aromatic amines are well known as occupational carcinogens and are also found in 

cooked foods, tobacco smoke, synthetic fuels, and agrochemicals . The metabolic 

N-oxidation of several primary arylamines, which is generally regarded as an initial 

activation step leading to carcinogenesis, recently has been shown to be catalyzed 

primarily by rat cytochrome P-450I F-G and by its human ortholog, cytochrome P-450 

We now report that human liver aic~osomal caffeine (CF) 3-demethylation, the initift 

major step in CF biotransformation in humans, is selectively catalyzed by P-450PA . CF 

3-demethylation was highly correlated with 4-aminobiphenyl (ABP) N-oxidation (r - 0 .99, 

p


Notes exposed to fractionated doses of 6000 1=raya (1 .7 Gy weekly and an accumulated 

dose of 6 .8 (}y) . Mice were sacrificed at various time during 1- 

195 days after irradiation . Some changes in histopathology, cytogenetics 

ultrastructure and trace element were observed . The results indicated 

that radiation could induce thymoma in treated mice (66 .3%), none of it 

occurred in control . By histopathological and ultrastructural studies, 

4 steps appeared clearly after irradiations thymic cortex atrophy, hyperplasia, 

precancerous and cancerous phenomena . Some round viruslike 

particles could be seen in lymphoma cells of exposed mice . The most frequent 

type of chromosomal aberration was reciprocal translocation and 

the abnormal clones appeared in some cells of thymus, spleen and tone 

marrow in exposed mice . But there was no significant difference on the 

frequency of SCE between two groups . Both the contents of Zn and Cu 

have risen after irradiation . The results suggest radiation itself is a 

potential mutagen, radiation induced mutation in the target cells may 

be responsible for the development of tumor and leukemia in an irradiation 

subject . 


METABOLIC ACTIVATION OF 6-NITROCBRYSENE IN ISOLATED RAT HEPATOCYTES DERIVED FROM 

UNINDUCED AND INDUCED SPRAGUE-DAWLEY RAT LIVERS . D .A . Casciano, K .B . Delclos, 

R.P . Walker, and J .G . Shaddock . National Center for Toxicological Research, 

Jefferson, AR 72079 . 

It has previously been shown that 6-nitrochrysene (6-NC) can be activated to 

electrophilic species capable of reacting with DNA through metabolic pathways that 

form N-hydroxy-6-aminochrysene (N-hydroxy-AC) or trans-1,2-hydroxy-1,2-dihydroxy-6aminochrysene 

(AC-1,2-dihydrodiol) . We have alsoemonstrated that when N-hydroxy-AC 

is reacted with DNA in cell-free systems, three major adducts are formed, N-(deoxyinosin-8-yl)-6-aminochrysene 

(I) . 5-(deoxyguanosin-N2-yl)-6-aminochrysene (II), and N- 

(deoxyguanosin-8-yl)-6-aminochrysene (III) ; while an as yet unidentified adduct (IV) 

is formed from the in vitro reaction of a microsomal metabolite of AC-1,2-dihydrodiol . 

In order to test the influence of microsomal enzyme induction on the activation pathway 

in an intact cell system, 6-NC was incubated with freshly isolated hepatocytes 

from rats that were either untreated or pretreated with phenobarbital (PB), 3-methylcholanthrene 

(3-MC) or Aroclor 1254 (ARO) . Hepatocytea isolated from untreated or PBtreated 

rats formed adducts derived from N-hydroxy-AC (predominately I . II, and III), 

whereas hepatocytes isolated from rats pretreated with 3-MC or ARO formed adducts 

derived from AC-1,2-dihydrodiol (predominately adduct IV) . Our results provide the 

first clear demonstration that exposure to inducers of drug metabolizing enzymes can 

profoundly alter the qualitative pattern of adducts formed in the DNA of cells treated 

with a nitro PAH . 

USE OF POLYMERASE CHAIN REACTION (PCR), DIRECT SEQUENCING, AND DNA 

COLONY HYBRIDIZATION IN THE CHARACTERIZATION OF SdIJKONBI .L4 

?YPHIMURIUM REVERTANTS. T.A. Cebula and W.H. Koch. Food and Drug Administration, 

200 C Street, S.W., Washington, D.C. 20204 . 

The S. typbLnuriuas reverse mutation assay Is widely used to assess the mutagenicity and 

potential carcinogenicity of a large variety of chemical compounds . Elucidation of the molecular 

events anderlying the reversion of the kLsG46, 6ttD30S2, and bisC3076 mutations by well 

characterized carcinogenic and mutagenic agents should provide valuable insights Into the 

mechanistic relationship between bacterial mutagenesls and oncogenic potential in higher 

organisms . Thus, we have developed a battery of synthetic DNA probes, discriminating single 

base-pair mismatches, for use in DNA colony hybrldiatbn to study spontaneous and mutageninduced 

revertants of S. &phimu.Lrsi strains harboring each of the aforementioned mutations . 

Using PCR, we have selectively amplified a single 400 bp fragment encompassing the bLsC3076 

locus from total genomic DNA of strain TA2637 . Direct sequencing of this fragment showed the 

mutation to be a GG Insertion in a run of four C residues ; DNA sequence near the hisC3076 

locus Is S'- ...TTAAAAGTGATCGCCCC=ATCCGCTIT...4'. Southern analysis, using synthetic 

probes (18 men) designed to discriminate between rrild-type and hisC3076 DNA, confirmed the 

sequence tindings . We are currently using PCR and direct sequencing to characterize mutant 

sequences of hisD3052 and hisC3076 revertants unidentitled by the probing analysis . Thus far, 

approximately 1000 A1s* revertants have been characterized using these combined methods . 

50869 3546 

93 

94


COMPARISON OF AMBIENT AIR MUTACENICITY DETECTED WITH TRADESCANTIA STAMEN Notes 

HAIRS AFTER CHERNOBYL ACCIDENT AND ONE YEAR LATER 

Antonina Cebulska-Vasilevska Radiobiology Department, Institute of Nuclear 

Physics, 152 Radzikovskiego, 31-342 Cracov, POLAND 

This paper presents the results of the research on mutagenic effect of ambient 

air in the Cracow area . Reported studies were condueted first in May 1986, 

after the Chernobyl accident . For comparison, second studies were performed in 

various sites within the city in the Spring of 1987 . Counts were made of 

stunted hairs and pink cells in the stamen hairs of Tradescantia clone 4430 . 

Mutation scored since llth day after the beginning of exposure vera used as a 

measure of mutation effect caused by the exposure . The mean mutation 

frequencies measured for investigated periods were 0 .41 and 0 .17 nut/100 

hairs respectively . Mutation frequency levels observed after the Chernobyl 

accident show a correlation with iodine-131 and cesium-137 activity measured 

in the air at that time . Although, results obtained in 1987 show on the 

average the significant decrease of the ambient air mutagenieity, but the 

variation betveen levels of mutations caused by chemical pollution and 

observed in the Spring 1987 in different sites of the Cracov area is rather 

high (0 .09 to 0 .31 mut/ 100 hairs) . It also indicates some correlation with 

S02 contents in the air . Comparison between biological effects observed in 

those two periods demonstrates the importance of chemical pollutants 

mutagenicity . 

96 

INFLUENCE OF SOME ENVIRONMENTAL FACTORS ON DOSE RESPONSE CURVE FOR SOMATIC 

MUTATIONS IN TRADESCANTIA . 

Antonina Cebulska-Vasilevska+, Robert Rorzeniovski***Radiobiology Department, 

Institute of Nuclear Physics,152 Radzikowskiego, 31-342 Cracov, 

**Civil Engineering Department, Technical University of Cracow, L-1 

24 Varszavska Street, 31-155 Cracov, POLAND 

Somatic mutations and cell lethality observed in the stamen hairs of the 

heterosygous for the flower color clones of Tradescantia have often been used 

as a biological plant test system . Due to its high sensitivity to radiation 

and chemicals the system is particularly suitable for environmental studies . 

The radiation dose response curve for the somatie sAtations in Tradescantia 

is vall described by linear quadratic model . Presented paper shows hov an 

alteration of the biophysical processes caused by some environmental agents 

may influence the mutation dose-effect relationship . There are presented 

different dose-response curves obtained by modification of the various 

parameters describing DNA repair process efficiency . Results obtained both, in 

previous studies with sodium fluoride as a possible repair processes 

inhibitor, and also in recent studies with magnesium contents changed through 

the use of dolomite sdded to the soil, are discussed and explained by 

presented dose response curves analysis . 

97 

COMPARATIVE STUDIES ON THE MICRONUCLEUS ASSAY IN CYTOKINESIS-BLOCKED BINUCLEATED AND 

CONVENTIONAL MONONUCLEATED METHODS IN HUMAN PERIPHERAL BLOOD LYMPHOCYTES . 

Channarayappa, J . Nath and T . Ong, West Virginia University and Division of 

Respiratory Disease Studies, National Institute for Occupational Safety and Health, 

Morgantown, WV 26505 . 

Bone marrow as well as peripheral blood lymphocytes and cultured manmalisn cells 

have been used in the micronucleus assay . However, if the measurement of micronuclei 

is to be used as a reliable indicator of damage due to chromosomal break or spindle 

dysfunction, scoring of micronuclei must be restricted to cells which have undergone 

division . Several attempts have been made to discriminate divided from nondivided 

cells . Among them, the cytokinesis-blocked binucleated cell (CB) method of Fenech 

and Morley (Mutation Res . 147 :29, 1985) is being used by several investigators . In 

this study, we determined the optimum concentration of cytochalasin B (CYB) for the 

induction of a maximum number of binucleated cells in human peripheral blood 

lymphocytes (PBLS) and compared the efficacy of CD method with the conventional 

mononucleus (CM) method for scoring micronuclei after human PBLS were treated with 

mitomycin C and cyclophosphamide . The results showed that 3 pg CYB/ml was an 

optimum concentration for the induction of binucleated cells without any significant 

toxic effect and that the frequency of micronuclei in the CB method was higher than 

that in the CM method for both chemicals . Thus, it appears that scoring of 

micronuclei in binucleated cells is a usefyl method for measuring chrosasomal damage 

in human PBLS . 


35



Notes MUTANTS DEFECTIVE IN POLY(ADP-RIBOSE) NETABOLISN EXHIBIT DIVERGENT PATTERNS IN 

SUSCEPTIBILITY TOWARDS DIFFERENT DNA DAMAGING AGENTS . S . Chatterjee, N .F . Cheng, 

S .J . Berger and N .A . Berger, Ireland Cancer Center, Case Western Reserve University, 

Cleveland, Ohio 44106 . 

We have developed two groups of mutant cell lines from V79 Chinese hamster cells 

capable of proliferating with impaired poly(ADP-rtbose) metabolism . One group 

consists of ADPRT 54 and ADPRT 351 which are defective in poly(ADP-ribose) 

polymerase activity while the other consists of N2, N3 and N4 which can grow stably 

in the absence of free nicotinamide in the mediun with total NAD levels of 1 .5 - 

3 .0% of that found in parental V79 cells grown in camplete medium . Thus, in the 

latter group of cells, poly(ADP- N bose) synthesis is restricted due to limited 

availability of the substrate MAD . Since poly(ADP-ribose) polymerase has been 

implicated in DNA repair we examined the susceptibility of these mutants towards 

various kinds of DNA damaging agents and topoisomerase II inhibitors . Our studies 

show that these mutants are resistant to topoisomerase II targeted drugs such as 

VP-16 and m-ANSA. In contrast, these mutants showed increased sensitivity to NNNG, 

bleomycin and X-rays . Preliminary studies show that ADPRT 351 cells repair X-ray 

induced DNA strand breaks more slowly than do V79 cells . This phenomenon could 

account for their increased sensitivity to X-rays . A detailed account of the 

susceptibility of the mutants and Y79 ce11s towards a variety of DNA damaging agents 

and possible role of poly(ADP-ribose) polymerase in DNA repair will be presented . 

MJhDCZ7iAR ANALYSIS OF HPRT MUTATICNS 3N Y79 CRINESE HAMSTFR CEL1S 

M. A . Chaudhry and Margaret Fbx, Paterson Institute for Cancer Research, 

Nanchester, M20 .9BX . 

To understand basic mectusnisms of mutation at the HPRT locus in Chinese 

hamster cells, spontaneous, Xray, MM14 and (F74S) nutants have been independently 

isolated . 9 Spontaneous, 36 MMS, 20 Xray, and 20 IIdS mutants were analysed by 

Southern blotting and more limited number Northern analysis . None of the 

spontaneous mutants showed changes detectable by Southern analysis . 14/36 M 

induced, 9/20 Xray induced, and 0/20 FMS induced mutants showed oomplete deletion 

of HPRT DNm sequences . The remainder showed no detectable changes . Northern 

analysis of the 9 spontaneous mutants indicated 6/9 produced no detectable HPT .T 

mRNA,, also in 3/7 MMS induced mutants no mFM was detectable . In a further two MNS 

induced mutants the level of niessage was much reduced oaipared with wild-type 

levels as indicated by similar levels of APRT message in all cell lines . sJmA frae 

further 14 induced mutants was analysed by dot blotting and was much reduced in one 

Xray and absent in one Eti4 induced mutant . HPRT mRNn frae 14 mutants has been 

amplified using the polymarase chain reaction . In three mutants in which sTM was 

undetectable on Northern analysis low levels were detectable after in vitro 

amplification . To date the site of the nutatien has been identified by direct UNA 

sequencing in four of these mutants . The sutations ware oonsistent with the 

lmowm mechanism of action of the autagens . The high proportion of deletions 

produced by Xrays and MPiS probably explains their inefficiency as mutagens . 

100 

LONG-DISTANCE TRANSPORTATION AND RAPID PROPAGATION TECHNIQUE FOR PLANTS 

OF TRADESCANTIA PALUDOSA 

Kui-2hang Chen and Gui-Ying Peng . Guangxi Institute of Botany, 

Yanshan, Guilin, Guangxij PEOPLE'S REPUBLIC OF CHINA 

Tradescantia Micznnucleus (Trad-DLS1) Assay, established by Dr . Te-Hsiu Ma, is a 

well lnown, simple and effective technique for monitoring mutagens in the envirocment . 

Sinoe it was introduoed into China in 1980, it has been used for in situ monitoring 

of many sites . In 1986, it was registered in the regulation of Erniir+amental Matiitoring 

Technology by the State Environrental Protection Bureau of China . Because 

Tradesoantia clone 03 can be repraiuoed only with vegetative propagation, the longdistance 

transportation of plants would be a barrier to increasing the appli,ed range . 

To overoome these difficulties and keep plants living during lang-distanoe transportation 

and to obtain a large number of plants with the same original micronucleus 

rate in the pollen mother cells in short time assay, a series of experimsnts have 

been doee . These show that selecting a plant whose pollen mother cells oontain low 

original micronucleus rate and adopt3ng rapid propagation methods of the test tube 

plants , the white buds are suitable for long-distance transportation 

99 

101 

A PRACTICAL TECHNIQUE FOR SHIPPING AND RAPID PROPAGATION OF TRADESCANTIA PLANTS UNDER 

POLLUTION-FREE CONDITION, Rulshaaa C_I1gG* and Guiying Peng*, Guangxi Institute of 

botany, Yanshan, Guilin, PRO (Introd . by T . H . Na) Departsent of Biological Sciences, 

Western Illinois University, Haoosb, IL (USA) 

50869 3548

Tradescantia-Micronucleus (Trad-MCN) bioassay is an efficient test for genotoxioity 

of environmental pollutants . It is currently under the validation prooess for the 

International Program on Chemical Safety, WHO, UN for possible world-wide adoption for 

chemical testing . Large population of Tradescantia clone #4430 has been vegetatively 

propagated in the field and greenhouse in Guangxi Institute of Botany where the 

climate and day length are suitable for the natural growth of this plant all year 

round . Our Institute could serve as the supply house of this plant material for all 

corners of the world . In order to maintain the genetic purity of this plant during 

long distance shipping and rapid propagation of a large quantity, we developed a 

practical technique to meet thie demand . First, young plant buds (usually whitish in 

color and about 0 .5 cm long) which are beneath the soil surface are selected from the 

low background stocks . The selected buds are then transplanted in a sterile 

pollutant-free medium in a container and kept in the dark for shipping . These plant 

buds can be kept alive in the shipping package for as long as a month duration . The 

plant buds will turn into normal green plantlets after exposure to the light and ready 

to be transplanted into field or greenhouse . The new plants will reach maturity Sn 

about a month . The plants shipped in this fashion could minimize the possible genetic 

change during shipping and gain easy passage of quarantine stations at the 

international boundaries . 

102 

A NEW TW0-DINENTIONAL ANALYSIS OF DNA FRAGMENTS : TEMPERATURE GRADIENT GEL 

ELECTROPHORESIS . Y .Chen, J .Fu, G .FAN, and B .Liu . Vest China University of 

Medical Sciences . Chengdu, Sichuan (China) 

A new technique, nased teeperature gradient gel electrophoresis (TGGE) . 

used for sequence dependent separation of DNA fragments has been developed . 

If double helical DNA is partially ∎elting in polyacrylamide gels, its 

electrophoretic ∎obility undergoes a sharp transition, resulting in a 

large reduction in ∎obility . In the present experiment, the transition that 

was effected at a uniform concentration of denaturant, formamide . in a 

temperature gradient formed during elect.rophoresis . Each restrictive 

fragment of pBR322-DNA and bacteriaphage lambda-DNA exhibited the ∎obility 

transition at a particular temperature . The sudden retardation of fragments 

moving forward in the gel depended upon nucteotide composition and sequence . 

rather than the length . When combined with length dependent electrophoresis 

in the perpendicular direction . TGGE provided a two dlsentional separation 

of DNA restrictive fragments . The resolving power of the new system was 

demonstrated by the clear resolution of over 80 fragments of the restrictive 

entyme Hinfl digest of lambda-DNA . This technique would find its appiic:ation 

in ∎any fields . 

103 

RADICAL-MEDIATED SITE-SPECIFIC CROSSLINKING OF NUCLEAR MATRIX PROTEIN AND DNA . S .M . 

Chiu, L .Y . Xue, L .R. Friedman, and N .L . ole nick . Departments of Radiology and 

Environmental Health Sciences, Case Western Reserve University, Cleveland, ON (USA) . 

Ionizing radiation-induced DNA-protein crosslinks (DPC), as assayed by 

nitrocellulose filter-binding, are formed preferentially between domains of mammalian 

chromosomal DNA enriched in active sequences and normal DNA-binding proteins of the 

nuclear matrix (Chiu et al ., Radiat . Res . 107, 24 . 1986). In an effort to explore the 

mechanism for the microheterogeneity, we have fractionated V79 cells and nuclei both 

before and after irradiation. When intact cells were irradiated, the same radiation 

dose response was obtained if the assay of DPC was subsequently conducted on cells, 

nuclei, or nuclear matrix, suggesting that all of the components of DPC are retained 

in the nuclear matrix. The same result was obtained for irradiation of nuclei and 

assay of nuclei or nuclear matrix . However, the formation of DPC reached an early 

maximum when nuclear matrix wae irradiated . Nuclear matrix was isolated by extraction 

with lithium diiodosalicylate (LIS) . A. analyzed by SDS-PAGE, a similar set of 

proteins was found in matrices prepared by LIS or by extraction in 2 N NaCl. In the 

case of LIS extraction, nuclei were first stabilised by incubation in 0 .5 ∎M Cu804 . 

The radiation dose-response for formation of DPC in these nuclei was 4-6 timea greater 

than in untreated nuclei . Tests for the presence of Cu++ or other trace metals and 

for the participation of Fenton-type teactions suggest a poseible explanation for the 

selective formation of DPC at nuclear matrix sites, i .e ., the generation of hydroxyl 

radicals or other oxidative radicals in the vicinity of preferential sites of binding 

of metal ions . (Supported by NIH Grants RO1CA15378 and P30CA43703 .) 

104 

ISOLATION AND PARTIAL CHARACTERIZATION OF RAD4 GENE OF Saccharomyces cerevlsiae THAT 

CAN BE PROPAGATED IN S .coli WITHOUT INACTIVATION . I .S .Choi, J .B .Kim, and S .D .Park, 

Department of Zoology, College of Natural Sciences . Seoul National University, Seoul 

151-742, South Korea 



Notes 

37


Notes The RAD4 gene requiied for incision of UV-induced excision repair in Saccharomyces 

cerevtsiae has been isolated by phenotypic complementation of a UV-sensitive rad4-4 

mutant with yeast genomic library amplified In E .coli (Yoon et al .,1985) . The yeast 

insert in recombinant plasmid designated as pPCl was isolated in the 2 .6Kb BglII-gamHI 

fragment in the aubcloned pPC100 and contained a functional RAD4, but not a suppressor 

tRNA gene as determined by restriction mapping and DNA-tRNA hybridization . In addition, 

the observation that all different plasmids harboring yeast insert conferred UV 

resistance to rad4-4 cells to a similar level regardless of their copy number, strongly 

suggests that our cloned gene does not contain a suppressor . Northern blot analysis 

with pPC100 as the probe indicates that the insert DNA containing the entire RAD4 gene 

can hybridize with transcripts of 1 .2Kb and 2 .3Kb in length . Stationary-phase cultures 

of E .co1i BL21(DE3) strain transformed with plasmids harboring the cloned RAD4 gene 

showed a considerable delay in the resumption of exponential growth and a dramatic 

reduction in the production of host proteins . Moreover, the overexpressed Rad4 protein 

estimated as 89Kd by 2-D gel analysis revealed a toxic effect In Z .coli, suggesting 

that the Rad4 protein interferes with normal growth control in 6 .co1t cells . 

105 

USE OF THE BALB/c-3T3 ACAR SUSPENSION ASSAY TO DETECT CHEMICAL-INDUCED TRANSFORMATION . 

M . A . Cifone, L . Custer and E .J . Matthews, Hazleton Laboratories America, Inc ., 

Kensington, Maryland . 

An alternate endpoint to morphological transformation In the BALB/c-3T3 transformation 

assay has been described . In this assay, the formation of colonies in agar was 

measured after chemical treatment . Approximately 50,000 cells were seeded on day 0 and 

treated on day 1 with a chemical for 24 hours . The cells were then kept in logarithmic 

phase for 11 to 13 days and plated in 0 .4% Noble agar . Four weeks later the colonies 

were fixed with 2 .5% glutaraldehyde and colonies with diameters greater than 0 .1 mm were 

automatically counted using an Olympus Q2 1mage analyzer . Fifteen chemicals were 

investigated In this system and activities were compared to the presence or absence of 

structural alerts, and published results obtained in the standard BALB/c-3T3 

transformation assay, several in vitro genotoxicity assays, and rodent bioassay . The 

chemicals studied included four genotoxic carcinogens, six genotoxic nonearcinogens and 

five nongenotoxic carcinogens . The results with the agar suspension assay closely 

correlated with the results obtained with the standard BALBe/-3T3 transformation assay . 

However, in some cases the agar suspension assay appeared to be les∎ sensitive . Some 

chemicals gave an equivocal or negative response In the agar suspension assay and ware 

weakly active in the standard transformation assay . While the BALB/c-3T3 agar 

suspension assay is labor intensive and not appropriate for routine screening, it 

measures an endpoint that is crucial to the transformation process and can be used to 

answer mechanistic questions . 

(supported by NIEHS Contract No . N01-ES-65150) . 

PARALLEL DETECTION OF SCE, DNA ADDUCTS AND PROTEIN ADDUCTS IN MICE DOSED WITH THE 

CARCINOGEN 2-ACETYLAMINOFLUORENE . 

106 

G .Citro, R .Zito, A .Verdina, R .Galati, R .Benigni, P .Leopardi, A .Zijno, and R .Crebelli, 

Istituto Superiore di SanitB and Istituto Regina Elena, Rome (Italy) . 

In order to explore the interrelationships among DNA adducts, protein adducts and 

SCE simultaneously induced in the complexity of in vivo situation, an investigation 

was carried out in Swiss mice treated with the carcinogen 2-acetylaminofluorene (2AAF) . 

Groups of 4 male mice were treated either by gastric intubation (at 25,50 and 100 mg/kg 

on 4 consecutive days) or by chronic dietary exposure (2 months feeding with a satura- 

ted 2AAF water solution) . For each animal the frequency of SCE in bone marrow cells, 

as well as the levels of binding of 2AAF to liver and spleen DNA and blood proteins we- 

re determined by means of cytogenetic techniques and competitive immunoassays, respect . 

The results obtained highlight a dose-related trend in SCE rates after acute exposure, 

with wide interindividual variance wich was significantly related to individual levels 

of liver DNA adducta . Conversely, the amount of covalent binding to spleen DNA proved 

to be related only to individual levels of binding to blood proteins . Such picture was 

modulated by the schedule of treatment : chronic 2AAF exposure in fact produced relati- 

vely higher levels of binding to spleen than to liver DNA (about 1,000 and 500 adducts/ 

million nucleotides, respect .) and did not increase significantly SCE rates in bone 

marrow . 


50869 3550

STRUCTURE ACTIVITY ANALYSIS OF AZO DYES AND RELATED CONPOUNDS . L .D . Claxtonl, D .B . 

Walsh2, J . Esancy3, and H . Freeman3, lEnvironmental Protection Agency, Research 

Triangle Park, NC (USA), 2Environmental Health Research and Testing, Inc ., Research 

Triangle Park, NC (USA), and 3North Carolina State University, Raleigh, NC (USA) . 

Azo dyes represent a significant group of industrial dyes . The carcinogenicity of 

some azo dyes have been known for decades . However, debates persist as to the role of 

metabolism and various substructural units in the genotoxicity of these azo dyes . For 

example, although one of the most prevalent metabolic pathways implicated in the 

activity of azo dyes is azo reduction, not all reductive cleavage products are 

mutagenic . A computerized SAR system, ADAPT, was used to analyze two separate data 

bases : a group of azo dyes and a group of reductive cleavage products . The data used 

for both data bases was the response when tested in the Salmonella reversion bioassay . 

A compound was considered mutagenic if there was a positive response in any of the 

standard five tester strains either with or without metabolic activation . However, a 

compound was considered nonmutagenic only if there was no response in any of the 

strains with or without metabolic activation . The azo dye data base consists of 25 

mutagens and 13 nonmutagens . A set of molecular descriptors, including substructural 

descriptors, was selected that successfully classified the mutagens from the 

nonmutagens . These molecular descriptors were used to predict the mutagenicity of a 

group of azo dyes not included in the data base . The reductive cleavage data base 

consists of 48 mutagens and 13 nonmutagens . Structure activity relationships of both 

data bases, as well as their predictive capability, will be discussed . This is an 

abstract of a proposed presentation and does not necessarily reflect EPA policy . 

108 

IN VIVO MICRONUCLEUS TESTS IN MOUSE . COPPARISON OF TFE SENSITIVITY OF THREE TARGET ORGANS 

(BONE MARROW, LIVER AND TESTIS) TO SIX CARCINOGENS . 

I .Cliet, E .Fournier, C .Melcion and A .Cordler . Dbpartement de Toxicologie, Centre de 

Recherches de Vitry, RhBne-Poulenc SantB, 13 Qual Jules Guesde, 94400 Vitry sur Seine, 

France . 

In vivo somatic chromosome mutation is usually carried out using the bone marrow 

micronucleus (BMM) test In mouse, which is considered of predictive value In the study of 

clastogenicity In the germ cell line . However, it has been reported that the sensitivity 

of the BMM test !s insufficient to detect unstable compounds or short-lived metabolites 

and the use of target cells with metabolic activity (hepatocytes and spermatids) has been 

questioned . In order to analyze In vivo micronucleus lnduction, particularly In celis with 

metabolic enzyme activity, we compared the sensitivity of somatic and germ cell lines 

towards six carcinogens In the bone marrow, liver and .spermatld micronucleus test In 

mouse . Five procarcinogens, with a complex metabolic pattern : dlmethylnitrosamine (DMN), 

diethyinitrosamine (DEN), 1-1-dimethyihydrazine (1-1-DMW), 4-aminophenol (4-APOL) and 

4-aminobiphenyl (4-ABPYL) and one direct unstable mutagen, a-proplolactone (BPL) were 

tested . DMN, DEN, 1-1DMH, BPL were not detected by the BMM test due to their lnstability, 

whereas 4-APOL and 4-ABPYL were clearly positive . All six carcinogens were detected In the 

mouse liver and spermatid micronucleus test and induced clear clastogenic effects . In 

conclusion this study demonstrates the importance of organ-specificity studies . Moreover, 

the results of the liver and spermatid micronucleus tests show that the BMM test cannot 

replace clastogenicity evaluation in the germ cell line . 

109 

MAMMALIAN CELL MUTAGENESIS: A MAJOR ROLE FOR NON-DNA TARGETS . D . Clive, 

Wellcome Research Laboratories, Research Triangle Park, NC 27709 USA 

Evidence is accumulating for major non-DNA targets for specific locus mutageniclty In mammalian 

cells involving heritable DNA alterations . Such a possibility (a) Is consistent with radial-loop models 

of chromosome structure In mammalian cells (Marsden and Laemmll, Cell17 : 849, 1979) ; (b) is 

based upon non-DNA structural components of the eukaryotic chromosome (e .g., topolsomerase is ; TP) 

(Gaulden, Mutagenesis 2(1987) 357 ; Evans et al., Mutation Res. 217 : 53, 1989) ; (c) invokes 

genetic alterations quantitatively larger than, and mechanistically different from, classical point 

mutations as a major class of gene mutations (Yandell et al ., Som. Cell Mol. Gen . 12 : 255, 1987; 

Applegate and Hozier, Banbury Report #28 : 213, 1987) which (d) are of the types seen In rodent and 

human tumors (All et al ., Science 236 : 933, 1987) ; (e) attributes the relative Insensitivity of some 

genotoxicity tests to the wrong chromosomal architecture (prokaryotes, including the Ames assay), the 

wrong chemistry (DNA repair synthesis incapable of detecting TP redimerization) or the wrong 

endpoint (dominant or X-linked loci fail to detect large scale recombination events) ; (f) explains the 

higher sensitivities of other assays (SCE assays, chromosome aberration tests, L517gY/fk +/- mouse 



Notes lymphoma assay [MtA)) ; and (g) Implies the existence of penotoxicJcarcinopenic chemical structures 

different from those Implicated in DNA reactivity . The cytopenet)c and molecular mutational spectra of a 

number of chemically diverse mutagens In the MLA Indicates that only a few mutagens (e .y., EMS, 

2-amino-6N-hyd(oxyadenine) Induce a significant proportion of subgenic DNA alterations at the 

heterozypous tk locus, whereas most Induce predominantly DNA alterations which are at least many 

kilobases (and probably multigenic) in extent (Glover et al ., these abstracts) . These results will be 

discussed In terms of penotoxicity models and mechanisms which Indude non-DNA targets . 


110 

MUTAGENIC ACTIVITY OF EXTRACTS FROM ROLLING MINERAL OILS AND LEATHER SAMPLES . 

E . Cionferot, P . Venier=, M . iordanz and A .G . Levis= . t Inst . Occup . Health 

University of Padova (ITALY) 2 Dept. of Biology, University of Padova (ITALY) . 

Unused crank case oils are norsally negative in the Salmonella/sicrosose assay 

but become strongly sutagenic after use due to the enrichment of their PAH content 

resulting from engine cosbustion processes . On the other hand refined rolling oils 

lack sutagenic activity even after prolonged use because of the low temperature 

working conditions . Finished leather probably contains naturally occurring tannins, 

but other chemical compounds used in the tanning and refining processes may also 

play a part in determining the carcinogenicity of soae leather dusts . We evaluated 

the sutagenicity (in the Saleonella/ .icroso.e assay) of extracts fro . a aineral oil 

esulsion used in a rolling ∎ill and from finished leather . ' The oil emulsion was 

suspended in DMSO and extracted with CHzCl= . The extracts fros the unused oil were 

negative, while after prolonged use sutagens directly active on Salmonella strains 

TA98 and TAIOO were found (8-10 ind . rev ./ .g eq . of extract) . Relatively low 

concentrations of oil (10 ∎g/plate) were found to inhibit totally the .utagenicity 

of 5 pg of benzo(a)pyrene assayed in the presence of S9 . " After treatment in a 

Soxhiet apparatus with toluene or ethanol the extracts from a leather widely used in 

the furniture and dresssaking Industries were mutagenic on strain TA98 of 3, 

j,yphisurius in the absence of S9 ∎ix . The analysis of the extracts from leather at 

various intersediate stages of processing showed that the mutagenic activity 

appeared after the coloration process . The responsible compound was identified to be 

an azodye (Color Index : Acid Brown 83, .utagenic potency about 4 

revertants/sicrogras) . SUPPORTED BY C .N .R . p .f . "ONCOLOGIA" . 

111 

THE MUTAGENICITY OF 2-AMINO-N6-HYDROxYADENINE (ASA) TO L517BY TK+/- CELLS MEASURED 

BY THE INDUCTION OF TRIFLUOROTHYMIDINE (TPT), 6-TBIOGUANINE (6TG) AND OUABAIN (OUA) 

RESISTANCE AND THE INDUCTION OF MICRONUCLEI . 

Jane Cole, Frances Richmond and Bryn Bridaes, MRC Cell Mutation Unit, University of 

Sussex, Brighton BN1 9RR, Gt . Britain . 

L5178Y TK+/- (3 .7 .2c) cells were treated with AHA at concentrations ranging from 

0 .005 to 2 .Oµq ml-1 (100 - 15% survival) . Samples were plated on day 0 after treatment 

to determine the toxic effect, on days 2 and 3 to determine TFT and OUA mutant 

frequency and on day 7 to determine 6TG mutant frequency . All platinqs were done by 

the microtiter method of Cole at al . (1986, Mutaqenesis 1, 157-167) . Slides were 

prepared 24h and 48h after treatment to determine the induction of micronuclai (MN) . 

Positive controls (ethyl or methylmethanesulphonate) were included in every experiment 

. Over the non-toxic range (0 .01 - 0 .04µq/ml, 80 - 100% survival) there was 

approximately linear induction of TFT, OUA and 6TG resistance . At toxic concentrations, 

TFTR continued to increase with dose, 6TG resistance plateaued at around 0 .5µq 

/ml, and a peak of OUAR mutants was observed at 0 .25µq/ml, at higher doses there was 

a marked decrease in OUA resistance . Only small increases in the percentage of MN 

were observed . 

112 

A NEW METHOD FOR PREPARATION OF METAPHASES FROM THE G .I . TRACT OF RODENTS . Barbara 

Coles, Leslie McSparrin, Judith Horvath, and William S . Barnes, Department of Biology, 

Clarion University of PA, Clarion, PA . 

Research has frequently been handicapped by the lack of a good experimental 

system to study the events of colon carcinogenesis . Carcinogenic activity can 

be identified by induction of SCE, but it remains difficult to obtain good yields 

of mitotic cells from the G .I . tract . In our protocol, 35 g . male C57BL/6 mice 

were implanted with a 25 mg agar-coated BrdUrd tablet . Twenty-three hours later, 

they were injected i .p . with 10 mg/kg coichicine . Twenty-five hours after BrdUrd 

adminsitration, animals were anesthetized and a loop of small intestine was exposed 

from the body cavity . The loop was cut from both ends, being careful not to cut 

any blood vessels, and fecal material was flushed out . The segment was then treated 

sequentially with two incubations of 0 .5mM EDTA, followed by two incubations of 

collagenase in 10mM HEPES (750 units/ml .) . The segment was removed from the animal . 

50869 3552 

,

everted, and vortexed vigorously in 3% glacial acetic acid to remove mucosal cells . 

Preparation of metaphase spreads and sister chromatid differentiation were done 

according to standard techniques . This technique yields many M~ metaphases with a median 

of approximately 30 chromosomes/metaphase . Various refine6Tenta are being investigated 

. We are also adapting this method to the large bowel of P344 rats . Supported 

by the Clarion University Foundation and a SSHE Professional Development Award . 

113 

EVALUATION OF THE GENOTOXICITY OF CARBOFURAN IN HUMAN PERIPHERAL BLOOD 

LYMPHOCYTES . Ilce Mara de S . Colus, Catarina S . Takahashi and Elza T . Sa 

kamoto-Hojo (FUEL-PR, FFCLRP-USP, IBILCE-SJRP-UNESP) . Brasil 

The pesticide Carbofuran is a carbamate specifically used for the 

treatment of seeds from grains such as wheat, corn and rice, among others . 

The genotoxicity of the compound was tested in vitro in terms of induction 

of chromosome aberrations and SCE in hutfan petip :eia~ blood lymphocytes 

submitted to concentrations of 4 .13 x 10 and 4 .13 x 10 jug/ml . The drug was 

added when the culture was initiated and allowed to act until fixation 

time, which was 48 hours later for chromosome aberration test and 72 hours 

for SCE test . The percentage of cells with chromosome aberrttions did not 

differ among groups : 3,62 4 or the control and the 4 .13 x 10 concentration 

and 5 .8% for the 4 .13 x 10 concentration of the product . Similarly, the 

mean number of SCE/cell did not diffber among the three groups : 10,65 for 

the control,-211 .20 for the 4 .13 x 10 pg/ml eoncentration and 11 .07 for 

the 4 .13 x 10 pg/ml concentration . The results agree with those reported 

in the literature, which indicate that Carbofuran is not mutagenic in microorganisms, 

in the SLRL test using Drosophila, or in human fibroblasts . Turba 

genic effects of Carbofuran have been reported in Ilordeum vuZgare and in 

AZZium cepa . Since the insecticide Carbofuran proved to be ineffective in inducing 

a genotoxic effect on human lymphocytes, and considering that the cultures 

usually exhibit a low metabolizing ability, we may conclude that the pa= 

rental compound did not show clastogenic activity on the test system used . 

114 

MOLtCOLAR MLCalUlIBM OF OZNLTIC NLCON7INatION : . 

rORMATION aND RISOLOTION Or IOLLIDIIY JOMCTIONS IN VISRO . 

Edward C . Conley, Berndt Milller and Stephen C . West 

Imperial Cancer Research flund, Clare Hall Laboratories, South Misms, Retts EN6 IID, D .R . 

A central internediate in the process of qenetic reconbination is a Holliday junction in 

which two DNA helices are int.rlinked by a reciproul strand crossover . Holliday junctions ray 

be formed in vitro by the enzymatic action of the F. coli RecA protein . Rach gains access to 

duplex DNA by nucleation from a short sinql.-strended gap, to form a spiral nucleoprotein 

fil>ment which is capable of interaction with homoloqous duplex DNII. Any part of the filamant, 

whether it contains single- or double-strandad DNA, is capable of pairing with the homoloyous 

duplex . Homaloqous contacts between reqions of duplex DNA lead to an increase in the initial 

rate and final extent of joint molecule formation . !]tparimants indicate that pairing is 

facilitated by the formation of nascent synaptic internrdiates between duplex DtA seQuences . 

Duplex-duplex pairing reactions involve underwindinq of the double helix relative to normal B- 

form DNA .In an ATP-dependsnt reaction, RecA protein drives the reciprocal exchanqe of D1A strands 

:n polar fashion from a gapped region into and along four strands of DNA,so forming a Holliday 

;unction . When T4 endonuclease VII i. added to the reaction mixture, the Holliday junctions 

formsd by RecA protein are resolved to form heteroduplex DNh . Cleavage occurred by cutting in 

either of the two possible orientations . Thus, recombinant DNA mol .cules may be formed in an in 

vitro system by the combined action of a recombinase and a r .solvase protein . 

115 

THE E .E .C . GENOMIC MUTATION PROGRAM : RESULTS WITH ASPERGILLUS NIDULANS 

R .Crebelli, G .Conti, L .Conti, and A .Carere, Istituto Superiore di Sanitli,Rome(Italy) 

In the framework of the coordinated E .E .C . Genomic Mutation Program for the evalua- 

tion of current methodologies for the detection of aneuploidy, a set of nine reference 

compounds (colchicine, econazole, chloral hydrate, hydroquinone, diazepam, thiabenda- 

zole, cadmium chloride, thimerosal and pyrimetamine) were assayed in Aspergillus nidulans 

diploid strain P1 . The following chemicals proved to increase significantly the 

frequency o,f:~hole chromosome segregants : econazole (active dose range : 0 .1-0 .2 /lg/ml), 

chloral hydrate (6-10 mM), hydroquinone (1-3 mM), thiabendazole (12-40 mM), thimerosal 

(0 .035-0 .40 Ng/ml) . In the case of chloral hydrate, thiabendazole end thimerosal no 


4 


Notes



Notes concurrent increase in mitotic cross-overs was observed, suggesting the disturbance 

of chromosome segregation as primary genetic event . Conversely, in the case of hydroquinone 

the secondary origin of chromosome malsegregants was demonstrated, whereas 

additional assays are required to disclose the nature of mitotic segregants produced 

by econazole treatments . Finally, colchicine, diazepam, cadmium chloride and pyrimeta- 

mine were assayed in a wide range of doses with completely negative results . (Work 

partially granted by the E .E .C . contract n . EV4C-0044-I(a) ) . 

116 

THE DEVELOPMENT OF A PANEL OF HUMAN CELL LINES EXPRESSING SPECIFIC HUMAN 

CYTOCHROME P450 cDNAS 

C . L . Crespi and B . W . Penman, Gentest Corporation, Woburn, MA (USA) 

We have previously reported a development of human lymphoblastold cell lines which express 

tranfected human cytochrome P450IIA3 and microsomal epoxide hydrolase cDNA's . We have 

now isolated additional cDNA's encoding human cytochrome P4501IE1, P450IIC9 and P450IIIA4 

using oligonucleotide probes . The cDNA's were sized and the DNA sequence of the 5' end of 

the isolate was determined. The cDNA's had the following properties : 

P450 Form cDNA Size DNA Sequence HomologV Coding Sequence 

fIE1 1 .7 kb 97% 11 bp short 

11C9 1 .9 kb 100% 32 bp short 

IIIA4 2 .1 kb 99% Complete 

The IIE1 and IIC9 Isolates which did not contain complete coding sequences were completed 

by the addition of synthetic oligonucleotides. The complete cDNA's are being introduced into 

a pHEBo based expression vector system under the control of the herpes simplex virus thymidine 

kinase gene promoter and polyadenylation signal . Recombinant cell lines expressing these 

cytochrome P450 cDNA's are being constructed . These cell lines, in addition to cell lines 

expressing human cytochrome P450IIA3 and P4S01A1, should provide a useful panel of cell lines 

evaluating the ability of specific human cytochrome P450 forms to activate chemical mutagens . 

117 

ASSESSMENT OF THE MUTAGENIC EFFECT OF MATERNAL FACTORS ON HUMAN CHORICNIC VILLI BY 

MICRONUCLEUS TEST 

Y .Q . Cui, Z .W . Dong, S .B . Liu, S .C . Chang, Y . Wang, X .Y . Ji, National Research 

Institute for Family Planning, Beijing, People's Republic of China . 

Rapid determination of DNA damage by micronucleus test is well accepted . Animal bone 

marrow cells or human peripheral lymphocytes used in most studies could not directly 

reflect the influence of the mutagenic effect on the offsprings by environmental 

factors . Although the cytogenetic effect of human chorionic villi chromaomes are used 

in prenatal diagnosis, human chorionic villi sdcronucleus test to detect directly the 

mutagenic effect of environmental factors has not been reported in the literature . 

Direct determination of human chorionic villi sdcronuclei (CVlIIi) was established by 

us, to study the mutagenic effect of mother's age, gravidity, abortion history, contraception, 

smoking and drinking on the offsprings . Cross investigation and micronucleus 

test were used in 507 couples undergoing artificial abortion . Micronuclei were scored 

according to Countryman's standard . 2, 000 interphases were observed in each subject 

for CVMN frequency . Aresine transformation {aresine (Sqr(P)1) was used in transforming 

CVMN frequency :and the analysis of variance were used for statistics . No correlation 

between CVM4 frequency and mother's age, gravidity, gestation age, abortion history, 

and contraception was found . Neither smoking nor drinking habit was found among the 

women of this study . The CVlA1 frequency of husband smoking was 0 .76+0 .06%., of husband 

non-smoking-drinking was 0 .55t0 .06X., of husband drinking was 0 .57t0 .20X ., of husband 

smoking and drinking was 0 .79* 0 .08X . . There was a statistical difference in CVMN 

frequency between husband smoking and non-saaking . No significant difference was 

found between husband drinking and non-drinking . 

118 

METHYLAZOMETHANOL ACETATE (MAMoAC), A STABLE FORH OF THE ULTIMATE MUTAGEN FROM 

CYCASIN IS ACTIVATED BY PORCINE LIVER ESTERASE . Michael L . Cunningham and Timothy F . 

McMahon, Systemic Toxicology Branch, National Institutes of Health, NIEHS, 

Research Triangle Park, North Carolina, 27709 

Nethylazomethanol (HAH) is the short-lived toxic and carcinogenic aglycone of cycasin, 

a natural component of the cycad plant . These plants may be consumed by people in the 

South Pacific islands either as foodstuff or in medicinal preparations . Thg 

spontaneous decomposition of MAN to reactive methyl carbonium ions above 0 C makes 

the study of its mutagenic effects difficult . Hovever, we have used the stable ethyl 

50869 3554


ester of I41M, 1L1M acetate (MAMoAC) in combination with porcine liver esteraee Notes 

(Carboxylic-ester hydrolase ; EC 3 .1 .1 .1) to study the mutagenic activity of NAM . 

MAMoAC produces a dose-dependant increase in the mutation rate in Salmonella 

typhimurium His G46 at concentrations of 2 .5-30 micromoles/plate following 

preincubation at 30oC for 90 minutes . Addition of porcine liver esterase increased 

the mutation rate from 14 to 2200 and from 375 to 5800 revertants per plate at 2 .5 

and 5 .0 micromoles/plate, respectively . This increased mutagenicity was dependant on 

esterase concentration (optimum 63 micrograms protein/ml) . The preincubation time 

was also critical for expression of the mutagenicity of the MAMoAC/esterasa reaction . 

The dose-response curve was shifted to steeper slopes by increasing the preincubation 

time up to 90 minutes . Our results indicate that the MAMoAC/esterase system is a 

convenient model with which to study the mutagenicity of 14AM . 

119 

ANALYSIS OF MULTIPLE CONGENITAL ANOMALIES /ICPEMC/ 

Andrew Czeizel 

par men o uman Genetics and Teratology, National Institute of 

Hygiene, Budapest, Hungary 

Three indicator conditions of offspring : sentinel anomalies, Down 

syndrome and unidentified multiple oongenital anomalies /UIICAs/ are 

being evaluated within the Hungarian population-based surveillance 

of germinal mutations . Ul[CAs are possible indioators of germinal 

dominant gene and ohromosomal mutations . The component oongenital 

abnormalities of UMCAs were classified into 45 groups . These 

component congenital abnormalities were reduced to pairs A pair is 

a set of two independent component congenital abnormalities in index 

patients with two or more congenital abnormalities . Baseline figures 

of all component congenital abnormality pairs in 3,722 UMCAs were 

determined in the study period 1973-1982 . The observed annual data 

after 1982 were compared with expected occurrences based on baseline 

figures . This pair-wise evaluation of component congenital 

abnormalities within U11[CAs seems to be an adequate surveillance 

method to detect any time cluster of congenital abnormalit' pairs 

due to environmental factors including germinal mutagens . The 

Hungarian data will be presented concerning tpe Chernobyl nuclear 

power accident . 

120 

SJMAN MEIUPIC CHRObDSCMAL IIANW(£ IN ItffERCIIB MhIIES AE To ImCPIQI . 

T .V . Darmdaran an9 K .M . Marinuthu, Department of Genetics, P .(; .Institute of 

Basic Me icaT-Sciences, University of Madras,Taramani, Medras 600 113,MIA . 

lwenty one infertile males were studied using mitotic, meiotic and 

histological techniques . Syphilis (2), lynphogranulana venerum (2) and 

nonspecific chronic epididymo orchitis (17) were the various infections 

observed . Meiotic studies fran normal healthy men (11) formed the control 

data . Changes in the meiotic percentage cell dietribution pattern (MPM) t .es 

correlated with meiotic chromeanal anamolies . There was no change in WD 

in 2 patients, wtro had shawefl micronuclei of the spermatids and increased 

frequency of mitotic chrormeane aberrations . Altered MI (msiotic met.aphase 

I) and M II (meiotic metaphase II) values were noted in 2 patients who have 

sho.ed separation difficulties of MI and M II . Slight increase in SPM 

(Sbermatogonial metaphase) values were noted in two patients . Highly 

increased SPM value vss noticed with loss of architecture of bivalents and 

M II elements were saen in a patient . Thus, the results abtained so far 

irdicate that though the actual mrJde of action could not be fully undertood 

the fact that the meiotic studies fro :n normal healthy control mmles did not 

shar any of these ananaliess oonfizm the possible clastogenic and autagenic 

action of these microbes on meiotic system . 

121 

MATERNAL UPTAKE AND TRANSFER TO EGGS OF A PROMUTAGEN 

(CYCLOPHOSPHAMIDE) BY AN ESTUARINE FISH, CYPRINODON VAR/EQATUS. C.B . 

Daniels, D . McMillin and J .C . Means . Institute for Environmental Studies, Louisiana State 

University, Baton Rouge, LA 70803 . 

The cytogenetic and mutagenic properties of cyclophosphamide (CP) have been shown to 

be a function of its metabolism to 4-hydroxycyclophosphamide . We have Initiated research to 

determine the availability of cyclophosphamide to developing eggs and to characterize the 


43


Notes major DNA adducts formed In an estuarine fish, C. varlgQAtus. as the result of exposure to 

radioiabeled i14CJ cyclophosphamide . Mature females were allowed to swim In tx10-3M CP 

for 5d then placed In tanks of clean water along with a mature male . The female was allowed 

to depurate for a total of 5d then sacrificed and analyzed for radioactivity(body w/o ovaries 

and ovarian tissue) by counting on a Packard TRI-CARB 1500 iiquid scintillation counter . All 

eggs spawned during this period were collected and assayed for radioactivity . A subsample 

of the eggs collected were subjected to cetyltrimethylammonlum bromide for the Isolation of 

DNA (Murry and Thompson, 1980) and the major adducts formed characterized by high 

performance liquid chromatography (Benson and Garner, 1987) . Kinetic data for the uptake 

into adults and transfer of cyclophosphamide to eggs will be presented and contrasted to the 

kinetics of uptake of CP in eggs directly from aqueous media . Data on the molecular binding 

of cyclophosphamide to cellular DNA and chromosome aberration frequencies will also be 

presented . 


122 

COMBINED jjj VIVO DNA BINDING AND TUMOR DOSE-RESPONSE STUDIES TO INVESTIGATE THE 

MOLECULAR DOSIMETRY CONCEPT . R . Dashwood, P . Loveland . A . Fong, J . Hendricks and 

G . Bailey, Oregon State University, Corvallis, OR 97331 . (USA) . 

Swenberg et al . (Prog .exp .Tumor Res .11 :42,1987) have discussed the possibility of 

using " . the concentration of DNA adducts . . as a better exposure index for quantitative 

risk assessment than . .the chemical's exposure concentration" . To evaluate this 

molecular dosimetry concept, we have undertaken concomitant jD vivo DNA binding and 

tumor dose response (d-r) studies in rainbow trout . In one approach, -10 000 animals 

were exposed both to the carcinogen aflatoxin B1 (AFB1) in the dose-range 10-320 ppb 

in diet, and to 5 doae levels of a natural anti-carcinogen known to reduce AFB1-DNA 

binding in trout (indole-3-carbinol, I3C) . When each data-point from the 12 mo . tumor 

d-r analyses was plotted with its corresponding AFB1-DNA binding data-point (logit % 

tumor response vs (log) AFB1-DNA binding), a linear relationship was observed at each 

13C dose-level . At I3C doses


high incidence of tumors in sun-exposed skin. The defect In early steps of excision repair of XP cells Notes 

leads to hypermutability towards UV-mimicking agents . DNA from 8 XP tumors were screened for 

activated transforming genes using 3T3 transfection . In 2 skin tumors Isolated from a XP child, an 

activated N-M oncogene was detected . Synthetic oligonucieotide probes were used to characterize the 

mutation in the La8 gene. Both tumors were found to be mutated In the 61st N-Lal codon from gin to his . 

The mutation was accompanied by an Increase in the level of N-tg6, specific mRNA and in one 

transformant, by the alteration of the p21 protein . In the same tumors, c-mTv .c amplification and over 

transcription, and Ha-LSS gene rearrangement and amplification were also detected . Amplified and/or 

rearranged Ha-LaS gene sequences were detected in 10 other XP tumors . The normal skin fibroblasts 

from XP patients show normal pattern levels of N-L&I, c-1,Dy0 and Ha-1aS sequences . It Is proposed that 

the presence of several oncogene alterations In the same tumor could be due to the high amount of UV 

-induced DNA lesions found in the exposed skin cells, In the absence of efficient repair . An activated KI- 

L,U was detected in a new XP tumor which contains a mutation In the 12 th codon, probably due to 

replication errors at a thymine dimer resulting in a substitution of glycine by valine . We are at present 

analysing several XP tumors for ras p21 expression which together with screening by PCR 

amplification should help further identify the activated oncogenes in XP tumors . 

MECHANISMS OF INHIBITORS OF GENOTOXICITY . RELEVANCE IN PREVENTIVE MEDICINE . Silvio De Flora, 

institute of Hygiene and Preventive Medicine, University of Genoa, via Pastore 1 . 1-16132 Genoa (Italy) 

Inhibition of genotoxic effects in germ and somatic cells provides a fundamental strategy, complementary 

to the control of environmental and lifestyle risk factors, aiming at extending life expectancy and at 

preventing mutation-related pathologic conditions, first of all cancer . Moreover, some measures against 

mutagens and carcinogens counteract certain agents, like those causing oxidative damage to the cell, which 

are also involved in the pathogenesis of other chronic-degenerative diseases . Unfortunately, due to the 

double-edged nature of many physiologic protective factors and to the multiple biologic properties shared 

by several inhibitors, it is very difficult to stimulate the host defense machinery without inducing adverse 

effects . This renders mandatory not only to assess the efficacy and safety of putative inhibitors In various 

experimental systems, but even more to understand and elucidate their mechanisms . Genotoxicity can be 

inhibited in extracellular environments, either by hampering the uptake of mutagens, or by preventing 

their endogenous formation, or by deactivating them before penetration into metabolizing or target cells. 

Thereafter, genotoxicity can be inhibited by blocking reactive chemical species and by modulating 

intracellular mechanisms, such as cell replication, metabolic activation- and detoxification pathways, DNA 

functions and repair processes . At later stages, inhibitors can -act within initiated celis by preventing 

tumor promotion and/or progression by a variety of mechanisms . Certain inhibitors work through multiple 

mechanisms, which warrants a larger spectrum of efficacy . Moreover, inhibitors displaying complementary 

mechanisms or acting in different cell compartments can be conveniently associated for a combined 

chemoprevention. In any case, the exploitment of protective dietary factors and/or pharmacologic agents in 

humans is advisable only on the basis of careful risk-benefit analyses . 

De Flora, S . (Ed .), Role and Mechanisms of Inhibitors in Prevention of Mutation and Cancer, Mutation Re,t . 

202 : 277-446 (1988) . 

126 

STIIOY OF PROCEDURES FOR THE DESTRUCTION OF FOUR ALRYLATING AGENTS . 

p, Moo . M .P .t, M . Castagnero2 . M . Lagett and G . Dumknilt 

I l,boratoire de Microbiologie, FacultE de Pharmacie . 27 ilct Jean Moulin, 13385 

1Ir,rp~eille Cedex 5, France . 

^_ tnil' 1es Canc.tro¢Anea do 1'Environnement et Facteurs d'Hote, IARC, 150 fburs 

Aihr•rf-Thomas, 69372 Lyon Cedex 8, France . 

nestrnction procedures of dimeth,vl sulfate (DMS), diethyl sulfate (DES), methvl 

w1hsnFealfonate (!P]S) and ethyl methanesulfonate (EMS) were studied . Destructinn 

.nr; t-rformed by adding the alkylating agents at once to a flask containing 1N 

N,nH, IN NHaOH, IM Na2C03 and IN NazSOa . Determination of the alkylating ngeuts: 

•Inrinc the destruction process was performed by the derivation of p-nitrophenoxide 

and the resulting p-nitroanisole and p-nitrophenetole were separatetl hy high 

pprfnrm-mce liryuid chromatography . Mutagenic activity of the destruction prodacts 

,.•,e eva].uated by the Ames test using Salmonella tester strains TA 97, TA 98 . TA 

100 nn .l TA 102 . The kinetics of destruction followed an time-deirrrolvnt 

F-:prnenlinl relationship with all the solutions . Solutions of 1M Na280 . shnwvd eh• 

Iiiph-st ^nt,RCity of destruction of the four alkyknting agents . Half-time lit•es ~if 

nHS . neS, !PdS and EMS were 0 .12 ∎in, 1 .2F ∎in, 0 .60 ∎iu and 5 .26 ∎in respert i••-1• 

in IM NnzSn3 . No mutnqenic aotivity eould be detected following t .he .v .ml•l+•re 

d-stru••lion of the qlk,-lstiog n :;ents in 1M Na2S03 . Destrttction of lhese nikYlstinn 

agents in the workplace is discussed . 


45



Notes HETEROZYGOUS EFFECTS OF X-RAY-INDUCEO SPECIFIC LOCUS MUTATIONS RESULTING FROM 

GENE/POINT MUTATION AND MULTILOCUS DELETION IN THE dQ,-3 REGION OF ye(irgspQEicEass_ . 

F .J . de Serresa• L .K . Overtona and B .M . Sadlerb aCenter for Life Sciences and 

Toxicology• bCenter for Bioorganic Chemistry . Chemistry and Life Sciences . Research 

Triangle Institute . Research Triangle Park . NC 27709 (USA) 

Previous studies (de Serres, Mutation Res . Z)Q . 281 . 1989) on X-ray-induced 

p_d9D_ine-3 (gQm3) mutants induced in a two-component heterokaryon (H-12) of y . cr_ .a_ssa 

showed that they were not recessive and had heterozygous effects in terms of markedly 

reduced linear growth rates . H-12 is heterozygous for mutants at 3 closely linked 

loci, gQ-3A• ad-JQ and .n_ig-3 (nicotinamide-requiring) ; thus, X-ray-induced mutants in 

this region can be of 5 different genotypes : Sg-IA . i1o.-3@ . .4.Q~A AQ $@• 9~~94.Q 3_@ 

n_ic-Z, and g .d_-36 pic-2 . All 5 classes can result from multilocus deletion (g_d,=$+R)o 

gene/point mutations (_#d_3R) are usually restricted to the first 2 classes . Present 

studiQs were pQrformed to determine whether single-locus mutations of either type 

(ad-3R or ad-3`R) sho~v heterozygous effecIA . For example . qi(-~A.IR mutants were 

combined with an ad_3a mutant or an pd- @ mutant, and the lin ar growth r tes of 

the resulting forced dikaryons (aq-jA7~ + q~ .jBR, or aQ;~I$ + ~) were 

determined . Ad-3AIR utants showed more pConounced heterozygous effects in combination 

with an ad-381~ mutant thin an ad_-9oR mutant ; similar results were found with 

_Q-381R mutants . Only 1/30 aQ_3AR showed a heterozygous effect• in contrast to larger 

numbers of __d_-_~B R_~BR mutants . Mutants at the AA-3B locus show allelic complementation, 

and the level of heterozygous effect is higher among noncomplementing mutants than 

among complementing mutants with either nonpolarized or polarized patterns . 

EVALUATION OF THE EFFECTS 07 CHERNOBYL IN WESTERN EUROPE 

by p . ~E WALS, H . DOLE, F . BERTRAND, M .F . LECNAT 

EUROCAT Csntral Registry and Department of Epidemiology, 

Catholic University of Louvain, Brussels, Belgium . 

128 

Following the Chernobyl accident there was widespread public concern over the possible 

mutagenic and teratogenic effects of the radiological contamination in the 

countries of Western Europe . Assesament of the radiological exposure of the population 

in the countries of the EEC .as made by the V .II . National Radiological 

Protection Board indicating excess average effective doses to adult individuals in 

the first year ranging between 0 .2 pSv in Portugal and 130-300 Y8v in Nest-•Gesmany . 

Italy and Greece . Data from the EIIROCAT sussillance system covering 19 re=ions in 

12 countries were used to compare tha frequency of chromosomal syndromds and central 

nervous system malformations in exposed and non-eYposed fetusas . The prelisinary 

results do not indicate any increase but a small cluster 'of apina bifida in Odenae 

(Denmark) . Other studies were carried out in Finland, Sweden and Hungary with negative 

results concerning indicators of teratogenic or mutagenie effects . A cluster of 

Down syndrome cases in West-Berlin and of neural tube defects in Bursa, Turkey have 

been reported but relationship with the contamination is not established . Indueed 

abortions and postponement of conceptions vere two measutable psychological conasquences 

of the accident . 

EVALUATION OF THE EFFECTS OF CHERNOBYL IN WESTERN EUROPE 

by P . DE WALS, H . DOLE, F . BERTRAND, N .P. LECNAT 

EUROCAT Central Registry and Department of Epideaiology, 

Catholic University of Louvain, Brussels, Belgium . 

129 

Following the Chernobyl accident there was ridespread public concern over the possible 

autagenic and terstogenic effects of the radiological contamination in the 

countries of Western Europe . Assessment of the radiological exposure of the population 

in the countries of the EEC was made by the U .K . National Radiological 

Protection Board indicating excess average effective doses to adult individuals in 

the first year ranging between 0 .2 pSv in Portugal and 150-300 vSv in West-Germany . 

Italy and Greece . Data from the EUROCAT surveillance system covering 19 regions in 

12 countries were used to compare the frequency of chromosomal syndromes and central 

nervous system malformations in exposed and non-exposed fetuses . The prelimioary 

results do not indicate any increase but a small cluster of spina bifida in Odense 

(Denmark) . Other studies were carried out in Finland, Sweden and Hungary with negative 

results concerning indicatora of teratogenic or mutagenic effects . A cluster of 

Down syndrome cases in West-Berlin and of neural tube defects in Bursa, Turkey have 

been reported but relationship with the contamination is not established . Induced 

abortions and postponement of conceptions were two measurable psychological consequenees 

of the accident . 

50869 3558

130 

ANALYSIS OF MICHAEL2ADDITION-TYPJ COMPOUNDS Ij MQUSE LYMPHOMA CELLS . K .L . Dearfieldl, 

K . Harrington-Brock , C2L . Doerr , M .M . Moore . U .S . Environmental Protection Agency, 

Washington, DC 20460 ; E5vironmental Health Research and Testing, Inc ., Research 

Triangle Park, NC 27709 ; U .S . Environmental Protection Agency, Research Triangle Park, 

NC 27711 . 

The mutagenicity and clastogenicity of some Michael addition-type compounds including 

acrylamide, several acrylates and vinyl sulfones have been evaluated . These compounds 

have terminal double bonds that suggest they may be substrates for epoxide formation ; 

however, unlike epoxides, they are apparently inactive in the Salmonella assay :'In mammalian 

cells, they appear to act via a direct acting clastogenic mechanism . Our work 

with L5178Y mouse lymphoma cells demonstrates that acrylamide, several acrylates and 

methacrylates and vinyl sulfones (vinyl sulfone and methyl vinyl sulfone) without exogenous 

activation induce increases in the number of small colony tk-deficient mutants . 

This suggests a clastogenic mechanism which was confirmed by demonstrating increases in 

aberration and micronucleus frequencies . With exogenous metabolic activation systems 

(S9), the activity of these compounds may be altered, e .g . activity increased . in 

methacrylates (probably due to recruitment of epoxide formation) and decreased in 

acrylates (alternate detoxification pathways via S9 may decrease the potent response) . 

Since these compounds are known to deplete glutathione, phorone, a model glutathione 

depleter, will be examined . The results suggest that the direct-acting Michael addition 

mechanism may have activity relevant to genetic toxicity . Since acrylamide is a potent 

germ cell mutagen, this has importance in terms of heritable risk . (This is an abstract 

of a proposed presentation and does not necessarily reflect U .S . EPA policy .) 

131 

POTEtr"I'IAL EPA/OPP uUfAGENICITY TESTING REQUIREMENTS-GUIDELINES REVISION . K .L . Dearfield 

U .S . Environmental Protection Agency, Office of Pesticide Programs, Washington DC 20460 

The USEPA's Office of Pesticide Programs (OPP) is currently revising its Pesticide 

Assessment Guidelines - Subdivision F dealing with the requirements for mutagenicity 

testing . Currently, to register a pesticide with the OPP, mutagenicity testing must be 

performed in a minimum of three tests, one each in the categories for gene mutation, 

structural chromosomal aberrations and other genotoxic effects . The OPP's purposes for 

mutagenicity testing are to detect, with appropriate assay methods, the capacity of a 

chemical to alter genetic material in cells and to incorporate these findings in i) the 

assessment of heritable genetic alterations of concern to humans, ii) the 

weight-of-evidence approach for an oncogenicity classification of a chemical when 

bioassay results are available, end iii) the decision to trigger an oncogenicity study 

if such a study is conditionally required . The auggestAd revised mutagenicity testing 

begins with an initial battery : Salmonella, mouse lymphoma (tk locus, small and large 

colony counts), and in vivo cytogenetic assays . Other tests appropriate to the test 

chemical may be substituted upon discussion with the OPP . Testing to confirm results 

from the initial battery, such as with another in vivo organ or tissue, may be 

necessary . A weight-of-evidence approach will be used to reach a decision for the need 

for further testing . For a heritable concern, tests such as dominant lethal, 

cytogenetics in germ cells, specific locus and/or heritable translocation assays may be 

required . It should be recognized that these are potential revisions and have not 

undergone complete Agency and policy review . As such, they are not to be considered 

current EPA/OPP policy . 

132 OXIDATIVE DNA DAMAGE : REPAIR AND INDUCIBLE CELLULAR RESPONSES 

g . Dgmplg, D.S . Chen, J.T . Greenberg, J .D . Levin, P. Monach and S .C . Popoff, Department of 

Biochemistry and Molecular Biology, Harvard University, Cambridge, Mass, 02138 . 

Escherichia coil can respond to different kinds of oxidative stress by activating multicomponent 

systems that can prevent damages, regulate cell division, repair DNA lesions, and Increase NADPH 

production, as well as a large number of proteins of unknown function . These irxluctions are triggered 

by nontoxic levels of certain agents (e .g ., H202 or superoxide-generating agents) and confer elevated 

cellular resistance to higher levels of the same agents, and are therefore termed 'adaptive responses" 

to reflect their possible roles in evolution . We have been studying the complex response of E. coli to 

low levels of oxidative damage . Redox-cycling agents such as the quinone menadbne oonvey single 

electrons from NADPH or NADH to molecular oxygen, generating intracellular superoxide radical 021, 

which can then be converted to H202 by superoxide dismulase . Redox-cycling triggers the elevated 

synthesis of >70 polypeptides, of which about half are inducible by H202 directly . Consequently, E . 

coli possesses at least two distinct reponses to oxidative stress, one triggered by peroxide and at least 

part of the rest activated by superoxide . We have isolated menadione-resistant bacterial mutants that 



Notes


Notes express constitutively 10 proteins that are usually indudbie by Or . These proteins Include three 

known enzymes : Mn-containing superoxide dismutase (to destroy Or), giucose-B-phosphate 

dehydrogenase (to generate NADPH), and DNA endonuclease IV, which acts on a variety of oxidative 

damages In DNA . These soxR mutants express Increased resistance to a variety of oxidative agents, and 

these elevated resistances are only partly dependent on the sodA-encoded superoxide dismutase and the 

n/o-enooded endonuclease IV . Consequently, the functions of the soxR regulon contribute In multiple 

ways to cellular resistance, reflecting the complex modes of ceil damage caused by free radicals . 


133 

HAVE THE COCA LEAVES AND BASIC COCAINE CONSUMERS A HIGH FREQUENCY OF CHROMO 

SOME MUTATIONS . J . Descallleaux, M .L . Guwaro, L .A . Rodrrguez,M . Paredes, and M . Abarca. 

Lab . Genet . Hum ., Fae . Ctenclas Biol6gioas, UNMSM, P .O . Box 14-0010, L(ma-PerG . 

The number of coca leaves (CL) and basic cocaine (BC) oonsumers in the peruvian andeon region (CL) 

and the capital ctty intiabitants (BC) (s In constant raise, (CEDRO,1987), the wnrks reported In relation 

with the pharmacology effects of these hobitudes are oonhoverstals In the CL case, but all of 

them hove appointed the BC as a very toxlc drug to the human health . We have selected two samples 

of routinely consumers of CL and BC with their respective controls, In all of them, we have obtalned 

4-6 ml of periptieral b lood to m ade a lymphory te culture and measure the chromosome aberro - 

tions (CA) In first-dlvision metaphase oalls, and 5CE In seoond-diviston metaphase cells ; and exfolia 

te cells from the oral m uoosa In order to estimate the frequency of mteronuclsi . 

The results obtained In CL consumers showed a minor frequency of CA and SCE than the control - 

group suggesting that the CL hove on enti-mutagenle property that may be a consequence of t he 

high level of vitamine A(Pio6n-Rebtegu1,1976) knowing that this vitamine has on anti-mutogenle pro 

perty (Grubbs et a1 .,1977) . But the mieronuelel frequency was higher In CL consumers, In this relation 

it'is important to consider tha• the "ehoecheo" habitude (CL chswing) enolose the use of the 

cal (CoO) alcaline and caustic substance, that would be the responstble of the aberront nuelel . In 

the BC consumers, although our first results m ust be eonsidered as prei(minary, mainly because of the 

s mall number of patients e xamined, they are showing an Inerese frequency of CA and SCE In the con 

sumers than In the control group . 

MUTAGENICITY OF TWO GIUCOCORTIC0I0St DEXAYiETFWSONE AND PREDNISOLONE 

H .S . Dhillon and J .R . Singh, School of Life Sciences, Human Genetics Laboratory, 

Guru Nanak Dev University, Amritsar (India) 

Glucocorticoids are therapeutically very important as they possess strong 

antiallergic and immunusuppressive properties . Keeping in view the mechanism of 

action of these medicines which involves the interection of the drug with DNA 

via specific receptor proteins, the mutagenic propertisa of two glucocorticoids - 

Dexamethasone and Prednisolone - were investigated using Ames Salmonelly/S-9 test 

and host mediated assay with mice and Salmonella tester strains (TA97e, TA98, TA 

100 and TA1535) There was no significant increass or decre4se in the nueber of 

His+ revertests/plate either with or without S-9 mix at the doses rangIng from 

1 ug4to 1x10 ug/plate in the Ames test . Ho"r at higher doses (Sx10 ug and 

1x10 ug/plete) the growth and number of His revertants/plate were inhibited 

significantly . In the host mediated assay doses of the drugs tested were 1, 10 

and 100 mg/kg bw . No significant positive results were obtained with either of 

the drug, indicating that observed non-mutagenicity in the Salmonella straina is 

not because of the insufficient metabolic degradation of the glucocorticoids in 

the in vitro system but due to their inability to interact with the prokeryotic 

DNA . 

COMPARATIVE EFFICACY OF ASCORBIC ACID AND EXTRACT FROM 

PHYLLANTHUS Mii BLICA FRUIT IN MODIFYING METAL CLASTOGENICITY 

H .Dhir, S .Ghosh, Sharmila Ghosh, A .ohosh, A .K . Ghosh, S . Palit . 

G . Talukder, A . Sharma, Human Genetics Unit, Centre for Advanced 

Study Cell rx Chromosome Researeh, Department Botany, University 

of Calcutta, Calcutta 700019, India . 

134 

135 

Fruits of Ph 1 an hus emblica L . contain Vitamin C and are Used 

extensively n many me c na preparations of Ayurvedic and Unani 

systems of medicine . Aqueous extravts of the fruit were fed to 

laboratory mice for different periods to observe if the clasto- 'P 

50869 3560

genicity of well known cytotoxic metals like lead, cadmium, tin, 

cobalt, nickel, aluminum, barium and cesium could be modified by 

the extract . Oral gavage of the mice with the extract for 7 days 

prior to intraperitoneal injection of the metal salts, significantly 

reduced the frequencies of chromosomal aberrations, gaps and 

rearrangements induced by the metals when compared with control 

mice given the metal alone and not previously fed the extract . 

substitution of the fruit extract with an equivalent amount of 

ascorbic acid as that presebt in the extract, also reduced the 

frequency of chromosomal aberrations induced by the metals, but 

to a lesser extent than the fruit extract . On the other hand, 

following treatment with some of the higher doses of the metals 

used, ascorbic acid increased the clastogenic'effects . The greater 

efficacy of the •fr1dt, .Axtrdct can .be ii:LL'.lbuted to_ .the combined action . 

136 

REVISED GUIDELINES OF UK COMMITTEE ON MUTAGENICITY (COM) 1989 

DR DIGGLE AND DR FIELDER (DEPT HEALTH LONDON) 

The COM is an expert advisory Committee, chaired by Professor B Bridges, set up 

to advise UK Government Departments on the mutagenic risk of substances . Their first 

guidelines on testing (1982) have now been updated, a revised strategy being 

recommended . Initial studies (Stage I) consist of in vitro screening designed to 

ensure a high probability of detecting mutagenic poten-fia-T .7-7ests should be done to 

the best available protocols, and results confirmed 1n an lndependent experiment . Two 

tests are routinely required, a bacterial assay for gene mutation and a test for 

clastogenicity tn mammalian cells, except where human exposure is expected to be 

extensive or sustained and difficult to avoid, when a test for gene mutation in 

mammalian cells is also necessary . This may be followed by 1n vivo studies (Stage 

II) . It is not felt justifiable to use in vivo studies for gene-screening and 1f 

the in vitro tests are negative no further lesting would normally be required . The 

excep-tTon--Ts substances where relatively high exposure, or moderate but prolonged 

exposure, is anticipated . An assay for chromosome damage in bone marrow would then be 

recommended . This may also be required for substances mutagenic tn vitro, to see 

whether the activity may be expressed In vivo . If negative one or more further 

in vivo assays tn a different tissue Teg Ztver, gut) may give the necessary 

reassurance (in the light of other toxicological data)•that the substance Is unlikely 

to be genotoxic to man . The most appropriate test(s) need to be determined on a 

case-by-case basis . Stage III consists of in vivo tests for germ cell effect, and is 

only necessary when a risk assessment of herTl :abTe effects ts justified . 

137 

CHEMISTRY OF DNA ALKYLATION AND ARAIXYIATION . Anthony Dipple, BRI-Basic Research 

Program, NCI-Frederick Cancer Research Facility, Frederick, MD 21701 . 

Since the mutagenic properties of many carcinogens depend upon their metabolic 

conversion to chemically reactive metabolites, chemical reactivity can be considered 

to be the 'essence' of mutagenic/carcinogenic activity for these compounds . Agents 

with different chemical reactivities modify different sites on DNA bases . For 

example, simple alkylating agents preferentially modify the ring nitrogen at the 7position 

of guanine residues whereas alkylnitrosoureas modify both the 0s- and the 7position, 

and polycyclic aralkylating agents modify the exocyclic N=-site on guanine 

residues almost exclusively . In order to understand the basis for these changes in 

site selectivity with changes in reactivity of the agent, extensive studies of site 

specificity in guanosine modification by benzylating agents, which attack the 7-, 0'and 

N2-positions of guanosine, have been undertaken . More recently, an optically 

active benzylating agent, styrene oxide, has been examined so that the 

stereochemistry of various guanosine alkylation products gives insight into the 

differences in mechanism through which alkylation at different sites occurs . These 

investigations indicate that both the degree of ionic character in the reagent (i .e . 

the Ssl or Ss2 character] and the nature of the ionic intermediate (i .e . whether the 

charge on the reaction center is localized (hard) or dalocalized (soft)) determine 

the sites of alkylation on guanine residues . Research sponsored by the NCI, DHHS, 

under contract No . NO1-CO-74101 with Bionetics Research, Inc . 

138 

ANALYSIS OF MUTATION INDUCTION IN VIVO AND IN VITRO WITH AN SV40-BASED SHUTTLE 

VECTOR. K . Dixon, J . Hauser, M . Carty, N . Zernik, E . Roilides, J . Carr, and A . S . 

Levine, Section on Viruses and Cellular Biology, NICHD, NIH, Bethesda MD 20892 

We have used the SV40-basad shuttle vector, p2189, to study mechanisms of 

mutagenesis in mammalian cells . When the vector DNA is treated with either UV 



Notes



Notes radiation or benzo[a]pyrene diolepoxide and then allowed to replicate in CV-1 monkey 

cells, mutations are induced in the mutagenesis target gene (the bacterial auoF gene) 

of the vector . DNA sequence analysis of these mutations reveals that the two 

mutagens induce very different spectra of mutations . By comparing the mutational 

spectra with the spectra of damage induced by the two agents, we have been able to 

deduce cellular mutational mechanisms . Recently, we constructed a series of 

polyomavirus-based vectors that replicate in rodent cells to facilitate the analysis 

of the role that cellular DNA repair processes play in mutational specificity . With 

these vectors we have begun to analyse the specificity of UV mutagenesis in repairdeficient 

mouse cells . We have also studied mutagenesis in an in vitro DNA 

replication system . The p2189 vector can be completely replicated in this cell-free 

system in the presence of SV40 T-antigen . Undamaged vector DNA is replicated with 

high fidelity in this system . UV-damaged vector DNA is also replicated, but less 

efficiently . It appears that mutagenesis occurs as a consequence of replication of 

the UV-damaged templates in vitro . Preliminary results suggest that the spectra of 

mutations induced by UV in vitro resemble those induced in vivo . Thus, the in vitro 

system may prove to be particularly useful in the molecular analysis of mutation 

induction in mammalian cells . 

139 

STABLE EXPRESSION OF P450IIB1 AND P450IA1 CDNA IN V79 CHINESE HAMSTER 

CELLS APPLIED TO MUTAGENICITY TESTING 

J . Doehmer, S . Satish, H .R .Glatt, and F . Oesch 

Institut fUr Toxikologie, Johannes Gutenberg-Universit8t, 

D-6500 Mainz, FRG 

cDNA encoding P450IA1 and P450II81 were recombined with the SV 40 

eukaryotic vector . The recombinant plasmids were transferred into V79 

Chinese hamster cells by the calcium/phosphate co-precipitation 

procedure using the neomycin phosphotransferase gene as selective 

marker . Several cell clones were obtained . Cell clones were 

characterized by Southern-, Northern-, and Western-blotting . The 

enzymatic acttivity was determined using P450 specific substrates like 

7-pentoxyresorufin in the case of P450IIB1 and 7-ethoxycoumarin in the 

case of P4501A1 . Specific activities were found to be in the same range 

typical for uninduced liver . Cytotoxicity and mutagenicity studies 

revealed that P450 producing cells respond to the exposure of 

cyclophosphamide, benzo(a]pyrene, and trans-7,8-dihydroxy-7,8dihydrobenzo[a]pyrene 

in a dose dependent manner . The mutation rate 

also correlated with the different levels of activity contained in the 

various cell lines . So far, the enzymatic activity remained stable in 

these cell lines for more than one year . 

The project is supported by the Deutsche Forschungsgemeinschaft (SFB 

302 "Kontrollfaktoren der Tumorentstehung") . 

140 

EFFECT OF SIMULATED ACTIVATED SLUDGE TREATMENT USING BENCH-SCALE BIOREACTORS ON THE 

MUTAGENICITY OF MUNICIPAL WASTEWATER . J . U . Doerger, J . R . Meier, R . A . Dobbs and G . 

N . Stelma, U . S . Environmental Protection Agency, Cincinnati, Ohio, 45268 . 

Previous studies of mutagens in municipal wastewater have shown that following 

activated sludge treatment the level of extractable organic matter had decreased . 

However, the level of mutagenic activity had not decreased substantially with treatment . 

Two possible explanations for this finding were postulated : the bioreaction process 

increased the number or potency of the mutagens, or the process removed non-mutagenic 

compounds more efficiently than it removed autagens . In order to have a controlled 

system for comparing pre- and post-treatment samples, bench-scale bioreaction studies 

were performed . Primary effluent was inoculated with aerobic organisms from the aeration 

basin and incubated at 23°C with aeration for 24 hrs . The extracts were evaporated 

and redissolved in DMSO for subsequent autagenicity testing using the Ames Salmonella 

assay with tester strain TA98 with and without S9 activation . The results of the 

bench-scale bioreactor were consistent with data derived from treatment plant studies . 

Total mutagenic activity was not significantly changed with incubation ; however, the 

specific mutagenic activity (his+ revertants/mg) of post-incubation extract did increase 

approximately 50% . The weight of the extracts decreased 35% . Viable cell counts of 

the inoculated wastewater indicated 40-fold increase after the incubation . These data 

suggest that the compounds responsible for mutagenicity in a municipal wastewater are 

less biodegradable by activated sludge treatment than the bulk of the extracted compounds 

. Consequently, improved biological and/or chemical-physical processes may be 

required to remove the mutagens . (This abstract does not necessarily reflect EPA 

policy) . 

50869 3562

141 

CORRELATION BETWEEN CHROMOSOME ABERRATION FREQUENCY AND SMALL-COLONY TK-DEFICIENT 

MUTANT FREQUENCY IN L5178Y/TK+/- -3 .7 .2C CELLS. C .L . Doerrl and M .M . Moore2 . 

lEnvironmental Health Research and Testing, Inc ., Research Triangle Park, NC 27709 

USA ; 2U .S . Environmental Potection Agency, Research Triangle Park, NC 27711 USA . 

The L5178Y mouse lymphoma assay quantitates gene mutation at ths thymidine kinase 

locus . Karyotypic and molecular analyses of mutants have shown an association 

between small-colony phenotype and chromosomal events . We have bean evaluating the 

association of small-colony induction with gross aberration analysis to determine if 

the mouse lymphoma assay can be used directly to determine potential clastogenicity 

of test compounds . Based on data from 36 compounds, we find a clear association 

between clastogenicity and small-colony induction . There is, as expected, no simple 

mathematical correlation between the two endpoints . Many gross aberrations are 

incompatible with cell survival and colony formation, and some small-colony TK 

mutants are products of events which cannot be scored visually as chromosome 

aberrations when small changes in some essential DNA sequence(s) results in slow 

growth . Based on these analyses and previous studies, we fsel that the small-colony 

TK mutant frequency is the most useful measure of the clastogenicity of test 

compounds . It reflects cytogenetic events compatible with long-term cell viability, 

and these are the types of events important in human disease . (This is an abstract 

of a proposed presentation and does not necessarily reflect U .S . EPA policy .) 

142 

SULFOTRANSFERASE AND SULFATE LEVELS VARY THE ACTIVATION OF2-P.MIW-3-METHYLIMIDA7A(4,5- 

f)QUINOLINE (IQ) 

P . Dolara, M . Lodovici, P . Grassi 

Department of Pharmacology, University of Florence, Viale Morgagni, 65, Florence,Italy 

The in vitro binding of 3H-IQ to herring sperm DNA was measured in the presence of 

S9 and microsomes from Wistar rat livers . Both S9 and microsomes from control rats 

catalyzed DNA binding of IQ, and a considerable increase in binding was observed using 

livers from PCB treated animals . DNA binding was totally blocked by pentachlorophenol 

and 2,6-dichloronitrophenol, a specific inhibitor of sulfotransferaae . A mod- 

est increase in DNA binding was observed with 3'-phosphoadenosine-5'-phosphosulfate . 

Incubation of IQ in media with high sulfate concentrations increased DNA binding in 

vitro . An increase of IQ binding was also observed in vivo by varying sulfate pools 

in rats with the administration of sulfite in drlnking'water . The data demonstrate a 

role for sulfotransferase in the activation of IQ, and suggest a possible role of 

dietary sulfite in modulating the activation process of IQ . 

143 

DETERMINATION OF 4-AMINOBIPHENYL HEMOGLOBIN ADDUCTS AS A MEASURE OF ACTIVATION OF 

AROMATIC AMINES IN HUMANS 

P . Dolara, G . Moneti, M . Salvadori . 

Department of Pharmacology, University of Florence, V .le Morgagni, 65, Florence, Italy 

The levels of hemoglobin (Rb) adducts of 4-aminobiphenyl (4-ABP) in smokers are a 

function of the exposure to cigarette smoke and of the activation of aromatic amines 

to reactive metabolites . We measured the Hb adduct of 4-ABP in humans with massspectrometry, 

after hydrolysis of Rb and extraction with methylene chloride . Methy- 

lene chloride is preferable to hexane, used in published methods, because of better 

recovery of 4-ABP . The exposure to cigarette smoke was quantified by measuring the 

24 h excretion of a long-lived metabolite of nicotine (cotinine) . Cotinine levels are 

poorly correlated with self reported smoking habits . A good correlation was observed 

between 24 h cotinine urinary excretion and the levels of 4-ABP adducts to Hb . The 

levels of 4-ABP in non-smokers were never inferior to 50 pg/ g Hb 

. The correlation of 

cotinine urinary levels and 4-ABP adducts to Hb is defined by a line, the slope of 

which is a function of metabolic activation of aromatic amines 

. The method was applied 

to smoking controls and urinary cancer patients and gave a good estimate of the 

capability of different subjects to activate aromatic amines . 



Notes


Notes 


144 

COMPARATIVE STUDIES* ON THE GENOTORIC POTENTIAL OF SIDESTREAN SMOKE FROM CIGARETTES 

WHICH BURN OR ONLY HEAT TOBACCO . D . J . Doolittle, C . K . Lee, G . T . Burger and A . V . 

Hayes, R . J . Reynolds Tobacco Company, Bowman Gray Technical Center, Winston-Salem, 

NC 27102 

The in vitro genotoxic activity of sidestream cigarette smoke (SSCS) from cigarettes 

which heat but do not burn tobacco was compared to that of SSCS from cigarettes 

which burn tobacco . SSCS from five cigarettes were compared . Three of the 

cigarettes [the Kentucky reference research cigarette (1R4F), a commercially available 

ultra-low tar brand (ULT) and a commercially available ultra-low tar menthol 

brand (ULT-menthol)) burn tobacco while two of the cigarettes [a regular (TEST) and a 

menthol (TEST-menthol)] heat tobacco . SSCS from all cigarettes was collected by 

identical techniques, which involve collecting particulate matter on Cambridge filter 

pads and combining with the vapor-phase materials collected by bubbling the smoke 

through DHSO . All samples were simultaneously evaluated at identical concentrations 

in the test battery . SSCSs from 1R4F . ULT and ULT-menthol cigarettes were positive 

in the Ames bacterial mutation assay while TEST and TEST-menthol SSCS was negative . 

SSCS from 1R4F, ULT and ULT-menthol cigarettes was positive in the CHO-chromosome 

aberration assay and in the CHO-sister chromatid exchange assay while TEST and 

TEST-menthol SSCSa were negative in both assays . 1R4F, ULT and ULT-menthol SSCSs 

were weakly positive in inducing DNA repair in cultured rat hepatocytes while TEST 

and TEST-menthol SSCSs were devoid of activity in this assay . These results demonstrate 

that sidestream smoke from the TEST and TEST-menthol cigarettes was not 

genotoxic under conditions in which sidestream smoke from 1R4F, ULT and ULT-menthol 

cigarettes were genotoxic in a concentration-dependent manner . 

145 

THE EVOLUTION OF MUTATION RATES : PROSPECTS FOR ANTIMUTAGENESIS . 

John W. Drake, Laboratory of Molecular Genetics, National Institute of Environmental 

Health Sciences, Research Triangle Park, NC (USA) 

Arguments can be adduced for the operation of forces that would either increase or 

decrease mutation rates in the course of evolution . In microbial systems, an equilibrium 

might be established reflecting the advantages of higher mutation rates that generate 

adaptive mutations and the disadvantages of lower mutation rates that generate deleterious 

mutations . In more intensively sexual systems, the balance might be between the costs in 

time, materials and energy between reducing mutation rates and the costs of bearing the 

load of deleterious mutations . Classical experiments bear on both of these possibilities . It 

is also likely that antimutagenesis by either genetic modification or pharmaceutical intervention 

is a tenuous goal. This notion is superficially contradicted by diverse reports of 

antimutator mutations and antimutagenic chemicals, most of which are helpful in understanding 

mutational processes but are illusory in promising ways to lower mutation rates in 

genetically optimized animal and plant germlines or in higher primates . 

146 

Comparison of the clastogenic action of mutagens in the presence and 

absence of bromodeoxyuridine . 

Joachim H . Dresp 

Pharmaceutical Research, Department of Toxicology 

F . Hoffmann-La Roche i Co . Ltd, CH-4002 Basel, Switzerland 

The incorporation of the base analogon bromodeoxyuridine (BrdUrd) into 

replicating DNA allows a distinction between cells which are in their 

first or second division after initiation of the cell culture . 

Up to now the question whether or not the presence of BrdUrd influences 

the experimentally induced rate of chromosomal aberrations in human 

peripheral lymphocytes cannot be answered unequivocally . 

Experiments with different xenobiotics were carried out . The results 

illustrate the benefits and the disadvantages of the Brdurd-labelling 

technipue . 

50869 3564

147 

SEMPERVIRINE : A NON-MUTAGENIC ANTI-TUMOR ALKALOID FROM GELSEMIUM 

ELEGANS 

Du,. .Y .-X .' Wu, Z .-L .', Liang, W .-Z .', Chen, H .-H .•, Liang, X .-Re• 

Departments of Hygienee and Microblologye, Guangzhou Medical College, 

195 Dongfeng Rd ., Guangzhou 510182, The People's Republic of China . 

Gelsemium Elegans Benth, a medicinal herb grown in South China and 

used locally as a roborans for pigs and sheeps, is seveNly toxic to 

humans . Recently however, Sempervirine, an alkaloid extracted from 

Gelsemiun elegans has shown promise as an antitumor agent in cancers 

of the liver, esophagus and stomach . In this study we have examined 

the in vitro effects of sempervirlne on tumor cell lines A-549, 

PLC/PRF/2 and CNE-2 from lung, liver and nasopharynx respectively . At 

drug concentrations between 100 to 400 yg/ml a significant decrease in 

cell proliferation was observed for all three cell lines examined . 

Whereas no effect was observed for in vitro mutagenicity assays using 

bacterial strains TAs7, TAss, TAioo and TAio : at drug concentrations 

between 2 to 500 Ng/ml in the presence or absence of a S-9 fraction . 

Chromosome abberation assays with CHL cells exposed for 24 or 48 hours 

at five drug concentrations between 5 to 100 pg/ml showed no difference 

as compared to controls. The micronucleus test with NIH mice at 

dosages of 1/2LDso, 1/5LDso and 1/10LDso showed no statistical 

difference (P>0 .05) in the ratio of polychromatic erythrocytes to 

normocytic erythrocytes as compared to controls . 

148 

TRAHSGENIC ANIMALS EXPRESSING THE BACTERIAL 06ALKYLGAUNINE-DNA ALKYLTRANSlERASE 

GENE : A MODEL TO STUDY THE ROLE OF DNA REPAIR IN THE CARCINOGENBSIS OF N-NITROSO 

COMPOUNDS. L .L . Dumenco, D .W . Clapp, I .K . Lim, S . Kesssn, C . Donovan, 3 . Warman, 

N . Gorodetzkaya, J . Yun, T . Wagner, R .W . Hanson, S .L . Gerson . Ireland Cancer 

Center, Dept of Biochem and Med, Cleveland, OH, 44106, and Edison Biotech Center 

Ohio Univ Athens, OH, 457016 

The DNA repair protein 0 alkylgaunine-DNA alkyltransferase is a critical 

factor controlling tissue specific tumor induction following nitrosamine and 

nitrosourea exposure in animals . We designed a chimerio gene consisting of 340 bp 

of the promotor-regulatory region of the rat phosphoenolpyruvate carboxy kinass 

(GTP) (PEPCK) gene linked to the ads gane coding for the ~~} alkyltransferase . 

The PEPCK promotor is highly inducible in transganic animals by a high protein diet 

or serotonin treatment and is primarily expressed in kldney and liver . Using this 

chimeric gene, two haterozygous transgenic founder animals were obtained . 

Offspring had two-fold increased alkyltransferasa activity in liver and kidney and 

ada gene expression as confirmed by Northern analysis . The ~ gene was inducible 

in the liver and kidney by a high protein diet and/or serotonin . ?olloving 

induction, total alkyltransf.rase activity was increased five-fold above background 

in liver and at least two-fold in kidne~r . These transgenic mice will allow us to 

determine whether efficient repair of 0 alkylguanine-DNA adducts by high levels 

of alkyltransferase activity can decrease the tissue specific carcinogenicity of 

N-nitroso compounds . 

149 

EXPRESSION OF XRCC1 IN HUMAN TUMOR CELL LINES . 

E . Dunphy, R .R . Weichselbaum, L .H . Thompson and J .L . Schwartz, 

University of Chicago, Chicago, IL and Lawrence Livermore National 

Laboratory, Livermore, CA (USA) . 

XRCC1 is a gene whose expression complements the defect in the 

mutagen-sensitive Chinese hamster cell line EM9 . The XRCC1 gene product 

is believed to be involved in DNA single-strand break rejoining and SCE 

induction . Absence of this gene product confers sensitivity to 

ionizing radiation, monofunctional alkylating agents and halogenated 

pyrimidines . In this study, the expression of XRCC1 was examined in a 

variety of human tumor cell lines . These cell lines have widely 

different radiosensitivities which might, in part, be a function of 

xRCC1 expression . Twenty-five human squamous cell carcinomas and 

sarcomas were examined . The radiosensitivities (DO) ranged from about 

1 .0 Gy to 2 .7 Gy . The expression of XRCC1 was variable, being either 

normal (compared to nontransformed fibroblasts) or somewhat elevated . 

One cell line had a much reduced level of expression . There was no 

relation between expression of XRCC1 and radiosensitivity nor-between 

XRCC1 expression and the repair of DNA single-strand breaks (as 

measured by DNA alkaline elution in a subset of the 25 tumor cell 

lines) . Therefore, variations in the expression of XRCCl do not 

underlie differences in human tumor radiosensitivity or the repair of 

radiation-induced DNA damage . We are presently analyzing SCE frequency 

and monofunctional alkylating agent sensitivity in these lines . 



Notes



Notes ANEUPLOIDY DETECTION BY ANALYSIS OF INTERPHASE NUCLEI USING CHROMOSOMESPECIFIC DNA 

PROBES . D .A. Eastmond and D. Pinkel, Lawrence Livermore National Laboratory . LNermore, CA (USA) 

Fluorescence In situ hybridization with probes for chromosome-specifio DNA sequences (usually 

centromeric) results In brightly fluorescent spots In Interphase nuclei at the position of the target DNA 

sequences . The number of spots In a nucleus therefore corresponds to the number of copies of the 

chromosome that are present . Eight thousand Interphase nuclei from untreated 72 hr lymphooyte 

cultures were examined following hybridization with probes for chromosomes 1, 7 . 9 or 17 . The 

frequencies of nuclei containing 0, 1, 2, 3 and 4 hybridlzation spots were 0 .0024, 0.079, 0.915, 

0 .0024 and 0 .001, respectively . Based on these frequencies, scoring 1000-2000 cells should allow 

detection of aneuplold cells with a 0 .013 frequency of hyperdiploldy or a 0 .11 frequency of hypodiploidy 

for a specific chromosome of interest (a - 0.05, S- 0.80) . This difference In test sensitivity Is related 

to the higher frequency of cells with one apparent spot . A comparison of the ratio of spot to nuclear area 

in the two dimensional Images used for this analysis Indicates that an overlap of the two spots probably 

accounts for the high frequency of apparent monosomy observed in normal cells . Using a chromosome 9 

probe, colchicine (0 .1 µM), vincristine (0.06 µM) and DES (30 µM) treatments were shown to Induce 

hyperdipiokiy for this chromosome at frequencies of 0 .05, 0.02 and 0 .36, respectively. The use of this 

technique should facilitate a more rapid Identification of aneupioidy-Inducing environmental agents . 

Work performed under the auspices of the U .S . Department of Energy by the Lawrence Livermore 

National Laboratory under contract number W-7405-ENCi-48 with additional support from the 

Alexander Hollaender Postdoctoral Fellowship (D .A.E.) . 

ANTIMUTAGENIC ACTIVITY OF VEGETABLES HARVESTED DURING DIFFERENT CONDITIONS . 

J . Ebata, and T . Inoue, Osaka City University, Osaka (JAPAN) 

151 

By employing streptomycin-dependent strain SD100 of Salmonella typhtmurtum (T .Kada 

K . Aoki, and T . Sugimura,Envtronmental Mutagenesis 5,pp .9-15(1983)),the antimutagenicity 

of homogenates of fresh vegetables has been examined for AF2 . In this paper, 

the influences of the growing conditions on the antimutagenicity of 5 kinds of 

vegetables cultured in open fields of Agricultural Experiment Station on Nara Prefecture 

were studied . The antimutagenic activities were compared with vegetables 

having suitable maturity and nearly of the same size . The antimutagenicity of 

spinach, 8ptnacia oleracea L .cv .'Orion' increased summer< autumn< winter . The activity 

of cabbage, Brassica oleracea L . var . capltata L . cv .'Shutoku' was more potent 

during the autumn harvest than in summer and more in the outer layer than the inner 

for both harvests . In the case of eggplants, Solanun aelnyena L . cv .'Senryou', the 

maximum activity was found in September during harvest time (July - October) . The 

activity of cucumbers, Cucumis sativus L . cv .'Ryokusui' and 'Sachikaze' increased in 

the order of low

153 

Clastogenic effects of ethyl-diamminedichbroplatinum in mice . 

1 .Induction of ehromosomal aberration in Bone marrow cells . 

A .E1-Tarras , A,N,Sharaf and N,A .Abdalla, 

Dept . of Genetics, Fac . of Agric ., Cairo University, Cairo, Egypt . 

The in vivo clastogenic effect of the anticancer drug ethyldiamminedich) 

.roplatinum was investigated as chromosomal abersations 

(CA) in the bone marrow cells of mice . Studies was carried out on F1 

mice of the cross C3HX101 aged 12-14 weeks and weighted 25-30 gm . 

The drug concentrations were 1,2 .5, 5 .0 .10,15 and 20 mg/kg at 

duration time of 6,12 and 18 hours and the number of cells tested per 

group is 500 cells . Results showed that 10 mg/kg dose was lethal 

between 3 and 6 days while 15 and 20 mg/kg doses lethality oooured 

within 24 hours . The percentage of cells containing CA without gap 

for the doses 1,2,5 and 5 mg for 12 hours were 2,25%, 5 .25% and 8 .8% 

respectively. However at 5 mg dose the CA persentage was 4 .2 at 6 

hour interval while it was 2 .2, at 18 hours . These results indicate 

that the clastogenicity of ethyl-platinum depends upon the stage of 

cell cycle (S .p?iase) and it had a dose-dependant . Calculting the 

doubling dose (DD) for clostogenic effects of ethyl-platinum in BM 

was 0 .5 mg/kg . (Y a 0 .8 + 1 .6 D) . 

154 

POTENTIATING EFFECTS OF CAFFEINE ON MUTAGENICITY AND TERATOGENICITY 

OF ALKYLATING AGENTS . M .M . E1-Zawahri and L .K . A1-Ghaith, Department of 

Zoology, Faculty of Science, Kuwait University (Kuwait) . 

A set of experiments was carried out to study the effect of caffeine 

on the mutagenic and carcinogenic activities of EMS and MMS . Males 

of D. melanogaster were fed or injected with 0 .5% caPfeine in sa11ne~~ 

before or after their treatment with a solution of 0 .1 mM EMS or M`15 

in 10% sucrose for a period of 48h . The effects studied were dominant 

lethals and sex-linked recessive lethals . Pre-treatment with caffeine 

injection revealed a significant increase in the frequency of dominant 

lethals but not of sex-linked recessive lethals induced by EMS or MMS . 

Post-treatment of males with caffeine injection did not alter the frequency 

of either of the two types of lethal mutations induced by MMS 

or EMS . uhen 0-10h old males emerged from lardae reared on agar containing 

0 .5% caffeine were treated with a solution of 0 .1 mM EMS or MMS in 

10% sucrose, the frequencies of both dominant- lethals and sex-linked 

recessive lethals significantly increased than of those untreated with 

caffeine . These results indicate that caffeine may inhibit the repair 

mechanism(s) of genetic damage induced by alkylating mutagens and this 

may increase the mutagenic and teratogenic potentialities of such chemical 

agents . 

155 

AUTOMATION OF SCREENING ASSAYS FOR DNA DAMAGING AGENTS WHICH INDUCE THE SOS 

RESPONSE . R .K . Elespuru . Basic Research Program, NCI-Frederick Cancer 

Research Facility, Frederick, MD 21701 . 

The induction of the SOS response in E . coli is a physiological response to 

DNA damage in which 20 or more genes are turned on . Unlike single gene 

mutation, SOS induction is not a rare event and may be sean to occur in the 

majority of an induced population . SOS induction may be monitored biochemically, 

therefore, in a matter of hours after the event . Monitoring of 

SOS-inducible genes has been facilitated by the fusion of these genes to jME, 

the product of which, B-galactosidase, is expressed upon induction and is 

easily monitored . Among SOS-inducible genes which have been fused to JM7, 

and used for screening assays are lambda phage, &U and yQy . Two types of 

colorimetric substrate-cleavage assays for B-galactosidase have been developed 

which may be automated for screening purposes . One uses a soluble substrate ; 

a miniaturied version of this quantitative assay may be performed in a∎ingle 

microtiter well and may be automated using dispensers and plate readers . The 

other, a spot test assay, uses a substrate generating an insoluble reaction 

product ; this assay may be run like a semi-quantitative •dot blot• on petri 

dishes to a dilution endpoint . For screening, several hundred samples may be 

processed simultaneously on 243mm bioassay plates containing lawns of bacteria 

and activating enzymes . Research sponsored by the National Cancer Institute . 

DHHS, under contract No . NO1-CO-74101 with Bionetics Research, Inc . 



Notes



Notes ALTERATIONS IN l41NG-INDUCED MUTATIONS AT SPECIFIC LOCI IN E• COLI i1U3610 UNDER 

CONDITIONS OF THE "ADAPTIVE RESPONSE" . R .K . Elespuru and L .L . Stupar . Basic 

Research Program, NCI-Frederick Cancer Research Facillty, Frederick, MD 21701 . 

156 

Treatment of EU coli with low concentrations of alkylating agents results in the 

induction of the "adaptive response", an alkylation damage-specific DNA repair 

response . Increased levels of two enzyses, an 0s-alkylguanine alkyltransferase, and 

a 3-aethyladenlne glycosylase, are associated with this response . Among guanine 

lesions, the transferase is specific for, and removes stoichiometrically, alkyl 

groups at the 0s position . We have exmined the spectrum of mutations lnduced by 

N-methyl-N'-nitro-N-nitrosoguanidine (NNNG) at 8 monitorable sites in E . coli IJU3610 

under normal and adaptive conditions . The level of mutations at three monitorabie 

GC-AT transition sites was reduced by 90-95% under adaptive conditions, implicating 

the 0s-methylguanlne lesion as the causative agent in the generation of these 

mutations . A substantial loss of mutants was also observed at two putative AT•GC 

transition sites, consistent with the existence of 04-aethylthyaine as a premutatlonal 

lesion at these sites (0'-aethylthyaine has been reported to be a 

substrate of the transferase) . Little or no change was observed in mutation 

frequency at two TA+AT transversion sites . Increases at these sites might have been 

expected via glycosylase removal of 3-aethyladenine lesions and generation of 

apurinic sites . However, unlike the transferasa, th.re is a high constitutive level 

of 3-MA glycosylase in normal E . coli . Action of the induced enzyme could have been 

masked by the constitutive activity . Research sponsored by National Cancer 

Institute, DHHS, under contract No . NO1-CO-74101 with Bionetics Research, Inc . 

157 

SOLl181LQATION OF PARTICULATE CHROMIUM COMPOIlnIDS AND tTS RELEVANCE TO CYTOTOXICITY 

AND MORPHOLOGICAL TRANSFORMATION OF SYRIAN FNMSTER EMBRYO (SHE) CELLS. 

Z . Elias, O . Poirot, M .C . DaniBre, F. Terzetp, O . Schneider and F . Baruthio, I .N .R .S ., 

54501 Vandoeuvre (France) 

From previous experiments concerning water-Insoluble or poorly soluble Cr(VI) compounds, 

we have found differences In cytotoxiclty and transforming potency among some of them . In the 

present study we examine the possible relevance of the extracellular solubilization and 

intracellular level of Cr accumulation to cytotoxicity end morphological transformation of SHE 

cells Induced by particulate Cr compounds . Ca, Sr, Zn and Pb chromatea, of inedium, slight and 

scarcely water solubilities, were tested . In two parallel experiments, the cells were treated with 

supernatants or with suspensions of the Cr compounds . The cloning efficiency and the 

transformation frequency for each compound were determined after 7 days of exposure . 

Measurements of Cr In complete medium alone . In cell culture conditions and In counted cells were 

made by electrothermal atomic absorption spectrometry . The results showed that : (1) Incubation 

In cell culture conditions significantly increased the solubilization process ; (2) Intracellular Cr 

concentration was directly related to Cr treatment concentration ; (3) oytotoxidty was dependent 

on the extracellular solubillzed Cr ; (4) transformation frequency induced by Ca, Sr and Zn 

chromates correlated to the intracetlular soluble Cr concentration ; (5) Pb=• lons could play a 

role In transforming activity of Pb chromate . The results suggest that the oytotoxicity and 

transformation are distinct processes and depend, among other factors, on the stte and the kinetics 

of particle dissolution . 

158 

EVALUATION OF THE BIOLUMINESCENCE ASSAYS AS SCREENS FOR GENOTOXIC CHEMICALS . 

Eugene Elmore, NSI Technology Services Corporation, P .O . Box 12313, Research Triangle 

Park, North Carolina 27709 

The need for rapid and cost efficient screens for muteyens and other toxicants has 

increased dramatically over the past few years . This increase is due In part chemical 

manufacturing and the need for monitoring of effluents and clean up activities at hazardous 

waste sites . The bioluminescence test was first proposed for screening 

genotoxic agents by Ulitzur et al . (Mutat . Res . 74, 113-124, 1980) . Bioluminsscence 

tests measure the ability of the test chemicals to restore luminescence to dark 

mutants of various Photobacterium species, probably by derepression of the luminescence 

operon . A variety of chemicals with known mutagenic or carcinogenic activity 

have been evaluated and the bioluminescence assay has been shown to be responsive to 

direct mutagens lncluding point and frameshift mutayens, DNA-damsplny agents, DNAintercalating 

agents, and DNA synthesis Inhibitors . The results correlate very well 

with the published data obtained with the Ames assay . The results of a coded validation 

study using chemicals provided by the National Toxicology Program In the 

Microbics Mutatox11 Assay, which uses dark mutants of the luminous bacteria, P . phosphoraeum, 

will be reviewed .


Notes 

VALUE-OF-INFORMATION ANALYSIS OF TESTING STRATEGIES . F .K . Ennever, H .S . Rosenkranz, 

L .B . Lave, and G .S . Omenn, Case Western Reserve University . Cleveland . OH (USA), 

Carnegie-Mellon University . Pittsburgh . PA (USA), and University of Washington . 

Seattle . WA (USA) . 

The goal of reducing the incidence of cancers caused by environmental exposures 

requires strategies for identifying hazards among >50.000 chemicals in commerce and 

industry . Our value-of-information framework analyzes strategies for classifying 

chemicals as human carcinogens or non-carcinogens . Any classification will have false 

negatives (FN) : falsely exonerating a human carcinogen ; false positives (FP) ; falsely 

indicting a human non-carcinogen ; true negatives ; and true positives . One extreme 

strategy treats all chemicals as non-carcinogena, as existing chemicals generally are 

treated ; FP = 0 and FN = c, where c is the proportion of carcinogens among those 

chemicals . A second extreme strategy treats all chemicals as carcinogens ; PN - 0 and 

FP = 1 - c . A third strategy classifies chemicals on the basis of results of 

structure-activity analyses, short-term tests, and/or rodent cancer bioassays ; PN - 

(1 - p)c, where p is the weighted sensitivity of the tests, and PP =(1 - q)(l - c), 

where q is the weighted specificity of the tests . Using reasonable estimates of c, p, 

q, the cost of testing, and the societal costs of PN (needless cancers) and FP (loss 

of the use of a chemical), our model has shown that in many cases the lifetime rodent 

bioassay is not the most cost-effective way to classify chemicals as human carcinogens 

or non-carcinogens . Our most recent work has focused on the influence of uncertainty 

in parameter estimation and on deriving a sequential testing strategy with decision 

rules for when to classify a chemical without further testing . 

160 

COVALENT BINDING TO MICROTUBULAR PROTEINS AS A POSSIBLE CAUSE OF THE ANEUPLOIDY AND 

MICRONUCLEUS FORMATION INDUCED BY CARCINOGENIC ESTROGENS AND OTHER CARCINOGENS . 

B . Epe, U .H . Harttig, D . Schiffmann, and M. Metzler, Institute of Toxicology, University 

of Wiirzburg, Versbacher Strasse 9, D-8700 Wurzburg, Fad . Rep . Germany . 

Certain estrogens, e .g . diethylstilbestrol (DES), transform Syrian hamster embryo 

fibroblasts neoplastically in vitro without producing detectable DNA damage or structural 

chromosomal abnormalities . Instead, induction of aneuploidy and micronucleus formation 

was observed and was associated with cell transformation . In order to elucidate 

the biochemical mechanisms of aneuploidy induction, we have studied the interaction of 

several radioactively labeled estrogens and their metabolites with tubulin in a cellfree 

system . We observed highly specific covalent bindiijg to the carboxy terminal 

domain of /1-tubulin of those estrogens which undergo peroxidase-mediated quinone formation 

(DES, indenestrol A and the catechol estrogens 2-hydroxyestradiol and 2-hydroxy- 

17a-ethinylestradiol) . Estrogens which do not form peroxidative quinone metabolites 

(hexestrol, estradiol, 17K-ethinylestradiol) did not bind covalently . Covalent binding 

was not inhibited by sulfhydryl reagents nor by colchicine, podophyllotoxin, taxol or 

vinblastine . Specific covalent binding to tubulin was also obtained with hydroquinone, 

a major metabolite of the non-mutagenic carcinogen benzene, upon peroxidase-mediated 

oxidation . Electron microscopy of the microtubulee formed from tubulin coupled to DES 

or hydroquinone shoved clear abnormalities indicating that covalent tubulin binding 

has functional consequences . Therefore, we propose covalent binding to tubulin as an 

early biochemical lesion in the mechanism of aneuploidy induction and neoplastlc cell 

transformation by non-DNA-damaging carcinogens . 

Supported by Deutsche Forschungsgemeinschaft and BMJFFG . 

161 

SELECTION OF AN EAPERIMENTAI. PROTOCOL FOR SCREENING TEST AGENTS IN THE MOUSE BONE 

MARROW MICRONUCLEUS TEST . G .L . Erexsonl, J .L . Hustonl*, R .M . Boehml*, D . Gulati2, 

and M .D . Shelby3 . lEnvironmental Health Rea . 6 Testing, Inc ., P .O . Box 12199, RTP, NC 

27709 and 22514 Regency Road, Lexington, KY 40503 ; 3NIERS . NTP, Box 12874, RTP, NC . 

Due to methodological variations in the mouse bone marrow micronucleus (!D1) test ; 

one universal treatment protocol for in vivo chemical exposure is desirable . A common 

protocol among laboratories would simplify comparison of MN data . Mitomycin C(14/C) 

and 7 .12-dimethylbenzanthracena (DMBA) were tested i .p . at doses of 0, 0 .5 . 1, and 1 .3 

mg MMC/kg and 0, 25 . 50, and 100 mg DMBA/kg using three different protocols . Male 

B6C3F1 mice (9 to 14 weeks of age, 5 mice/dose) were treated with either (1) one 

exposure with harvests for bone marrow polychromatic erythrocytes (PCEs) at 24, 48, or 

72 h post-treatment ; (2) two exposures separated by 24 h with harvests at 24 or 48 h 

after the second treatment ; or (3) three exposures separated by 24 h with one harvest 

time at 24 h after the third treatment . For comparison, DMBA was administered also by 

gavage but only for the three-treatment protocol at the same doses used for the 1 .p . 


57


Notes injections (0, 25, 50, and 100 mg/kg) . Bone marrow smears were stained in acridine 

orange (pH - 7 .4) and 2000 PCEs/souss were scored for NN-containing PCEs . The percent 

PCEs was determined by counting 200 consecutive PCEs and normochromatics . Statistical 

analyeas revealed that the three-treatment protocol vas superior . Therefore, this 

protocol was selected by NIP for use in in vivo rodent bone marrow MN testing . 

Additional experiments were done using this protocol to select a positive control dose 

for both MMC and DNBA to be used in testing coded compounds . The positive control 


doses chosen are 0 .2 mg l4lC/kg and 12 .5 mg DNSA/kg which lnduce about 8!Q4-PCEs/3000 

PCEs . .[This research was supported by a contract fro's NTP, /NOI-ES-85208) . 

162 

DEVELOPMENTAL AND GENETIC TOXICITY OF SHORT-CH4IN CARBOXYLIC ACIDS . 

A . Esposito, G . Corsale, G .G . Giordano, and 0 . Pagano 

Istituto Nazionale Tumori, 1-80131 Naples, Italy 

The genetic and developmental toxicity of acidic pollutants was reported previously 

(Pagano at al ., 1985a,b) ; a weak acid-induced teratogenic action was reported by Nau & 

Scott (1986) on mammalian embryos, whereas the amidic analogues tested were ineffec- 

tive on embryogenesis . A series of short-chain carboxylic acids (as sodium propionate, 

malonate, and valproate), and their amidic analogues have been investigated for their 

action on sea urchin (P .lividus) early development and fertilizatlon . Embryos were exposed 

to acids (or amides) at levels ranging from 10-'M to 10-'M, without any detect- 

able shift of medium pH ; the endpoint consisted of changes in the frequencies of : 

a) developmental defects, and b) cytogenetic abnormalities . Sperm pretreatment experi- 

ments led to the following outcomes : 1 . changes in fertilizstion success ; ii . alte :a- 

tions of offspring quality, including mortality, developmental defects, and cytogenet- 

ic abnormalities . The results showed that the acids exerted developmental toxicity at 

levels ranging from 10-'to l0-°M, whereas their amidic analogues were ineffective up 

to 10-= M . Cytogenetic analysis and offspring quality confirmed acid-induced genetic 

damage, as reported previously . The data provide further evidence for the role of 

acidic pollutants and drugs in affecting cell division and differentiation . (Supported 

by the Italian Ministry of Health) . 

(;ESiETIC REST FOR 7[PM P%WI'E17S ATID COCAACIIIO(04.S 

Rudolf Fahrig, Department of Genetics, Frauntafer-Institut f(ir Tbotikologie ia ;d 

Aerosolforschung, Ha:ne%rer 61, F .R. Gezmany 

163 

Escpariments with yeast and with mioe show that tumor prcmoters and cocarcinogens are 

genetically active when given in eombinatien with a nutagen . The activity is indeperr 

dent of the nutagen/carcinogen used but specific for a given eocarcinogen or tumor 

praroter . 4fiere are striking similarities between the eacurrenoe of specific genetic 

effects and specific effects in carcinogenicity tests : 

1 . Cocarcinogens were ornutagenic . 

2 . 2lanor promoters which are anticarcinogenic if given simultaneously with the carcinogen 

were oorecaabinogenic and antinutagenio . 

3 . Rtiamr promoters which can bA transforns+d into eocur.irwgcets reverted their genetic 

effects fsrom corecambinogenicity to oortutagenicity upon metabolic activatien . 

4 . Substances which are t:mor promoters as well as cocarcinogens were also comecaebinogens 

and cenutagens . 

The hypothesis presented offers plausible explanatiens for the meny divergent effects 

of these substances in carcinogenicity tests . It therefare seems desirable to establish 

the test procedure deacribed as stazt-tean tests for turor promoters and eocancisrogens 

. 

MULTIPLE ENOPOINT MJTATIONAL ANALYSIS IN TFE MOUSE 

Jack Favor, GSF-Institut filr Slugetiergenetik, D-8042 Neuherberg, Germany, F .R . 

164 

Two main classes of mutational event from the wildtype allele are possible, loss 

or gain type mutations . The type of mutational event is important in determining the 

mutation rate observed and the effects of such mutant alleles when they occur in a 

diploid, randomly mating population. A loss event represents the loss of functional 

gene product . Such a mutation may result from a wide spectrum of DNA alterations 

ranging from deletion to point mutation, and should have no phenotypic effect when 

occurring as a heterozygote . A gain type mutational event represents an abnormally 

functioning gene product, is likely due to a defective gene product or a misregulated 

50869 3570 

t


normal gene product, and would result in an altered phenotype when occurrirg as a Notes 

heterozygote . Comparative mutagenicity data in the mouse are available for four 

genetic endpoints in which recovered mutations are genetically confirmed : recessive 

mutations at seven specific loci, dominant cataract alleles, enzyme electrophoretic 

variants and enzyme activity alleles . Results indicate the mutation rate to be an 

order of magnitude higher for those genetic endpoints which screen for loss events 

than for those genetic endpoints which screen for altered gene products . Further, the 

ratio gain/loss mutational yield is increased for ENU mutagenic treatment as compared 

to radiation, which is due to a shift in the spectrum of DNA alterations to point 

mutations as compared to mainly deletions by radiation . Thus, the observed 

sensitivity to mutation induction is dependent upon the type of mutational event 

screened as well as the type of mutagenic treatment employed . 

165 

THE ROLE OF PHOTOSYNTHESIS IN PROMUTAGEN ACTIVATION BY PLANT SYSTEMS . G . Fedorvicz, 

J . Day, G . J . Gentile and J . M . Gentile . Hope College, Holland, MI ., 49423 (USA) . 

We investigated the ability of photosynthetic and non-photosynthetic cotton 

suspension cell cultures for the ability to activate 2-aminofluorine (2AF) and a 

contaminant of 4-nitro-o-phenylenediamine (NOPX) into forms mutagenic to Salmonella 

tyhpimurium using the plant cell/microbe coincubation assay . The activation potential 

of each cell line was compared under varying light conditions both in the presence and 

absence of a potent inhibitor of photosynthesis (3-(3,4-dichlorophenyl)-1,1-demethylurea 

(DCMU) and as a function of the plant-cell growth cycle . NOPX was activated 

to the same degree by both cell lines under both light and dark regimes, and late-log 

phase cultures proved most effective for activation . DCMU, which is not mutagenic to 

Salmonella, had no effect on NOPX activation by either cell line under any test 

conditions . Experiments with 2AF indicated that this compound was preferentially 

activated by non-photosynthetic cells which were harvested from early to mid-logphase 

of growth, independent of DCMU treatment . In general, photosynthetic cells 

proved non-responsive under all treatment conditions . We are continuing to intestigate 

the activation potential of our photosynthetic cell line under more stringent 

photosynthetic-inhibitory conditions . If is feasible that calls which are actively 

photosynthesizing, or cells that maintain an intact photosynthetic apparatus, may 

not rely on certain enzymes complexes for general metabolic functions (e .g .,P450related 

enzymes) . Therefore, substrates which require such enzyme complexes for 

activation (2AF) would not be metabolized while substrate+e which require other enzyme 

complexes (NOPX) would be activated by these cells . Support from NSF Grt .BB5-8712566 . 

166 

MUTAGENICITY OF BURNT GUN PROPELLANTS. J.S . Felton, P. Lewis, M .G . Knize, 

and G . Millerl, Biomedical Sciences Division and lHazards Control Dept ., Lawrence Livermore 

National Laboratory, P .O . Box 5507, Livermore, CA 94550 . 

The use of the Ames/Salmonella assay as a workplace monitoring method is a long-standingpractxx at 

LLNL. This practice has led to the discovery of very mutagenic soot in and around a 4 inch test gu 

Examination of a rag used to clean the barrel of the gun revealed 87,000 TA98 (+S9) revertants/30 cm~ 

of the cloth. Analysis of the residue in the gun breech after fuing 2140 g HPC95 and 34 g H870 

propellants showed 24 x 106 rev/g residue . Open-air burning of HPC95, H870, M-1, and M-6 

propellants (all of which are not mutagenic before burning) gave 3800, 3500,16,600, and 5700 revhng 

(-S9) of residue . The presence of S9 reduced the response by -50% . Samples from cloth2ttsed to clean a 

22 caliber pistol and rifle and a 12 gauge shotgun showed 289, 1494, and 2375 rev/6 cm (TA98, +S9), 

respectively . Two S9 requiring and 1 direct acting mutagenic components have been separated by 

HPLC . Subsequent mass spectral anal sis and NMR analysis have not given additional structural 

information due to small amounts of purified material available after the final clean-up . Estimated specific 

mutagenic activity is >100,000 rev/µg . Chromatographic behavior and specific Ames/Salmoneila 

response in TA98 suggest aromatic amutes are responsible for the mutagenic activity . It appears that the 

propellant components, nitrocellulose, nitroglycerine, diphenylamine, potassium nitrate, phthalates, and 

graphite (present in various concentrations), when heated may produce nitroarornatics in the open-air 

burning and aromatic amines in the reduced atmosphere of the guns ; not unlike production of potent 

mutagenic aromatics from diesel exhaust and cooked meat . (Work done under auspices of the U .S.DOE 

by LLNL under contract No . W-7405-ENG-48) . 

167 

THE CYTOKINESIS-BLOCK MICRONUCLEUS ASSAY : BIOLOGICAL DOSIMETRY IN CANCER PATIENTS 

FOLLOWING IN VIVO FRACTIONATED EXPOSURE TO IONISING RADIATION . Fenech M .*, Denham J .** 

Francis W .** and Morley A .A .*** . *Bianedicine and Health Prog ., Australian Nuclear 

Science and Technology Organisation, New Illawara Rd, Lucas Heights, NSW, Australia ; 

**Dept . of Radiotherapy, Royal Adelaide Hospital, Nth . Terrace, Adelaide, SA, 

Australia ; *** Dept . of Haematology, Flinders Med . Centre, Bedford Park, SA, Australia . 


59


Notes A prospective study was campleted on micronucleus (FP() induction in cytokinesisblocked 

(CB) lymphocytes in eleven cancer patients undergoing radiotherapy . This 

study was performed to evaluate the CB micronucleus assay as an in vivo dosimeter . 

Measurements before .during and at the end of therapy showed that-TFere was a clear 

dose-related response in MM induction in all the patients and that the extent of 

induction (between 59 .0 and 578 .0 MN/1000 CB cells) was directly proportional to the 

estimated equivalent whole body dose . Measurements were also performed after completion 

of therapy to estimate the rate of decline in MH frequency . These values were 

expressed as a percentage of the values at the end of therapy with the results 

showing that MN frquencies (mean + 1 s .e .) dropped to 91% (+ 11) after 3 months, to 

72% (+ 13) after 6 months, to 57~(+ 10) after 12 months . Measurements made 24 

months post-treatment showed that MR frequencies only returned to base-line levels 

in two of the four patients studied . The other two patients retained very high 

MN frequencies (212 .9 and 223 .3 MN/1000 CB gells) . These reults suggest that a state 

of chromosome instability (loss or breakage) may have been induced In the surviving 

lymphocytes of the latter patients . 

168 


ANTIb1LfA0ENIC eCTIVITISS OF CHLOHOPHYLLIh 

Pko-ohaen 'rèn,YLn-Jiob Chand,Ji ..g-A1 ..6 Chu,Xiao-yinS Cha_~, and Luaaa 

Fac~,Shar.g~i ..i lusL :Lui:e or Oecupa, .oual health in Chem .oal Indusi,ry 

Shsnghai(PK Chi.na) 

6hlorophyllin,the sodium and copper salt of ohlorophyll,was tested 

for• its ability to Ii,hibit the mutagenic activitv of soc•s chericsl 

pr•oducts(2-1 :oroartobenzimidezol, 0-Nitrouniline, 0-Phsnvlenedismine), 

extrations of fr :ed beef and and red wine ooneentratfons of tap 

wai~r #:nd knov.n mutagens(Duunomyoin, 2-AFj in TA98 of SaiTonslla 

typhimurium . Complete inhibition of these were obtainel with l .cSm_¢ 

cf chlorophyllin per plate . The antimutaRenlc activit,y of chlorcphyllin 

was heat-stuble . The results indicate that cn3orsphyllin is 

potentially useful as sin ant .'mutagenic agent . 

169 

NEW Mf7D(1LAIVRS OF MMOXICITY IN YFAST CIIJ .S 

L.R . Fbrguson and B .C . Baguley, Cancer Jbsearch Laboratory. University of Auckland 

Medical Sohool, Private Bag, New Zealand . 

We havee previously shown that verapsmil, a calciun antagonist which is )nown to 

reverse multidzug resistance in memnalian oe11s, reduoes the ability of a nnrber 

of DNA intercalating agents to induoe mitochondrial "petite" nutations in yeast 

eells . We have developed an assay system enploying Sacchn:nrtyces oeaevisiae D6 and 

a strongly basic analogue of the antileukemia agent amsacrine to search for other 

conpounds which reduce mitochondrial nutagenesis . 'Petite' induction by the amsacrine 

analogus can be reduced fram 80% to less than 104 by the oo-addition of appropriate 

concentrations of some cartpounds . ZMieen 80, chloroquine and cyclosporin A 

were found to be highly effective . The most likely axplanation for the andulation 

of mutagenic activity is through the inhibiticn of m,itocriondrial nptake of the DNA 

intercalating mutagen . This principle could be of inpox'tanoe in liroitiag mitochondrial 

mutagenesis in rtemmalian cells . 

170 

DETECTION OF GENE MUTATIONS IN MOUSE SPERM WITH POLYMERASE CHAIN 

REACTION (PCR) . G . Ficsor, L . C . Ginsberg J . F . Klepetka and T . P . 

McManus . Western Michigan University, Kalamazoo, MI (USA) 

To detect base-pair substitutions and small deletions in sperm, PCR 

was used to amplify a 228 base-pair segment of the PGf:2 gene of mice . 

The amplified DNA ran as a single baud on a 1 .4% agarose gel 

corresponding to its expected size . Dot blot hybridization demonstrated 

that we could detect a single base pair difference between the PGK-2a 

and PGK-2b alleles by binding of the appropriate 21 mer probe under 

stringent conditions . To detect a rare event such as a new gene mutation 

in a single sperm amongst thousands and even millions of non-mutant 

sperm the PCR alone is of limited help since it amplifies both the 

mutant and non-mutant DNA resulting in more DNA, with the mutant 

sequence still a minor component . We attempted to solve this problem 

by restriction digesting sperm DNA before amplification with Hinc II 

which cuts the normal DNA sequence to be amplified in two . If a basepair 

substitution mutation or small deletion is present in the }(incll 

sequence, that target DNA will not be cut by HinclI and will be 

available for amplification by PCR . As a consequence the amplified DNA 

will be enriched for mutant DNA sequences . The amplified DNA then can 

be analyzed for the presence and amount of mutant DNA sequences . 

Supported by NIH grant 1 R15 HD21171-O1A1 and by a Western Michigan 

University Faculty Research Fellowship and Grant . 

50869 3572

171 

CYTOLOGICALEFFECTS OF ALUMINIUM IN PLANT ROOTS 

G . Fiskesjt9, Institute of Genetics, University of Ltatd, Sweden 

Structures of a new type ("A1-stnictures") have been discovered in the oyEoplasta of 

root cells of certain plants treated with altzainitmt ions (A13+) . Material leaches out 

frotn the nuclei particularly of root cap cells, forming oblong structures one in each 

cell, eventually dividing into two equal-sized structures, one at each end of the cell 

with the nucleus in between . Feulgen/Light Green staining of the Al-structures indicates 

that they possibly are connected with IaiA and rwcleoli(Fiskesjt5 1983) . Lightand 

transtnission electron microscopy of sectioned material of Al-treated cells 

yielded new aspects on the structure of cytoplasm and cell arambrane(Fiakesj8 et al . 

1989, in press) . The current acidification of forest soils induces increase of Al in 

solution in the soil . A13+ ions in concentrations around 20 mg/L cause severe damage 

to Allitnn roots, and concentrations of this strength have actually been fotatd in forest 

soil solutions when pH decreases towards 4 . The specific Al-structures which 

were first found in Alliua roots after A1-treatments, have later been found also when 

Alliun roots were grown in Al-rich forest soil solutiens(Berggren & Fiskesj8 1987) . 

Al-structures have been found in two Allium species(oepa and schoemphrasum) and in 

growth-restricted forest roots after A1-treatments(e .g . Picea abies, Fagus silvatica) 

(Fiskesj8, in preparation) . Thus, A13+ ions undoubtedly ootttribute to forest damage 

by interfering with root cell na:tabolism . The Al-structures may be a general response 

of plants to Al . kbether the Al-structures actually oontain Al, and whether there is 

a oonnection between the formation of the structures and the function of the nucleoli 

are questions which remain to be answered . 

172 

DNA-DAMAGE INDUCIBLE GENES IN MAMMALIAN CELLS, Albert J . Fornace Jr., N .C .I ., 

N .I .H ., Bethesda, MD 20892 . 

Based on the results of others in bacteria and yeast, many genes would be expected to be DNA-damage 

inducible (DDI) in mammalian cells ; some may represent specific responses to DNA damage, while others 

may be general stress responses to cell injury . A variety of mammalian genes, such as metallothionein, 

collagenase, c jos, ubiquitin, and B-polymerasel, have been found to be DDI by our group and/or other 

investigators . Most of these examples probably represent general stress responses since they were induced 

by unrelated agents such as heat shock and/or activators of protdin kinase C. However, B•polymerase 

mRNA was found to be specifically induced only by alkylating agents and similar agents that produce DNA 

damage repaired by a mechanism involving B-polym erasel . The 8-polymerase gene had several properties 

in common with bacterial genes that are specifically DDI : low abundance, rapid induction of 2-10 fold, and 

induction specific for DNA damage . An approach to isolate cDNA clones of other such DDI genes was 

developed using hybridization subtraction at low ratios of RNA :cDNA2. 49 different cDNA clones were 

isolated that coded for transcripts rapidly induced 2-28 fold by UV radiation in Chinese hamster cells2 . 

Many of these transcripts were induced only by DNA-damaging agents ; these DDI cDNA clones were 

divided into 2 classes. In Class I, only UV radiation and other UV-mimetic agents were effective inducing 

agents, while in Class II other base damaging agents such as alkylating agents were also inducing agents . 

Characterization of individual DDI cDNA clones will be presented including evidence that a Class I 

member (DDlA18) encodes a nucleic acid single strand binding protein, and that several Class II genes are 

coordinately regulated and may represent members of the same rqulon . These results support the 

conclusion that multiple transcripts in mammalian cells are specifically induced by DNA damaging agents, 

Ind that their protein products may be involved in the cellular response to such damage . 

Fornace AJ. Jr ., Zmudka, B .Z., Hollander, M .C., and Wilson, S .H .: Molec. Cell . Biol . 9: 851-853,1989 . 

2 Fornace AJ. Jr ., Schakh, H., and Alamo, I. Jr.: Proc . NaO . Acad . Sci . USA 85 : 8800-8804,1988. 

173 

ACCUMULATION OF DNA SINGLE-STRAND BREARS AND POSSIBLY ARTIPACfUAL IIDS RESPONSE IN RAT 

HEPATOCYTES BY DIFLOXACIN . F . L . Fort, X . A . Cifone, and R . 0 . Curren, Abbott Lahe, 

Abbott Park, IL, Razleton Labs, Rensington, MD and Microbiological Associates, 

Rockville, MI 

Difloxacin is a quinolone antibacterial which has been found positive for 

unscheduled DNA synthesis (UDS) activity in rat hepatocyte cultures . Skare, et al . 

(Mutat . Res . 172 : 77-87, 1986) reported an artifactual UDS response with sodium 

fluoride presumably due to precipitahle complex formation involving fluorine ion and 

[3N]thymidine triphosphate . Since difloxacin has low aqueous solubility and is difluorinated, 

it is possible that the positive UDS response might be artifactual by a 

similar mechanism . As a preliminary test of this hypothesis, difloxacin was tested in 

a modified UDS protocol in which the culture medium containing drug was filtered prior 

to treatment of hepatocyte cultures . Under these conditions difloxacin did not induce 

a UDS response even though the drug concentration after filtration was equal or higher 

than that when a UDS response was obtained without filtration . Therefore, the UDS 

response may be artifactual ; further studies are necessary to prove this . To further 

test the mechanism for the UDS response, hepatocyte cultures treated with difloxacin 



Notes



Notes were analyzed for DNA single-strand breakage by alkaline elution . A dose-related 

positive response for DNA single-strand breaks was obtained . These results indicate 

that difloxacin causes an accumulation of single-strand breaks in hepatocyte DNA . It 

is possible that accumulation of DNA single-strand breaks may result from the action 

of this drug as a topoisomerase inhibitor, but this accumulation may be unrelated to 

the apparently artifactual DDS response . 

ATRAZINE AND THE GENOTOXICITY OF ITS METABOLITES 

Franekic . J . . G . Hulina . J . Kniewald and M . Alabevic 

Faculty of Food Technology and Biotechnology . Zagreb . Yugoslavia 

174 

Tne aim of our study was to examine genotoxicity of atrazine and its metabolites 

deethylatrazine (2-chloro-4-amino-6-isopropylamino-s-triazine) and deisopropylatrazine 

(2-chloro-4-ethylamino-6-amino-s-triazine) detected by t .1 .c . and gas chromatography, 

and structurally identified by mass speetrometry in the kidney . brain and 

liver of male rats treated with atrazine . 

Experiments were performed with microbial test-systems with Salmonella t himurium 

strains TA100 and TA98 (plate-incorporation assay and preineu a ion met an 

with Saccharomyces cerevisiae D7 . 

In the plate incorporation assay atrazine was negative in both strains (TA100 and 

TA98) : deethylatrazine was positive in strain TA100 and deisopropylatrazine was positive 

in strain TA98 . Results of preincubation method indicated that all examined 

substances are genotoxic and toxic . In S . cerevisiae D7 only deethylatrazine showed 

recombinogenic effect. - 

175 

FIVE COMPOUNDS WITH ANTIVIRAL ACTIVITY TESTED FOR THEIR RESPECTIVE 

GENOTOXIC POTENCIES IN THE DROSOPHILA SOMATIC MUTATION AND 

RECOMBINATION TEST (SMART) . H . Frei and F .E. Wiirgler, Institute of 

Toxicology, ETH and University of Zurich, Schwerzenbach, Switzerland . 

The two potential anti-AIDS drugs azidodeo:ythymidine (AZT) and 

dideoxycytidine (DDC) were tested for their respective genotoxicity 

in the Somatic Mutation And Recombination Test (SMART) of Drosophila . 

Both nucleoside analogs apparently interfere with DNA synthesis since 

mutant twin spots (IDtdh and f1ta) as well as single spots (mFih or 

fjZ2) were induced in the wing disc cells of animals which were 

trans-heterozygous for the two recessive markers . ThA compounds were 

fed for 48h to 3rd-instar larvae . Sibs from the same cultures which 

were heterozygous for the marker QKh and the recombination-suppressing 

balancer-chromosome TM3 allowed to evaluate mutagenicity separately 

in the absence of recombination . Overall, AZT was 30-50x less 

genotoxic than DDC . With DDC, recombination clearly predominated, 

whereas with AZT, recombination was only moderately more frequent 

than mutation . Three other antiviral agents, i .e . ribavirin, phosphonoformic 

acid, and particularly acyclovir also had stronger genotoxicity 

than AZT . Thus, SMART is not only useful for rapid screening, 

but also allows to assess genotoxicity quantitatively and to take 

into account basically different endpoints . 

176 

CHARACTERIZATION AND EXPRESSION OF EURARYOTIC GENES REQUIRED FOR NUCLEOTIDE EXCISION 

REPAIR :YEAST AS A MDDEL SYSTEM . Errol C . Friedberg, Lee Bardwell, A . Jane Cooper, 

Itzik Harosh, Wolfram Siede and Jae-Mahn Song, Department of Pathology, Stanford 

University School of Medicine, Stanford, CA 94305 . 

In the yeast Saccharomyces cerevisias at least 10 genes are involved in the 

removal of bulky base adducts . Five of these (RAD1, RAD2, RAD3, RAD4 and RADIO) 

are absolutely required for damage-specific recognition and incision of DNA . These 5 

genes have been cloned by phenotypic complementation . Their nucleotide sequences 

predict expression of proteins with calculated molecular weights of 126 .2 kDa 

(Radl), 117 .7 kDa (Rad2), 89 .7 kDa (Rad3), 87 .1 kDa (Rad4) and 24 .3 kDa (Rad10) . The 

cloned genes have been tailo4ed into vectors for overexpression in yeast and E . 

coli . The RAD3 gene is multifunctional . In addition to its role in nucleotide 

excision repair RAD3 is an essential gene . Furthermore, certain rad3 mutant 

alleles confer a phenotype of increased spontaneous mutation and increased mitotic 

recombination . Rad3 protein has been purified to apparent homogeneity . The purified 

50869 3574

protein is a DNA-dependent ATPase with DNA helicase activity . The role of this 

ATPase/helicase in the various RAD3 functions identified remains to be established . 

The RAD2 gene is DNA damage-inducible . Following exposure to a variety of DNAdamaging 

agents steady-state levels of RAD2 mRNA increase -3-6 fold . Induction is 

positively regulated . Cis-acting sequences required for induction have been 

identified by deletion mapping and mutants have been isolated that are defective in 

induction of RAD2 . The amino acid sequences of the RAD10 and RAD3 genes share 

homology with the human excision repair genes ERCC1 and ERCC2 respectively . 

Additionally, the RAD10 gene partially camplements the phenotype of mammalian cells 

defective in the ERCCI gene . Thus, genes for NER are apparently conserved in 

eukaryotes . 

177 

IN VIVO EVALUATION OF CYCLOACTIVE AND CLASTOGENIC EFFECTS OF BEET ROOT COLORS . N .C . Froes and N . V. Garcia, 

TEILC-'UNESP, Seo Jose do Rio Preto, S ;o Paulo, Brazil . 

The recent development of food industry has made food additives a relevant cause of human cancer, 

particularlydue to the autagenic activity of several synthetic food colors . The knowledge of mutagenic 

effect of natural food colors gives scientific support for the replacement of synthetic by natural ones . 

Two different forms of beet root colors were tested, the form I without nitrate residues and the form II1 

with nitrate residue . The coloring active principle of both extracts is the betanin pigment which gives the 

characteristic beet root purple color . llatagenic effects were tested in vivo . The experiments were carried 

out with bone marrow cells of males and females Rattus ~norvergi~cus var . star six-seven weeks old, 90-100g 

of weight, orally treated with both forms for one wee , w-3tTi- 0 .02 mg and 5 .0 mg of betanin per 100 g of body 

weight per day ; the bone marrow material was collected at the 8th day . For the bone marrow cell experiments 

two different control groups were established : a negative group of untreated animals and a positive group 

of animals treated with 5 .0 mg of cyclophosphamide per 100 g of body weight 24 hours before sacrifice . 

Mitotic index (MI), chromosome anomalies (CA) and micronuclei frequencies were recorded . Slides of bone 

marrow were analysed in blind tests . There were no differences in MI, micronuclei, and CA frequencies, when 

the treated samples were compared with the negative control . The positive control presented a gross MI 

reduction and an increased micronucleus and CA frequency. These results suggest that aiie both forms of the 

compounds were metabolized and inactivated in non-mutagenic derivates, justifying the absence of S vivo 

cycloactive and clastogenic effects . The present results seem to support that beet root color is a sa er 

alternative food additive . Economic and technical restraints were not considered . 

178 

PREDICTION OF POSSIBLE CARCINOGENS, TUMOR-PROMOTERS AND ANTI-TUMOR 

PROMOTERS IN THE GLANDULAR STOMACH . C . Furihata and T . Matsushima, 

Institute of Medical Science, University of Tok~o, Tokyo (Japan) 

An in vivo short-term assay method was developed for prediction of 

carcinogens, tumor-promoters and anti-tumor promoters in the glandular 

stomach . In this method, test chemicals are administered to male F344 

rats . Then tumor-initiating activity is assayed by measuring inductions 

of unscheduled DNA synthesis (UDS) and DNA strand scission (by alkaline 

elution method), and tumor-promoting activity is determined by measuring 

induction of ornithine decarboxylase (ODC) activity and stimulation of 

replicative DNA synthesis (RDS) in the glandular stomach mucosa. By 

this method five possible glandular stomach carcinogens, glyoxal, 

methylglyoxal, diacetyl, 3-diazo-N-nitrosobamethan and 1-nitrosoindole- 

3-acetonitrile were identified . Hickory smoke condensate, with or 

without treatment with nitrite, was found to be a possible glandular 

stomach carcinogen . In addition, 20 possible glandular stomach tumor 

promoters were identified, including various sodium salts, potassium 

salts, and an ammonium salt of food additives and bile acids . CaC12 was 

found to be a possible anti-tumor promoter in the glandular stomach : its 

administration inhibited stimulation of replicative DNA synthesis 

induced by subsequent administration of NaCl, a tumor promoter in the 

glandular stomach . 

179 

IN VIVO MUTAGENICITY TESTS ON POLYPLOID INDUCERS 

Furukawa,A ., Ohuchida,A . and Wierzba,K .* 

Drug Safety Research Lab . and * Biological Research Lab ., Taiho Pharmaceutical 

Co .,LTD . . Kawauchi,Tokushima 771-01 Japan . 

The micronucleus (MN) and a chromosomal aberration (CA) tests were used to 

study in vivo the mutagenicity of polyploid inducers in mouse bone marrow cells . 

Five chemicals, e .g . ethyl vanillin, narcotine, p-nitrotoluene, 

diethylstilbestrol, thiabendazole, were used as specific in vitro polyploid 

inducers (1) . These chemicals were suspended in olive oil and were injected 

intraperitoneally to BDF1 male mice (8 veek-old, 23 .2-28 .5g) . In pilot PQN-test, 

frequencies of micronucleated polychromatic erythrocyte (MNPCEs) varied from 0 

to 0 .4% depending on the dose and sampling time . The main test was carried out 



Notes



Note s 24 hour after treatment using 5 animals per group . The test compounds did not 

increase the frequencies of MNFCEs . The preliminary in vivo CA-tests were 

carried out at 6, 24, 48hours after administration . The frequencies of polyploid 

were 0-3 .5% and were not different from control . Furthermore, the effect of 

thes chemicals on polymerization of tubulin was examined . These compounds did 

not inhibit specifically tubulin polymerization to microtubules . Therefore, in 

vitro induced polyploid can not be mediated through the microtubular system . 

(1)Ishidate,M . et a1(1988) Mutation Rea . 195 151-213 . 

180 

MUTAGENIC ACTIVITY, DNA BREAKAGE AND SOMATIC MUTATION OF ARENEQUINONES . 

H . Furukawa, K . Kavai, T . Miyazawa and T . Sato, Meijo University, Nagoya(Japan), Tohoku 

University, Sendai(Japan), and Inaba Biophoton Project, Research Development 

Corporation of Japan, Sendai(Japan) . 

it was reported that arenequinone was synthesized photochemically by sunlight 

irradiation from corresponding arenas . The mutagenic activity of 3,6-banzola)pyrenequlnone, 

1,6-benzota]pyrenequinone, 1,6-pyranequinone and 1,8-pyrenequinone are 44, 77, 

91 and 298 revertants on Salmonella typhimwrdun TA100 without S-9mix . . It was 

considered there must be the other mechanism with the exception of epoxide formation . 

Futhermore breakage of DNA such as pBR322 or A phage DNA by 1,6-pyrenequinone or 1,8pyrenequinone 

were suppressed by active oxygen scavenger such as buthylhydroxytoluene, 

glutathione, a-tocopherol, sorbitol and L-aacorbic acid in vitro . Therefore we 

considered that both mutagenic activity and DNA breakage activity were caused by active 

oxygen molecules and the like . Small single spot frequency in Drosophila wing spot test 

by 1,6-pyrenequinone, 1,8-pyrenequinone, 1,6-bento(a]pyrenequinone and 3,6-benzo(aJpyrenequinone 

were risen about two fold of control's frequency . Then it was considered 

active oxygen molecules caused the somatic mutation of Droeoph{la melanOgaster . For the 

reason above mentioned phenomena, we considered that the planar ring surface of arenequinone 

molecules are x-electron deficient, and toxic arenequinones contain 

considerable radical structures in the resonance formulae . And we would like to 

emphasize the urbane airborn particulate containes a few arenequinones and toxic 

arenequinones should be formed by sunlight irradiation from corresponding arenes . 

TRANSPOSITION : EFFRCTg AND MECtlAMISMS 

David J . Galas Molecular Biology, University of Southern California, Los Angeles, CA 

90089 

Transposition of mobile genetic elements is apparently a universal phenomenon 

occurring in the genomes of all organisms . Certainly in all the experimental organisms 

common to the genetics laboratory, they have been extensively characterized . The list 

begins with maize, of course (McClintock, 1956), and includes Drosophila, 8aecharomyces 

Caenorhabditis and several different types of bacteria . The genetic effects of mobile 

elements are diverse and include the induction of insertion mutations, deletions, 

inversions and other rearrangements, and the turning off and on of gene expression . In 

this talk, I will discuss several examples of the ways in which the mobile elements can 

cause these effects . I will draw examples from observations and experiments in several 

systems, but will focus on bacterial systems in discussing specific mechanisms . The 

bacterial insertion sequence, IS1, has been studied to determine the molecular 

mechanisms of the transposition process, the component parts of the molecular 

apparatus, and to dissect the specificity determinants of the protein and DNA 

components . 

GFW=CITY OF VANADIUM CCt1QC[[RNID6 

A . GaLLI, L . GIHCKDR, R . DEL CARFA2WE, C . DELIA CPDCE and G . BI+DNZETTI 

Instituto di Mutagenesi e DifferenziamenYa CIIIR PISA ITALY 

181 

182 

Acmnnium Metavanadate and Vanadyl Sulfate were tested for their ability to induce mitotic 

gene oonversion and point reverse uutatian in the D7 strain of S . oerevisiae . Metavanadate 

increased the convertant and revertant frequencies and the highest ac v ty was observed 

without metabolic activation . This indioates that S9 hepatic fraction and cells fron logphase 

oantainirg a high level of cytochreme P-450 biotransform vanadate probably reducing 

it to vanadyl . Vanadyl did not induce any genetic effects in the same experimental conditions 

An increase of gene conversion and point mitation was induced by vanadyl in cells 

fraa log-phase, suggesting that these cells are able to axidate vanadyl to vanadate . To 

explain our results, we hypothized that matavanadate is the genetoxically active form and 

the monooxygenase system is involved in biotransfotmntion of varadiun, particularly reduoing 

vanadate or oxidatiry vanadyl . To confirm this hypothesis, years cells harvested fram 

lod-phase with high level of cytochrane P-450 were treated with metavanadate and vanadyl 

50869 3576

in the presenoe of specific inhibitors of cytochrome P-450 . In these eorditions, vanadyl 

genotwcicity renaiz>aa unaffected, while the presenoe of inhibitors determined an increase 

in mitotic yene conversion an3 point reverse mutation irr3uaed by vanadate . Therefore, 

tcrmooxygenase system cytochrome P-450 depertflent is probably involved in the vanaditm tretabolism 

reducirg vanadate, but not oxidating vanadyl .. 

183 

DIFFEREhTIAL MUTAGENIC RESPOOiSE CF A SYtVCi-ic .~l'IC pYR::THRCID, DELTAMETHR4tv, 

IN SUB-MAN:MALIAN AND MAMNALIAN TEST SYSTfVIS . G . Gandhi, J .b . Chowt9'tury, 

P .K .Sareen and I .S .Grover, Scliool of Life Scit•aces,GNDU,Amritsar,India . 

Deltamethrin is one of the most commonly used pesticides in North 

India . Mence,•-tts genoto•ricity was studied for germ-cell mutagenesis in 

Dromvhil~ and for clastogenicity in the mouse bone-marrow !• :0 test . LM 

(solvent) exhibited mutation rates comparable to the norn :al control v luas 

while EMS elicited a positive response in all the test assays . il.iA 

were exposed to media containing varying concentrations of the insecticide 

(0 .2, 0 .4, 0 .6, 0 .8 and 1 .0 ppn) for the induction of dominant 

lethals . A steady but non-significant increase in lethality fr(m 32 .58:4 

at 0 .20 ppm to 5O .05X at 1 .00 ppm was ob served . Also none of the concentrations 

i nduced significa nt SLRLs (2 .45%) even at the highest eoncent ration 

tested (0 .80 pgo) . Cytogenetic damage in mice was screened for three 

ip administered doses (32 .50, 162 .50 and 300 .00 tng/kg b .w), selected from 

estimated LD50 (325 mg/),-.g b .w) . significant genetic damage (1 .26 and 

'1 .35% micronucleated PCES) was observed at the two higher doses . The PCE/ 

tCZ 

ratio demonstrated a significant ir.crease in the percentage of pCES, 

at lower doses sig nifying a stimulatcry eff ect of deltamethrin . Though, 

deltameth rin showed no germ-cell mutagenesis in ro• :nnhd .11, yet it acted 

as a strong clastogerl/sPindle ir4fibitor in the mouse . Its differential 

response has rather made it desirable to investigate its ; genotoxicity in 

oth er systems too . 

184 

RFGULA'TION OF NUM,AN DNA GLYCOSYLASFS 'rdAT INT'fTA'TF gAgfy FXCISION RFPAIR OF OXii)ATTVE 

PYHf,lIjL2F MODIFICA'TTONS . Tapan Cang-il y and aahum J . Duker, Temple University 

School of Nndicine, Philadelphia, PA (OSA) 

DNA oxid,rrive damap,ea are among the most frequPOt~ tyoes encountered in the 

lifetime of a cell . TheaPe can result from ionizing or gamma radiation and froaa 

activated oxygen species Renerated from metabolic procesees . Fxciaion of oxidized 

bases from DNA of hurnan cella ia initiated by DNA qlycosylaees . The oxidized 

t7yminrc moiety 5-hydroxyrethyluracil is removed frorn DNA by 5-hydroxyerethyluracil- 

DNA e,lycosylase . A redoxyendonuclease, active against a wide variety of substrates, 

has ;lycosylic activity aF,ai .ist many modified DNA pyrimidiiea . We studied 

re.gul .ation of these enzymes in proliferating human cella . Both glycosylases were 

assayed by measurement of direct telease of modified free bases from their DNA 

substrates . Serum-atimulated JI-38 Sunan cells were the sources of enzyme 

a .tiviries assayed in crude extracts as a function of cell division . 5hydroxyaret.hyluracil-DNA 

qlycosylase activity did not vary significantly during the 

cell cycle . ny contrast, the glycosylic activity of the redoxyendonucleaee 

increased four-fold as a function of cell growth . Maximum stimulation was obtained 

during peak DNA synthesis . Tnis enzyme activity increased once again aa thn cella 

entered a second growth cycle . This ia similar to the stimulation observed for 

uracil-UNA glycosylase in a aynchronous cell population . Theae results indicate 

that the glycosylasea that initiate base excision repair of oxidised DNA are not 

coor3inately induced during the cell cycle . 

185 

LATE EFFECTS OF FEMALE SEX HORMONES 

han Fengeing, )laog Hsnyfug, and Yu Yannao, Dept . of Blological Effect of 

Radislion, Laboratory of tnduslrial Nygiens, Ministry of Public Health . I 

Xinkang Street, Desheng.enr,l, Beijing, (China) 

Inj . Hydroxyprugesterone Co . (containing hydroxyprogesterons capruste (P) 

250 •g and estradiol valerate (E) S sfgi .l of vehit•ls, .EP) was Isjecled 

intrseuscularly into fe .ale Vister rats and different strains of rtee of both 

sexes with doses of 5 to 100 ti .es that used Is huans ones or twice a month for 

10 to 24 ti .es . In so .e experl .ents EP was co .bined with •hole-body 3 Gy ga. .a 

radiation once or twice . The purpose of this work was to detsr .ine whether F•P 

would possess carcleogenleity or not and whether synergistic cercinogeeicily 

would exist when EP had been ad .lnistered In coebination of ga . .a radletion . 

Results show that EP has ubvious carcloogenicity/tu .our lncidence was siegnifi- 



Notes



Notes cenlly increased . HI'• also has the sffect of laducing relrugrade infeclion or 

urugenital sysles . F.Y enhanced the gassa ray carcisogenieily, thereby 

increasing the tumour incidence or the ratio of sa1)gnant to benign tuaosrs . 

Mhen added into the sedium, 1iY,fi or P aloae •as able to directty lnduce 

matignant transformation of souse esbtyo cetls in vitro . it caa partly explain 

the reasan •hy the aajur lyps of tusors ladueed by 8Y in various atralae of 

sire ua : qaitr difforeat as a result of observing ihe distribution of 

trltialed estradlol receptor cosplexes in various t)ssues and organs by 

nutoradlography . 

186 

HUMAN GENOTOXICITY IN PHOSPHINE-EXPOSED APPLICATORS . V . Garry, T . Danzl, J . Griffith, 

R . Nelson, E . Whorton, University of Minnesota, Minneapolis, MN (USA), and U .S . EPA, 

Research Triangle Park, NC (USA) . 

Fumigation of grain is a world-wide agricultural practice . In this effort, we 

examined the health histories of more than 400 persons who may have come in contact 

with fumigants and pesticides . We identified a small group of workers who perform 

fumigant application . We then undertook an integrated human study of fumigant 

applicators exposed to phosphine, one of the most common fumigants . Evidence for 

qenotoxicity was expressed in terms of sister chromatid exchanges and chromosome 

aberrations in banded and non-banded preparations . These data were coupled with 

personal breathing zone sampling for phosphine . As a group, applicators (n-24) show 

significantly increased chromosome aberrations compared to control subjects (n-24) . 

Workers exposed to phosphine alone (n-9) have increased chromatid gaps/deletions 

compared to all other groups . Workers earlier exposed to phosphine or to phosphine 

and other pesticides have significantly increased chromosome rearrangements, 

including chromosome translocations (11/12) compared to controls (2/10) . Chromosome 

breakpoints identified in the exposed workers seem to cluster in certain oncogene 

regions of specific chromosomes . In vitro studies of phosphine suggest that 

phosphine or a phosphine-generated product(s) crosslinks DNA as studied by alkaline 

elution procedures . Further work indicates that the mechanism of qenotoxicity may 

be related to peroxidase inhibition . 

187 

A IL TRDUSTRIAL AND UK PERSPECTIVE ON SHORT TER_M TEQTING . 

DG Gatehouse, Genetic and Reproductive Toxicology Department, Glaxo Group Research 

Lta ., Ware, Herts . 

The appropriate use of short-term tests for the screening of carcinogenic agents 

is currently under review . In the UK, the DHSS Committee on Mutagenicity (COM) has 

been revising its guidelines with a view to publication later this year . To 

compliment this the UKEMS are revising their recommendations for the conduct of the 

main categories of short-term tests . In these revisions it is recognised that a 

limited number of in vitro tests carried out exhaustively but with a degree of 

flexibility is the most effective strategy for priaary screening . Mammalian gene 

mutation assays have been retained in the revised DiiSS CON Guidelines, aa some 

"unique" mammalian cell mutagens have been identified by the recent R!P study . 

However, the credibility of these data is being contested and further studies are 

required to resolve this . There is still considerable debate on the role of 

short-term in vivo tests ie . confirmatory or screeningt . Their use as screens may 

still be essential when complex metabolic activation processes are required . If used 

in a confirmatory role, there is accumulating evidence that organ-site specificity 

requires that more than one tissue should be examined to eliminate false negative 

results . It is possible that species-specificity might also be an important 

consideration when designing experimental protocols . Finally the need for an 

additional test(s) to detect "aneugenic" agents has still to be decided . Some 

potential aneugens may be detectable using the existing assays (eg micronucleus teat 

and in vitro cytogenetic assays) . Further validation data are required before any 

firm decisions can be made . 

188 

DATA AND RATIONALE FOR A MODEL THAT EXPLAINS THE VARYING FREQUENCY OF ANEUFLOID CHILDREN 

WITH MATERKAL AGE (THE J-SHAPED CURVE) . M .E . Caulden, Radiology Department, University 

of Texas Southwestern Medical Center, Dallas, TX 75235 

The majority of aneuploid children are born to older women and result from nondisjunction 

at first meiotio division in the ovary . What ovarian condition promotes 

aneuploidy induction? I propose that it As decreased miorooiroulation around follicles, 

leading to deficient 0p supply and a concomitant inorease in C02 and anaerobic produots 

such as lactic acid, whioh lower pH . We have found that exposure of somatic cells in 

vitro for 1 h, 38°C to C02 or acid medium (pH


causes dissociation of 1-2 chromosomes, comparable to that caused by 0 .025 ug/ml Coloe- Notes 

mid, producing aneuploid daughter eelle . The follicle ie avascular ; the oooyte in an 

immature follicle is probably hypoxic because the capillaries in the surrounding theca 

are few . Development of capillaries around a maturing follicle is determined by sex 

hormones and angiogenlo factors, whose levels may be lower in the very young and the 

older woman, and also occasionally in one of intermediate age . Exchange of gases 

and other substances must take place through granulosa oelle and follioular fluid, so 

the oocyte 02-C02 balance is dependent on an ample blood supply . Thus, a compromised 

microciroulation could account for aneuploidy incidence in women of any reproductive 

age, the frequency varying with the probability of events leading to reduced development 

and/or funotion of the critical perifollioular capillary bed . The testioular tubule is 

also avascular, so small localized regions of reduced circulation could result in• 

aneuploidy . Lagging angiogenesis has been shown to cause bypoxic regions in tumors, so 

reduced pH could be responsible for some of the aneuploidy seen in practically all 

advanced tumors . Experiments in progress with mouae oooytes will be reported . 

189 

SYMPOSIUM : CEOtOMOSOME ABERRATIONS :IMECHANISMS 

MICRODOSIMETRY, LET AND CkQt0t4DSOMAL ABERRATICNS . 

Charles R . Geard, Radiological Research Laboratory, College of Physicians and 

Surgeons o Co umbia university, New York, N .Y . U .S .A . 

Microdosimetry deals with the statistical fluctuations of energy deposition in 

small volumes of irradiated matter . When applied to the cell nucleus or parts 

thereof, the frequencies and intensities of different ionizing radiations can 

be related to the induction of individual chromosomal changes . That is, a 

relationship can be established between track based energy deposits and the 

probability of lesion induction and interaction . It has been long established 

that as linear energy transfer (LET) increases so does the likelihood of 

biological effect . Currently this is particularly pertinent for the alpha 

particle cellular traversals from radon daughters . At environmental levels of 

radon, individual cells are very rarely likely to encountermmore than one 

alpha particle . Therefore it is necessary to evaluate thromosomal changes in 

individual cells on a per particle basis . To attain this end microdosimetric 

evaluations of track events and of energy transfers in sub-nuclear volumes are 

necessary in conjunction with morphometric assessments of cellular targets . 

Over the LET range consistent with radon daughter alpha particles there is a 

non constant probability of aberration induction both in terms of frequencies 

and types of aberations . Hence assuming an equivalent status for these alpha 

particles which are of profound societal concern is inappropriate . 

190 

USE OF CYTOGENETIC STUDIES IN ASSESSING THE INVOLVEMENT OF MUTAGENIC AGENTS IN 

PRELEUKAEMIC SYNDROMES AND ACUTE LEUKAEMIA . A .D .Geddes and A .Jacobs . Department of 

Haematology, University of Wales College of Medicine, Cardiff, U .K . 

The involvement of mutagenic/carcinogenic agents in the induction of preleukaemia 

and acute leukaemia has been known for some time . Exposure may occur as a result of 

chemo/radiotherapy or from occupational or environmental sources . Secondary leukaemias 

are generally rapidly progressive with poor response to normal therapeutic regimes and 

short survival . Cytogenetic characteristics include a high incidence of clonal 

karyotypic abnormalities in the bone marrow (>85a) ; a high level of aneuploidy, 

particularly chromosome loss ; high incidence of complex rearrangements including 

unstable configurations such as rings, dicentrics, double minutes, etc and specific 

involvement of certain chromosomes particularly 5 and 7 (66-90% of cases) . Cytogenetic 

studies are therfore useful in the identification of mutagen induced disease and have 

been used in Cardiff over the last 3 years to assess the potential involvement of 

mutagenic agents in new cases of preleukaemia and leukaemia with no apparent history 

of therapeutic or occupational exposure as well as monitoring those patients with a 

history of prior therapy for other disorders or of occupational exposure to potentially 

mutagenic agents . Studies included investigation of clonal karyotypic abnormalities 

and aneuploidy in bone marrow and assessment of chromosome aberration levels in both 

bone marrow and peripheral blood . Although such studies cannot prove a direct causative 

role for mutagens in the development of preleukaemia/leukaemia they can provide 

indications of mutagenic involvement and they may also assist in defining groups and 

individuals at risk . 


67

; 

68 



Notes MOUSE MODELS FOR UNDERSTARDING HUMAN DEVELOPNffirfAL ANOMALIES 

Valderico N . Generoso 

Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-8077 (USA) 

191 

Mutaganesis research in mice has a long tradition of addressing the problems of 

human genetic risk and of enriching our knowledge of basic mammalian biology . In line 

with this tradition, we are using mouse models in order to understand the origin and 

pathogenesis of certain classes of human developmental anomalies . One such model 

involves the study of the developmental defects that are caused by chemically induced 

chromosomal rearrangements and imbalances . Of interest are the specific chromosomes 

involved, the nature of breakpoints, the teiotic segregation that produces unbalanced 

segregants that survive to late gestation, and the sequence of changes that are 

observed in the pathogenesis of the defects . Another model involves the study of 

developmental anomalies that are produced subsequent to exposure of zygotes to certain 

mucagens . The fetal malformations produced in these studies are generally similar to 

the majority of human malformations, for which the etiology is largely unknown . The 

evidence suggests a genetic basis for the mouse fetal anomalies, but of a type that is 

different from conventional gene mutations and ehromosome aberrations . 

Research sponsored jointly by the National Toxicology Program under NIEHS Interagency 

Agreement Y01-ES-20085 and the OHER, U .S . DOE under contract DE-AC05-840R21400 with the 

Martin Marietta Energy Systems, Inc . 

192 

COMPARISON OF THE GENOTOXIC EFFECTS OF 1- AND 2- NITROPROPANE IN THE RAT 

E . George, B . Burlinson and D .G . Gatehouse, Dept . Genetic and Reproductive Tox ., 

Glaxo Group Res . Ltd ., Ware, Herts, England . 

2-nitropropane is a potent rat liver carcinogen, whilst the 1-isomer is 

non-carcinogenic in rodents . Although the 2-isomer induces UDS in the rat liver, 

uniformly negative results have been obtained in the mouse micronucleus test . The 

inability of the latter to discriminate between the carcinogenic and non-carcinogenic 

isomers may reflect either species-specific genotoxicity in vivo ; an organospecific 

effect of the 2-isomer or an end-point specific effect . 

To determine which of these factors precluded detection of 2-nitropropane in the 

mouse micronucleus test, studies were carried out in the rat in which micronucleus 

induction (bone marrov and liver) and UDS induction (liver) were measured after oral 

treatment with each compound . 2-nitropropane was found to induce UDS in rat liver, 

whilat the 1-isomer was negative, thus confirming earlier studies using the 

intraperitoneal route . In the bone-marrov micronucleus test, small increases were 

obtained with individual animals, however group means fell within the historical 

control range and the results were considered negative . In the liver micronucleus 

test, 2-nitropropane induced a highly significant response . Therefore the negative 

mouse micronucleus test results reported for 2-nitropropane were not due to species 

specificity, or end-point specificity ; but instead were a reflection of the 

organospecific genotoxicity of this compound in vivo . These data provide further 

evidence that bone marrov assays are insufficient for the detection of all genotoxic 

carcinogens in vivo, indicating the need for a second tissue, although the choice of 

end-point may be of less critical importanae . 

193 

A CHROMOSOME STUDY OF 387 REFERRED CASES HITH VARII3D GENETIC DISORDERS . 

M .A.Ghalib, G .S.Issac, A .Jyothy and O .S .Reddy, Institute of Genetics and Hospital 

for Genetic Diseases, Osmania University, Begumpet, Hyderabad . A .P . (INDIA) . 

This paper describes the result of chromosome study carried out on 387 cases 

suspected of chromosomal abnormalities . They were referred during the period 

from January 1987 to July 1988 to the Cytogenetics Division of the Institute of 

Genetics and Hospital for Genetic Diseases from the various districts of the State 

of Andhra Pradesh, India . They Include congenital anomalies (134), am biguous 

external genitalia (29) . Down's syndrome (101) . Klinefelter's phenotype (17) . 

Turner's phenotype (12), and primary and secondary amenorrhea (94) cases . 

Out of these 387 cases . 119 were found to have chromosomal abnormalities, a 

frequency of 30 .75% of abnormal karyotypes, leaving 268 with normal keryotypes . 

Autosomal aberrations were detected in 99 cases and the remaining 19 cases had 

sex-chromosome abnormalities . The frequency of 30 .75% in a referred population 

for chromosomal aberrations is considered high when compared to similar reports 

from other countries . Factors related to the parents that might have a predisposition 

in the aetiology of the aneuploidies will be discussed . Acknowledgements . 

The first author would like to thank the Rector . University of Aden, Government 

of P .D .R . of Yemen and the Education Officer . Ministry of Human Resources Development 

. Govt . of India fo~ financial support . The authors acknowledge the Director 

of the Institute for providing facilities . 

50869 3580


Notes 

II+cLUEhCE OF iiALIDI%IC ACID ON THE KILLING AND h1GTATION IiJDliCED BY UV 

LIGiIT AP+D M~tiNG IN DENSITY INHIBITED V79 CELLb 

Rita Gaosh(Datta), S. at a ^ and G . Bnaumik, 8aha Institute 

of Nuclear ?nyeics, I ~, alt ake, Calcutta-700 064 . 

Density inhibited plateau phase V79 cells after treatment with UV 

light or N-metnyl-N'-nitro-N-nitrosoguanidine(13NNG) exhibited improvement 

in survival accompanied by lowering of mutant yield (resistance 

to 6-tnioguanine) on delay in trypsinization (20h) . If nalidixic acid, 

an inhibitor of topoisomerase activity was present during tae period of 

delay, survival levels were similar to tnose obtained on immediate 

trypainization For mutational analysis, UV fluence was va 1ed from 

4J/m` to 20J/m! ; corresponding mutant frequencies (per 10~ viable 

cells) were 3 .0+0 .5 and 16 .9+0 .8 at these two fluences on immediate 

trypsinization . On delayed trypsinization, tnesp values decreased to 

1 .8+0 .6 and 13 .0+0 .5 respectively and again increased to 5 .5+0 .8 and 

21 .a+0 .8 when nalidixic acid was present . In case of JSYtteG, tpe doses 

varied from 0 .5µg/ml to 2 .Oµg/ml f3r lh treatment time ; the ccrresoonding 

mutant frequencies (per 10 viable cells) were 7 .5+0 .5 and 

32 .5t2•5 at these doses on immediate trypsinization . If tnB trypsinization 

was delayed, the corresponding values were 4 .5±1 .0 and 23 .3+2 .2 

respectively . However, wnen nalidixic acid was present during thle 

delay period, tue values were 8 .5+1 .2 and 38 .8+2 .0 . The results indicate 

the involvement of topoieomerase in repair of potentially lethal 

damage . 

195 

IN SITU EVALUATION OF POTENTIAL GENETIC HAZARDS FROM CHEMICAL WASTE SITES . 

B .S . Gill, J . Rice and S .S . Sandhu . EHRT, Research Triangle Park, NC 27709 and EPA, 

Research Triangle Park, NC 27709 . 

In situ monitoring of biological effects from chemical waste sites provides hazard 

assessment under the complexities of natural environmental conditions . For such studies, 

plant assays are cost effective and are ideal for preliminary investigations . 

The studies reported here were initiated at The Fairway Six pesticide site in Aberdeen, 

North Carolina and Palmetto Wood Preserving site in Dixiana . South Carolina using Tradescantia 

micronucleus and maize vaxy locus assays . The chemical analyses of soil 

samples from these sites indicate concentration of lindane (17385 ug/kg), beta BHC 

(12645 ug/kg), and heptachlor (378 ug/kg) at four feet dbpths at the Aberdeen site and 

arsenic (1292 mg/kg), chromium (1444 mg/kg), and copper (924 mg/kg) on the surface at 

the Dixiana site . Results of Tradescantia micronucleus assays shoved significantly 

higher frequencies of micronuclei from the contaminated plots as compared to the control 

plots . Toxic effects for maize growth were observed on contaminated plots at 

Dixiana site . The soil samples collected from these sites were analyzed in the laboratory 

for their biological effects using Vicia root tip, wheat aneuploidy, and 

Tradescatia micronucleus assays . Preliminary evidence confirms the in situ findings . 

The sites have now been cleaned . The in situ and laboratory experiments vill be 

repeated to evaluate the efficacy of the remedial operations . This is an abstract of 

a proposed presentation and does not necessarily reflect EPA policy . 

196 

OXIDATIVE STRESS-INDUCED GENETIC INSTABILITY IN CULTURED CHINESE HAMSTER CELLS 

J .J .P . Gille, C .G .M . van Berkel, F .A .J .M van de Klundert, and H . JoenSe . Institute of 

Human Genetics, Free University, P .O . Box 7161, 1007 MC Amsterdam, The Netherlands . 

Two different inducera of oxidative stress, i .e ., normobaric hyperoxia and H202 

were compared with respect to their ability to induce strand breaks and gene muta- 

tions . 

Normobaric hyperoxia (1 atm ., 984 02) was found to be a very weak inducer of DNA 

single-strand breaks and alkali-labile lesions, whereas H202 was found to induce large 

amounts of strand breaks . Iron-chelators were unable to afford protection against the 

clastogenic and SCE-inducing effects of hyperoxia, whereas it is known from the 

literature that they do protect Chinese hamster cells against the induction of DNA 

strand breaks and SCEs by H202 . From these results, together with the knowledge that 

most OH radical-induced DNA lesions are detectable by alkaline elution, it is suggested 

that the induction of genetic damage by hyperoxia is, in contrast to H202, probably 

not OH radical mediated . 

Data on the mutagenicity of hyperoxia and H202 on three loci (aprt, hprt, and 

xprt) will be presented . Conclusions with respect to the type of mutation (base 

substitutions and small deletions v .e . large deletions) induced by the different 

sources of oxidative stress will be discussed . 

Supported by grant 87-10 from the Netherlands Cancer Foundation . 



Notes 


197 

LIVER PROTEIN EXPRESSION IN FETAL MICE HOMOZYGOUS OR RETEROZYGOUS FOR CHROMOSOMAL 

DELETIONS AROUND THE ALBINO LOCUS . C .B .Giosetti, M .A .Geazsell, S .L .Tollaksen, and 

S .Gluecksohn-Waelsch, Argonne National Laboratory, Argonne, IL (USA) and Albert 

Einstein College of Medicine, Bronx, NY (USA) 

Liver protein expression was examined in fetal mice homozygous or heterozygoua 

for the c3N or c14CoS deletions in the albino locus region of chromosome 7 to identify 

apecific gene products that correlate with the dedeted ene s,~ ences . Liver 

proteins from vildtype (ceh/cch)~ heterosygous (co /c3 or cc14 ~), and homozygous 

(c3H/c3H or c1~'CoS/c14C0S) fetal mice (18/19 days gestation) were separated by 

two-dimensional electrophoresis (2DE) . The Coomassie Blue-stained protein patterns 

were screened for both qualitative and quantitative protein differences by using the 

Tycho analysis system . In the 2DE patterns from c3H homozygotes, 20 proteins were 

missing that were present in equal abundance in patterns of c3H heterosygous and 

h4~o~gous wildtype littermates . However, the same 20 proteins were present in the 

C1 homozygotes . In addition to the 20 qualitative protein differences, five 

proteins were effected quantitatively in the c3H homozygotes (four decreased and one 

increased in expression) relative to both heterosygous and homozygous vitdtype 

littermates . The identity of quantitative expression in deletion heterozygotes and 

wildtype homozygotes supports previous interpretations of experimental findings 

suggesting that the albino deletions involve loss of one or more regulatory genes . 

This work was supported by the U .S .DOE, ONER, contract W-31-109-ENG-38, NIH grant 

GM27250, American Cancer Society grant CD38 . 

COMPARATIVE MUTAGENICITY AND GENOTOXICITY OF THE ANTIPARASITIC DRUGS . 

MEBENDAZOLE . FLUBENDAZOLE AND AN OXIME DERIVATIVE OF FLUBENDAZOLE 

A .K . Girl, S . Osorio, J .E . Sinsheimer, D .S . Mise and L .B . Townsend . 

College of Pharmacy . University of Michigan . Ann Arbor . MI 48109 . USA . 

As part of a program to develop compounds with macrofilaricidal activity . we 

have tested the ∎utagenicity in Salmonella, and the In vivo genotoxicity to 

bone- marrow cells in ∎ice of the commercially available anthelaintic drugs 

Mebendazole and Flubendazole as well as an oxiae prodrug of Flubendazole . This 

information was sought to develop a base line for the further preparation of 

∎ore efficient .acrofilaricidal compounds without eenotoxiclty . Mutagenicity 

was established only for TA 98 with the presence of $9 at high doses for 

Mebendazole and at lower levels for the oxime . Mutagenicity was not observed 

for Flubendazole under the same conditions . A dose related increase in sister 

chroaatid exchanges (SCE) was found In vivo for Mebendazole and Flubendazole . 

There were comparable significant (pNPO>NPGEtTCPO> control . When the genotoxic 

effects of two of their metabolic precursors, 1-allylnaphthalene (AN) and 

trichloropropylene (TCP) were also carried out In vivo . AN was genotoxic in bone 

marrow but not TCP . The ∎uch shorter half life of TCPO is a factor in 

explaining the lower SCE and CA results than would be predicted by chemical 

alkylation rates or by the preincubation version of the Ames test . DNA strand 

breaks in liver were used as a test requiring less In vivo exposure ti .e for a 

genotoxicity comparison of the four epoxides and the two precursors . A 

significant increase in x of unwound DNA was observed for all the compounds 

except AN at high doses . TCPO, the least gsnotoxic in bone marrow . Is indicated 

to have the greatest liver toxicity after 4 b exposure while NOE shows the most 

toxicity after 6 h . Supported by grant RO1 ES0334S . NIENB . 

50869 3582 

198 

199


Glickman, B .W., MUTATIONAL SPECIFICITY AS A WINDOW ON THE MECHANISMS Notes 

OF MUTATION, Biology Department, York University,4700 Keele Street, 

Toronto, Canada M3J 1P3 

The development of cloning and sequencing technology has permitted the examination 

of the nature of mutation at the molecular level . These studies have in many cases 

confirmed the targeted nature of mutation and hence, provided insights into the lesions 

responsible. Mutational spectra obtained following treatment with a variety of agents 

also confirm that mutation is non-random . In some cases this appears to be related to 

the initial deposition of damage, in others, it likely reflects differences in the efficiency 

of repair at different sites . Spectra may also reveal a component of mutation that 

reflects the influence of the local DNA sequence on the accuracy of repair or replication 

past a DNA lesion . Studies with a diverse series of agents over a broad ran*e of doses 

in several repair deficient backgrounds have contributed to our current view of the 

mechanisms of mutation. This lecture will concentrate on the kinds of lessons that 

might be )earned from the study of mutational spectra . In particular, we will examine the 

mutational specificity of a series of alkylating agents and discuss the basis of their 

mutational specificity in light of both the expected lesions and the role for DNA repair 

in the avoidance and/or fixation of mutation . 

201 

MOLECULAR SPECTRA OF L5178Y/tk'/' MUTANTS INDUCED BY DIVERSE MUTAGENS . P . 

Gbver, R . Krehl and D. Clive, Wellcome Research Laboratories, Research Triangle Park, NC 27709 

U5A 

Southern blot analyses were performed on DNA from at least 10 large and 10 small colony tk-Imutants 

induced by each of 10 mutagens [2-amino-N6-hydroxyadenlne (AHA), EMS, MMS, 2-AAF, 

methotrexate (Mtx), caffeine, methapyrilene (MP), m-AMSA, hycanthone methanesulfonate, 

procarbazine (Proc)) . Two molecular mutant genotypes were recognized upon digestion with Nco-1 and 

subsequent probing with 1 .1 kb cDNA Insert from plasmid pMtk 4 (ATCC N37556) : (1) no detectable 

alteration and (2) absence of the newly mutated tk allele as Indicated by the absence of the 6 .2 kb 

fragment (Applegate and Hozier, Banbury #28 : page 213, 1987) . In combination with the previously 

established chromosomal nature of most small colony tk -I' mutants (Moore et al ., 1985), this 

pemritted the classification of these 10 mutagens according to the relative proportions of each of 4 

classes of genetic damage they Induced . AHA and EMS gave mutational spectra consistent with their point 

mutational effects In other systems . The other 8 mutagens Induced mostly small colony mutants, most of 

which had lost the entire tk allele . Mtx Induced high frequenciei of large colony mutants at the rk 

locus, mostly lacking the tk allele, and was weakly or nonmutagenic at the hemizygous hprt locus . Four 

mutagens--Mtx, caffeine, MP and Proc--lack structural alerts for DNA reactivity (Ashby and Tennant, 

Mutation Res ., 204 : 17-115, 1988) Implying a major class of non-DNA targets for mutagenicity in 

mammalian cells (Clive, these abstracts) . The mutagenicity spectra for 2-AAF . Hyc and caffeine are 

quite similar, raising the possibility that the DNA adducts of 2-AAF and the Intercalating activity of Hyc 

may not be their principle mechanisms of mutagenicity In mammalian cells . 

202 

EVALUATION OF THE CLASTOGENIC AND MUTAGENIC POTENTIAL OF DIETHYLENETRIAMINE (DETA) 

B . Bhaskar Gollapudi, V . Ann Linscombe, and Anil K . Sinha, The Dow Chemical Company, 

Health and Environmental Sciences, Freeport, TX 77541 

DETA (C .A .S . N111-40-0), a colorless/yellowish hygroscopic liquid, is widely used 

in the production of paper wet-strength resins, epoxy-curing agents, chelating 

agents, lubricating oil and fuel additives, surfactants, and corrosion inhibitors . 

The clastogenic potential of DETA was evaluated in vitro by treating Chinese hamster 

ovary cells in culture for 4 h with 250, 833, and 2500 pg/ml DETA in the presence 

and absence of a metabolic activation system (Aroclor 1254-induced rat liver S-9) . 

The treatments did not induce significant increases in the chromosomal aberration 

frequency in cells harvested 18 h after termination of the treatment 

. DETA, when 

ad .inistered by oral gavage to CD-1 mice at dose levels of 85, 283, and 850 mg/kg 

body weight, did not significantly increase the frequency of micronucleated bone 

marrow polychromatic erythrocytes at any of the three sampling times (24, 48, or 72 

h) . The mutagenic potential of DETA was evaluated in the male germ cells of 

Drosophila melanogaster by the sex-linked recessive lethal (SLRL) assay . Adult 

Canton-S males were allowed to feed on treatment solutions containing 60 mM DETA for 

22-24 h . The treated post-meiotic male germ cells were sampled in three broods of 

3, 2, and 2 days each . The treatment did not significantly increase the frequency 

of SLRL mutations . Thus, DETA failed to induce a genotoxic response in any of the 

test systems employed . 



Notes GENETIC TOXICITY TESTING OF CHLORPYRIFOS AND ITS MAJOR METABOLITE 

B . B . Gollapudi, J . A . Zempel, A . L . Mendrala, V . A . Linscombe, M . L . McClintock, 

and A . K . Sinha, The Dow Chemical Company, Health and Environmental Sciences, 

Freeport, TX 77541, and Mammalian and Environmental Toxicology Research Laboratory, 

Midland, MI 48674 . 


The genotoxic potential of the organophosphate insecticide chlorpyrifos [Q,Qdiethyl-Q-(3,5,6-trichloro-2- 

pyridyl)phosphorothioate, C .A .S . ! 2921-88-2j and its 

major metabolite, 3,5,6-trichloro-2-pyridinol (C .A .S . I 6515-38-4) were evaluated in 

a battery of short-term assays consistiny of Ames, UDS, CHO/HGPRT, and the mouse 

bone marrow micronucleus tests . In the Anes test, the chemicals did not elicit a 

positive response in the Salmonella typhimurium tester strains TA98, TA100, TA1535, 

TA1537, and TA1538 either in the presence or absence of an externally supplied 

metabolic activation system (Aroclor 1254-induced rat liver S9) . Hepatocytes 

collected from male Fischer 344 rats (11-15 weeks old) were treated in vitro with 

the test chemicals in the presence of H-thymidine and the extent of UDS was 

quantitated by autoradiography . Neither chemical induced a positive UDS response . 

No significant increases in the frequency of TGr colonies were observed in the 

CHO/HGPRT assay after treatment of CHO-K' -8H4 cells with the test chemicals either 

in the presence or absence of S9 . Administration of the test chemicals by oral 

gavage to CD-I mice did not increase the frequency of micronucleated bone marrow 

polychromatic erythrocytes . These results suggest lack of genotoxic potential for 

the test chemicals . 

204 

MOLECULAR ORBITAL AND GEOMETRICAL STRUCTURE DESCRIPTORS IN GSAR STUDIES : MUTAGENICITY 

OF SOME AMINO- AND NITRO-BENZENES 

V .K . Gombar, K . Enalein 

Health Designs, Inc ., 183 East. Main Street, Rochester, NY 14604 

Topology-based structure quantifiers are commonly employed In QSAR studies for the 

ease and speed of their computation . They, however, by definition, do not encode 

structural information pertaining to the spatial arrangement of atoms . A study has been 

conducted to investigate the importance of descriptors derived from three-dimensional 

atomic coordinates in QSAR models . A set of 43 amino- and nitro-benzenes (X- 0-NYZ) 

tested for mutagenic response of Salmonella tester strain TA100 (rat liver S-9, Aroclor 

1254 induced) has been selected for the study . Twenty-aix of these were labeled +ve and 

17 -vP . The geometry-based parameters investigated Include shape parameters viz . 

molecular surface area and volume, principal moments of inertia, etc . and electronic 

parameters such as Atomic charges, dipole moment, orbital energies, etc . Topological 

shape descriptors K (kappa) give a poor correlation (n = 40 ; m- 5 ; r z 0 .486) . A 

marginal improvement is observed with group surface area and volume (n : 40 ; m c 5 ; 

r = 0 .583) . However, the two types combined yield a significant improvement (n a 40 ; 

m = 8 ; r = 0 .738) . Similar comparison of QSAR equations obtained using other electronic 

and geometrical descriptors will also be presented . 

205 

DEVE)APMRNT OF' AN IMMUNOASSAY TO MONITOR EXPOSURK TO REN7.O(n)PYRENH BY MHASUREMF.NT OF 

URINARY METABOLITES M . Gomes, T . Vo--Dinh and R . M . Santclla, Oak Ridge National 

Laborat.ories, Oak Ridge, TN (USA) and Columbia University, New York, NY (USA) 

The aim oi' this study was to develop an i.munoassay to monitor human exposure to 

benzo(a)pyrene (Bt') by meaaure.ment of BP and its metabolites in urine . A monoclonal 

antibody was produced from the spleen cells of Balb/c mice immunized with 6-amino-BP 

covalently coupled to bovine serum albumin . The most sensitive antibody, 4U5, was 

characterized in terms of sensitivity and specificity by competitive enzyme-linked 

immunosorbentt assay (ELISA) . The antibody recognizes BP (50% inhibition at 4 pmolo) as 

well as a number of BP metabolites . With BP phenols including 1-OH, 3-OH, 4-OH and 5- 

OH, 50% inhib .ition occurs at. 20 . 90, 60, and Hpmol, respectively . With BP-1,8--diol 

and 9,10-diol and 7,6,9,10-tetraol, 50% Lnhibition is at 1 .4, 4 .5 and 1 .0 paolo, 

respectively . Crossreactivity was also seen with several other polycyclic aromatic 

hydrocarbons (PAHs) including pyrene (60% inhibition at 1 .6pmol), 1-aminopyrene (60% 

inhibition at 0 .49pmo1) and 7,12-dimethylbenz(n)anthrecene (50% inhibition at 67pmol) . 

These results indicate that this assay will detect multiple PAH metabolites . 

Validation of the assay was carried outt on ∎ice treated with [3H) RP . Urine was 

treated with beta--glucuronidase and arylsulfatase and BP and its metabolites isolated 

by affinity chromatography on ScpPek cartridges . Samples were counted and analyzed by 

competitive ELISA using BP in the standard curve . These studies indicated that the 

imaunoassay detected about 54% of the adducte measured by radioactivity . This assay 

will be used to analyze urines from populations with high exposure to PAHs including 

BP . Because of the broad specificity of the antibody, absolute quantitation of PAH 

metabolites may not be possible . This work was supported by a grant from NIOSH-02622 . 

50869 3584

206 

METABOLISM AND ACTIVATION OF FOOD MUTAGENS 

N .J . Gooderham, B.P. Murray, S. Murray, A .R . Boobis and D.S. Davies. Department of Clinical Pharmacology, 

Royal Postgraduate Medical School, London, W12 ONN, UK . 

There is considerable evidence that diet plays a major role in the aetiology of cancer in man . In addition 

to naturally occurring carcinogens present in food, a number of heteroaromatic amine carcinogens are known 

to be formed during the cooking process . These include imidazoquinoline/ lmidazoquinoxaline (IQ 

compounds) which are of particular interest due to their high mutagenic potency . Consumption of cooked be te 

containing the IQ compound 2-amino-3,8-dimethytitnidazo[4,5-fiquinoxaline (MeIQx) at levels of 0 .8 - 

2 .sng/g beef, resulted in systemic exposure, evidenced by urinary excretion of 1 .3 - 4 .9% of the dose as 

unchanged amine . Studies in animals have shown that MelQx is almost completely absorbed but then undergoes 

extensive metaboli m, with only small amounts of unchanged amine excreted in urine . After administration 

(oral, ip or iv) of [i4C)Me1Qx (2 - 60mg/kg) to mice, 25 - 35% of radioactivity was excreted In urine and a 

further 35-45% was eliminated in faeces within 24 h . Urinary metabolites of MelQx included N-acetylsted, 

N-sulphated, N-glucuronidated, ring N2-demethylated, C8-hydroxylated and ring CS-hydroxylated 

derivatives . IQ compounds are readily activated by liver microsomal fractions to derivatives which are 

genotoxic in the Ames Salmonella typhimurium test . Human liver microsomes are particularly efficient at 

activating these amines. The primary activated product of microsomal oxidation is thought to be the exocyclic 

amino N-hydroxy derivative . Studies of hepatic microsomal activation of IQ compounds, employing 

monoclonal antibodies and specific chemical inhibitors, have shown that they are converted to mutagenic 

derivatives by a monooxygenase system predominantly involving the cytochrome P-450 isozyme orthologous 

to rat P-450d . 

207 

MUfAL2I1S IN 1QNYAN '1RADITIQiAI. MICIId: PtEC3IIATiQS . 

H .N.B . Copalan and A.N . WairimA, University of Nairobi, Department of Botany, P .O. Haet 30197, Nairobi 

(Kenya) 

Several plant praducts are used in Kenyan traditianal medicine which is popular both in rural and 

urban centres . 7his form of inedicine is gaining raamenum due to goverrnrntal support and active 

interest in it from both physicians acd pharmacologists . Several of the plant producta are in camen 

usage . Houever, their effects have not been vigorausly tested . In the current study, the fresh sap fram 

Aloe graminicola, ard the sethanol extrazts of Amona aenegelensia, Centella asiatica, Msess lanceolat :a, 

sine africana ~d I rica salisifolia Which are all extensively used in Benyat tr~aditional medicine, 

were tested for their garotoxicity using the Nmes test and the Vicia faba root meristem assay . TA 104 

yielded the highest nuaaer of revertants vith soet extracts . TA 102 detected aame but not as 

effectively as TA 104 . A reduction in nuober of revertants at higher does uas noticed . Apart from 

M. lanceolata, all other plant extracts induced abrotael metaphaaes in V . faba root taeristem cells, 

after 2 hrs of treatrrent . After recovery, only the extracta from C.aas'iatTc-a-, M . africata and 

M . salisifolia caused statistically significant increase in chromosoae aberrations . Fcesh extract of 

A . graminicola induced micracuclei formation after recovery . 'ilau several plants used in itenyan 

craditional medicine appear to erfiibit m¢agatic potential . Further atudies using pirified etaaacts 

are needed before recanendations regarding eotuinued use can be made• 

* lbrk foras part of the requirarents for the degree of Master of Science of the University of 

Nairobi . 

208 

A FORWARD Ml1TATIONAL SYSTEM TO DF.TECT FRAMESHIFTS WIT1i1N A IRO BASB-PA1R TARdI:T 

OF F.schcrichia cnlr. Alasdair J.F C)ordon, Jennifer A. Halliday, Michael J . Horsfall and Barry W . 

Olickman . Dept. of Biology, York University Toronto Canada M3J 1P3 . 

A forward mutational system is described which is based on the acquisition of lacl negative 

dominant properties as the resolt of secondary frameshift mutation (of either sign) within that region of 

the lacl gene that encodes the DNA-binding domain of the lac repressor . Strains containing both I'd 

(dominant-negative) and wild-type alleles of the lacl gene exhibit constitutive synthesis of Qgalactosidase 

and thereby grow on agar plates in which the non-Inducing sugar phenyl-i3•D-galaetoaide is 

the sole carbon source . A consequence of -1 frameshifts in the initial 180 base-pain of lacl is the inframe 

creation of translation termination signals. Translation reinitiation at codons Va123, Met42 or 

Leu62, results in repressor fragments that confer the 1'd phenotype since the ability to aggregate fa 

function of the core domain) is not impaired . However, the first in-frame stop codon which results from 

a+ I frameshift is at buse-pair 277 t1legl/Lyag4Y and therefore translational reinitiation will not produce 

the intact core domain required for an 1'd phenotype . Utilising an F7acl factor with a +A frameshift at 

position 46 (recessive negative) we have devised a test which specifically selects irattteahifts as events 

that yield the I-d phenotype. Within the 181) base-pair target frameahifts of rither sign 1+ I or -1) cnn be 

detected ; -1 events restore the reading frame of the core domain ; +1 events in conjunction with +A46 

exhinit the con.equences of a-1 frameshift. In either aae, some portion of the DNA-hinding domain will 

be non-functional and hence the repressor will be 1-d . This selection system In combination with routine 

cloning and sequencing technologies will facilitate the rapid colliection and characterisation of the large 

numbers of mutations required for studies of mutational specificity . 



Notes 

73



Note s METHYL ISOCYANATE'EXPOSURE IN BHOPAL HAS DAMAGED PLANT AND HUMAN CELLS 

H .K .GOSWANI, PUNEET, RAJEEV GOSWAMI, NANOJ CHANDORKAR AND L .K .SENGUPTA, 

DEPARTMENT OF GENETICS, BHOPAL UNIVERSITY, BHOPAL 462 026, INDIA . 

A muttidLsciplinaay etudy conducted 6ince the ga4eou4 expoeuae on the 

2nd Dec .1984, haa pnoved beyond doubt that methyl ieocyanate (I/IC) hae b4ought about 

biotogical damagee . The aeeed.ament o6 the potential damage6 by SCE 6aequencEee and 

chaomoeomal abe4aatione in human lymphocyte cultuaea have been eetimated a6tea etudying 

200 peneona by Giemoa banding . In oadeR to eompute4ibe the data on epeeisic metaphade 

chROmoAome4, 100 micaophotogaaphe, each baom ga4 expoeed and otheRwiee no4eal donoita 

weae uaed . Thie gave a highly etatietiealty aigni6ieant aatio that chaomoeome 9, 

11 and 16 aae mo4t paedominantly ab6eeted . 

The MIC expoauae have baought about ala4ming changea in haemogtobin . 

Exten,6ive .6tudiee on moae than 1,400 peaeon4 by cellogel electaophoaetic eepenation 

have aevealed the 11ae o6 Foetat haemoglobin (HbF) in adult aamplea . 

Tideue cultuae etudie4 os Datbea ia aieeoo, have ehown dib6eaential gaowth 

in MIC expoeed explant when compaaed to eon ao undentical eultuae condEtion3 . 

One o6 the modt impoatant in6eaeneee, theae6oae, appeaad to be that Nethyl 

idocyanate might be mutagenic undea ceatain conditione . 

210 

MUTATION ASSAY FOR PERSONAL AIRBORNE PARTICULATE SAMPLES BY A HIGHLY SENSITIVE ULTRA- 

MICRO FORWARD-MUTATION METHOD. S . Goto ; Y . Takagi ; H . Murata ; J . Levtas ; and 

H . Matsushita ; 1National Institute of Public Health, Minato-ku, Tokyo (Japan), 

zSchool of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa (Japan), and 

3 Health Effects Research Laboratory, Research Triangle Park, NC (U .S .A .) . 

Actual sensitivity of forward-mutation method using SaUnonsZEa typh*merium TM677 

was improved remarkably by the modification of preincubation step, where 10 yil of the 

solution containing tester strain and a test chemical was incubated at 37'C for 2 hr . 

Actual sensitivity of this ultramicro forward-mutation method was about 10 times higher 

than the micro forward-mutation method (volume of the mixed solution in the preincubation 

step : 100 p1) and about 100 times higher than the original method (1000 ) :1) . This 

method can measure mutagenicity of extracts from airborne particulates obtained by only 

3 cubic meter air sampling . This method was applied to the survey of mutagenicity of 

airborne particulate samples collected by personal air sampler at 1 .5 - 2 1/min for 24 

hr . Mutagenic activity of these personal air samples was in the following order ; smoker 

(10 cig/sampling), smoker(6 cig .), passive smoker(5 cig .) and non-smoker/non-passive 

smoker during sampling, irrespective of presence and absence of S9 mix . It was also 

found that mutagenic activity of airborne particulates collected in Washington D .C ., 

U .S .A . was higher in the test condition with S9 mix than without S9 mix . On the other 

hand, mutagenic activity of airborne particulates collected in Tokyo, Japan was nearly 

same or higher in the test condition without S9 mix than with S9 mix, suggesting 

difference in species of mutagens in both the airborne samples . 

211 

EFFECT OF METHYL ISOCYANATE ON PROLIFERATICN AND DI6'FEREIJfIATICN OF MOUSE MUSCLE 

CELLS IN VITRO 

Shobha GoTyFe-, -School of Life Sciences, Jawaharlal Nehru University, New Mehrauli 

road, New Delhi-110067, India . 

One of the striking features of the Bhopal disaster in which several thousand 

people were killed or injured was the lack of toxicological information on the 

causal agent methyl isocyanate (MIC) . This incident has raised new concern 

over MIC as a toxicant . To ascertain the morphological and functional sequalae 

of a single MIC exposure to the mamealian skeletal muscle, the neonatal rat 

muscle in vitro was exposed to different concentrations of MIC ranging from 

0 .025 -6.5~~ ml medium . The drug was suspended in the growth medium . It 

was added to the cultures at the end of days 1 to 6 . The effects of MIC were 

dose dependent . Fusion of myoblasta was inhibited at 0 .125 and 0 .25 yl, and 

at 0 .5 u1 cells were completely destroyed . Lower concentrations of MIC allowed 

cell fusion to occur, but the total cell count was significantly low . This 

would suggest either an effect on muscle differentiation or a selective toxicity 

towards myoblasta . 

212 

ANALYSIS OF STRUCTURALLY RELATED COMPOUNDS IN THE DROSOPHILA WING 

SOMATIC MUTATION AND RECOMBINATION TEST (SMART) . U . Graf, A . Alonso 

Moraga and N . van Schaik, Institute of Toxicology, ETH and University 

of Zurich, Schwerzenbach, Switzerland, Department of Genetics, 

Faculty of Sciences, University of Cordoba, Spain, and Department of 

Genetics, University of the Witwatersrand, Johannesburg, South Africa . 

50869 3586


The test for somatic mutation and recombination in cells of the Notes 

wing imaginal disc of Drosophila melanoyaster makes use of the two 

recessive markers Dltitl (multiple wing hairs) and = (flare) on the 

left arm of chromosome 3 . In cells trans-heterotygous for the two 

markers, mutant clones can be induced which differentiate into somatic 

spots on the wings of adult flies . Twin spots, consisting of an MWh 

and aLIX clone, result from mitotic recombination ; mwh (and f-U) 

single spots are due to point mutation, deletion, or mitotic recombination 

. Compounds are tested by acute (2 to 6 h) or chronic (48 h) 

feeding of 3-day-old larvae . Three different groups of structurally 

related compounds have been tested : Alkylating agents (nitrogen mustard 

and half mustard, three N-nitroso-guanidines), 5-nitrofurans 

(nifurtimox and eight related compounds), and tricyclic antidepressants 

(amitriptyline, desipramine, imipramine, nortriptyline, protriptyline) 

. The results obtained demonstrate that the Drosophila wing 

spot test is ideally suited for this type of study due to its fast and 

easy performance . 

213 

T:fE USE OF TRADESCANTIA AND VICIA FASA BIOASSAYS FOR THE IN p7 DETECTIO1 OF 

MUTAGEdS IN AN AQUATIC ENVIRONMEiVT . Ff . F . Grantl, H . G . Lee , D .M . Logan and M .F . 

Salamone3, IMcgill University, Montreal (Canada), 2York University, North York 

(Canada), and Ministry of the Environment, Toronto (Canada) 

Most studies with plant bioassays have been carried out in the laboratory, or 

under atmospheric in situ conditions . The primary purpose of this study was to gain 

some experience with plant mutagenicity bioaasays under in situ aquatic conditions . 

The assay systems teated were the Tradescantia stamen hair and micronucleus assays 

for the detection of gene mutations and chromosomal aberrations, respectively, and 

the Vicia faba bioassay system which detects chromosomal aberrations in root tips . 

The assays were used to test the effluent from a pulp and paper mill locatqd on the 

north shore of Lake Superior . Assays were performed in a creek containing raw 

effluent and in a bay of Lake Superior into which the creek emptied . All in situ 

treatipents were carried out for 24 hours . On the creek, 11 .5 km from the source, the 

effluent was toxic to the Vicia faba roots as expresse# by a reduction in the mitotic 

index . The effluent in the creek induced a significant increase in the number of 

stamen hair mutants, micronuclei, and chromosome aberrations . Likewise in the bay, 

witn the exception of the two most distant sites, the creek effluent also induced 

increases in stamen hair mutants, micronuclei, and chromosome aberrations . 

Tradescantia tested in the laboratory several days later with water from the test 

sites, demonstrated decreased mutagenicity compared to the in situ tests . This 

suggests that at least part of the mutagenic fraction is unstable and indicates the 

need for aquatic testing either in situ or as soon after sampling as possible . 

214 

THE CARCINOGENICITY INFORMATION DATABASE OF ENVIRONMENTAL SUBSTANCES (CIDES) AND THE 

ASSESSMENT OF POSSIBLE HUMAN RISK . Mildred R . Green, Technical Database Services, 

Inc ., 10 Columbus Circle, New York, NY 10019 

The Carcinogenicity Information Database of Environmental Substances (CIDES) contains 

numeric data on the effects of approximately 1000 known or suspected environmental 

carcinogens : cancer-relevant data covering a wide variety of long- and short-term 

tests and physical/environmental property data . The carcinogenicity data are organized 

into six categories - whole animal carcinogenesis, mammalian call lines, mammalian 

tissues or cells, bacterial tests, all other assays and human epidemiological 

studies . Searches can be directed to specific areas of interest, e .g ., species, route 

of entry or assay type . A unique feature in CIDES is that the collected studies are 

analyzed to provide a rating, the Carcinogenicity Ratio(CR), summarising the likelihood 

and the weight of the evidence(w) that a substance is a carcinogen . The CR is the 

means of the percent positive studies weighted by a factor that varies with teat category 

; the w is based upon the extent of the testing . When thie approach was applied 

to known human carcinogens, the CR was consistently above 50 . For example, for bensidine 

and 2-naphthylamine the calculated CR/v ratios summarizing all the evidence included 

in CIDES were 84 .9/94 .0 and 65 .6/83 .0 respectively . However, CR/v values may 

vary as new studies are added to the database . This analysis can be used to identify 

compounds for which data on human exposure are not available but whose test results 

indicate that they are likely to be carcinogenic, and also, compounds which should be 

tested furthtfi to improve the weight of the evidence . CIDES will be available through 

the Numerica online service of Technical Database Services in the summer of 1989 . 


75


Notes 


215 

THE CARCINOGENICITY PREDICTOR : AN ONLINE ANALYSIS BASED UPON THE RESULTS OF SHORT-TERM 

GENOTOXICITY TESTS . Mildred R . Green, Technical Database Services Inc ., 10 Colusobus 

Circle, New York, NY 10019 

The Carcinogenicity Predictor is a computerized package based upon the CPBSTM research 

model developed at Case Western Reserve University(CWRU) by H . S . Rosenkranz and 

others . The program calculates the probability that a chemical is carcinogenic from 

results of short-term genotoxic assays . The user identifies one or more tests in a 

battery, indicates the results (positive or negative) for each one, and specifies the 

prior probability of carcinogenicity for the chemical in question . The prediction of 

probability results from a Bayeslan analysis of the information input and of data quantifying 

the sensitivity and specificity of each assay which is stored in the package . 

The sensitivity and specificity values reflect the performance of each genotoxic test 

in distinguishing between known carcinogens and noncarcinogens . Currently, the Carcinogenicity 

Predictor contains a database of sensitivities and specificities for 32 different 

assays ; updated values will be added as a result of further research at CSiRU . 

However, users have the opportunity to create their own databases if they want to use 

other values for a particular test . Calculations may be obtained at a single value of 

prior probability (the default of 0 .5 or some other value chosen by the user) or as a 

graph and a table of predicted probabilities of carcinogenicity at 11 values of the 

prior probability from 0 .0 to 1 .0 . Recent enhancements to the package include the development 

of a user interface which permits the evaluation of genetic toxicity via remote 

accew The new version of the Carcinogenicity Predictor is available on the 

Numerica online service of Technical Database Services . 

216 

COMPLEMENTATION ANALYSIS OF X RAY SENSITIVElARA-C RESISTANT HAMSTER CELL 

(AraC 213) - HUMAN ATAXIA TELANGIECTASIA HYBRIDS . Gloria Greer and R. Julian Preston, 

Univ. of Tennessee Biomed. Grad. Sch . and Biology Division, ORNL, Oak Ridge, TN 37831 . 

We have recently isolated an ara-C resistant X ray sensitive cell line, AraCa 213. Initial studies had been 

performed with the cell line Ara 2 .1 (derived from a CHO-KI cell line) which was screened for X ray 

sensitivity after selection for ara-C resistance at a concentration of S x 10' M . This mutant cell line was 

mutagenized a second time with EMS and selected at a concentration of 10° M ara-C in an effort to isolate 

a mutant which would exhibit an increase in X ray sensitivity . Thiss was found to be the case . This hamster 

mutant cell line (AraC 213) shows an increased sensitivity to X ray-induced chromatid-type aberrations for 

G, exposures, as is also the case with ata :aa telangiectasia (AT) cells. Other characteristics of AraC" 213 

are similar also to those of AT. Therefore, cell fusion etperiments were performed using K1-CHO hgprt' 

or AraC` 213 with a wild type human lymphoblastoid cell line (GM606), or an AT lymphoblastoid cell line 

(GM717). Complementation analysis of the hamster-human hybrids was assessed by colony forming ability 

after exposure to X rays. Preliminary experiments show that the CHO-AT hybrid has the same sensitivity 

as the normal hamster cells, while the AraC` 213-AT hybrid showed no complementation ; it was still X ray 

sensitive . These results suggest that there might be a common repair deficiency in the ara-C resistant mutant 

and the AT cells. Additional experiments will further characterize the defects such that AraCR 213 can be 

used to isolate the human repair gene that reverts the ara-C resistance phenotype and also the AT X ray 

sensitivity . (Operated by Martin Marietta Energy Systems, Inc . under contract DE-ACOS-840R21400 with the 

U. S. Dept. of Energy. GG is supported by NIH-CA08104-13) . 

SITE-SPECIFIC MUTAGENESIS . Arthur P. Grollman, SUNY at Stony Brook . Stony Brook. NY 

11794. 

Chemical methods have been developed by which nucleoside adducts and related lesions 

may be incorporated at defined positions in DNA. Biological systems have been devised which 

allow mutagenic spectra to be determined, site-specifically, in bacteria and mammalian cells as 

follows : A duplex oligodeoxynucleotide containing an adduct or other lesion is ligated into an 

SV40-based shuttle plasmid vector, then used to transform bacteria or transfect simian kidney 

(COS) cells. Mutations are fixed during plasmid replication . Progeny DNA. recovered from a 

COS cell lysate, is amplified in F. coG, then digested by a restriction enzyme known to cleave within 

a unique nucleotide sequence that includes the site of adduct modification. Circular DNA, obtained 

from mutant plasmids, is amplified preferentially by transforming competent strains of E coJl . 

Specific mutations are identified by oligonucleotide hybridization techniques. The advantages of 

this experimental system include the homogeneity of input DNA, lack of bias in the procedure used 

for detection of mutations and the potential for systematically altering base sequence in the vicinity 

of the adduct . This approach has been used to establish the mutagenic specificity of abasic sites, 

selected bulky and exocyclic DNA adducts and to explore molecular mechanisms Involved in 

chemical mutagenesis . 

217

218 

CYTOGENETIC ACTION OF METHYLMERCURY CHLORIDE IN HUMAN LYMPHO- 

CYTES, 

Helena Groot de Restrepo and Luz A . Carvajal de Gil . Laboratorio de Genbtica 

Hurnana, Universidad de los Andes, A .A . 4976, Bogot3, Colombia . 

An in vitro study was carried out to analyse the effects of inethylmercury chloride 

(MM) in hurnan lymphocyte txaltures . Analyses of cell cycle kinetics, chromosome 

aberrations and sister chromatid exchange (SCE) were performed . The results 

show a dose effect relationship in tlie cell proliferative processes . No 

statistically difference was observed for the frequency of SCE in the cultures exposed 

to different concentrations of MM (dose-response curve) non in the exposed 

and control cultures (blood from 10 donors) . However the dose-response curve 

at one of the concentrations of MM used (1 .26 ug/ml) show a significant increase 

of SCE and a higher replication index (RI) . A significant increase in the frequency 

of chromatid type aberrations was found in the MM exposed group . 

219 

INSERTIONAL MUTAGENESIS OF HUMAN CELLS USING RETROVIRUS SHUTTLE VECTORS 

Andrew J . Grosovskyt, Linda S . Rosst, Barry W. Glickmanz and Adonis Skandalis2, tUniversity of 

California, Riverside, California 92521 and 2York University, Toronto, Ontario M3J 1P3 

Insertional mutagenesis is a powerful approach for identifying novel genes with an identifiable phenotype 

because genes of interest may be simultaneously inactivated and tagged for cloning as a consequence of the 

insertion . Although this strategy has been widely used in prokaryotes and lower eukaryotes, its applicability 

in mammalian systems has been limited by the lack of suitable insertion elements . Retrovirus based shuttle 

vectors are, however, well suited for this role since they infect a broad range of host cells at high efficiency, 

integrate into the host genome at low copy number and at random, and encode selectable markers within the 

vector genome. 

We present here a characterization of insertional mutagenesis of the human B lymphoblastoid cell line 

TK6 following infection with the retrovirus shuttle vecto pZipNeo . TK6 cultures were exposed to pZipNeo 

infection for periods ranging from 12 to 72 hours . A population which contained stably integrated provirus 

was selected by exposing the infected population to G418 ; G418 resistance is encoded by the neo glne carried 

in the vector genome . The infection efficiency in the exposed population, as estimated by determining the 

frequency of G418 resistance in a clonal assay, ranged up to 3% in cells exposed for 72 hours. Insertion 

mutation frequency was monitored using several endogenous selectable markers available in TK6 cells (aprt, 

hpri, tk, Na'/K- ATPase) . An exposure time dependant induction vyas seen for the first three markers . 

Induction ranged from 6 to 30 fold following a 72 hour infection period . A small induction was also 

observed at the Na'/K" ATPase following 24 hours of infection ; this may be attributable to effects on gene 

expression or gene amplification associated with retroviral integration . ' 

Experiments are now being conducted to determine the percentage of mutants recovered following 

retrovirus exposure which are directly attributable to disruption of the coding sequence 

by a provirus, and to determine the provirus copy number after various infection periods . 

220 

G---7CTOXIC :T'_ Or FESTICL0:S N•TD P :J :a'T_^ SY31'»2ii 

: . :;, Gt~)v:: .2, School of Life Sciences, 3uru Nanak -'ev University, 

q,-,rit ;ar-143005, In3ia . 

Several short term assays encompassing all the maior tax>nomic groups 

hav' been rsaommen3e3. Although plant 3ystems are being use3 

successfull_v by a ntmtber of workers, yet the scepticism about it 

Persists . A comparative account of the cp_notnxicity of 20 pestici3es 

workz3 out by cytogenetic assays in root meristems and pollen mother 

cells in &li_~rn ceDa/Hor3_e_tlm W19 ra , Ln 

chromosomal aberration 

and microzuclel assays in bone marrow calls in rats an .9 histidine 

r=version assa :• in j~1lmonel_l:a_ tyaUmu~j


Notes 


221 

SEL3CPIVE ANTIMUTAUENIC ACTZVITY GF TERKNALIA 1C~E9J~L! IN SALhiONEL14 

TyyUlhUk2= . I .S .GRLVER and Saroj bAl,p . School of Lite Sciences, Guru 

Nanak Lev University, Amritsar-143005, India . 

Tecmiralin chebula is a eommon medicinal plant whose fruit is used in 

"Ayurvedic" and "Unani' system of medicine as caaninative, exFectorant 

and gastric disorders . Antimutagenicity of its water and chloroform 

extracts of dried f wits was determined against two direct acting 

mutagens, sodium azide and 4-nitro-o-phecylenediamine (NPD) in strains 

TA1o0 and TA1535 and TA97a and TA98 respectively and S9 dependent 

mutagen 2-aminofluorene (2-AF) in TA97a and TA9S of ~ .S,ynhimurium . It was' 

obse rved that water ext ract s reduced NrL i nduced hi a revertant s i n both 

TA97a and TA98 significantly but did not have any perceptible effect 

against sodium az ;de induced his+ revertant in TA100 and TA1535 of 

.Z,tvl)hlm yrium, The pre-incubation studies where the extract was incubated 

at 370C for 30 minutes with the said mutegen prior to plating enhanced 

the inhibitory effect . Autoclaving the water extract reduced its effect 

but the reduction was not significant . The water extracts inhibited signi . 

ficantly 2-AF reduced his+ revertants too in all the strains tested but 

its effect inTA98 was striking as it reduced the revertants to spontaneous 

level . No inhibitory effect was observed in any of the strains and against 

all the test mutagens with chloroform extract . It appears that inhibitory 

factor (antimutagen) is water soluble, heat stable and desnutagen . 

COMYARISON OF TF1E ANTIMITTAUENIC EFFECT ( .F WACER Ey.TRAGTS OF TWU 

DIFFEFtEhT VARIETIES OF INLIAN CGUSEbF :NIQf . I .S .GRGVER 6, AjpLMD liaur, 

School of Life Sciences,Guru Nanak l :ev University, Amrit :.;ar, 1NDIA . 

Indian gooseberry (Llnbiica Officinalis) is a very comnon medicinal 

plant and its fruit is thv richest source of Vitamin C . Nntimutagenicity 

ot the wate r extracts ot its two difterent varletiess was 

tested against two direct acting.mutagens, sodium azido and 4-nltrcL 

o-phenylenediaminr in TA1535 and 'l'A98 respectively using thn Ames 

test . water extract of var .-I reduced the his+ revertants in TA98 

upto 5r/. but its effect was not significant in TA1535 . Pre-incubation 

of the extract with the mutagen at 370C for 30 min did~not have a 

significant eff--ct in TA98 but reduction'of his+ revertants in TA1535 

was . enhanced by pre-incubating the extract with sodium azide . Auto-oclaving 

of ext ract reduced its inhibitory effect in TA98 but promoted 

its effect in TA1535 . Water ext ract of var .-II showed a maximum of 

52% inhibition in the sodium ezide inLluced frequency of his+ 

revertants in TA1535 but its effect was corvparatively less in TA98 . 

P re-incubation of the extracts with the mutagen increased the 

inhibitory effect in both the strains . 1Heating of the extracts did 

rnt have -a significant effect on its inhibitory activity . Thus it 

may be concluded from the results that both th ese varieties may have 

different nutagenic facto rs . 

222 

A GENETIC ASSAY FOR TNE DETECTION OF INEUPLOIDY AN? CLASTOGENICITY USING A 

NONOCBRONOSONAL HYBRID CELL LINE . R . 6ud1 , R .S . Athwal . and S .S . Sandhu2 . 1Nsw 

Jersey Medical School, Newark, NJ 07103 USA ; 2U .S . Environmental Protection Agency, 

RTP, NC 27711 USA . 

We have developed an assay for induced chromosomal anomalies based on the 

quantitation of loss of a chromosome or chromosomal segment by growth of cells in 

selection media . For this purpose, a mouse/human hybrid cell line containing human 

chromosome 5 as the only human component has been produced . This chromosome S 

carries two dominant selectable markers, Ecogpt, and a gene for sensitivity to 

diptheria toxin (DTs) . Cultivation of these cells in DNEN containing mycophenolic 

acid and xanthine (NX medium) selects for Ecogpt and thus for the retention of 

chromosome 5 . The growth of these cells in the medium containing 6•thioguanine and 

diptheria toxin (DT) gives the frequency of chromosome loss . Similarly, growth of 

hybrid cells in MX medium containing DT (10'10l1) selects for the cells that have lost 

the segment of chromosome 5 carrying DTs gene while retaining Ecogpt . This provides 

a method to quantitate induced chromosome breaks . Preliminary results using model 

compounds show a dose-related response for induced aneuploidy and chromosome breaks . 

The same cell line can also be used to quantitate induced point mutations by growth 

in the medium containing ouabain (10-3M) . A unified genetic assay for multiple 

endpoints will enable the evaluation of test chemicals at doses relevant to human 

exposure . (This is an abstract of a proposed presentation and does not necessarily 

reflect U .S . EPA policy .) 

50869 3590 

223


ACTIVATION OF MUTAGENS BY HUMAN CYTOCHROME P-450 ENZYMES . F .P . Guengerich, T. Notes 

Shimada, M . Iwasakl. and M .V. Martin. Dept. of Biochemistry and Center in Molecular Toxicology . 

Vanderbilt University, Nashville . Tennessee 37232 

Cytochrome P-450 (P-450) enzymes have been implicated in the In otbro bloactivation of mutagens 

and carcinogens (Cancer Res . 48. 2946, 1988) . The specificity of a series of the major human liver P-450 

enzymes towards a series of pro-mutagens was examined tn vitro using the approaches of enzyme 

reconstitution, correlation with marker activities in liver samples . selecUve inhibition and 

sttmulation, and Immuno-inhibition . The endpoint used was activation of a chimeric umuC'lacZcontaining 

plasmid (pSK1002) in Salmonella typhtmurtum TA1535, which is Indicative of DNA 

alkylation. P-45OpA, the low Km phenacetin O-deethylase . is the major enzyme involved in the 

activation of 2-aminoanthracene . 2-acetylaminofluorene . 4-amtnobiphenyl. 2-amlrtoAuorene, and the 

food pyrrolysates Glu-P-1, Glu-P-2, IQ, Me1Q, MEIQx, and Trp-P-2 . P-45oNF . the nifedipine oxidase, is 

the major enzyme involved in the activation of 6-aminochrysene, trls(2,3-dibrompropyl)phosphate, 

aflatoxins B1 and G1 . stertgmatocystin, benzo(a)pyrene-7,8-diol . benzo(b)tluoranthene-9 .10-diol, and 

7,12-dlmethyl-benz(a)anthracene-3 .4-diol. The umu system is not responsive to N-nitroaamines but 

other studies (Cancer Res. 88, 1499 . 1988) suggest that P-450J is involved in the activation of 

N.N-dimethyl, N-benzyl-N-methyl-, N-butyl-N-methyl- . and N,N-diethylnltrosamine. Our studies 

suggest that human P-45ODB (debrisoquine 4-hydroxylase) . P-450Mp (mephenytoin 4'-hydroxylase, and 

P-45OTB (tolbutamide hydroxylase) do not contribute to the activation of the compounds studied . Rat 

P-450 enzymes are generally not unreasonable models for human orthologs, although a number of 

results suggest cauuon in making interspectes comparisons among P-450 gene products with regard to 

catalytic specificity. (Supported in part by USPHS grants CA44353 and ES00267) 

225 

MUTAGENICITY OF NITROSCANATE AND ITS PUTATIVE METABOLITES IN 

SAIA4JNELLA MJfATION ASSAY 

_R .L . Gupta, I .P. Kaur and T .R . Juneja, Department of Pharmaceutical 

Sciences, Panjab University, Chandigarh-160014, INDIA . 

Nitroscanate, 4-Nitrodiphenyl ether, an anthelmintic drug, belongs 

to nitroarenes which have been reported to possess mutagenlcity 

and carcinogenicity and their toxicity is considered to be mediated 

through metabolic reduction of the nitro group . We have investigated 

nitroscanate (1) and its hydrolytic product amino (2) and the corresponding 

acetamido (3) including its reduction products formohydroxamic 

acid (4), nitroso (5), hydroxylamine (6) and its N-acetyl 

(7) and N,O-diacetyl (8) for their mutagenicity in TA98,TA98NR 

(nitroreductase deficient) and TA98/1,8DNP 6 (arylhydroxylamine 

esterifying deficient) . Nitroscanate was direct acting mutagen to 

only TA98 suggesting that bacterial nitroreductases and esterifyinenzymes are necessary for its activation in vitro 

. However, 4,9 

and 6 showed direct mutagenicity to TA98 and . TA98NR and the activity 

increased after mammalian S9 activation . In contrast, strain TA98/1,- 

8DNP6 was markedly resistant to these compounds . It would, therefore, 

appear that arylhydroxylamine esterifying bacterial enzyme Is 

necessary for their activation . To conclude that nitroscanate and 

its hydrolytic product are mutagenic only after reduction and subsequent 

esterification . 

226 

Mtl1'ATIONAL SPECIFICITIES OF N-NITRC50 COMPOUBIDS• J .B. t;uttenplan ., Dept . of 

Biochemistry ., N .Y . University Dental School,, and Dept . of Environmental Medicine,, 

N.Y . University Medical School,~ New York,, NY (U .S .A .) . 

The mutational specificities of a group of N-nitroso compounds were examined in 

Salmonella using the system of Levin and Ames (Environ . Mutag .#, 8., 9-28 (1986) which 

assays the six different point mutations . All strains were uvrB and harbored pia1101 . 

Direct acting N-nitroso-N-alkyl derivatives of ureas anitroguanidines .. with 

increasing chain length ., were compared . Additionally, N-nitcosodimethylamine (NDMA) 

and nitrosomethylphenylamine [nitrosanethylaniline (NMA)j were studied . Consistent 

with previous reports, methylnitrosourea (IRiU) induced predominantly GC--AT 

transitions . Ethylnitrosourea induced mainly three base changes, . GC-AT,, AT--GC and 

AT-GC with only the latter two detected at low doses . Both the propyl and butyl 

con4ounds also induced these three mutations,, but the fraction resulting from AT--CG 

transversions was somewhat reduced . The fraction of GC-CG tcansversions ., although 

small, also increased with chain length . The fractions of GC-TA, and AT-TA 

transversions increased with chain length and were quite significant for the butyl 

compound . NMA induced exclusively AT--CG transversions . NDMA surprisingly exhibited 

a much different profile than MNU at the doses examined . It induced both AT-TA and 

GC-AT base changes when metabolized by rat or hamster S-9 fraction . Bowever ., when 

metabolized by microsomes, it exhibited the same specificity as lRNU . This study 

demonstrates (1) that even relatively similar compounds have their own unique 

mutational profiles and (2) that the comparison of mutational profiles can be 

valuable to a tool in determining whether similar compounds induce mutations by 

coamon or different pathways . Supported by Grant #ES03332 . 




Notes AN OPTIMIZED PROCEDURE FOR CULTURING RAT LYMPHOCYTES FOR IN VITRO CYTOGENETIC 

SCREENING . P.J. G azie and Y. OaWro, O.D. SearM & Co .. Skokle, IL . 

Treatment prooedures and culture conditbns were optimized for the tn vitro treatment of peripheral blood 

lymphocytes (PBLs) of rats for oyto0enstb evaluation . Approxktttlaly 12-16m1 of blood was aolMded by cardisc 

puncture Into heparinl2ed VACUTAOJERTN tubes . Whole blood cultures and FboR4epaated ytnphooyla wan 

cuUured at 37'C for 48 h In RPMI-1824 medium supplemented with 20%fetal bovine sennn, L-pkAamMn (2 mM) . 

peniciuiNstreptomydn and the mitogen, ConcanavaNrt A(Con A : 6.7 Npht* . Various a>aure aondtbns were 

tested Including the addition of agents whidt have been reported to Increase blaslopenaN of ymphooytes . 

The addition of UCI (0.3 mM) to cultine medium aotltatNtq Con A did not slpNfioarMy Yanaa the ntlbfb Index 

relative to the use of Con A alone . However, the addition of 2-mathoxyelharal (2-ME ; 0 .9 µM) to medium 

contalninp Con A produced an 8-told karease In the mitotio Index compared to that observed with Con A alotw . 

M approxhnate 6-fold krorase tn numbent of mltotio oaNS was observed with FbDDN-teparaled cultures compared 

to whole blood cultures oodaininp both 2•ME and Con A . The effect of c.u density on tM mltadc kdex was 

studied by culturing Flooll-separated PBL's at densltNs of I X 108, 1 .6 X 106, and 2 X 106 oa6s per culture . 

Maximum mitotic Indices (6-7%) were achieved when 1 .6 X 108 PBLS were cultured In RPMI medium 

supplemented with Con A, and 2-ME for 48 h . In addition, cell cycle kinatkn were defined using 

5-bromodeoxyuridine In the culture medium to dineretttlale tha propottbtr of cells that had undarpona 1, 2 or 3 

cen .divisions over a defined period of 6me . TheN sabNs Indicated an avlrape oeM division tlme of approxknatey 

14-16 h after an InRial delay of 24-28 h . TrlelhylensrnslatNne (TEM ; 0.06 mphnq, a dUect-actUq daslopen, was 

added to the culture medium for 4 h after a 24 h cuMura period . Lymphocytes were harvested after a total of 48 h 

(final 3 h with coloemid) In culture and evaluated for chromoaome damage . Approximately 37% of the 

TEM-treated cells were aberrant compared to oottirol levels of 3-4% . Additional known dastopans and 

non-clastogens are being tested to evaluate thk test aystem for roupne aonankp of potential dastooens . 

POINT ttUTATIOMS IM THE K-RAS ONCOGRa,tt IY DMA-SAHPLSS FROIt LUNG CANCER PATIENTS 

228 

Hackman, P ., Husaafvel-Pursiainen, K ., Mttila, S ., KalliomYki, P .-L ., HeikkilY, L ., 

Hattila, S . & Vainio, H ., Institute of Occupational Health, Topeliuksenkatu 41 aA, 

00250 Helsinki, Finland 

Activation of the cellular onco6enes in the rn-Sene family has been implicated 

in many types of human piali6nancies . In this work we are studyina the mutational 

activation of K_ras-onco6enes in lunS cancer patients . Peroperational lung tissues 

as well as blood samples from 21 lunb cancer patients, were obtained from the 

Helsinki Universlty Central Hospital . DnA was isolated from tumour tissue as well as 

from normal peripheral lung tissue, and from wfiite blood cells and stored freeze 

dri•ed . All of these patients were smokers or ex smokers . Of the 21 lung cancers 

twelve were squamous cell carcinomas, six were adenocarcinomas and three small cell 

carcinomas . Fourteen of these patients had a history of occupational exposure to 

asbestos . DNA-sequences from samples of human DNA were amplified using the 

PCR-technique (Polymerase Chain Reaction) . The amplimers used in this reaction were 

identical with sequences situated on both sides of the human K-ras-oncoSene exon 1 . 

Codons 12 and 13 which are located in this exon have been reported to be amon6 the 

critical points for the mutational activation of the K_ras-onco6ene . Sxperiments are 

in proaress to analyse the amplified DHA for specific point mutations by 

hybridization using specific 20 bp oliònucleotldes . Wild-type oli6onucleotides as 

well as at least 6 "mutated" oli6onucleotides differinS in one base pair/codon are 

used in these experiments . 

229 

THE ORIGIN OF MIlTA[JT3 . Bany 0 . Hall . Dept. of Biology, University of Rochester, Rochester, NY . 

Spontaneous mutations are assumed to occur randomly, condntxxtsly, and without regard to their utility . 

Two studies from my labaatory suggest that some mutations occur more often when they are advantageous 

than they do when they are irrelevant to the well being of the cell . In both cases these spontaneous ad hoc 

mutations occur in aged, nutritionally depleted, colonies of BschertchJa coll . In the first study excision of a 

mobile DNA element, IS103, from within the bglF gene is required for expression of that gene which then 

permits utilization of the 8-glucoside sugar salicin . The excision event is undetectable (Q x 10-12 per cell 

division) in cultures of growing cells, but it occurs at frequencies as high as 10'1 in aged colonies on 

MacConkey plates if, and only if, salicin is present in the plate . In the second study reversion of point 

mutations within the trp operon of E . coli occur 15 to 25 fold morefrequently In aged colonies on 

tryptophan depleted medium than they do in growing cells . A variety of trivial explanations for these 

observations are ruled out, and it is concluded that the vast bulk of the mutants recovered in these 

experiments are the result of mutations that would not have occurred under non-selective conditions by 

chance alone. It now seems likely that spontaneous mutation rates are not inherent properties of organisau, 

but instead that they are subject to modulation by normally encountered envtronmental conditions . 

Furthermore, the probability of a particular mutation arising may depend strongly upon the selective 

advantage conferred by the mutation. 

50869 3592


TFIE EFFDLT OF DIETAFB! B1diSSIMS SPAWiS CN 7~ GENOTWQCTIY OF AFLi~T03mi, Notes 

D AND 2-ACLZYLAIIDFiDl7RFIJE . C .M . Hatniltcnt, R .M . Iblmest . ~ P . 

Bakke~, K .E . Garinf , K.L. Steirmetz', G .S . Steawsand2, and J .C . llirsalist . SRI 

International, Menlo Park, CA, and 2Corne11 University, Geneva, NY . 

Dietary oortstmgtion of civcifervus vegetables, especially lmmsels sprouts, have 

been shown to decrease hepatocarcinogerticity of aflatAxin 13~ (AFBi) in rats, but the 

medhanisn of this effect is tatlvfam . We e~mined the ef ect of di bzussels 

sprouts (~ Q, L . ) on tha gertotwdci of senrezal krtv.m carcirogens in 

male Fisdaer-344 zats . Rats wer+a fed a basal diet~80) or a brussels sprouts amerded 

diet (SSAD ; 20% freeze-dried btussels ) for apQrwtimately 3 weeks prior to 

testinq . Ur~3iedu .led ON71 is was aieesured in ilepatocytes expesed to 

AFSi, dimethylnitrosamine ~j~ ~-acetylaminofhsoresfe (2-AA ) . BD rat 

exposed to 0 .0005, 0 .001, and 0 .005 µq/ml yielded -0.9, 6 .2, and 22 .0 

~g~rau~is/twcleus (NG) . respectively, with 24, 54, and 92% of eells in repair (tIIt, 

t


Notes Research supported by National Institute of General Medical Sciences (GM 09901) and by an 

Outstanding Investigator Award from the National Cancer Institute (CA 44349) . References: Bohr VA, 

Phillips DH, Hanawalt PC, Cancer Res 47 :6426 . 1987 ; Hanawalt PC, Environ Health Pers 76 :9, 1987; 

Mellon I, Spivak G, Hanawalt PC, Cell 31 :241, 1987 ; Vos J-M, Hanawalt PC, Cell 30 :1789, 1987; 

Scicchitano DS, Hanawalt PC, Proc Nail Acad Se! USA : in press; Ho L, Bohr VA, Hanawalt PC, Mo! 

Cellu 8iol: in press. 


233 

ANTAGONISTIC EFFECT OF ASPARAGIIS ON DPIIi-INDUCED NICRONUCLEI AND APOPTOSIS IN THE COLON 

CRYPT CELLS OF NICE . S . Hanxiao, Z . Qingfan, A . Yuhui, F . Shuli, D . Guirong, Y . Bin, 

and W . Yongjun . Dept . of Pathology, Henan Medical Univ ., Zhengzhou, Henan, P .R . China . 

The effect of asparagus on micronuclei and apoptosis in the colon crypt epithelial 

cells of mouse induced by Dimethylhydrazina (Dt41) was studied . Four groups (5 mice/group) 

were intraperitoneally injected with D141 solution, among which three groups were treated 

with oral liquid asparagus in the volume of 0 .1, 0 .5 or 1 ml per mouse, respectively, by 

stomach intubation one hour prior to injection . The fifth group was injected with 1mM 

EDTA intraperitoneally as the control . Twenty four hours after injection, all animals 

were sacrificed . The maximum number of micronuclei and apoptosis were observed in the 

DMH alone group . All three groups of oral liquid asparagus mice had lower incidence of 

micronuclei and apoptosis than Dt4t alone treated animals, and showed a positive doseeffect 

relationship . The results indicated that asparagus has antagonistic effect on the 

mutagenicity of colonic carcinogens . 

TABLE . Number of Micronuclei and Apoptosis in Colon Crypt Cells (p < 0 .01) 

Mean M of lt .A_/ Mean N of lK .A ./ 

Cx=t of Each Nouse Crypt of Each Grouo 

Crouv 1 2 3 4 5 

DMH 4 .05 3 .80 3 .50 4 .15 4 .45 3 .99 

1 ml 0 .80 0 .70 0 .70 1 .00 0 .60 0 .76 

0 .5 ml 1 .90 1 .85 1 .15 1 .35 1 .50 1 .55 

0 .1 ml 2 .85 2 .30 3 .20 2 .55 2 .65 2 .71 

EDTA 0 .45 0 .30 0 .65 0 .35 0 .45 0 .42 

234 

MUTAGENICITY STUDIES ON FENITROTHION IN BACTERIA AND MAMMALIAN CELLS . 

M . Hara, S . Kogiso, F . Yamada, M . Kawamoto, A . Yoshitake and J . 

Miyamoto, Biochemistry and Toxicology Laboratory, Sumitomo Chemical 

Co ., Ltd ., Osaka, Japan . 

The mutagenicity of fenitrothion was determined in strains of 

Salmonella ~t h~imurium and Escherichia coli . Fenitrothion was found to 

be non-mutagenic in Salmone la t mur~um strains of TA98, TA1535 and 

TA1537 and in Escher c WP uvrA both with and without S9 mix, 

while weak mutage t~y was observ-eT-only in TA100 strain and enhanced 

by the addition of S9 mix . The mutagenicity observed in the TA100 

strain was not expressed in a nitroreductase-deficient strain, TA100 

NR, and decreased in a transacetylase-deficient~strain TA100 1,8-DNPG 

The mutagenicity of fenitrothion was also examined by a gene mutation 

assay using the gene for hypoxanthine-guanine phosphoribosyltrangferase 

(hgprt) in V79 Chinese hamster lung cells . Fenitrothion 

did not induce any increment of 6-thioguanine-resistant mutant cells 

at doses ranging from 0 .01 to 0 .3 mM regardless of the presence or 

absence of S9 mix . The results suggest that reduction of fenitrothion 

by a bacterial nitroreductase of TA100 to an active form is essential 

for the expression of the mutagenicity of fenitrothion in TA100 and 

that a bacterial transacetylase of TA100 also has an important role in 

the process of mutagenic activation . 

235 

EVALUATION OF SEVERAL ANTINEOPLASTIC DRUGS IN THE IN VITRO UNSCHEDULED DNA 

SYNTHESIS ASSAY. P.R. Harbach, S .K . Wiser. A .L. Smith and C .S. Aaron . The Upjohn Co ., 

Kalamazoo, MI 49007 

The in vitro unscheduled DNA synthesis (UDS) assay in rat primary hepatocytes is an 

essential part of the genetic toxicity at The Upjohn Company (TUC) . This assay detects the 

repair of damage to rat hepatocyteDNA caused by a wide variety of mutagens and 

carcinogens. Primary hepatocytes were dissociated from the liver and placed into 

monolayer culture which was incubated with the test and control compounds and 

labelled thymidine for 16-20 hours . The cultures were fixed, and the incorporation of 

thymidine was measured by autoradiogrsphy . The results were expressed as net 

grains/nucleus (NG) . We have tested several antineoplastic dru s in the UDS assay : 

tetraplatin (U-77,233) cis-platinum, m-AMSA, CC- 1065, U-73,97~and menogaril . The 

latter three compounds were developed by TUC . U-73,975 is an analog of CC-106S which 

50869 3594

has been reported as strongly $enotoxic in other systems . Both compounds were 

extremely potent DNA-damaging agents, inducin8 > +29 NG at 30 and 10 

nanograms/mI, respectively . Tetraplatin was strongly positive : + 6.47 to + 58 .26 NG at 3- 

30 ug/ml . Cisplatin also induced UDS with similar cytotoxicity to tetraplatin . Menogaril 

and m-AMSA were tested up to toxic or near toxic doses and were both negative . Based 

on these observations, anticancer drugs differ dramatically in their ability to induce 

unscheduled DNA synthesis . 

236 

CORRELATION OF RESULTS FROM THREE GENETIC TOXICOLOGY TESTS WITH RESULTS 

FROM ONCOGENICITY ASSAYS . M . C . Harnois*, T . Sofuni, and M . Ishidate, 

Jr ., National Institute of Hygienic Sciences, Setagaya-ku, Tokyo 

(Japan), *Fellow, Japan Foundation for Promotion of Cancer Research . 

Literature data (1965-85) from the in vitro clastogenicity (IVC) 

test (951 chemicals) were compared with databases on the S~ .~tSr.p~himurium 

point mutation (STPM) test (316 chemicals),the bone marrow micronu~eus 

(BMM) test (105 chemicals) and oncogenicity (0) tests (177 chemicals) . 

Of the IVC+, 41% were STPM- and 55% were BMM- . Of the few IVCsubstances, 

5/18 were STPM+ and 2/10 BMM+ . There were no 0- chemicals 

when all three mutagenicity tests were + . Chemicals negative in all 

three tests were either 0- (pyrene, methionine) or had an unresolved 

oncogenic status by the criteria used (diazepam, phenanthrene) . 

Chemicals IVC+ and 0+ but STPM- in our data were benzene, 

hexamethylphosphoramide, mitomycin C, urethane (BMM+) and actinomycin 

D, diethylstilboestrol, griseofulvin, lead acetate (BMM) . 

Dibutylnitrosamine and ethylene thiourea were BMM- and IVC- but STPM+ 

and 0+ . The data indicate that no one test was adequate to demonstrate 

genotoxic effect or predict oncogenic effectl the chromosome assays 

and STPM test were complementary in both instances . (Ref . M . Ishidate, 

Jr . et al . : Mutation Res ., 195, 151-213, 1988) 

237 

GENOTORICITY OF 2-AMINO-N6-NYDROEYADENINE (AMA) T6 LS178Y/TK+/- -3 .7 .2C OUSE 

LYtlPHOMA CELLS . K . Narrington-Brockl P . Glover2 P . Poorman-Allen2 C . Doerr~, R . 

Krehl2, D . Clive2 J . Nozier3 and M .N . Moore4 . lEnvironmontal Health Research and 

Testing, Inc ., RTP, NC 27709 USA ; 2Wellcome Research Laboratories RTP, NC 27709 USA ; 

3Florida Institute of Technology, Melbourne, FL 32901 USA ; 4U .S . Environmental 

Protection Agency, RTP, NC 27711 USA . 

Studies in a two-component heterokaryon of Neurofpora Srassa have shown AMA to be a 

potent inducer of adenine-3 point mutations ; none of the induced mutants were found 

to be multilocus deletions (Overton et al ., Environ . Mutagen ., 5•501-502, 1983) . An 

international collaborative study has been initiated by do Serres to understand the 

types of mutations induced by AHA in higher eukaryotic organisms . Using L5178Y/TK+/- 

-3 .7 .2C mouse lymphoma cells we have found AAA to be a potent inducer of TK-/- 

mutants (70 ng/ml induced approximately 1600 mutants/106 survivors ; survival - 19X) . 

The majority of the colonies were large colonies ; however, a significant number of 

small colonies was also induced . AMA induced a moderate (as compared to ethyl 

methanesulfonate) response at the heort locus (40 ng/ml induced approximately 200 

mutants/106 survivors) . The induced mutant frequency at the ouabain-resistance 

marker was slightly lower (70 ng/ml induced approximately 135 mutants/106 survivors) . 

Consistent with the induction of small-colony TK mutants, AAA was found to be 

clastogenic . From these studies it appears that in addition to point mutations, AMA 

may be capable of inducing chromosomal events in mammalian cells that are not 

possible in the two component heterokaryon of Neurospora . Banded karyotype and 

molecular analysis will be used to evaluate the type of genetic events induced by 

AHA . (This abstract does not necessarily reflect U .S . EPA policy .) 

238 

ROLE OF ONCOGENES AND TUMOR SUPPRESSOR GENES IN HUMAN LUNG CARCINOGENESIS . Curtis C . 

Harris, Laboratory of Hunan Carcinogenesis, National Cancer Institute, Bethesda, MD 

20892 . 

Five families of proto-oncogenes, ras, raf, fur, un and c have so far been 

associated with human lung cancer . Human ~nch~ al ~thelicells in vitro are 

being used to investigate the functional role of these specific oncogenes growth 

regulatory genes in carcinogenesis and tumor progression . Using the protoplast 

fusion method for high frequency gene transfection, the v-Ha-ras oncogene initiates a 

cascade of events in the normal human bronchial cells leading'To their apparent 

immortality, decreased responsiveness to inducers of squamous differentiation, 

aneuploidy, and tumorigenicity with metastasis in athymic nude mice . Transfection of 



Notes



Notes the SV-40 T antigen gene leads to nontumorigenic cell lines that have a nearly normal 

pathway of terminal squamous differentiation and can be readily transformed to 

malignant cells by transfected Ha-ras . N-ras or K1-ras . The combination of 

transfected c-myc c and c-raf-1 will aTso cause neoplastic transformation of human 

bronchial epitheTial cetls that exhibit some phenotypic traits found in small cell 

carcinomas . These and other results indicate that proto-oncogenes dysregulate 

pathways of growth and differentiation of human bronchial epithelial cells and play 

an important role in human lung carcinogenesis . Allelic deletion and somatic cell 

genetic analyses suggest that tumor suppressor genes may be present on chromosomes 

3p, llp, 13q and 17p . These studies also indicate that tumor suppressor genes may 

play a dominant role in lung carcinogenesis and provide in vitro model systems for 

isolating these genes by subtraction library and insertlonaT-mu-Tagenesis approaches . 

INVESTIGATION OF SPONTANEOUS TRANSFORMATION OF BALB/c-3T3 CELLS . S .B . Harris and 

E .J . Matthews, Hazleton Laboratories America, Inc ., Kensington, Maryland . 

239 

Spontaneous transformation (ST) was investigated using the A31, I-13 clone of chemical 

transformable BALB/c-3T9 cells . All experiments were performed with one lot of 

fetal bovine serum, and one cryopreserved pool of cells . ST was shown to include a 

continuum of Type I, 11, and III foci of diffarent sizes and morphologies, and the 

appearance of foci in individual dishes in an experiment was not normally distributed 

. A log, mathematical transformation of the ST data was shown to convert it to 

a normal distr1bution . Detection of ST was determined to be influeneed by several 

different experimental parameters . First, ST was expressed in proportion to the 

tog „ of the surface area of the culture vessels over the range of 36 to 160 mm in 

diameter . Second, ST was significantly different for cells derived from different 

ampules of cells . Third, pre-existing spontaneous transformants were occasionally 

observed in experiments, and these foci were detected in cultures seeded at cell densities 

> 3 .2 x 10• ce11s/60 mm dish . In contrast, two experimental parameters did 

not effect ST . First, the level of ST was shown to be expressed independently of the 

initial seeding density of the cell cultures . Seeond, ST was relatively constant for 

10 passages and up to 60 population doublings . Taken together, these data permitted 

an estimation of the frequency of spontaneous transformants of 0 .64 x 10-6 for a 

contact-inhibited monolayer of cells in a 60 mm dish . Investigations were supported 

by NIEHS Contract NO1-ES-66160 . 

240 

NATURALLY OCCURRING . IMIDAZOLE-CONTAINING ANTIMUTAGENS : ERGOTHIONEINE AND CARNOSINE . 

Philip E . Hartman, Department of Biology, The Johns Hopkins University, Baltimore, 

MD . 

Ergothioneine (2-thiol-L-histidine betaine) has properties both of a thlol and 

of a thione ; it is present in millimolar ooncentrations in fungi synthesizing it and 

in particular tissues of animals ingesting it . Ergothioneine !s oxidized and 

catalyzes the oxidation of reduoed glutathione to the disulfide in the presence of 

H=0, (reviewed in Hartman PE 1989 Meth Enzymols in press) . Ergothioneine protects 

baeteriophages from Y-irradiation (Hartman et al 1988 Radiat Rea 114 :319) and 

decreases mutagenicity of t-butylhydroperoxide and products of nltrosated spermidine 

for Salmonella (Hartman Z and Hartman PE 1987 Environ Moleo Mutagen 10 :3) . 

Ergo one ne lessens lipid peroxidation in animal systems (Kawano at al 1983 Chem 

Pharm Bull 31 :1676, 1682) -- Carnosine (6-alanyl-L-histidine) is present in 

millimolar amounts in striated musolea of humans and other animals (Crush 1970 Comp 

Biochem Physiol 34 :3 ; Parkhouse at ai 1985 J Appl Physiol 58s14) . Carnoaine quenches 

singlet oxygen and traps lipid peroxyl radicals more effeotively than does free 

L-histidine (Dahl at al 1988 Photochem Photobiol 47 :357 ; Kohen at al 1988 Proo Natl 

Aead Sei USA 85 :3175 ; Hartman Z at al these prooeedings), proteots baoteriophages 

from Y-irradiation (Hartman at al 198~ op oit), and prevents induction of lipid 

peroxidation in muscle sarcoplasmio retioultso (Dupin at al 1987 Biochemistry 

52 :672) . Honocarnosine (Y-aminobutyryl-L-hiatldine) exhibite similar prQteotive 

actiona (Kricheskaya et al 1985 Vopr Med Khim 31(4) :75 : Kohen at al 1985 0 oit) . 

--These prevalent and naturally occurring imidazole compounds warrant furtRer 

investigation as to their possible protective actions against a range of chemioal 

mutagens . 

241 

C-TERMINAL HISTIDINE DIPEPTIDES AS EFFECTIVE SCAVENGERS OF SINGLET MOLECULAR OXYGEN . 

Zlata Hartman, Philip E . Hartman and Katherine T . Ault, Department of Biology, The 

Johns Hopkins University, Baltimore, MD 21218 USA . 

Exogenous singlet oxygen ('0,) ia highly oytotoxic for Salmonella (Dahl at al 1988 

Photochem Photobiol 47 :357) but non-mutagenio (Dahl at al-T986'Mdt Res 201 :127) . 

Free deoxyguanosine is degraded by ainglet oxygen but ia about 40-fold less sensitive 

when in single-stranded DNA and 1000-fold less sensitive when in duplex DNA (E 

Hildebrand 1989 PhD Thesis, Johns Hopkins University) . The imidazole, L-histidine, a 

50869 3596

scavenger of singlet oxygen (Bellus 1979 Adv Photoohem 11 :105), reacts with singlet 

oxygen about 8-fold more rapidly than does free deoxyguanosine (Hildebrand, op cit) . 

Comparative studies were carried out on over 30 imidazole-containing compounds using a 

modification of the RNO bleaching assay for ainglet oxygen of Krali6 (of Verlhac et al 

1984 Nouv J Chim 8 :401) . L-Carnosine (B-alanyl-L-hiatidine), the imidazole compound 

prevalent in human striated muscle (Parkhouse et al 1985 J Appl Physiol 58 :14), 

scavenges singlet oxygen 2- to 3-fold faster than does L-histidine (also see Dahl et 

al, op cit) . We find similar high efficiencies for other dipeptidea with oarboxyterminal 

histidyls . These dipeptides are roughly twice as effective as analogous 

dipeptides with histidine at the amino terminus . A number of other imidazole 

containing compounds either have low solubility, do not exhibit increasing efficiencies 

with increasing concentrations over the range tested (1 .5 - 15 mM), or lose their 

measured capacity for quenching of singlet oxyaen uDon Grolonged ex ofure to oxidants . 

L-Carnosine may be a near-optimal molecule that doubly serves as a gu fer and as a 

defense against oxidative damage . 

242 

ENHANCEMENT OF GENOTOXICITY BY LEAD . 

A . Hartwig, R . Schlepegrell, and D . Beyersmann, University of Bremen, Bremen (F .R .G .) 

Inorganic lead compounds are considered as suspected carcinogens ; however, the mode 

of action is not well understood . Part of the contradictory results in short-term 

assays might be due to differences in bioavailability, since we found a'marked dependency 

of lead cytotoxicity and uptake on cell type and medium composition . Because 

mutagenicity and transforming ability are not accompanied by direct DNA damage (1), we 

investigated whether the genotoxic action of lead is due to rather indirect effects by 

interfering with genetic control and repair mechanisms, using UV as a standard mutagen . 

Pb(II) is comutagenic with UV in the V79 HGPRT-assay . Even though there is only a weak 

induction of SCE's in the same cell line by lead alone, we find a pronounced enhancement 

of UV-induced SCE's . Pb(II) did not produce DNA strand breaks itself but increased 

the number of breaks generated during repair of UV damage, indicating an inhibition of 

the polymerisation or the ligation step of excision repair . We conclude that the genotoxicity 

of lead compounds is in part due to interference with repair processes . 

Reference : . 

1 . Zelikoff, J .T ., Li, J .H ., Hartwig, A . Wang, X .W ., Costa, M . and Rosaman, T .G . (1988) 

Carcinogenesis 9, 1727 - 1732 . 

243 

FLOW CYTOMETRIC MICRONUCLEUS TEST WITH MOUSE BONE MARROW AND PERIPHERAL 

BLOOD ERYTHROCYTFS 

Makoto Hayashil .2, Hannu Norppa2, Toshio Sofunil and Motoi Ishidate. Irl 

1Divisioo of Mutagenesis, Biological Safety Research Center, National Institute of Hygienic Sciences, 

1-18-1 Kamiyoga, Setagaya-ku, Tokyo, 158 Japan 

2Mutagen Laboratory, Department of Industry Hygiene and Toxicology, Institute of Occupational Healtb, 

Topeliuksenkatu 41 aA, SF-00250 Helsinki, Finland 

Flow cytometry was applied to the micronucleus test with mouse bone marrow (BM) and peripheral 

blood (PB) erythrocytes. BM cells were fixed with 1% glutaraldehyde in 1/20 M phosphate buffer at pH 

6 .8 and stored in 70% ethanol . PB erythrocytes were sphered and fixed with 1% glutaraldehyde containing 

0 .03 mg/ml of sodium dodecyl sulfate in 1/20 M phosphate buffer at pH 6 .8. PB cells were also treated 

with RNase to reduce noise from RNA-eoataining erythrocytes . The cells were stained with DAPI, and 

50000 erythrocytes were analyzed in an EPICS V flow cytometer using a UV laser operating at 150-200 

mW. Data from the flow cytometer were further analyzed by a computer program Including model fitting 

to estimate the frequency of micronueleated erythrocytea . For each sample 1000-2000 srythrocytea were 

also analyzed from Giemsa-stained smears microscopically . There was a good correlation between the 

flow cytometric and microscopical measurements after treatment with mitomycia C (BM,PB), benzene 

(BM,PB), 6-mercaptopurine (PB), benzo[aJpyrene (PB), N-ethyl-N-nitrosourea (PB), bromodichloromethane 

(PB), and potassium chromate (PB) . A repeated experiment with mitomycin C also showed good 

reproducibility for the flow cytometric measumeat of mieronuelai In BM . Generally, standard deviations 

were smaller by the flow cytometric method than by the manual method probably because of the different 

sample sizes (50000 erythrocytes for flow cytometry and 1000-2000 erythrocytes for manual analysis) . 

244 

MONITORING OF FOOD MUTAGENS 

Hikoya Hayatsu 

Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700, Japan 

Monitoring of food for mutagenicity requires a simple, practical way of preparing 

testable samples . We have been using the blue cotton method for this purpose . Blue 

cotton is absorbent cotton bearing covalently linked copper phthalocyaaine trisulfonate . 

Since this ligand has a selective affinity to polycyclic compounds, adsorption of mutagens 

having polycyclic structures takes place when food extracts in aqueous media are 


4 


Notes 

/



Notes treated with blue cotton . Elution of the cotton with organic solvents, most efficiently 

with ammoniacal methanol, results in recovery of the adsorbed polycyclics . With this 

method, a high concentration extent is generally obtainable, and the entire work needs 

only short periods of time and little labor . Since mutagenic heterocyclic amines and 

polycyclic aromatic hydrocarbons are efficiently adsorbable to blue cotton, the use of 

this method allows monitoring of these classes of mutagens in food . It has been shown 

that many proteinaceous foods that had undergone heating processes contain mutagens . in 

most cases the heterocyclic aminee ; e .g ., katsuobushi, a heat-dried bonito-fish meat, 

which is one of the everyday food items in Japan, has been shown to contain MeIQx . We 

have also observed that some shell fish in their raw state contain mutagens . We have 

recently improved the method by introducing blue rayon, an adsorbent more powerful than 

blue cotton . The blue-cotton and -rayon techniques can also be used for isolating foodmutagen 

metabolites in excretions and body fluids, and for monitoring mutagens in 

environmental waters and airs . (Supported by grants from the Ministry of Health and 

Welfare, Japan) 

245 

GENOTOXICITY OF INITIATING CARCINOGENS IN THE SKIN . 

S . He and R .S .U . Baker, National Institute of Occupational Health & Safety, Building 

A27, University of Sydney . NSW 2006 (Australia) . 

Nicronucleus(MN) induction can be evaluated in keratlnocvtes followinR application 

of chemicals onto the dorsal skin surface of hairless HRA/Skh mice . Previous studies 

have indicated that animals of this strain are sensitive to chemical induction of 

akin cancer, and also that the active alkylating carcinogen, triethylenemelamine, can 

be readily detected as a genotoxicant in keratinocytes using this procedure . For 

chemicals requiring in vivo metabolism, it was first necessary to establish an 

appropriate sampling time between chemical treatment and removal of skin for in vitro 

studies . With 7,12-dimethylbenz[a]anthracene (DNBA), MN induction reached maximal 

values at 12, 24 and 48h post-treatment . A sampling time of 24h was therefore 

selected for all chemicals . Dose-related increases in HN were detected with the 

initiating carcinogens DMBA, benzo[a]pyrane (BP), chrysene and urethane while pyrene 

failed to induce MN at subtoxic doses up to 2 .5mg/mouse . Significant MN induction 

(p

247 

GESVMXICITY OF MRGANIC MEFiLURH 

HEIMI, S . ; EIrSFEFII, M. ; EL-ZYAT H . & M76TAFA M.H . Institute of Graduate Studies 

and FEsearch, University of Alexandria, A exa a, Egypt . 

Genotoxic effects of HgC1 were tested using a battery of tests . In Vicia faba 

the predaminant cytogenetic iffects in msiosis ware the formntion of ~~i, 

structural chranoscme aberrations, and abnormal ctuamsc :ne distribution . In 

Saccharanyces oerevisiae, strains were different in their sensitivity to HgC1 , the 

nor JD strain was 4 and 1 .4 ppn when ex.oosure was for 3 and 5 hours res~ectively, 

Qe the ID5p values for strain D7 were 1 .4 a,rri 0 .6 pZ m. Log-phase cells were more 

sensitive th3n those of stationary phase . Mitotic gene conversion at HIS4 locus in 

strain JD1 and TRP5 locus in strain D7 was suppressed by HgC1 . In AsMElus imnersus 

(Pasadena strains the older the strain the more seensitive R was to HgCr'-CUFUrrg 

in distilled water, oomplete liquid , axrl cortQlete solid media supplementW with HgC12 

gave different results . Meiotic gene eonversion frequency in + x m crosses was not 

affected, only direction of eonversias showed significant change in the favour of 

wildtype allele . It was proposed that HgCl influenced mis-match repair mechanism 

rather than hybrid DNA formation freduenc .y? However, HgCl increased crossing-over 

frequency - except at 1 pfm - significantly. ZWo hypothe2s were proposed to explain 

such effect :one is based on the fate of half-chiasmata and their resolution, the 

other eonsiders the physical arrangement of chranatids . With respect to forward 

mutation frequency, HgC1 significantly lowered the spontaneous frequency of asoospore 

colour mutations . Probab~y, the inhibition of enzymes involved in the repair of 

premutational lesions by FigC12 caused such reduction . 

248 

EFFECT OF PHENOLIC ANTIOXIDANTS ON BENZO(A)PYRENE METABOLISM, GENOTOXICI- 

TY AND ITS METABOLITES BINDING TO BACTERIAL DNA IN SOS CHROMOTEST . 

E .E . Hennig, K .K . Demkowicz-DobrzaAski, and L . Dock, Medical Academy, 

02-097 Warsaw (Poland), and The National Institute of Environmental Medicine, 

S-104 01 Stockholm (Sweden) 

The effect of butylated hydroxyanisole (BHA) and butylated hydroxytoluene 

(BHT) has been studied on benzo(a)pyrene (BP) metabolism, .genotoxicity 

and BP metabolites binding to bacterial DNA . BP activation has been 

performed using S9 fractions from the liver of mice fed a diet containing 

BHA or BHT . Both BHA and BHT treatment slightly enhanced total BP metabolism 

and markedly increased water-soluble BP nyetabolites formation as was 

indicated by the estimation of the distribution of organic extractable 

and water-soluble metabolites formed in the presence of S9 fractions . The 

HPLC analysis of BP metabolic profile, in the presence of antioxidantsmodified 

fractions, shows significant decrease of 9-hydroxyBP formation . 

The bacterial test SOS Chromotest was used to study the genotoxicity of 

BP . Formation of BP metabolite adducts in Escherichia coli DNA was analysed 

by HPLC procedure . There was indicated a strong inhibition of BP genotoxicity 

when the S9 fraction from BHT-fed mice was used for BP activation 

. BHA had only a moderate, not significant, inhibitory effect . Our 

results indicate that there existed a clear correlation between antioxidants 

effect on BP genotoxicity and total BP metabolites binding to bacterial 

DNA . 

249 

STRUCTURE-ACTIVITY RELATIONSHIPS INVOLVED IN THE GENOTOXICITY OF ANALOGS OF PYRVINIUM 

IN YEAST AND BACTERIA . U .G .G . Hennig and R .C . von Borstel, Department of Genetics . 

University of Alberta, Edmonton, Alberta (Canada) T6G 2E9 

The structural requirements for the mutagenic action of pyrvlniue were investigated 

with structural analogs . The genotoxicity of these analogs was studied in diploid (D5 

and 07) and haploil (XV1B5-14C, XY718-1A, and •1854-1A) strains of Saooharomyoee 

cereviaiae . Substitution with a methyl group at the 6-position of pyrviniuln did not 

affect the mutagenicity ; the 6-methyl-analog induced frameshift and base-substitution 

mutations just as readily . However, the 6-methyl-analog induced both transitions and 

transversions, whereas pyrvinium induced only transitions . With the 6-chloro-analog, 

the toxicity and mutagenicity were diminished but detectable levels of frameshifts and 

transitions were observed . Pyrvinium and the 6-chloro-analog induced frameshifts and 

transitions, whereas the 6-methyl-analog induced frameshifts, transitions, and 

transversions . The hydroxylation of the methyl-substituent of the 6-methyl-analog 

probably results in a mutagen that is as active as pyrvlniun itself . A second DNA 

binding site is the cationic site at the methylated ring nitrogen of pyrvinium . This 

is evident fram the diminished, yet not abolished, genotoxicity of the chloro-analog . 

A methyl- or dimethylamino-substituent at the 6-position and the cationic site at the 

methylated ring nitrogen are required for mutagenic activity . The reversion spectra 

for SaZmoneLLa typh{muri.um (strains TA97, 98, 100, and 102) indicate that the chloroand 

methyl-analogs are mutagenic in all strains (-S9 and +S9) . Pyrvinium pamoate and 

pyrviniun iodide are frameshift mutagens (-S9 and +S9) but activation is required for 

them to induce base-substitution mutations . (Research supported by NHRDP and AHFMR) . 



Notes



Notes EFFECTS OF PROGFSTERONE AND ESTRADIOL ON PROLIFFRATION OF HUMAN L1'MPFDCYTFS IN VITRO . 

L .A . Herrera M ., R . Montero M ., and P . Ostrosky-Wegman . Instituto de InvestTgacones 

Biom6dicas, UNAM, Apdo . Postal 70228, 14sxico 04510 D .F . Mexico . 

During a study of lymphocyte cell-cycle kinetics we found variability between individuals 

and between samples of the same individual taken at different times . Trying to 

understand some of the factors that could be involved in those variations, we thought 

over the relationship between menstrual cycle and cell vroliferation . In vivo studies 

on blood from 6 women, sampled every week during 3 months, didn't show any cTar effect 

that could be directly correlated with their menstrual cycles . Since many other factors 

could be interfering in our studies, we evaluated in vitro effects of estradiol 

and progesterone over cell proliferation kinetics (CPK) ard'Ft'totic Index (MI) on 

human lymphocytes both from men and from women on day 1 of menstrual cycle (lowest 

concentrations of respective hormones) . The two hormones were added to the cultures 

individually or simultaneously, 2 h after stimulation with PHA . Lowest doses of each 

hormone showed an inhibitory effect on CPK and MI . When the hormones were added 

simultaneously, the t of cells in 3th or more divisions decreased at doses similar to 

those found in menstrual and lutheal phases, whereas it increased at doses similar to 

the ovulatory phase . The MI of lymphocytes from women donors decreased at all doses 

of the hormones, whereas those of men donors decreased only at doses similar to menstrual 

phase . The effects of hormones on lymphocyte cell cycle should be further 

studied mainly in relation to the recently described increased sensitivity to genotoxic 

dama?e on lymphocytes from both pregnant women and women taking oral hormonal 

contraceptives, and to the well known increased sensitivity to infections of pregnant 

women . 

PROTOCOL EVALUATION FOR THE MICRONUCLEUS TEST . C . Hilliard, R . Tice, and M.D . 

Shelby*, Integrated Laboratory Systems, P0 Box 13501, Research Triangle Park, NC 

27709 and *NIEHS . P0 Box 12233, Research Triangle Park, NC 27709 . 

251 

Using dimethylbenzanthracene (DNBA) and benzidine (B2D), the efficacy of three 

different in vivo micronuclei (MN) protocols were evaluated, using polychromatic 

erythrocytes (PCE) sampled In both bone marrow and peripheral blood . B6C3F1 male 

mice (9-12 weeks of age ; 5 mice per group) were Injected IP with DMBA (25, 50, 100 

mg/kg) or with BZD (50 . 100, 200, 300 mg/kg) on 1, 2, or 3 consecutive days, with 

bone marrow and peripheral blood smears being made at 24, 48, and/or 72 hours after 

the first injection . Slides were stained with acridine orange and 2000 PCE scored 

per tissue per animal for the presence of MN . DMBA induced a significant increase in 

MN-PCE at all sample times in both tissues using all three protocols, with the yield 

of MN-PCE at 48 and 72 hr being independent of treatment protocol and tissue . BZD 

induced a positive response in bone marrow and peripheral blood only when injected 2 

or 3 times and sampled at 72 hr after the first injection . When administered by 

gavage, a significant increase in MN-PCE was induced by BZD in bone marrow 24 hr 

after a single treatment, with increased activity being demonstrated after 3 daily 

injections . These data indicate that a treatment protocol based on multiple 

injections eliminates the need for multiple sampling times, minimizes the number of 

animals and scoring time, and simplifies the statistical analysis . Also, the lack of 

a significant difference in MN levels between bone marrow and peripheral blood 

suggest that either tissue can be used following this protocol . Supported by NTP 

contract N01-ES-85209 . 

252 

RELATIONSHIPS BETWEEN STRUCTURE OF NITRATED ARENES AND THEIR MUTAGENICITY IN SALMONEL- 

LA TYPHIMURIUH ; 2- AND 2,7-NITRO SUBSTITUTED FLUORENE, PHENANTHRENE AND PYRENE 

T. Nirayama, T . Watanabe, Y . Fujioka, S . Ozasa, and S . Pukui, Kyoto Pharmaceutical 

University, Kyoto (Japan) 

In order to elucidate the mechanisms of autagenic activation of nitroarenes, we 

studied the relationships between the autagenic potency and chemical structure of 2nitro- 

and 2,7-dinitro-arenes including nitrated fluorene (F1), dihydrophenanthrene 

(DNPh), phenanthrene (Ph), tetrahydropyrene (TBPy), dihydropyrene (DHPy) and pyrene 

(Py) together with 9-N02-Ph, 1-N02-Py and 1,3-diN02-Py . The autagenicity tests were 

carried out on Salmonella typhimurium TA98, TA98NR and TA98/1,8-DNP6 in the absence of 

S9 mix . The order of mutability of mononitro- and dinitro-arenes in TA98 is as given 

below : 2-NOy-THPy


EFFECT OF THE RELATIVE HUMIDITY ON THE FORMATION OF NITROPYRENES BY TME PHOTOCHEMICAL Notes 

REACTION OF PYRENE WITH NITROGEN OXIDES 

Yoshiharu Hisamatsu, Kazutoshi Sugita, and Hidetsuru Matsushita The Institute of 

Public Health, 6-1 Shirokanedai 4-chome Minato-ku Tokyo 108, Japan 

The 2-nitropyrene, which have not been identified in direct emissions, in collected 

ambient particulate organic matter have been idetified and quantified . We have investigated 

the effect of the relative humidity (r .h .) to study the atmospheric transformation 

of pyrene to 2-nitropyrene, which is direct-acting mutagen, by the photochemical 

reaction with nitrogen dioxide . 

The photochemical reaction products of pyrene with nitrogen dioxide under various 

humidity of the air which is used to dilute nitrogen dioxide gas were fractionated to 

analyze for mononitro-pyrene by HPLC . The partial fractions corresponding to mononitro 

pyrene were analyzed using GC-MS, and the molecular ions m/z 247([M]+) together with 

the characteristic fragment ions, m/z 217([M-NO]+) and 201([M-N02]+), were monitored . 

The 2-nitropyrene has been identified in the photochemical reaction products of 

pyrene with nitrogen dioxide below 20% r .h . of air, but it has not been identified 

in the reaction products of them under no light irradiation . The photochemical 

reaction products were the most mutagenic below 20% r .h . of air for Salmonella typhimurium 

strain TA98 in the absence of S9 mix , and the mutagenic activities of them 

decreased with the increase of air . 

254 

DIRECT MEASUREMENT OF CHROMOSOME REPAIR BY PREMATURE CHROMOSOME CONDENSATION . 

Walter N . Hittelman . Department of Medical Oncology . University of Texas M . D . 

Anderson Cancer Center, Houston, Texas, USA 77030 . 

Chromosome damage in interphase cells is generally visualized when the cell 

reaches mitosis and the chromosomes condense . However, several events might have 

occurred between the time of genetic insult in interphase and chromosome 

visualization at mitosis, including repair of chromosome damage and delay of•damaged 

cells from reaching mitosis . In addition, chromosome damage in non-dividing call 

populations cannot be visualized by conventional techniques due to the lack of 

mitoses . The technique of premature chromosome condensation overcomes these 

problems by allowing the direct visualization of interphase chromosomes . Thus 

chromosome damage and its repair can be directly measured in interphase cells . We 

and others have used this approach to better understand . .the underlying basis for 

chromosome damage and its repair as well as to characterize repair capabilities of 

various radiation and drug-sensitive and resistant cells . For example, chromosome 

repair in normal fibroblasts and lymphocytes exhibit a fast and a slow component of 

chromosome repair, while mature granulocytes show little chromosome repair . The 

fast component can be inhibited by hypertonic salt while the slow component is 

inhibited by inhibitors of protein or DNA synthesis . Radiation-sensitive cell lines 

such as LS178Y-S and Ataxia telangiectasia exhibit partial deficiencies in both the 

fast and slow components of chromosome repair . The ability to directly measure 

chromosome repair by the technique of premature chromosome condensation can now be 

applied to better understand the in vivo interaction of environmental mutagens with 

tissues within the body . 

255 

THE EBV-Xy1E SMJ111E : ITS OODE'D2111RI0ii MD MJfATIaiAL SPDCIFICITY IN Hu7AN CELL . 

X . K. Hong, X. L . Wang,X .F . Qiu and J .L.Hsueh, Institute of Genetics, Fudan University,Sha*si (Qdna) 

A n.rber of studies recently have been reported in which shuttle vector plasmids were used to study 

mitational specificity in memmalian cells . Shuttle vector systems have becc.re an inportant tool in the study 

of nutational mechanisms in higher organisms . We have developed a shuttle vector system for studying mr 

tational specificity, as a mutational target, the shuttle vector contains the EB virus origin and E . coli 

Xy1E gene, which codes for the enzyme Catechol : oxygen 2,3-oxidoreductase . '!he vector which we constructed 

nared pFV891, is derived fram plasmid p1CfA2, pTG503, p'iCfiO'+ and pACi3 . The bacterial gene XylE, carried on 

a shuttle vector, is introduced into human cultured cells by transfection and allowed to replicated autonomously 

in the cell nucleus . During the replication period of the vector, the cells are exposed to a sut .gan, 

an increased in nutation frequency above the spontaneous background is readily obtained . Mitations form in 

the shuttle vector DNA in the memrelian rucleus . DNA is extracted and introduced into bacteria . Colonies 

that express Xy1E becan; yellow within seconds after selection plates are sprayed with Catechol, while cells 

were transformed by the shuttle vector in which the XylE gene were mutated didn't change the colour . Therefore, 

induced sutants can be rapidly isolated and characterized . Tne sensitive colour assay offers an approch 

to develop this shuttle vector for a wide variety of host organisms . Use of the EBV-Xy1E shuttle vector 

should permit determination of the mrtagenic specificity of a wide range of muagens and carcinogens in hr- 

rmn cells . 


89


Notes 


256 

THE EFFECT OF THYMIDINE ANALOGUES ON RESTRICTION ENDONUCt .EASE INDUCED 

CHROMOSOME ABERRATIONS. OJ. Hook. R.J . Preston, University of Tennessee Biomedical Graduate Sclwol, 

and Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 . 

The thymidine analogue CIdU has been used to analyze the Importance of reoombination-like repair on the 

frequency and type of chromosome aberrations (CAs) inductd by X rays . The results of these experiments 

demonstrated that the level of CAs induced by X rays eould be enhanced and the proportion of exchange type 

aberrations Increased when CIdU was present in the template during 03 repair . CIdU Incorporation b a useful 

method of analyzing the Importance of excision repair on the formation of CAs Induced in Or 7ype 11 restriction 

endonucleases (REa) have Iken shown to induce CAs in mammalian cells in all parts of the cell cycle Including O, . 

We propose to test the bypotbeses that repair of RE induee double strand cuts involves more than simple ligation 

and the extent to which induced CAs are the result of lack of repair of the Induced double strand cuts as opposed 

to misrepair. CHO cells grown in the presence of CIdU were treated with Sau 3A1 at concentrations of 10 and 25 

units per 100 pl using the 'osmolytic shock' protocol . The cells were grown In the presence of CIdU for 26 hrs 

before treatment and harvested 4 hrs after treatment . Astoradiography has shown that the majority of cells 

harvested at 4 bn post-treatment were In O= or M when treated . The presence of CIdU (concentration 30yM) 

resulted In a reduction in the level of deletion type CAs and a doubling of the number of exchange type CAs above 

that expected from additivity. No Increase over additivity was observed when CHO cells were grown in the presence 

of BrdU. In conclusion, RE induced double strand cuts are, in part, repaired by a mechanism that Involves a 

resynthesis step . By altering the likelihood of misrepalr the damage that would have been converted Into deletion 

type aberrations was converted into exchange type aberrationt . (Research sponsored by the Office of Health and 

Environmental Reaearch, U. S. Department of Energy under contract DE-ACUS-840R21400 with the Martin Marietta 

Energy Systems, Inc .) 

257 

RESTRICTION ENZYMI


THE INDUCTION OF CHROMOSOME ABERRATIONS IN MOUSE BONE MARROW AND CH0 CELLS BY THE DNA Notes 

TOPOISOMERASE INHIBITORS CAMPTOTHECIN AND m-AMSA . D .R . Hovardl L .C . Backerl, J .A . 

Campbelll, D .M . DeMarini2, J .W . Allen2, 1EHRT, RTP, NC 27709 ; 2U .S . EPA, RTP, NC 27711 . 

Topoisomerases are enzymes that control supercoiling, breakage, and reunion of DNA 

strands . There is evidence to suggest that two antitumor drugs, camptothecin (CAMP) 

and amsacrine (ID-ANSA), inhibit topoisomerase activity by binding to the DNAtopoisomerase 

complex and preventing reunion of the broken DNA strands . a-AMSA binds 

to topoisomerase II to induce double-strand breaks in DNA . CAMP inhibits the activity 

of topoisomerase I, inducing single-strand DNA breaks . Although y-AMSA induces 

chromosome aberrations (CAs), CAMP has not been characterized for this effect . In the 

present experiments, CAMP and m-AMSA were compared for their capacities to induce 

chromosome- and chromatid-type aberrations in mouse bone marrow and CH0 cells . Male 

mice were exposed by i .p . injection to 0, 0 .5, 1 .5, or 3 .0 mg/kg of CAMP or a-AMSA 

dissolved in DMSO . Four animals/dose and 100 cells/animal were scored for CAs . Both 

chemicals induced approximately 50 chromatid-type and 10 chromosome-type aberrations 

per animal at the highest dose . CHO cells were incubated in medium containing either 

CAMP or g-AMSA at 0, 10, 50, or 100 ng/ml in 0 .5% DMSO . Cells were harvested at 16-18 

h and 100 cells/dose scored for CAs . At 100 ng/ml, a-AMSA induced 0 .56 chromatid-type 

and 4 .95 chromosome-type aberrations/cell ; CAMP induced 0 .44 chromatid- and 1 .33 

chromosome-type aberrations/cell . In summary, jD vivo analyses did not reveal 

qualitative or quantitative differences in clastogenic activity between a-AMSA and 

CAMP ; both induced predominantly chromatid-type aberrations . In contrast, both 

chemicals induced more chromosome-type aberrations jn vitro ; ID-AMSA was more potent 

than CAMP in producing this effect . (Seis .bsts .et do.s aet n.e.. . .rily r .fl.ct U .S . eM youey .) 

260 

METABOLISM AND MUTAGENICITY OF 1-NITROPYRENE, 3-NITROFLUORANTHENE AND 

1,8-DINITROPYRENE IN SELECTED STRAINS OF SALMONELLA TYPRIb1URIUM. Paul C . 

Howard and Elena C . McCoy, Department of Environmental Health Sciences, Case 

Western Reserve University School of Medicine, Cleveland, OH (USA) 44106 

The metabolism of three nitrated polycyclic aromatic hydrocarbons (1nitropyrene, 

3-nitrofluoranthene, 1,8-dinitropyrene) were determined in selected 

derivatives of the Salmonella typhimurium bacteria used in the widely used 

reversion assay (TA98, TA98/1,8DNP6, TA100, TA100NR, Tn5-1012), and contrasted to 

the mutagenicity of the chemicals in these bacteria. As expected, only 

nitroreductive metabolism was detected with the three chgmicals . In all cases, the 

nitroreductive metabolism of 3-nitrofluoranthene and 1,8-dinitropyrene were twice 

the rate of the metabolism of 1-nitropyrene. The lack of mutagenicity of several 

of the chemicals in some of the bacteria do not correlate with nitroreduction, and 

can be attributed to the loss of the bacterial arylhydroxylamine O-esterificase . 

However, in several other cases, the loss of mutagenicity of chemicals is apparently 

caused by differing expressions of multiple nitroreductases . Supported in part by 

grant ES-03648 from the NIH . 

261 

IN VITRA ASSAYS OF IN VIW E}PCSURE TO CY=PHOSPFm!-IIDE AND BENZO(a)PYItENE : INDCK,TIGN 

OF SISTER-C3ffZ0IWZD E}OQHANGE4 CF HUMAN PERIPIERAL LYMPHOCYRFS BY CHElffCT+L E7lPOBID 

NOUSE BIOOD 

You-Chiu Hu, M .D. and Lia Ping 

Cancer Research Lab ., Hunan Medical University, Charr3sha 410078, P .R. China 

A method was devised to assay the genotoxic potential of cycLophosphmnide (CY) 

and benzq7yrene (BP) . Mice were exposed to different doses of CY and HP, blood from 

the drug-exposed mice was added to human ly::phocyte cultures and the sister clu-emstic 

exc'ianges (SCE) of 1ymPlxx.ytes were assayed . CY at doses of 0 .2, 0 .4, and 0 .8 mM, 

BP at doses of 0 .08, 0 .16 and 0 .32 mM were ip injected to mice . 20 minutes post ip 

injection, 0 .4 ml blood fran each exposed m0use was added to a phyt9henagglutinin 

(PHA) stimulated human lymphoc.yte culture and the SCE's of human 1ynQhocytss were 

assayed . Normal saline or D6ND was ip injected as zero dose . In the control group, 

CY and BP at different doses were added directly to the PHA-stimulated human lytsphocyte 

cultures oontai .cting Brdurd . Exposed mouse blood induoed a dose-dependent SCE 

increase in hunan lymphocytes at all tested doses . The CY tested group, the increase 

of SCE/cell over base line were : 0 .2mM, 19 .81 ; 0 .4 mN1, 41 .97 ; 0 .8mM, 66 .74 . BP 

treated group showed similar results : 0 .8mM, 23 .06 ; 0 .16mN., 34 .30 ; 0 .32 mM, 55 .62, 

indicating the presence of direct-acting SCE-in8uciry metabolites of CY and BP in 

r.ice blood . in both control groups, the increase of SCE were nsgligible . This 

In Vi',tb assay-In VIvO exposure technique may be a simpl,e and useful system for 

assaying the In Vivo genotoxicity of a chetnical . 


91


Notes 


262 

SHURT-:r9Ji TES"C FCR FRF:DICTICN OF CARCINUGF24S AND/UR YAU11UT8RS 

?(uanq Fianjun, S .M .Chen and F .Lin 

Nat . S.ar.t . Contr . Phara . & Biol . Prod . lseijing, China 

e T.a7sr7 sch9me of mutagrnicity has boen established for investigation of thirty 

Eninaa.e herbs and pbarmaceutieals . Tnroe amrays applied were within the schemes 

c :,.te :tinle gene mutation in Awes tsntt detent:ng ohroa,oso .al aberration in CHL 

(Ch :nese hamster fibroblast cell line) teat :n vitro and in oiaronuoleus in vivo . 

Sy aix nas been applied in vitro . Mutaganioity of unknovn drugs in CBL teat, 37 .5% 

druge (9/24) induc-d chromosomal aberrations, 4•17yi drugs (1/24) shown inconcluaive 

(sle 58 .3% drugs (14/24) didn't induce chro®osoeal aberrations . In aieronueleus 

teatn, V,3% drup,e (6/2 ;) induced mioronuoleuted yolychrooatie erythroeytes (MNYCE) 

incrvr :ng, 73 .5% dritn (,17/23) shown Y.N" vas in nosmal . in /wea teats, the ∎utation 

nus-~bere of 4 strains varied within nosmal fur 24 drup.e . In wutagenicity study on 30 

druf*s, cytogenetic study has ehom that suta&nr, and/or caroinogens induoed 

chroc!osowal aberrations in mammalian eells in CHL and wioronuelsue tests . Howver, 

mutap+ns and/or carcinogens dldn't induee gene autation in AsNa testa, especially, 

horm,ne and antinotaboliea druRe . The battery ecneote of mutagenioity, especially, 

:wong GNL and mloronuoloua test, hae been prov .d being effeotive, sensitive, accurate 

and rpecific, but, Ames test seem *neffective, unaensitive and tnacourste . 

263 

MV:ACFaS 111 DRUGS 

Na.ang Nianjun, J,it .Neng, S .ll,Chen and D .q .bi 

Nat . Snst, Contr . Thans, & Biol . Prod . ]bijing, China 

an our elrtwg+nicity study on thirty drugs, cytogenetio study has shown that 

mute,w9ca arxl/or caroinogens induced chroaosomal aberrations in sesuealian oells in 

C3IL and sicronucleus teste . Nine drugs have been proved to be sutagsns . Among nine 

auta."•-is, one indirect mutagens and eight direct sutagene and/or promoters were 

discovered . 8561 (Coumarin from Ruta, graveoleus L), no ohromososal aberrations in 

C~:;, in culture when the cells vere directly treated for 24h and 48 h . While 

chror.ozenal aba*rations wers ∎ore prominently with S9 six and showed clearly dosedepenier.t 

relation . Tr^ break and exchange at .errations reached at 93% and 69% 

(72 . ;nc,/nl) respectively . Folloving five airt-ot .utsgena induced chromoaomal 

s.berritions withcut 69 mix and also showed the dose-dependent relation . The rate of 

c5roonaosal aberrations and nieronueleated Yolychromatio erythrocytes in Canciolovir 

vne ?t& (0 .5u ./al) and 16.67J4 . (Bi~Ong/kr'), iiarrsngtonine 47% (o .1y5ug/al) and 18 .05xe 

10 .4j :ng/Ir.g), Boanharringtonine 25% (n .1y5ug/v:i and 10 .940 (0 .625mg/kg),Desciclwir 

+1% (2 . ;?mt;/m1) and 10 .5%e (50W'g/kg), and Sulbactum NV)L (4 .59®g/ml) and 14 .9% . 

(± .04r:kg) . AnQther three drugs induc-d polyploid aberrations in CHL test,The rat .e 

of po::yplo.id ar,orrations for three druo reached very high levels Flubandosole 95% 

ea 0 .?bug./vl, 8stinylestrsdial 8YY. at 12 .5ug/sl, Hezoesterol 40A at 15ug/ml, The 

ei£~e :i .e to ind-n po2yploid aberrntiooc wan like dlnthyl,tilbestral . 

264 

TI$ $BFfiCT OF HR7fACHLOR09@iZ6NE AND DDT ON RfiPlODIQCTIPi OUT00lS :4 OF RURAL M0lRN. 

X . HUARG, S . VANG, X . sAN, BNTIRCIIlWlAL 1MAL'!8 I118TI1111Ti, ZtVI4liC lEDICAL DPIYSRSITY 

,RANGZHOa, CBINA 

It was founded that 11BC and DDT levels in paddy field soil,surfaoe water and foods 

in xizin village dropped rapidly after stopping use of these pesticides at the end of 

1982 . The present study was to investigate whether the reproductive outoors of vos»n 

in the sera wre related to the use of IdiO and DDT . During 1981-1986 a total of 995 

pairs of mother and child tnre underwent medical surveilanoe . 'me detailed questionnaire 

and examination mainly concerned rternal reproductive events and congenital aalforsrtione 

. The paraosters adopted in the investieation weret inoidenoes of spontaneous 

abortione,of gestation period ( 37 weeks or > 42 veska,of neonate birth wight < 2500 g, 

congenital malfor .ations and perinatal deaths. 'me results obtained in 1981 end 1982 

while llHC and DDT were being used didn't differ statistically from those in 1983-1986 

while ZXC and DDT had been banned(P> 0 .56) . 4fter l'41C and DDT vas banned wC level in 

breast milk of the village woman dropped fso∎ 0 .8639 pps W982 to 0 .5671 ppa in 1985 

and DDT from 0 .4279 ppm in 1982 to 0 .0731 ppn in 1985 Hspeotitnly, Fnrthesmore,regreseion 

lines analyses between each inoidence of the abov* reproduotive events in the area 

and 1qiC and DDT levels in breast milk in the same year were conducted respeotively . No 

signifioant difference was found . 'lhat is to say,the inoidences of abnorrl reproduotive 

outooss in Xi=in village remained the sar level after the reduction of ths 

. lkiC and conclusion iand abnotr~l this 

avestigatioxsi is that no association between the~ use of 

reproductive outoomss in Lixin village vas observed .


MUTAGENICITIES OF THAI FOLKLORE MEDICINES AND THEIR NITROSATION PRODUCTS Notes 

C . Ieamworapong, K . Kangsadalumpai and W . Rojanapo, National Cancer Institute, Bangkok 

and Institute of Nutrition, Nakornpathom, Thailand . 

Crude ethanol extracts from 10 commonly used Thai folklore medicinal plants namely 

Cassia alata Linn, Casstia angusti.foiia Vahl, Carthamua tinctoriue Linn, Centeila asiatica 

Urban, Andrographis paniculata Nees, Cassia fi.stula Linn, Curcwna domeattica Val, 

Curcumazedcartia Rosc, Cyperus rotundus Linn and Orosylum indiown Vent were prepared and 

tested for their mutagenic activity using the Salmonella/microsome mutagenicity tast . 

The first 4 plant extracts, i .e . C . alata, C . angusttifoltia, C. tinotorius and C . asfattiea 

exhibited mutagenic activity preferably to strain TA 98 only in the presence of 

S-9 mix . However, an extract of C . angustifolia showed highest mutagenicity and it was 

also mutagenic to strain TA 100 when tested in the presence of S-9 mix . After nitrosation 

of the above 10 plant extracts with nitrite under acidic condition, extracts from 

A . paniculata, C . rotundus and Oro2ylum indioum became mutagenic towards both strain 

TA 98 and TA 100 either tested in the presence or absence of S-9 mix . In addition, 

extracts of C. alata, C . angusttifolia and C . ttinctortius in which all of them were found 

to be mutagenic were also mutagenic towards both tester strains after nitrosation . The 

mutagenicities of these nitrosation products were markedly stronger than those of 

extracts before nitrosation . Results in the present study demonstrate that some commonly 

used Thai folklore medicinal plants contains mutagen especially C . angusttif0ltia 

which is now widely used as laxative drug . More interestingly, these results indicate 

that some of these plant extracts especially C . alata and 0 . tindiOLm contain chemicaUs) 

capable of being nitrosated to become strongly mutagenic compounds . 

266 

LUMINOL . AN INHIBITOR OF POLY (ADP-R I BOSE) POLYMERASE, IS A STRONG INDUCER OF SISTER 

CHROMATID EXCHANGES (SCEs) . 

T . Ikushima, Research Reactor Inst .,Kyoto Univ ., Kumatori, Osaka 590-04 (Japan) 

SCEs provide one of sensitive short-term tests for screening environmental 

mutagens . The disturbance of the programmmed replication timing of replicons in the 

processes of DNA replication is thought to be involved in the SCE formation 

(Ikushima, T . 1980, Annu . Rep . Res . Reactor Inst ., Kyoto Univ .13, 67) . Its has been 

suggested that there is a positive correlation between the inhibition of 

poly(ADP-ribose) polymerase and SCE induction . The specific loss of the oncogenes 

induced by inhibition of the enzyme has been recently shown in the transformants Jq 

vitro. Here, SCE inducibility of luminol . (5-atino-2,j3-dihydro-l,4-phtalazinedione : 

Sigma), a potent inhibitor of the enzyme, has been tested in cultured Chinese 

hamster V79 cells . The actively growing cells were treated with luminol for the 

two cell cycles, the last one, or the pre-one cycle at various concentrations during 

bromodeoxyuridine-labelling, and SCEs were detected by FPG method in 

colcemid-arrested metaphase chromosomes . Very high SCE frequencies were obtained 

after treatments with low concentrations (e .g . 82 SCEs/cell at 0 .5 mM) . Luminol was 

more than four times as effective to induce SCEs as 3-aminobezamide, a NAD site 

inhibitor of the enzyme . No enhancement of SCE level was observed after the 

pre-cycle treatment, in contrast with other strong SCE-inducers such as cis-platin 

or mytomycin C . SCEs might be formed during DNA replication by inhibition of 

poly(ADP-ribosyl)ation, accompanying the gene amplification or elimination . 

267 

SUPPRESSIVE EFFECT OF VANILLIN ON MICRONUCLEI INDUCED BY MITOMYCIN C OR X-RAYS . 

T .Inouye, Y .F .Sasakl, H .Imanishi, M .Wetanabe, T .Kato, K .Matsumoto, T .Ohta and 

Y .Shirasu, Institute of Environmental Toxicology, Kodaira, Tokyo 187, (Japan) 

Vanillin is a component of vanilla essence flavour . We previously reported the 

antimutaQenlc effect of vanillin on mutagenesis in bacteria(1) and its anticlastogenic 

activity in a cytogenetice study ueing cultured cells(2) . In order to 

find out in vivo anticlastogenic effect of vanillin, bone marrow micronucleous test 

was conducted. BDF1 male mice at 7 weeks old were treated with mitomycin C(MMC) or 

ionizing radiation . Thereafter vanillin at 500 mg/kg was given orally . Bone marrow 

cells were sampled 24 h after injection of 2 mg/kg MMC . In the experiments the time 

interval between MMC injection and vanillin treatment was varied, a significant 

reduction in the frequencies of micronucleated polychromatic erythrocytes(MN-PCEs) by 

38-50/ was observed from 6 to 9 h after YlAC injection . The frequencies of IQ)-PCEe 

induced by X-rays (200R, 150kV, 5mA) plus vanillin treatment was less than those of 

X-rays only (mean.SD :3 .1a1 .1/ vs 4 .97+0 .95), when bone marrow cells were sampled 24 h 

after X-irradfetion . When sampled at 15 h, however, the similar frequencies were 


93



Notes observed (3 .1+1 .75'vs 3 .6+1 .1) . These results indicate that vanillin is also 

effective in vivo syeteme and that vanillin may act at and before the S period of the 

cell cycle. 1 Ohta et al ., Food Chem . Toxicol . 24,51(1986) . (2)Sasaki at al ., 

Mutation Res ., 191,193(1987) . 

268 

IN VITRO CLASTOGENICITY OF 951 CHEMICAL SUBSTANCES . M . Ishidate, 

Jr ., M . C . Harnois*, and T . Sofuni, National Institute of Hygienic 

Sciences, Setagaya-ku, Tokyo (Japan), *Fellow, Japan Foundation 

for Promotion of Cancer Research . 

A literature review of 1964-1985 publications on the in vitro 

clastogenicity of chemical substances resulted in data from 240 

reports on 951 substances . Of these 951, 447 were consistently 

positive either with or without activationj 417 were negative 

without activation but not tested with activationi 30 were 

consistently negative when tested both waysi and 53 gave variable 

results . The variability was related to cell type, presence of 

activation mechanisms and treatment schedule . Addition of an 

activation system reduced the variation among different cell typesr 

no one cell type appeared to be superior for testing all clastogens . 

The conce%tration at which substayes tested positive ranged from 

4 .3 x 10 (trenimon) to 6 .9 x 10 mM (acetone) . Generally, there 

was an inverse relationship between the concentration required 

to induce aberrations and the frequency of exchange-type aberrations 

produced by a chemical at its maximum effect dose . The relevance 

of tests conducted at high concentrations is considered, and caution 

urged in the interpretation of test results obtained under 

physiologically stressful conditions .(Mutation Res .,195, 151, 1988) 

MOTAGENICITY AND ANTIHUTAGENICITY IN AIR-BORN PARTICULATES 

269 

Hiroko Iwado1'2 . Mitsuko Naitol Hikoya Hayatsu2, lOkayama Prefectural Research Center 

of Environmental and Public Health, Uchio, Okayama 701-01, 2Faculty of Pharmaceutical 

Sciences, Okayama University, Tsushima, Okayama 700 (Japan) 

Air-borne particulates collected by a high-volume air sampler in a suburban area of 

Okayama City were extracted with methanol under ultrasonic vibration . The residue 

obtained on evaporating the solvent was dissolved in a small amount of dimethylsulfoxide 

and the solution was divided into two portions . One was submitted as such to 

the Ames test for assaying mutagenicity (sample 1) . Another portion was suspended in a 

large volume of water and treated with blue cotton to adsorb polycyclic compounds to 

the cotton . A methanol-ammonia eluate of the blue cotton (sample 2) and the aqueous 

portion that remained after the blue cotton treatment, after evaporation to dryness 

(sample 3), were also examined . 1 shoved mutagenicity towards S . typhimurium TA98 in 

the presence of S9-mix in a dose-responding linearity up to 27 m3 air-volume equivalent ; 

however, the colony number stayed constant in the dose range between 27-270 >l3 . In 

contrast, 2 gave a linear dose-response up to 270 m3 . Although the numbers of 

revertant colonies found were about 2-times greater with 1 than vith .2 in the range 

lower than 27 m3, the numbers obtained with 2 were much greater than those with 1 in 

the higher dose range . These results suggest that antimutagenic factors were present 

in 1 . In fact, when 3 was mixed with 2, a strong suppression of the mutagenicity was 

observed . The antimu[agenic factors pieaent in 3 were capable of inhibiting the 

mutagenicity of benzo(a)pyrene and that of 2-nitrofluorene . (Supported by Nippon Life 

Insurance Foundation .) 

270 

INFLUENCE OF UMUC ON EMS-INDUCED YUTAOEBESIS IN E . COLI DEFICIENT IN 

MISMATCH REPAIEE- C . Janion, and E . Orsesiuk, InetiTaTe- of Biochemistry 

and Biophysics, Polish Academy of Sciences, Warsaw, Poland . 

we have compared mutagenio aetivit and speeifieity of EMS (ethyl 

methaneaulfonate) in ;gc~ 2497( +~~ ) and its derivative 

strains : EC2401(mutS+~um v _~ Y1(mu uC') and EC2402( ut~S'umuC-) . It 

has been found tE~"-t s ite of mu at on (based on ezam3nation of the 

phenotypes of Arg+ revertants) and the dutagenio specificity (based on 

ability of the Arg+ revertants to support growth of aaber and ochre T4 

bacteriophages) depends on um C, but only in the mutS-- mismatch repair 

de icient strain . The maJor+it~'of EIB-induoed Arg'r'~3vertants of AB 

24~7, EC2401 and EC2402, show the phenotype - Arg+His-Thr ; whereas 

the majority of EMS-induced Arg+ rewertants of Y1 show the phenotype 

50869 3606

Arg'His*Thr* .Examination of the pattern of amber and oohre T4 baoteriophage 

suppression indicate that the reversion to Arg+His Thr in AB 

2497 may be the result of su B and ~eu Eoc suppressor formation (whioh 

ân arise by a GC 4 AT tranion)•re~as the reversion to Arg*Hie• 

rhr* may be the result of g1tY or fxs-4 suppressor formation (which 

can arise by a GC (or AT) -~- TA traneversion) The frequency of El13-induced 

Arg* reversion is little dependent either on umuC or mutS . 

The mechanism of the different specificity of EUS-in ueed m-0s$ione 

will be discussed . 

271 

MULTIPLE END POINTS FOR SOMATIC MUTATIONS IN HUMANS PROVIDE 

COMPLEMENTARY VIEWS FOR BIODOSIMETRY, GENOTOXICITY, AND 

HEALTH RISKS 

R .H . Jensen, W .L. Bigbee, and R.G . Langlois, Biomedical Sciences Division, 

Lawrence Livermore National Laboratory, Livermore, CA 94550 

Recent technologic developments now allow us to measure genotoxic effects of human 

exposure to mutagenic phenomena In four different ways--HPRT mutations in lymphocytes, 

HLA mutations on the surface of lymphocytes, hemoglobin variant mutations In 

erythrocytes, and glyoophorin A mutations on the surface of erythrocytes . Each of these 

assay methods shows advantages and disadvantages In being used to monitor for 

exposure of individuals to mutagenic phenomena such as toxic chemicais or Ionizing 

radiation . For example, the techniques used for the lymphocyte-based assays can be 

extended to isolate nuclear DNA and characterize the mutagenic changes that have 

occurred, while erythrocytes contain no DNA for such analyses . On the other hand, the 

erythrocyte-based assays are probing a tissue in which differentiation Is straight forward 

and clearly delineated, whereas lymphocytes display complex and muRi-organ 

developmental pathways . Thus, using combinations of these assays (and others as they 

develop) for each individual should furnish a set of monitoring data that can be broadly 

interpreted to provide biodosimetrks Information and potential estimates of cancer risk . The 

impact of obtaining such information is high enough to justify the challenge of performing 

mufti-end point analyses on populations at high risk for mutagenic exposure . 

272 

GpNOTOXICI7Y OF 23 C}ICMCAIS TO S GFREVLSIAE 

li ang Z„oshu et at 

A .-parennt of Biology. Sooond Mitlifary Medical thriwsity, Shanghai, CMna 

23 kinds or danicals vNne used to evaluate the edidettcy of S ans4sias as a toot in the assay of genotopns 

The growing cells ar S aaevimac D7 wero treatod with thatdcels at 28°C for 6 houtz As te~oAOd eonte known 

mutagens such as MNNG, 4-NQO and hydroxytmea oould inoease the firqtta>cy of ttp oarvertants _ to 

3-10 t6rts, and we also found that same pvnulagw such as cyclopl~osp}rvnidc andtrypon btue could iitacase 

the froqua~y to s-10 times without any exogatotn adivation 'iltis itd'~caled that the yeast may 

cndogawuclY me~aboli~e prcmutagens into teadlre intanndiatea lt wat sepottod that mme eatdttopens had a 

false-ncgaGw iesportst in Amca trst, twl in our experinrnt 3l evas shatm lhat Q 1-Q 3•/% anihne muld lncrease 

Ux frcqua~y ot gene oonNersion in1tp Ioats to 1-3 4 tirrrs, 15-30 tng/ml aiodnic anhydride 1 .4-A 1 tanea, 

10-80 mM trypan biue 1-4 Gntca, Q 12-Q 4 M thioutt8l S-7 . S timx atd a2 -Q S•/. prhott Iettachlatide 

2 . 5-10 tirnes AdditionaRy , the intertupt prooodt~ was tssod to aaoen the indttoaa or ehrornason~e 

mahr~a fion The growing odls of S oaetidae D6L M t+ere irtated with diatticah at 28'C for 4 hottn, then 

at 0 C for 16 hours and again ,at 28°C for 4 twun brloee piatod wilh the tmditun oontaininnY Q S µg/m) 

cydoheuamide The fioqtrawy ot the while lettdne-ra1 oolonra tepresetttod Ihat of chrarnosatte loss It 

was shown that some chanicais such as methyl nntitae,jlate~oould catne ctunsttasorne loes We aostdudod that 

the S annisiae has a unique tac~~ulrcss in the aaay of gs,otapts 

273 

ANTIMUPAGENICITY OF CHINESE MEDICiNES . 

Tiang Zuoshu, WangUngh ua and Chen Zhongfu 

Dcpartmrnt of Biaiogy. Seoord Military Modiesl Lhtiversity and Institute of Gcnctics, Fudan L)niversity, Shan ;ha't 

Chma 

In order to study the antimutaganaty of Cldnese modidncs, the Inductest was uud to saern the inh'hiton of SO6 

re;ponsc homar~ang 601rnds of Clmiete rnodidr~, as it has tzen known that SOS response ptays an important role 

m mutagrnese. A IItu paper disc with 10 yt of 0. 1 mg/mt of Mitomydn C (MMC) and another one with the estract 

of Qiinese momcnie wce put 1 an apart on the surfem of the top agar containing both the Absogc* E[xa6 

acII ( GY5027) and the udieator E aiticd (GY4015) . Aller the plate was incubated at 3NC for 8 hours, phage 

plaques appeared around the MMC diu, and a partial afipse of SOS hile'hition ( with roplaque or darcased ntmber 

of plaque) would appear bdwern the two disc if the Ci :nae iiiediaie estract in tho disc had an inhibitory edax on 

SOS response The results showod that 11 kinds of Chineae nmbdnes had such an eQoct. Sane of than were further 

studiod with SOS dvamotest. In this test, the growing od af E ca6 PQ37 was exposed to U K and incvbated in LB 



Notes


6 

Notes 


:d witli the atract cf C]i6rae emdidne at 3TC for 2 boun, thepn ~eh(ye ~activity 11 -Balactoaidau was tottad which 

L~daa~ea~ : the S06~set~pon io t~~ Sv : e~t td I0a/ft af tlrc~aonlr~d rapo~ivebr. aythe +~icneo o- 

OW 1 (rec~A441) te o~'% SOe~ and 60e/. ~ the~aaurob but had not eda(on SOB rctw~a~k gene app osion i~ E6 

a.df QW1107 ( laulSl )(arore tlrn 9SK of tbe controi) . So it tu+mtm that thtae l~e nn&ars coetaui the 

ialr~iton of RxA proteaoe and nay be utod u antinutayr~u and/a antxarrirwgau . 

274 

CNRONOSORE ABERRATIORS IR NUNAR LYNPNOCYTES AS AR IROICATOR OF RADIATIOR DANACE 

Jin Cuithen, Institute of Radiation Nedicine, Beijing, China 

The analyses of chroaosoae aberrations in huean lymphocytes have shown their 

usability for the estioation of individual doses of over-exposed persons in radiation 

accidents and for the assesseent of their late radiation effects . Our laboratory 

has been involved in the cytogenetic studies for radiation accidents in 

China . The aain results can be suosarited as follows : 1) Under the circumstances 

of single acute exposure, the vast oajority of aberrations observed soon after 

the exposure were of unstable types . Among thes, the incidence of dicentries 

plus rings could be used to ∎ake reliable dose estisation . The results were eonsistent 

with the clinical courses of the vioties . 2) If the blood culture ras 

done s .veral eonths after the exposura, the aberration frequencies could not 

be used as a biological dosimeter, but it still showed sooe general correlation 

vith radiation dose previously received . 3) Follor-up studies on over-exposed 

peraons indicated that most of the remaining aberrations were of stable types . 

4) The chroeosoae analyses for victims following protracted exposure lasted half 

year exhibited evidence that the frequencies of unstable types were coeparable 

vith that of stable types . 5) G-banded chromosome analyses carrying out on 5 

victies, vho received accidental over-exposure several years ago, showed a nonrando∎ 

distribution of break-points . They vere eainly clustered in soae chroeosoees 

and vere preferentially located in the regions Sq3, 6q2, 9q3, 14q3, etc . Soae 

break-points were near or at the map sites of the known oncogenes . 

275 

TEMPERATURE EFFECTS ON RADIATION-INDUCED CHROHOSOHAL ABERRATIONS IN THE PRESENCE OR 

ABSEN*E OF DIMETHYL SULFOXIDE . E .E . Joiner, L . G . Littlefie~d, S .P Colyer* and E .L . 

Frome 1, Oak Ridge Assoc . Univer ., and Oak Ridge Nat 1 . Lab . , Oak Ridge, TN (USA) 

Studies of the dose response relationship for dicentrics in human lynphocytes 

exposed to X-rays at 37°C or 4°C demonstrated that radiation temperature acts as a 

dose-modifying factor by influencing the for .ation of chromatin lesions that lead to 

aberrations (Gumrich st al ., Int . J . Rad . Eiol ., 49 :665-672, 1986) . We employed DNSO 

as a model OH radical scavenger in experiments to determine to what extent radiation 

temperature modifies the proportions of aberrations resulting from DNA damage indueed 

by direct ionisations vs OH radical attack . Human whole blood was maintained at 

temperatures of 4°C vs 37°C in the presence or absence of 1M DHSO during exposures to 

1 .04, 2 .08 and 4 .17 Gy X-radiation . Lymphocytes were washed and cultured for 48 hr ., 

and aberrations were scored in lst division metaphases . Dicentric yields in cells 

irradiated at 37°C in the presence of Dtt30 shoved reductions of 650 compared to 

yields observed in unprotected cells . For lymphocytes irradiated in the absence of 

DNSO, dicentric yields were 411 lover following exposures at 4°C than the yields 

observed at 37°C . In contrast, when cells protected by Dt4S0 were irradiated, 

dicentric yields at 4°C were only 211 lover than those observed at 37°C . These 

preliminary results suggest that the effects of cold temperature (4°C) on the yields 

of radiation-induced chromosomal exchange aberrations in human whole blood 

lymphocytes are primarily due to the inhibltion of the indirect radiochemical actions 

of OH radicals on DNA targets . Supported by U .S . DOE/ORAU Contract 

DE-AC05-760R00033 . 

276 

MOLECULAR ANALYSIS OF RADON-INDUCED MUTANTS R .F. Jostesl, R .A . Giesi, 

T .L . Morganl, E .N . Fleck:, K .P . Gasperi, and F .T .Crossl . lPacific Northwest 

Laboratory, Richland, Washington and $WhitRian College, Nalla Walla, Washington . 

An in vitro system for exposing RutRtiaalian cells to radon gas and its daughters has 

been developed in our laboratory . Cells are exposed in spinner flasks and the doses 

reported here are to the cell culture medium . Radon-induced mutations at the HGPRT 

locus in Chinese hamster ovary cells show a linear dose response with an induced 

frequency of 1 .0 x 10-o mutations per viable cell per c6y . To date we have isolated 

35 mutants for molecular analysis . Southern blot analysis of DNA from 21 radoninduced 

HGPRT- cell lines indicated that 11 (52k) were caused by a complete deletion 

of the gene . Three mutations (14%) showed altered bandin~ patterns indicative of 

subtotal deletions and the remaining 7 mutations (34%) conta ned changes undetectable 

by this analysis . (Work supported by the U .S . Department of Energy under Contract 

DE-AC06-76RL0 1830) . 

50869 3608


Notes 

DEVELOPMENT OF A SENSITIVE HUMAN EPITHELIAL CELL LINE FOR MUTAGEN SCREENING . Y .S . 

Jou, C .C . Chang, and J .E . Trosko, Department of Pediatrics and Human Development, 

Michigan State University, East Lansing, MI 48824 . 

An ideal assay for human mutagens should be relevant (related to mutations in human 

cells), sensitive (able to detect low-dose mutagens) and informative (revealing genetic 

alterations at the molecular level) . Toward developing a cell line with these attributes, 

we have reactivated the hypoxanthine-guanine phosphoribosyl transferase 

(HGPRT) gene on an inactive x-chromosome by 5-azacytidine treatment in a 6-TGr human 

teratocarcinoma cell line (46 ch . ; xx ; t 15/20) . In contrast to the normal human cell 

line, the reactivated HGPRT gene is on a non-essential x-chromosome . Therefore, deletions 

or mutations of essential genes on the non-essential chromosome associated with 

the HGPRT gene mutation would not affect the survival of a 6-TGr mutant . After exposure 

to mutagnes, a higher frequency of induced 6-TGr mutants would be expected . After 

treatment with 5-azacytidine, HATr colonies were readily recovered . Most of these 

clones are revertible to 6-TGr cells at hi gh frequency . Few stable clones, however, 

were also recovered . Our preliminary studies using one of these stable clones indicate 

that the cell line had a 50-100 fold increase in x-ray induced 6-TGr mutant compared 

to the parental HATr cell line . In conjunction with molecular analysis using 

polymerase chain reaction amplification and DNA sequencing techniques, the cell line 

might become an ideal assay for human mutagens . (Research supported by a NCI grant 

CA21104-11) . 

278 

A CYTOGENcTIC STUDY ON SUBJECTS PRESENTING AIENORRNEA . STERILITY AND RPROOUCTIVE FAILURE . 

A . Jvothv, G .S . Isaec, A,Shobhe Rani . C. Kususa Kumeri and P.P .Raddv . 

Institute of Genetics . Hospital for Genetic Diseases . Owanie Universitv, Beouaoet . Hvderebad-S00 016. 

A .P . India. ' 

A B s T R A C T 

Chromosoae analysis plays a vital rola in spaculatinq the etioloqY of cases presanting s .enorrhsa . ster!- 

litv and reproductive failure . The present study is tharsfore aieedrto lnvestipate the role and distribution 

of chromosose abnormalities in causing thssa conditions . A total of 1575 twale subjects presenting 

enenorrhea . sterility and reproductive fsilure usre investigated for .chrososae abnormalities . These 

include cases of primary awenorrhea (37S) secondary wenorrhw ( 100), sterility (75) and reproductive 

failure ( 325) . 

The subjects were thoroughly examinsd and the history of the patient and her family rera recorded . Chroaosae 

preparations wre ∎ade according to the ∎odified ∎sthod of Moorhead atal (1960 ) and banded 

according to Seabright (1971) . Other staining techniques (CBG), Ag NOR etc .) wre employed wherever 

necessary . The typs of abnormalities detected include 45X ; 4SX/46XX ; 45X/46XY; 43X/47X)0(i 4V/46X, frsgi 

45X/46XX/46XY ; 46XX/46 XY ; 4SX/46xx/47XXX ; 46XY ( Phenotypic females) ; 47xXX ; 46XX, 13 p and 46 XX, 

16p* The abnormalities ldentified suggest the need for routine chrososose survey among patients with 

the above clinical syapta s . These studies will help !n the accurate diagnosis and eanapesent of such 

clinical entities . 

279 

BOTRAN AND BLEOMYCIN INDUCED CROSSING OVER AND ANEUPLOIDY IN ASPERGILLUS NIDULANS 

WHICH RESULTS IN DIFFERENT PATTERNS OF MITOTIC SEGRECANTS . E . Kifer, Department of 

Biology, McGill University, 1205 Dr . Penfiald Ave ., Montreal, P .Q . Canada H3A 151 . 

Botran and bleomycin reduce growth and increase mitotic segregation of recessive 

colour markers in diploid heterozygous tester strains of Aspergillus . In both cases, 

segregants are mainly diploid crossovers and haploids . The latter were especially 

frequent on botran media, and at low concentration aneuploids, mainly disomics for 

chromosome III, also formed discernible patches of coloured conidia . In addition, 

diploid segregants showed high levels of coincidence of crossing over, segregation in 

three or even four chromosome arms being not uncommon . On the other hand, treatment 

of germinating conidia which were plated onto complete medium resulted in few large 

crossover sectors but produced abnormal colonies in both cases . With increasing 

concentrations of bleomycin, these were especially evident, increasing from 25 to 75% 

among survivors . On replating, up to two thirds of them could be identified as 

aneuploids, the majority being genuine trisomics . These results suggest that both 

botran and bleomycin induce crossing over and malsegregation . (Supported by 

Strategic Grant 0032242 from the Natural Science and Engineering Research Council of 

Canada) . 


.



Notes MOLECULAR SPECfRUM ANALYSIS OF FRAMESHIFT MUTATIONS IN YEAST D . Kalirawcki, 

KM. Mottus, M.J. Plewa, and RW. Larimer', Institute for Environmental Studiea, University of Illiaois, 

Urbana, IL 61801, 'Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 . 

The purpose of this study is to recover sequence information at the HIS4A region on chromosome III 

from a large number of independently arising, htr• spontaneous and 4 nitroquinoline-N-oAde (4NQ0) 

induced revertants of Saccharomyces temirk+e . We constructed a shuttle vector, pMP4, that contains a 

438 bp deletion encompassing the +G framahift mutation, hls4 :38 . A large number of hit4- .38 spontaneous 

revertants were obtained ; a random sample of 200 spontaneous and 4NQO induoed revertants 

were screened for suppressor mutations and are being analpsed . Each revertant is separately transformed 

with linear pMP4 that contains the 438 bp gap and the Infocmation from the yeast chromosome is copied 

onto the plasmid by a gene conversion process known as double,strand gap repair. Since the gap extends 

3' beyond the original +1 frameshift mutation, a new 1.3 kb SaII restriction site appears in the repaired 

pMP4 . We have recovered and sequenced chromosomal information from over 40 spontaneous his4-38 

revertants . The 4NQO induced revertants were produced at a concentration of 2.3 µghnl and are being 

screened using the same procedure . This approach permits the isolation of DNA sequence information 

of a mutation from an unmodified eukaryotic chromosome. We will define the distribution of bases 

involved in mutation leading to reversion at his4- .Id The use of double strand gap repair allows the 

analysis of the molecular spectrum of these revertants and will provide insight to the mechanisms 

underlying spontaneous and 4NQO•induced frameshift mutation In eukaryotes . Funded by the 

Interdisciplinary Program in Environmental 7bbcology . 

281 

S'PuLY UiV 1'cir . .:c.. .dJiOk OF MUTA(3ENS FROM MJNICIPAL INCINP:RATORS BY MhnfiS 

OF AMES ASSAY . 

A~.Ka~miya,:~agoya City Environmental Pollution Research Institute,Chudochc 

nNTiamiku, Nagoya City(Japan), Y .Ose and T .Sato,Gifu Pharmaceutical 

University,Mitahira-higashi, 3ifu City(Japan) 

The mutagenicity and mutagens of fly ash,emission gas and wastewater 

in municipal incinerators have been investigated by Ames assay and chem•ical 

analysis . A negative relationship was obtained for complete combustion 

and a positive one for incomplete combustion . About 90% of all 

mutagens produced in a incinerator are released into atmosphere as emission 

gas,and 10% are disposed in the electrostatic precipitator and 

wastewater treatment plants . 1,6-Dinitropyrene(DNP) as direct acting 

mutagen was detected in emission gas from incomplete combustion . The gac 

phase photochemical reaction of oyrene with (vOs gas was examined in a 

quartz vessel with various reaction times and temperature . 1-NP was 

readily formed from pyrene in the absence of light irradiation and low 

temperature, but the formation of 1,6-DNP is dependent on light irradiation, 

temperature and nitric acid . Most of mutagens in wastewater 

treatment olants are not decomposed by normal aeration times, but are 

removed by adsorption onto suspendid solid . Total revertants per minute 

from the emitted gases corresoonded to that from the exhaust of 1700 - 

3000 motor vehicles . The mutagens emitted from total municipal incinerators 

in Japan 1985 were estimated to be 16 .3ton as benzo(a)pyrene . 

282 

ON THE VALIDATION OF TME SYSTFM OF ASPER(iILLUS FOR TESTING IINIFAM1FNfAL ANEIIGIIdS 

A . Kappas 

National Research Center "Aeaacritus", Athens, Greece . 

Chemically-induced malsegregation of chranosones is detected in two types of diploid 

strains of the ascamycete As r illus nidulans . One detects euploid mitotic 

segregants as products of either ma segregat on non-disjunction) or mitotic crossing- 

-over . The other detects aneuploids as unstable abnormal segregants and can distinguish 

between primary aneuploidy of whole chrat,oscmes and secondary spontaneous aneuploidy 

resulting from primary chrotaosome breakage . We developed a system which 

belongs to the first type of strains in which we can also detect chromosome breakage 

and it is possible to identify whether segregants can be the result of secondary spon 

taneous malsegregation of chraaosemes with terminal deletions . In this system we have 

been able to use the metabolic activation technique with an S9 mix with microsamal 

fraction from rats . In a coordinated progratmne of the EBC countries with the aim of 

developing and validating proper test system(s) for the regulation of environmental 

chemicals which can induce genaaic mutations, a variety of chemicals selected on the 

basis of their ability to interact with several cellular targets have been investigated 

in A . nidulans and many of them, including chloral hydrate, hydroquinone, thiabendazole 

grise'o uTvin, benomyl, were detected as aneugens . 

Supported by Grant EV4V-003S-GR from the EBC . 

50869 3610


SPECIES COMPARISONS REGARDING COMPARATIVE METABOLISM OF TWO STRUCTURALLY SIMILAR Notes 

NITROPHENYLENEDIAMINES (HC BLUE 1 AND HC BLUE 2) . F . Kari, S . Driscoll, C . Parker, 

K . Rudo, K . Tomer, and R . Langenbach ; National Inst u e of Environmental Health 

Sciences, Research Triangle Park, NC 27709 . Chronic evaluations reveal that HC Blue 

1 causes hepatocellular carcinomas in mice, but a structural analog, HC Blue 2 is not 

carcinogenic in mice . Neither of these chemicals are carcinogenic in rats . The 

bioassay results of this carcinogen/non-carcinogen pair mirror the species- and 

organ- specificity obtained with 23 congeners which have been evaluated under 

similar conditions . Accordingly, comparative metabolism studies were done to elucidate 

mechanisms for their discordant carcinogenic potencies and species-specificity . 

Urinary recovery of radiolabel following oral administration of the parent compounds 

was equivalent for both compounds in mice and rats . Conversion of parent compounds 

to metabolites was quantitatively comparable indicating systemic availability is not 

responsible for the discordant potencies . Metabolism of these two compounds (200pM) 

by hepatocytes isolated from mice and rats yields metabolic profiles similar to their 

respective in vivo profiles . HPLC separation shows that in mice HC Blue 1 Is metabolized 

to five major metabolites while the non-carcinogen HC Blue 2 yields only one 

major metabolite . Thermospray LC/MS analysis of HC Blue 1 metabolites from mice provides 

tentative evidence for nitroreduction, demethylation and conjugation 

(acetylation and glucuronidatton) . In the non-susceptible rat, HC Blue 1 produces 

three metabolites similar to those found in mice, but two metabolites produced in 

mice are notably decreased or absent in rats . Qualitative differences in metabolism 

of these compounds may contribute to their different carcinogenic potencies and 

species specificity . 

284 

INDUCTION OF CHROMOSOME-TYPE ABERRATION AT ZYGOTE FOLLOWING CHLORAMBUCIL ADMINISTRATION 

IN MALE MICE, M .Katoh and R .P .A .Valdivia, Food and Drug Safety Center, Hadano, 

Kanagawa (JAPAN), and University of Chile, Santiago (CHILE) 

Katoh et al . (1986) reported that most alkylating chemicals which are shown dominant 

lethals in postmeiotic male germ cells induce chromosome-type aberrations at zygotes 

and these agents associated with the inductions of heritable tranalocations~in offspring 

. Chlorambucll, which is an alkylating agent of the nitrogen mustard type, 

induced the high frequencies of dominant lethals in male germ cells except late spermstids 

(personal communication by W .M .Generoso) . This dominant lethal's pattern is the 

complete opposite of that observed by acrylamide which 7bhoved sperm protamina alkylation 

but not of DNA alkylation (Shelby et al .1986, Saga et a1 .1988) . Also, cytogenetic 

analyses by acrylamide demonstrated chromosome-type aberration (this result 

is presented by R .P .A .Valdivia in FIFTH ICEM 1989) . In the present study, cytogenetic 

analyses by chlorambucil were undertaken to clarify the mechanisms for chromosome 

aberrations in male germ cells of mice . Male mice were given single intraperitoneal 

injections of 25, 12 and 6 mg/kg of chlorambucil and then cytogenetic analyses at 

zygotes were carried out for 5 weeks after injection . The frequency pattern of zygotes 

with chromosome aberrations was found to correlate with that of dominant lethal . The 

sensitive germ cell stages were demonstrated in late spermatocytas, early .permatids 

and late spermatozoa . Late spermatid stage was ruled out . In these sensitive stages, 

chlorambucil induced chromosome-type aberrations . These evidences show that the induction 

mechanisms of chromosome-type aberration by chlorambucil is different from that 

of sperm protamine alkylating agents such as acrylamide and methyl methanesulfonate . 

285 

MUTAGENICITY TEST FOR GASEOUS AND VAPOUROUS COMPONENT OF COMBUSTION EXHAUST . A .Kawai', 

S .Goto', O .Endo', H .Matsushita=, 'Japan Automobile Research Institute, Tsukuba, 

Ibaraki,(Japan), 'National Institute of Public Health, Minato-ku,Tokyo (Japan) . 

Many reports have been published for the sutagenicity of particulate component of various 

combustion exhausts, but few for the mutagenicity of gas/vapour cosponent of these exhausts . 

We devised apparatus for sutagenicity assay of gas/vapour co.pounds and obtained a suitable 

test condition for this assay . Particle-free test gas was prepared by passing a diluted 

combustion exhaust through quartz fibre or similar filter, collected in a Tedra bag (200 l), 

and diluted with fresh air . The test gas was introduced into a Pyrex glass chasber(3 or 8 L) 

in which 10 or 16 test plates having tester strain on the surface were placed inversely . 

Following test condition was selected fros various kinds of test results as a suitable one 

for autagenicity assay ; 0 .251 /ein for flow rate of test gas, 2-8 hr for exposure time, and 37 

'C for the temperature of chamber . Mutagenicity test was carried out for the particle-tree 

exhaust gas from diesel engine,kerosene heator and side-streas of cigarette smoking,using 

Salmonella typhisurius TA98 and TA100 . E .Coli WP2uvrA/pKM101 was also used for the test of 

cigarette smoke . Gas/vapour component of diesel exhaust gave positive sutagenic responses for 

TA100 with and without S9 ∎ix and for TA98 with 39 six . The component of kerosene heater 



Notes exhaust gave positive responses for both the tester strains with and without S9 ∎ix . The 

component of side-stream saoke of cigarette gave positive responses for TA100 and 

WP2uvrA/pKM101 with and without S9 ∎ix, but negative for TA98 . It was also found that 

∎utagenic potency of all these gas/vapour co .ponents were higher than those of particulate 

components of these combustion exhausts, respectively . 


286 

: yylencke3ERD>NIST~ ~f E~ V BTE~e~E ~l~ of 

CHRON C ETONAL OF KTNKelseyt~~M . B eN-t. J 

Radiobidopy and 2Occupationai OSUR Health Program, Harvard School of Public Heafth, Boston, MA; 3Lab. of Radbbioioqy 

ind Environmental Health and Dept of Epidemiology and International Heahh, UnN . of CelMornia, San Francisco. CA, 

NIOSH, Cincinnati, OH . 

Ethylene oxide (ETO) Is a potent, m0r0% ;hcftW DNA alkylating agent that Is commonly encountered in the 

workplace . Cytopenetfo studies of an"s and human workers exposed by kihelatkxt to ETO have found elevated 

levels of cytopenetic damage In their peripheral blood lymphocytes . We have studied a cohort of 38 adult male 

monkeys that were exposed to 0, SOpprn and 100ppm concentrations of ETO by Infbiation from 1979 to 1981 . The 

lymphocytes from these animals had elevated levels of sUter chromatid exchange (SCE) knmediately upon cessation 

of exposure which persisted 8-7 years after cessation of exposure . Ths persistence of the SCE was attributable to a 

subpopulation of cells whlch were prefererkiatty detected at early t:ytopenstlo harvest times. To determine q the 

presence of this subpopuiatkxi of eeUs with elevated SCE was assockted with karyatypio change In stem cells we 

have banded and karyotyped 20-25 bone marrow ceAs from 2 aNmab exposed to 100ppm, 2 exposed to 60ppm and 4 

controls. In each animal, all of the karyotypes were normal 42 X Y, suggesting that there b not an exposure-Induced 

common stem ceil karyotypic alteration in the csits from exposed animals . 

This work has been supported by CCV902885 from the CDC . 

HETEROCYCLIC ANINE MUTAGENS IN ROASTED COFFEE BEANS AND BREWED COFFEE 

Kiyomi Kikugawa, Tetsuta Kato and Shinya Takahashi 

Tokyo College of Pharmacy, 1432-1 Horinouchi, Hachioji, Tokyo 192-03, JAPAN 

287 

Roasted coffee beans (hot air-roasted and charcoal-roasted) contained at least six 

heterocyclic amine mutagens generated during roasting coffee beans . They were extracted 

by methanol/ammonium hydroxide from the beans, and purified by partition in 

chloroform/hydrochloric acid and in chloroform/alkaline water, and finally by blue 

cotton adsorption . They were separated into six mutagenic fractions by high pressure 

liquid chromatography . One of the mutagenic fractions was identified as 2-amino-3,4dimethylimidazo[4,5-f)quinoline 

(MeIQ) . The other five mutagenic fractions were 

unknown heterocyclic amine-like mutagens . The MeIQ contents in roasted coffee beans 

were 0 .16 ng/10 g (hot air-roasted) and 0 .32 ng/10 g (charcoal-roasted) . These 

heterocyclic amine mutagens were tightly adsorbed to coffee bean fiber containing 

hemicellulose, and the mutagena could be hardly eluted into brewed coffee by general 

percolation with boiling water . Substances that could destroy these mutagens were 

found in brewed coffee . The substances were found to be water-soluble high molecular 

weight polyphenolics . They could destroy the mutagens in brewed coffee in the 

presence of dissolved dioxygen . Thus, even if a small amount of the mutagens were 

eluted from roasted beans into brewed coffee, the mutagens could be destroyed by 

these intrinsic substances in brewed coffee . 

288 

MUTAGENIC ACTIVITY OF IiAILLARD REACTION PRODUCTS FROM CARBOHYDBATES AND PROTEINS . 

N .Kinae, li.Yamashita, S .Kamiya, and S .Esaki. School of Food Nutritional Sciences, 

University of Shizuoka, 395 Yada, Shizuoka 422(Japan) 

It has been demonstrated that several reaction mixtures of carbohydrates and amino 

compounds such as ammonia, amines, amino acids, show mutagenic and/or antiaatagenic 

activity toward certain bacteria . Howver, there is few reports concerning the mutagenicity 

of the reaction mixtures of carbohydrates and paptides, or proteins . 

We tried to determine the mutagenic activity of the reaction products from carbohydrates 

and proteins . A mixture of 50-fold carbohydrate(D-arabinose, D-ribose, D-xylose, 

D-glucose, D-fructose, lactose) and protein(bovine serum albumin :BSA, human serum 

albumin :HSA) was dissolved in phosphate buffered saline(PBS, pH7 .4) and incubated at 

37-50'C . After 1-2 months, the browning solution was dialyzed and then lyophilized 

against distilled water . The residue was dissolved in PBS or dimethylsulfoxide and 

submitted to the Ames test . Hydrolysis of the lyophillsate was carried out in 6N HC1 at 

110'C for 12 hrs . The hydrolyzate which has fluorescence was also examined the mutagenic 

activity . 

Several reaction products(BSA-Glu, HSA-Glu, HSA Ara, HSA-Lac) and their hydrolysates 

showed weak mutagenic activity(80-1370 revs/10 mg) toward ji .t»himuriun TA100 

without metabolic activation . Some of them may be contained in daily foods and in our 

tissues . 

50869 3612

289 

CHARACTERISTIC PEROXIDASE-MEDIATED DNA-ADDUCTS OF BENZO(a)PYRENE AND OF DIESEL 

EXHAUST PARTICULATE EXTRACT . 

R . Kind, D . Wild, D. Henschler, Institute of Toxicology, University of WOrzburg, 

Wurzburg, Fed . Rep . of Germany 

It is our aim to elucidate the potential of peroxidases for the metabolic 

activation of Diesel motor exhaust components . While the monooxygenase (MO)dependent 

activation of PAHs and Diesel emissions and the resulting genotoxic 

effects have been investigated frequently, little is known about the peroxidasedependent 

activation . We have used benzo(a)pyrene (HaP) and Diesel particulate 

extract (DPF~)2and 2 techniques to assay peroxidase-mediated reactive metabolites 

: 1 . P-postlabeling assays for DNA binding metabolites and 2 . Salmonella/peroxidase 

assays for mutagenic metabolites . Horse radish peroxidase and 

bovine lactoperoxidase were used. For the adduct studies, calf-thymua DNA was 

exposed to BaP or DPE, peroxidase and H202. The DNA was analyzed for adducts by 

postlabeling, 2-dimensional TLC on PEI-cellulose and autoradiography . For comparison, 

analogous experiments were performed using MOs (PCB-induced rat liver 

S9 and NADPH) . Peroxidase-dependent BaP adduct spots were clearly demonstrated . 

Similarly, DPE was activated by peroxidases and produced prominent adducts and a 

diffuse adduct zone . A different adduct-pattern was seen following activation by 

MO . On the other hand, peroxidase-mediated mutagenic effects were not found . We 

conclude that peroxidases produce characteristic DNA-binding metabolites which 

are probably short-lived and cannot reach the Salmonella DNA target . These 

metabolites may however be relevant for cells with endogenous peroxidase activity. 

* Supported by BMFT and FAT * 

290 

DIPSffiNTLBZDANTOIM IS NEGATIVE IN A bATTER2 OF SHORT SBM ClTOGBN6IIC ASSAYS . 

D . Kindig, J . Beyers*, J . Erunny, J . Parton, and M . Garriott, Lilly Research 

Laboratories, Eli Lilly and Company, Greenfield, IN 46140 

5,5-Diphenylhydantoin (DPH) is an antiepileptic drug associated vith an 

increase in malformations in offspring of vomen vho took DPH during pregnancy . When 

DPH has been tested in genetic toxicology studies, the•results have been varied . 

Positive and negative results have been reported for in vivo and in vitro chromosome 

aberration (CAB) assays, in vivo and in vitro sister cTiromatid exc7isngi TSCE) 

assays, and in vivo micronucieus tests (W. This report presents results obtained 

from a battery oF Lests performed in a single cytogenetics laboratory . DPA vas 

tested in the in vitro CAB assay using Chinese hamster ovary cells at doses of 200, 

250, and 300 ug7mwfih and vithout an S-9 activation system . The percentage of 

aberrations ranged from 2 to 4 and from 0 to 1 vith and vithout activation, 

respectively, and there vas no significant increase in aberrant cells vhen compared 

vith the solvent control . The SCE assay used intraperitoneal doses of 5, 10, 20, 

and 40 mg/kg and bone marrovi cells from CD-1 female mice vere harvested 21 hours 

after dosing . The incidence of SCEs ranged from 3 .7 to 4 .5 SCEs per metaphase . 

There vas no increase in the number of SCEs in DPB-treated animals vhen compared to 

solvent controls . For evaluation in the MNT, intraperitoneal doses of 10, 20, and 

40 mg/kg vere administered to ule and female CD-1 mice . Bone marrov vas extracted 

from the femurs 24 hours after dosing . The frequency of micronucleated 

polychromatic erythrocytes ranged from 1 .4 to 2 .6 and did not reflect an inerease 

over the solvent controls . The results from this battery of tests indicate that the 

knnvn tPintnqen, DPH, is nonmutagenic . 

291 

INVESTIGATIONS ON THE EXTENT 0! TESTING REQUIRED TO EXCLUDE NON-CL)1STOGEtiB IN ROUTINE 

GHROMOSOHAL ABERRATION TESTS . D .J . Kirkland, M . Ishidate Jr, D . Gatehouae and 

C . Richardson . Microtest Research Limited, York, UK ; National Institute of Hygienic 

Sciences, Tokyo, Japan ; Glaxo Group Research, Ware, Herts, UK and ICI Central Toxicology 

Laboratory, Macclesfield, Cheshire, UK . 

A number of clastogens seem to require 48 hr treatments - 8-9 in CFRL cells to produce 

a positive response . 4 chemicals from Ishidate's Data Book (Elsevier, 1988) which 

were negative at the same doses after 24 hr - 8-9, were retested at the same or higher 

doses, but also with 6 hr treatment (- and + S-9) followed by 18 hr recovery (6-18), 



Notes



Notes to assess the importance of toxicity and variations to protocol . 3 of the chemicals 

were also tested in similar protocol variations (except 3-21 inatead of 6-18) in human 

lymphocytes (HL) . The fourth, maleic anhydride, became positive in a 6-18 protocol 

+ S-9 . Sodium dehydroacetate became positive at higher dosea in CHL cells after 24 hours 

-S-9 . In HL it was only positive after 24 hours -3-9, when doses giving > 50% toxicity 

could be found . Cocaine hydrochloride became positive in all 4 phases in CML cells . 

In HL it required doses > 10 mM to get similar positive results . 6-mercaptopurine 

became positive - and . S-9 in 6-18 protocols in CHL cells on a second retest when some 

doses giving > 50% toxicity were used . In ML, it was such swre positive over 48 hours 

-S-9 than 24 hours but was negative at much higher doses in 3-21 protocols . The results 

show that (i) "difficult" clastogens can be detected in HL as well as CHL cells ; (ii) 

prolonged treatments are not always necessary if ad .quate toxicity (>50%) is achieved ; 

(iii) higher doses are generally needed in pulse treatment/recovery protocols, and may 

not achieve adequate toxicity even at doses > 10mM . 

292 

X-RAY MUTAGENESIS OF A GPT TARGET IN CHINESE HAMSTER V79 CELLS YIELDS SMALL COLONY 

MUTANTS . C .S . Klein and T .G . Rossman (Presented by E .T . Snow) . Institute of Evironmental 

Medicine, NYU Medical Center, P .O .Box 817, Tuxedo, NY 10987 (USA) . 

The E . coli ct gene encoding xanthine-guanine phosphoribosyl transferase has 

been stably transfected into HPRT' Chinese hamster V79 cells . One cell line (g12) 

maintains a low spontaneous mutation frequency (2 x 10-5), and is useful for comparative 

mutagenesis studies (ykt vs . hprt) . Alkylating agents such as N-methyl-N'nitro-N-nitrosoguanidine 

(MNNG) and P-propiolactone (BPL) are equally mutagenic to 

the ypt and hprt loci at doses (MNNG, 0-2 )ig/ml ; BPL, 0-2 mM) which yield greater 

than 50% survival of the cells . UV (0-8 J/M2) is 3-4 times more mutagenic at 2pt 

than hprt in V79 cells at relatively non-toxic doses . The y~t locus of g12 transfectants 

also appears to be more (3-4x) sensitive than the endogenous hprt to x-ray 

(0-750 rads) induced mutations . This data is in agreement with previously reported 

x-ray mutagenesis in gpt+ CHO (Stankowski and Hsie, 1986, Radiat . Res . 105 :37) . An 

abundance of small qpt mutant colonies were found in g12 cells at high x-ray doses 

which are not seen in gpt mutants of CHO . Small colony mutants in mouse lymphoma 

cells are believed to arise from cells which have sustained chromosomal and deletion 

mutations at the tk locus . Deletions, especially multilocus deletions, are difficult 

to study in most mammalian assays since they are generally lethal events . The data 

suggest that 912 cells may be useful for studying clastogen-induced mutagenesis . 

Further studies are underway to define the mechanisms of small colony formation in 

these cells . 

293 

S9UDIES ON '1HE II-ID(1f.TICN OF CY10Qt4ZE P450 LSO@Pl)R['S BY 2-AMIPD-1 (4 .5bJP1QtIDI)4E 

(phIp) Alm 2-MQP43 .8-0D6DOQ.IIOAllip(4,5-f)%RNID(ALIM (MeIQc) IN VARIOUS OHfM RY!( 

MME RAIS . 

M. Kleman, E. (iservik . P. LinderJ:os ard J .-A. Qstafasoo. DepartmaK of Medical Nutriticn, Raxolinmka 

Institute, Stockhulm, SYweden . 

7he mrtasenic ard caninaserdc hetemcyclic amires fonaed in proteinrich food upan eookin6 are 

all deperdent an metabolic activation for the formation of their ultismtely active fonre . It was 

shown by previom imestisators, that rat cytoduvne P450 forms C . d ard the canstitutive P450 msle 

in vitiv performs this activation. fie aubJect of the present imestibatien 4ms to stt* if the 

food proMutasens phlP and MeIQc have the ability to irduce cytochrome P450 isoe :aymes and thus to 

ird:re their an activation . 'Rmee dnses of 50 uR MeIQc or phlP per kb b .w . were siven to sale 

Wistar rats on three consecutive days . Betairphtoflawne (BNF) aas given aa a positive eontrol at 

40 ms per lg b.w . and 0 .9% NaCI as negative control . On the fonrth day all ani :rels were sacrificed 

by decapitation ard lurg, forestomach, stomach, smsll intestine, liver ard kidney aere talmt . 

MicYosrnrs vrre prepared fiam all orsems ard inmdiately froaen at -70'C . 9000 & sapernatant (S-9) 

was also prepared from the livers . 1fie microsants were used In liestern inmrnblots a6aiist antibodies 

raised in rabbit to cytochYam P450,,,o srd cytorlaarc i450PR. A positive response uss 

obtained with liver microsares frao the MeIt* treated ardxsls a6ainst the anti 

Measurn~ents of EUrncy~on :fin4deetlrylase activity cordinped the irductiVe effect o Me• 

cytoch- KS%s in liver and sh4wed a sigdflcantly increased activity of these cytoc]:roee PW 

forms also in the iddneys of the MeIQc irduced rats . 7he liver S-9 aanples were used in hms test 

an strain TA98 a6ainst MelW and Aflatodn BI . Slaprisirgly. the phlp induced livers ah4wed a 

significantly increased capecity to activate MeIQc, ahile the MeIQc liver S 9 :s were fnt different 

fiom the controls . 

50869 3614


THE PERSISTENCE OF DICENTRIC CHROMOSOMES IN MURINE SPLENIC AND PERIPHERAL BLOOD Notes 

LYMPHOCYTES (PBLs) FOLLOWING IN VIVO ry-IRRADIATION . A .D . K1lgermanl E .C . Halperin2 

G .L . Erexson3, and G . Honor42 . lU .S . Environmental Protection Agency, Research 

Triangle Park, NC ; 2Duke University Medical Center, Durham, NC ; 3Environmental Health 

Research and Testing, Inc ., Research Triangle Park, NC . 

In developing extrapolation models for human genetic toxicology studies, it is 

important to determine the persistence of the damage in the target cells in both the 

human and the animal model . The persistence of lesions is primarily determined by 

three processes : DNA repair, the rate of cell cycling, and cell lifespan . In this 

study, we determined the persistence of dicentric chromosomes with paired fragments in 

splenic lymphocytes and PBLs following y-radiation as a measure of the lifespan of 

these target cells . Thirty-six male C57B1/6 mice were whole-body ry-irradiated (WBI) 

with 3 Gy using a linear accelerator . Blood and spleens were removed from each of 4 

mice within 30 min, and then on days 1,3,7,14,28,56, and 112 after WBI . Both B- and Tlymphocytes 

from the spleen and T-lymphocytes from the peripheral blood were cultured 

for one cell division for analysis of dicentric prevalence . Decay curves for Tlymphocytes 

from the spleen and peripheral blood were similar describing the average 

lifespan of a T-cell to be 25 and 18 days, respectively . The shape of the B-cell decay 

curve was very different from those of the T-cells, showing little change from day 0 

through 7(40X of the cells contain at least one dicentric with a fragment) . At day 

14, 90% of the cells with dicentrics have disappeared . However, for both B- and Tlymphocytes, 

there exists a small population (approximately 2%) of long-lived cells 112 

days after WBI that contain dicentrics with fragments . [This abstract does not ratl .ot 6vA policyl 

295 

COMPARING THE STRUCTURAL DETERMINANTS OF MUTAGENICITY IN THE GENE TOX AND NATIONAL 

TOXICOLOGY PROGRAM DATA BASES . Gilles Klopman and Herbert S . Rosenkranz, Departments 

of Chemistry and Environmental Health Sciences, Case Western Reserve University, 

Cleveland, Ohio 44106. ` 

The Gene-Tox (GT) and National Toxicology Program (NTP) Salmonella mutagenieity 

data bases have been widely analyzed with respect to their performances as predictors 

of carcinogenicity . The two data bases differ greatly : in GT, mutagens predominate 

(80%) prevalence), and 88% of the subgroup also tested for carcinogenicity, was 

carcinogenic . In NTP, on the other hand, only 54% of the ehemicals are mutagens . The 

prevalence of carcinogens is 67% but 40% of them are not mutagenic . The two data sets 

were each analyzed by CASE, the Computer Automated Structure Evaluation method . It 

was found that there was a highly significant overlap among the structural 

determinants responsible for the mutagenicity in each data set, even though there was 

insignificant overlap of the chemicals in GT and NTP . 

These findings indicate that each data set can be used for mechanistic studies 

and that in future studies they can be merged . The results, using two independently 

obtained data bases, prove that there is a unique structural basis for mutagenicity . 

296 

DE"rERmnoTION OF DAlfAGFNIC EFFFICiS IN LIVER .S, SPIFMr INfESmaI. CZxA'FSRS, BzACD SERA 

AIID URIHE OF NIItIDA7AIE TRF7ITID fYQCE WITH E . OOf,I K-12 STRAINS 

Siegfried Knascdiller 

Institut of Experimental Carx :er Research, University of Innslaruck 

Fritz-Pregl-StraBe 3/VII, 6020 Innsbruck, AUSTRIA 

Intrasanguirteous Host Mediated Assays were performed to ereasure the induction of valine 

and nalidixic acid resistant imutants in E .eoli K-12 oslls reeovered fran livers and 

spleens of mice treated orally with the antischistoeanal drug Niridazole (dose range 

500 - 125 mg/bo8y weight. Following an exposese tine of 180 minr a substantial dose 

dependent mutagenic respalse was fotimd in both or+gans with tYte nitr+oz'eductase pa'oficient 

parental strain E .eoli K-12 343/113 whereas with a niridazole nitrodreductase 

deficient derivative 343/113 NIR 200 (which resists the toocic action of 200 pg Niridazole 

per ml agar median), no c]ear sutagenic effects were detectable under identical 

experimental ootditions . In additienr urine samples, extracts of intestinal eontents 

and blood sera of niridazole treated animals were tested in liquid suspension assays• 

with the reductase proficient strain pronounced activities were fatald in all camsr 

whereas with the reductase deficient strain, positive results were restricted to experiments 

with urine smnples . These latter findings stx3qest that nutaqenio nletabolites 

formed by nkTmlalian enzymes and/oz nitsnreductiea by intestinal mieroorqattisire at ooncentratiens 

wich are to low to enable their detectien under the present in vivo oonditicns 

are concentrated by ultrafiltration and excreted via the renal system . 


103


Notes CARCINOGENESIS OF FOODS . lb Knudsen, DVM, Institute of Toxicology, 

National Food Agency, Msrkhmj Bygade 19 . DK 2860 Smborg (Cph .), 

Denmark . 


Food is a major determinant of human health and disease, not only from 

a nutritional point of view, but also from a toxicological point of view . 

Thus food toxicology is not only a scientific discipline to deal with the 

toxicology of food additives but also a ma jor source of knowledge about 

impact of major and minor food constituents on human health . The 

Institute of Toxicology has established a testing programme to deal with 

these matters covering natural fibers in the protection against colon 

cancer, possible interaction between urethane and alcohol in carcinogenesis, 

food mutagens as a human health factor . The paper will discuss 

our recent progress within these areas of research . 

INDUCTION OF CHROMOSOME CHANGES BY METAL COMPOUNDS 

IN CULTURED CHO CELLS . 

TS. Kochhar, B . Leonard, K . Harris, W . Moody 

Kentucky State University, Frankfort, KY 40601 

Several metal compounds have been identified as potential carcinogens through 

occupational exposure as well as in the laboratory . Since chromosome changes such as 

abberations and sister-chromatid exchange have been linked to DNA damage, the 

influence of certain metal salts on these parameters was assessed in cultured CHO cells . 

The cells were treated with CdCI2, Cr0„ HgCI= and N1CIz for 24 h . The concentrations 

used for aberration assay were 10'7 -10" M and for SCE assay it was 10-s-10's M . It 

was observed that all compounds enhanced . various types of chromosomal abnormalities 

compared to the controls . CdCl2 seemed to have the maximum effect as the cultures 

treated with 10-7 and 10-s M of this compound revealed a considerable number of 

chromosomes with two or more centromeres . Other increase in abnormalities were the 

ring chromosomes, chromatid breaks, chromatid exchanges and the stickiness of the 

chromosomes . CdCI„ Cr03 and NIC1 also enhanced SCE frequencies . In higher 

concontrations of these compounds (10'~ and 10-s M) SCE/cell Incrpsed more than twofold 

compared to the BrdU controls . HsCI= was relatively ineffective in raising the SCE 

rates . These studies indicate a definite link between the metals and the mammalian 

chromosomep-which calls for further detailed studiq . (5upported by NIH-MgRS RR 

08124 grant .) 

297 

298 

299 

DNA SEQUENCE ANALYSIS OF MUTATIONS INDUCED IN YEAST BY EXCESS THYMIDYLATE : EVIDENCE 

FOR NEXT•NUCLEOTIDE EFFECTS . S .E . Kohalmi and B .A . Kunz, Microbiology Department, The 

University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2 

In thymidylate (dTNP) auxotrophs of the yeast Saccharomvcas cerevisiaa, intracellular 

dTTP levels can be modulated by varying the concentration of dTMP in the growth 

medium . Provision of excess dTMP is mutagenic in yeast . Recently, yeast DNA polymerase 

III was found to have a 3' -> 5' exonuclease activity capable of proofreading in 

vitro . Thus, mutations induced by excess dTMP could be due to misincorporation of the 

nucleotide and the misincorporation frequency might be enhanced by next-nucleotide 

effects reducing the efficacy of the 3' -> 5' proofreading activity . On this basis, 

and since dTTP is a positive effector of GDP reduction In yeast, excess dTMP would be 

expected to cause increases in substitution by T and in the proportion of events at 

sites flanked by a 3' T or C . To test these possibilities, we are using DNA sequence 

analysis of mutations induced by excess dTMP in the SUP4-o gene of yeast . To date . 

100 induced mutations have been characterized and the spectrum and distribution of 

changes have been compared to those for 306 spontaneous mutations . We have determined 

that : 1 . 86% of the induced substitutions involve replacement by T whereas only 64% 

of the spontaneous changes do ; 2 . approximately 90% of the induced mutations occur 

at sites with a 3' flanking T or G vs . 73% for the spontaneous mutations . The results 

argue that dTMP misincorporation is responsible for the induced mutations and suggest 

that a proofreading function is part of the yeast DNA replication complex In vivo . 

Currently, we are analyzing additional mutants . (Supported by NSERC Canada) 

300 

THE USE OF LAbIDDA PHAGE SHUTTLE VECTORS IN TRANSGENIC MICE FOR 

DEVELOPMENT OF A SHORT TERM MUTAGENICITY ASSAY 

Kohler, S .W ., Provost, G .S ., Kretz, P .L ., Sorge, J ., Huse, W .D . and 

Short . J .M . Stratagene, 11099 North Torrey Pines Road, La Jolla, CA 

92037 . 

A short term mutagenesis assay has been developed which will permit 

analysis of suspected genotoxic substances in whole animals . 

Transgenic mice containing a stably integrated bacteriophage lambda 

shuttle vector can be rescued at high efficiency (>1000 pfu/µg of 

genomic DNA/integrated copy) from isolated genomic DNA with In vitro 

lambda packaging extracts . This rescue efficiency allows the recovery 

of over one million lambda phage genomes per tissue, a level 

50869 3616

1989 EMS Abstract s 

sufficient for measurement of the spontaneous mutation rates in whole Notes 

animals . Mutations are detected by monitoring the activity of a 

P galactosidase target gene contained within the lambda genome . The 

high rescue efficiency is accomplished through the use of methylated 

cytosine restriction deficient (mcr ) lambda packaging extracts and 

plating strains. This mutagenesis assay will permit comparison of 

mutation rates between different tissues in whole animals . In 

addition, the shuttle vector is being modified to include the in vivo 

excision features of the lambda ZAP cloning vector. This will 

facilitate the DNA sequencing for identification of the sequence 

specificity of genotoxic substances . 

30 1 

SCHISTOSOMIASIS DRUGS : AN ICPEMC STUDY 

P .G .N. Kramers (1), J .M. Gentile (2), B.J .A .M. Cryseels (3), P . Jordan (4), N. Katz 

(5), K .E . Mott (6), J .J . Mulvihill (7), J .L. Seed (8), and H. Frohberg (9) ; (1) 

National Institute of Public Health and Environmental Protection, P .O. Box 1, 3720 BA 

Bilthoven, (2) Holland, MI, (3) Leiden, The Netherlands, (4) Ware, UK, (5) Belo 

Horizonte, Brazil, (6) Geneva, Switzerland, (7) Bethesda, Md, (8), Frederick, Md, (9) 

Darmstadt, PRG . 

One of the interests of ICPEMC is to identify situations in which the possible 

induction of inherited defects in man by mutagen exposure could actually be studied . 

The large-scale use of mutagenic drugs in field programmes against Schistosomiasis, 

mainly during the seventies, was considered a possible case . An ICPEMC task group 

approached the problem by (1) updating the genetic toxicology data base for the 

various antischistosomal drugs known, and (2) searching for possible study areas . 

Expertise was combined from genetic toxicology, mutation epidemiology and tropical 

medicine . It was considered that : (a) hycanthone is the best candidate drug, if any ; 

(b) local demography would render the reliable tracking of substantial numbers of 

offspring of treated persons an almost impossible task (c) in most endemic countries 

proper diagnosis and registration of inherited defects is largely lacking ; (d) as 

estimated from animal experiments the sample sizes needed to show an effect, if any, 

would be very large; and (a) since non-mutagenic antischistosomal drugs are now in 

use, the problem is of low priority in endemic countries that are usually short in 

medical resources . Thus, studying offspring of hycanthone-trsated people to 

demonstrate the mutagenic potential of the drug in manais not a viable enterprise . 

302 

THE ROLE OF PROTEIN KINASE C IN SIGNAL TRANSDUCTION AND CELLULAR TRANSFORMATION . R.S . 

Krauss, G .M . Housey . W .W.-L. Rsiao, M .D. Johnson, S .A. Rotenberg and I .B. Weinstein . 

Comprehensive Cancer Center, Columbia University, New York, N .Y. 10032 . 

We have utilized a retroviral expression vector to construct a series of rodent embryo 

fibroblast cell lines that stably overexpress a full length cDNA encoding rat protein 

kinase Cgj (PKC) . Rat 6(R6) cells that contain 20-53 fold higher PKC enzyme activity 

than control cells show several disturbances in growth control, including the 

ability to form small colonies in soft agar and tumors in nude mice . R6-PKC-3, a line 

with a 53-fold increase in PKC activity, in hypersensitive to transformation by transfection 

with an activated ras oncogene, as measured by the formation of large colonies 

in soft agar . Recently, we have developed lines of C31i/10T1j cells that stably overproduce 

PKC . 10Tk-PKC-4, a line with an 11-fold increase in PKC activity relative to control 

cells (but with a specific PKC activity similiar to the R6-PKC lines), is morphologically 

altered, grows to 4-fold higher saturation density and has reduced adhesiveness 

. When grown in 2 .5% calf serum and 100 ng/ml TPA, these cells form numerous large, 

dense foci . However, unlike the R6-PKC cell lines, neither 10TIS-PKC-4 calle nor calls 

from the TPA-induced foci are capable of growth in soft agar, and they are non-tumorigenic 

in nude mice . These R6 and 10711 cell lines should be useful for investigating 

molecular events in tumor promotion and multistage carcinogenesis . We have also utilized 

these cell lines as a source for obtaining enzyme preparations that are highly enriched 

for PKCS1, thus facilitating biochemical analysis of this PKC isoform . The implications 

of these studies with respect to the role of PKC in carcinogenesis will be 

discussed . Supported by NIH grants CA02656 and CA08346 . 

303 

REASSESSMENT OF SELECT NTP COMPOUNDS IN THE L5178Y/tk*/- MOUSE LYMPHOMA 

ASSAY. R. Krehla and D . Clivea, aWellcome Research Laboratories, Research Triangle Park, NC 

27709 USA 

Strong conclusions have been drawn about the performance of 4 short term tests based on summary 

(+, -) evaluations of the NTP-sponsored studies on 73 rodent carcinogens and noncarcinogens (Tennant 


105


Notes et a!. , Science, 236 : 933, 1987) . Thus, despite an earlier critique of these studies (Ray et al ., 

Environ . Molec . Mutagen., 511 : 85, 1988), Ashby (Mutagenesis, 3 : 483, 1988) has suggested the 

existence of 19 nonclastogenic mutagens based on these evaluations . To date (February, 1989), the 

L5178Y/tk +/- mouse lymphoma assay (MLA) data on most of these 73 compounds have not been 

published . For these reasons an effort was made to retest In a single laboratory two subsets of those 

chemicals : those which had yielded marginal positive or negative results, and those proposed as possible 

nonclastogenic mutagens . Six of the former (allyl Isovalerate, allyl Isothiocyanate, benzene, ethyl 

acrylate, eugenol and geranyl acetate) and 7 of the latter (benzoln, benzyl acetate, chlorobenzene, 

cinnamyl anthranilate, 1,2-dichlorobenzene, geranyl acetate (also on the marginal response list) and 

sulfisoxazole) have been completed to date . Our results differ In 4 ways from those of the NTP : (1) we 

saw little of the Inter- and intra-experimental variability seen In the original NTP studies ; (2) our 

active dose ranges differed markedly for several compounds from those obtained in one of the contract 

labs ; (3) we generally obtained significantly stronger mutagenic responses than did that same contract 

lab, consistent with their weak MMS positive control responses : and (4) NTP's positive result for one 

chemical (benzyl acetate) In the absence of S9 was not seen In these studies . The significance of these 

results for test protocols, data evaluation, quality control, and assay correlations will be discussed . 


304 

KINETICS OF DIMETNYLNITROSAMINE(Dl4d)-INDUCED MICRONUCLEUS (IQ1) FORMATION IN MOUSE BONE 

MARROW AND SPLEEN CELLS . G . Krishna, M .L . Kropko, and J .C . Theiss, Parke-Davis Pharm . 

Res . Div ., Warner-Lambert Co ., Ann Arbor, MI (USA) 

The MN aasay has been extensively used in routine mutagen/carcinogen screening 

programs to detect agents which cause chromosomal breakage and spindle dysfunction . 

DMN is a known mammalian carcinogen and has been tested in both mouse and rat bone 

marrow for its ability to induce MN . For D!'Qd, both positive and negative MN assay 

results have been reported which may be due to differences in bone marrow sampling 

times . The present study was disigned to obtain information on the kinetics of MN 

formation following DMN treatment In mice . Groups of 3 to 5 CD1 male mice were 

injected once i .p . with 50 or 100 mg/kg DMN . Both bone ∎arrow and spleen cells were 

isolated at various sacrifice time-points (6, 12, 24, 36 . 48, and 60 h post-treatment) 

and processed for MN analysis according to established procedures . Cyclophosphamide 

(CP, 40 mg/kg) was used as a positive control and saline served as the vehicle 

control . In the study, the vehicle control group had 0 .6 and 0 .9 !Q1/1000 

polychromatic erythrocytes (PCEs) in bone .arrow and spleen cells, respectively . DMN 

at 50 mg/kg, caused 3 .8, 7 .8, 8 .5 and 10 .2 !Qi/1000 PCEs in bone marrow and 8 .0, 9 .2, 

19 .3 and 32 .8 AQJ/1000 PCEs in spleen at 12, 24, 36 and 48 h sacrifice times, 

respectively . A similar time-related elevation of MN frequency was noted for 100 

mg/kg DtQI . At each sacrifice time-point, spleen cells had a higher MN frequency 

than bone marrow cells . In general . DMN caused a decrease in the proportion of PCEs 

to normochromatic erythrocytes, suggesting toxicity . Thus, this study demonstrates 

the clastogenic activity of DMN in both bone marrow and spleen cells of mice and shows 

a time-related pattern in the elevation of DMN-induced MN frequency . 

MI1rAGFr18, CARCItOMNS AND ANfI'1SkDUR AMTI5 IN '14tE INDIAN DIET 

ARtM KRISf3171K[k1AR and V .M .SIVARN+Al62I 

(Isotope Division, Cancer Institute,t ryr, Madras 600 020, INDIA) 

The role of spices and other plant products wide consumed in India either 

in the diet or in indigenous medicine, in relation to the alimentary cancers (oral, 

gastric and oesophagal cancers) prevalent here, has been investigated with laboratory 

animals . Most of the spices are found to be very well tolerated, even when 

fed in large doses, with little change in physical appearance, growth rate or behaviour 

.Cinnamon actually increases the growth rate . Out of the 25 plant products 

screened,nine induced the carcinogen-detoxifying enzyme, glutathione-S-transferase, 

in the st.omach, liver and oesophagus sufficiently high to be considered protective 

agents . Ttrey are cunin (cLminum num) and poppy (Papaver aonniferrum) seeds ; 

drumstick (Moringa oleifera), mana li (§21anun ni rwn) and (Ocinum basilicum) 

leaves ; neem (Azadirachta indica) flowers ; asafoetida (Ferula asafoetida), 

kandanthippili (pi lon ) and turmeric ((lrrctnna 1a a)9 Eight o t~.e . , 

excepting neem flowers, suppress the geno c e ects of 3,4-benzo(a)pyrene, when 

simultaneously fed, cumin and poppy seeds being most effective . Feeding emblica 

(phyllanthus emblica) decreases glutathione-S-transferase possibly a tumour-promoter 

. Ames test reveals fried mustard, perandai (Cissus anr laris) and oil 

of calamus (Acorus calartus) to be mutagenic ; tamarind (Tamar s indica) non-mutagenic 

; while poppy s are antimutagenic . 

~ 

m 

OD 

01 

W 

01 

F+ 

00 

305


TFLE USE OF EGDCTORATE FOR M7NITDRING oCCUPATIQQ L P70?OBURE 

Notes 

A . Krmkje, Department of Botany, The University of Trondheim, Norway 

The aim was to investigate if mutagens are biologically available . The workplace 

investigated is the top of the batteries in a coke plant, where relatively high 

concentrations of polycyclic arrnwtic hydrocarbons (PAHs) are usually measured in the 

air. The study is aimed at group exposure . The 4 groups studied are control nonsmkers, 

control smokers, battery naumnkers and battery smoke.rs . The workers gave their samples 

within half an hour of finishing the workshift and after the night's sleep - "morning 

expectorate" . The samples from each group were pooled, hydrolysed with alkaline 

methanol and extracted with cyclohexane . The S . typhitturium strains TA98 and TA100 

rx:re used in the Salmonella/micro®ane-test . Liver and lung hanogenates were used as 

metabolic activation systems . The expectorate fran exposed workers - mostly fran 

smokers, but also to a certain extent fran nonsmkexs - were mutagenic ; however, the 

control samples fran both smokers and nonamokers were not . The positive results 

produced by expectorate samples from the exposed smokers suggest a synergistic 

relationship between exposure to air pollution of the working attrosphere and smoking . 

The analysis of the "morning expectorate" shnws that inhaled pollution with potential 

nrtagens is not effectively removed from the respiratory system during the night . The 

salmonella-test used on expectorate samples may be an effective assay in certain types 

of industries, viz . where the atmsphere contains partieulates acting as carriers for 

mutagens apt to be eluated by the body fluids or cellular membranes . 

307 

BACTERIAL MUTAGENICITY ASSESSMENT OF STRUCTURALLY-RELATED QUINOLINE 

THIAZOLAMINE COMPOUNDS, POTENTIAL ANTIPSYCHOTIC DRUGS . M .L . Kropko, J .C . 

Jaen, S .A . Wold, B .W . Caprathe, L.D . Wise, and J .C . Theiss, Parke-Davis Pharm . 

Res . Div ., Warner-Lambert Co ., Ann Arbor, MI(USA) 

Quinoline thiazolamines have potential for antipsychotic activity . In a 

routine Ames assay (TA98, TA100, TA1S35, TA1537, TA153B, S9t), PD 123403 was 

found to be mutagenic to TA98 (peak response of 14-fold background at 32 

ug/plate) and TA1538 (peak response of 19-fold background at 100 ug/plate) 

in the presence of S9 only . This frameshift mutagen is a quinoline 

thiazolamine comprised of fused pyridine, cyclohexane and aminothiazole rings . 

To provide structure-activity information for development of a non-mutagenic 

antipsychotic drug in this chemical class, structural analogs were synthesized 

and screened for mutagenicity using TA1538 with S9 . Methylation of the 

cyclohexane ring gave a weak mutagen (peak response of t .4-fold background at 

316 ug/plate) . Mutagenic activity was completely lost (maximum dose tested of 

10,000 ug/plate) either upon removal of the amine from the aminothiazole ring, 

upon ethyl substitution of the nitrogen in the partially reduced pyridine 

ring, upon saturation of the pyridine ring with hydrogen, or upon 

removal of the cyclohexane ring . These results suggest that oxidation of 

PD123403 by S9 may take place at the cyclohexane ring, producing a mutagenic 

fully aromatic amine compound . Thus, modifications to this quinoline 

thiazolamine which prevent oxidation to a tricyclic heteroaromatic amine, 

while at the same time not interfering with pharmacological activity, should 

result in a non-mutagenic antipsychotic . 

308 

TOXICITY OF ENDRIN ( A CHLORINATED HYDROCARBON ) ON THYROID HORMONE 

FORMATION OF A TELEOST . DHYANENDRA KUMAR,Department of Zoology,Jagjiwan 

College,Magadh University, ARRAH,Bihar,INDIA . 

Hypofunction of thyroid due to environmental pollutants may be mediated 

through the inhibition of thyroid peroxidase enzyme as this enzyme from 

pronephric (head) kidney and pharyngeal sources is readily inactivated 

by pesticides . Environmental pollutants such as organochlorine and 

carbondisulphide considerably depress thyroid function in fish . 

5'lhen Anbas testudineus,Bloch (a teleest) was exposed to 48 hr TLM 

concentration (0 .05 ppm ) of endrin, formation of MIT (Monoiodotyrosine) 

and ,IT (Diiodotyrosine) by head kidney soluble supernatatnt fraction 

were markedly depressed . Foemation of T(Thyroxine) was found to be 

similarly inhibited due,to endrin pollution . Per cent of inhibition 

due to exposure of 48 hr TLM to endrin was found to De 53 .6,56 .06 and 

50 .4'; for the formation of MIT,DIT and T4 respectively . Inhitory 

effect of endrin could effectively reversed by Cytochrome c . 


107


Notes 


THE AMPHIBIAN (BAI4A pjpgNS ) LENS AS A BIOLOGICAL MONITOR OF WATERBORNE 

XENOBIOTICS . J.S. Kung, C,$,Geard and B.V. Worgul, Eye Radiation and Environmental Research 

Laboratory and Radiological Research Laboratory, College of Physicians and Surgeons of Columbia 

University, New York, NY 

309 

$gpa p,jpj= shows promise as a biological monitor of environmental mutagens . As vertebrates, they 

are able to enaymatically activate promutagens to their genotoxic metabolites . Being aquatic, they inhabit 

waters that are polluted by domestic sewage and industrial waste containing waterbotne xenobiotics which 

maY chronically concentrate in their tissues . Since the products of all kns epithelial divisions are 

matntained within the lens for the entire lifespan of the animal, the lens may serve as a useful long-term 

indicator of genotoxicity. Frogs were exposed through the ambient environment to Benzo(a)pYrene (BP), a 

promutagen, 2 .5, 5.0, 10.0 and 20.0 µg/ml, and to Ethyl methanesulfonate (EMS), a direct-acting 

clastogen, 25, 50,100 and 200 µg/ml, by partial immersion for a period of one week . Following sacrifice 

of the animals, lens epithelial whole-mounts were prepared. The epithelia were stained by the Feulgen 

technique to permit visualization and quantitative analysis of nuclear DNA content . An increase in the 

premitouc 02 population was found with increasing concentrations of either mutagen . An inverse dose 

relationship with mitoses suggests that these mutagens may exert a premitotic block in the cell cycle . 

Micronuclei frequency, a measure of genotoxicity, was found to be elevated with both mutagens in a nondose-dependent 

fashion. A block in the cell cycle may well account for this since micronuclei production is 

a mitosis-dependent event . $= may prove to be valuable in the study of environmental mutagens since 

they are easily handled, adaptable to laboratory conditions, ubiquitously distributed and amenable to short 

term genotoxicity assays . 

Supported by Grant EY 02648 from the ?dEI . 

310 

THE SUp4-o SYSTEM FOR ANALYSIS 0F MUTATIONAL SPECIFICITY IN YEAST . B .A . Kunz, J .D . 

Armstrong, M . Clattke, S .E . Kohalmi and J .R .A . Mis, Microbiology Department, The 

University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2 

Knowledge of the DNA sequence alterations produced by mutagens, as well as their 

frequency and location within a gene, can provide valuable clues about the DNA damage 

responsible and how it is processed into mutations . To obtain detailed mutational 

specificity data, we developed a shuttle vector system wherein mutations in 

the tRNA suppressor gene &UP4-o of the yeast Saccharomvcas cereviaiae can be easily 

analyzed to determine the DNA sequence alterations involved . In this system, the 

SUP4-o gene is carried on a plasmid that mimics the behavior of yeast chromosomes . To 

date, more than 2,000 SUp4-o mutants have been characterized . The spontaneous mutation 

frequency is typical for chromosomal genes in yeast and all 6 possible base-pair 

substitutions (single and double changes), deletions of various lengths, base-pair 

insertions, duplications, transposon insertions and more complex events arise spontaneously 

. Single base-pair changes have been found at 68 of the 75 exon sites, and 

at 2 of the 14 intron positions, within the gene . The system can be used in the 

forward mode to analyze mutational specificity or as a reversion assay to provide 

additional information on site-specific mutagenesis . In addition, isogenic error-free 

repair deficient derivatives have been constructed to allow assessment of the role of 

DNA repair in mutagenesis . The $Up4-o system and its use will be described and 

results obtained in SUP4-o will be compared with data available for mammalian shuttle 

vector systems . (Supported by NSERC Canada) 

311 

CHARACTERIZATION OF THE YEAST rad52 MUTATOR EFFECT BY DNA SEQUENCING . B .A . Kunz, S .E . 

Kohalmi, J .D . Armstrong, and M . Clattke, Microbiology Department, The University of 

Manitoba, Winnipeg, Manitoba, Canada R3T 2N2 

Defects in the $aD52 gene of the yeast Saccharomvices carevisiae confer a mutator 

phenotype . This effect was characterized by sequencing a collection of 238 spontaneous 

SUP4-o mutations arising in a gDd52 strain . The resulting mutational spectrum 

was compared to that derived for an isogenlc wild-type strain (222 mutations analyzed) 

. This comparison revealed that the mutator phenotype was associated with an 

increase In the frequency of base-pair substitutions . All possible types of substitution 

were detected but there was a decrease in the fraction of A-T -> G•C transitions 

and an increase In the proportion of G-C -> C-G transversions . These changes 

were large enough to cause a two-fold greater preference for substitutions at G•C 

sites In the rad52 strain despite a decrease In the fraction of G-C -> T-A transversions 

. Base-pair changes occurred at fever sites In the tgd5Z strain but the mutated 

sites included several that vere not detected In the $OQ52 background . Only two of 

the four sites most freqdently mutated In the tyM strain were also prominent In the 

wild-type strain and mutation frequencies at almnst all sites common to both strains 

were greater for the X{¢n derivative . Although single base-pair deletions occurred 

at similar frequencies in the two strains, several classes of mutation that were 

recovered in the wild-type background including multiple base-pair deletions, insertions 

of the yeast transposable element Ty, and more complex changes, were not recovered 

in the tgdn strain . Our results suggest that the XAM mutator effect is not 

due defects in repair of abasic sites or base mismatches . (Supported by NSERC Canada) 

S0869 3620

312 

ANTIMUTAGENIC EFFECTS OF GLUTATHIONE-S-TRANSFERASE INDUCTION IN E . COLI . 

S . Kuo and D .M . Shankel, Department of Microbiology, The University of Kansas, 

Lawrence, Kansas 66045 . 

The glutathione-S-transferases are dimeric enzymes which mediate the detoxification 

of potentially mutagenic electrophiles via conjugation to the tripeptide 

y-glutamyl cysteinyl glycine (glutathione) . Glutathione-S-transferases are also 

present in a variety of bacteria . Using the E . coli K12ND160 system, it was observed 

that pretreatment of E . coli with butylated hydroxy anisole (BHA) results in a 

decrease in the frequency of induced revertants caused by ethylmethanesulfonate, 

nitrofurazone, quinacrine and hydrogen peroxide . This reduction in induced mutants 

occurred concomitant with an increase in activity of glutathione-S-transferase in 

the organism . Thus this enzyme may play an important role in the protective arsenal 

of E . coli against potentially genotoxic agents . The significance of these results 

and related findings will be discussed as they relate to the mechanism of antimutagenesis 

. 

313 

BIOLOGICAL SIGNIFICANCE OF INDUCED ENDOREDUPLICATION . A . Lafi and J . M . Parry, School 

of Biological Sciences, University College of Swansea, Singleton Park, Swansea SA2 8PP . 

Several authors have demonstrated that exposure of cultured cells to a number of 

chemical agents results to an increase in endoreduplication . Here we present 

comparative data on the ability of tobacco particulate matter (TPM), derived from 

three cigarette types, to induce endoreduplication . A dose related increase was 

observed but there were no consistently significant differences related to the tar/ 

nicotine levels per cigarette used . In cells allowed to recover from TPM treatment 

there was an increase in polyploidy and a decrease in endoreduplication suggesting 

that the increase in polyploidy observed was at least partly due to the recovery of 

cells from endoreduplication . Chromosome aberrations were induced in all three types 

of cells (diploids, endoreduplicated and polyploids) . However, the levels of all 

types of aberrations were higher in the endoreduplicating cells when compared to 

diploids and polyploids . This was a very unexpected resuit since it has previously 

been shown that endoreduplicating cells (when compared to diploid and ordinary 

tetraploid metaphases) are seen to contain a decreased frequency of SCEs (Speit . G ., 

Vogel, W . and Mehnert, K . 1985, Chromosoma, 89, 79-84) ; The presence of cells with 

different ploidy levels may have a significant effect upon the capacity of some 

chromosome abnormalities to be transmitted to progeny cultures . 

314 

IN VTW G[fEPIC =C1TY FFOIUCfl[S FYR TfQ3tAP1 ;iRTC HOOlW PCFMAAITQIS. Robert S . Lake, Safety 

Evaluation Center, Schering Corporation, Lafayette, NJ 07848 . 

Therapeutic proteins (cytokinas, gtvvth factors and imniwnndulatory polypeptides) pose tw special 

problem for routine in vitro safety testirg . (1) Mode of action is usuall,y species and cell-type 

specific such that the protein itself is rot acutely toxic and (2) clinically used dng fomailation 

excipients can modify test system regpor~ses . Dng substanoe is usuall;y ptrsented for testing as 

lyophilized active in for+iulatio ;tis as siaple as saline or as oanplex as mixtures with buffers, bulking 

agents, stabilizers, antioxidents, ard wettitg ageits . Ideally, the first ptoblem can be dealt with by 

the use of primate tissues or oetl types laaun to retain appropriate receptors . For qualiq+ control 

pucposes, sub4 .msn system (such as the Mrs Test) are atill weful for detectirg potential activity of 

excipients, contaminants, or biottac.tfonmatien-degtadation products . The seoatd problem neoassitates 

exteruive pilot trials to establish tolerated dose wlueas of fotsilatien vithout the active protein 

(placebo control solution or control vehicle) and subsequently cantrolling for toequal vehicle effects in 

all dose ard control groups. This latter approach involves adjusting all treataent carditiom to the 

level (v/v) of vehicle control solution used in the high-dose gtroup . If the vehicle is t .duly toxic to 

backgroud endpoints or interferes with positive control respas es, thet the foraulated therapeutic 

protein canot be tested . In effect, since the active protein is nort-tcodc, the ssscian tolerated dose 

level is set or deterndned by the level of vehicle control solution tolerated by negstive aod/or positive 

control groups . Failure to andify study desigrts can lesd to false negatives if the formulation 

conpone.nts suppress S9 sietabolisn or expcesaion of sutation . False positives could ocas- if spontaneam 

(backgrand) levels are erimoed or cytotcedcities from other systea emipaknts are eliminated . Such 

forau]ation effects have been observed in bacterial wtasgenaais, huaan l,ysptwcyte qtagenetics, CfA-tCPRP 

and rat hepqtocyte UDS studies . 



Notes



Notes MDTATION3 IIJDI= IN = 3WJ GEIiE OF E . ooli BY l-Nr1Sa08O-8-YiI=PYft@lE : T}IE 

INF11JF2KE OF PTA4IID 01 AND EXCISICN RtRAIIt CN THE *IPATICNAL SPECIS3BI . I .B . 

Inmbartl, A.J.E. Gatd, B.W. Gliclaaatl2, D .R . MoCalla, 11dcMastet' University, 

Namilton, Ontario and 2York University, Toionto, Ontario (Canada) . 

1,8-Dinitropyrene is an envizotmental oonRaminar :t MR :id : is mutageni.c in bacteria 

and cultured mmuaalian cells, ard aast;inogenic in rodents . This oacpo:atid is 

activated in vivo by nitsoraduction to 1-nitxneo-8-nitrvpyrei:e (1,8-NONP), and then 

to the hydroxylamine vhich, followinq soet.ylaticn, reacts with D2ZA . The major 17la 

adduct fozmed is 1-N-(2'- T :A txatwrrexsions oonsistent with preferential 

misinoorporatian of adenine during error pmne bypass of a bulky lesion . Deletions 

are detected in all strains, but are itduoed above the spontaneous frequency only 

in the e UvrB, p1C+D01 baclognound . 

316 

M'M(AF2~ 1~1TAGQIESIS ~ RtIE l~I ~ QF~',,,gJi . I .B. Imnbertl, T .A . 

Chin1, D .W . Bryant , B .W . Gliclcna~, D.R. MoCalla yMddaster University, Hamilton, 

Ontario (Canada) and 2York University, Toronto, Ontario (Canada) . 

AF2 is a 5-nitrofaran derivative vhich is :nrtagenic in baateria followitg 

activation by endogenas nitx+areduataoes. Pr+avious studies in our laboratory have 

suggested that AF2 mrtagenesis is depeneent on the irduction of SOB repair 

ftaretiens . In oider to gain greater in4iqht into the mec3 :aaimn by Vhidi AF2 induces, 

mutations, we have examined the nutaticnmi specificity of this oaipo :rd in the 11aI 

gene of E,gol . Traatment of a ntNrB, P1CU01 host with 1 UM AF2 yielded a nutatian 

frequency 300 X that of tastreated oontrols at 20% survival . Mztatiore whicli 

occurred in the first 180 base pairs of the episaml JWI gene were ctiaracterized 

by L'ta sequenoe analysis . Of the 144 autants in the oollectian, 125 are base 

substituticns with trareversicns a :trunberinq txansitions by about 2 to 1 . Ninetytwo 

peroent of the base substitutions oaas at G :C beas pairs (62 G :C -> T :A 

traneversions, 43 G :C -> A :T transitions, 10 G :C -C :G transveraions) . Base 

substitutions ooatr 3 .1 times more frequently at 5'-Pyrimidine-G than 5'-Parine-G 

sites . A mec3anism consistent with these results would be the error psnne 

imorporation of nucleoti8es across ftnm an apurinic site with an order of 

preference of adenine > thymine > guanine . Of the 11 framec+hift mrtatians recovered 

in this study, 10 ooour at one hotspot whicA irNolves the loes of the G fraa the 

sequence 5'-TTIGCDO-3' 

. A further characteriatio of this collection is of several oanplex mutatians irnrolvirg multiple closely spaced ( pn 

apart) mrtationnl events . 

317 

INVITRO DETOXIFICATION OF MX (3-CHLORO-6-(DICHLOROMETHYL)-5-HYDROXY-2-(5H)-F1IRANONE) . 

S . Lampelo, T . Vartiainen and S . LSt,jBnen, National Public Health Institute, Dept . 

Environm . Hyg . Tox . P .O .B . 95, Kuopio and University of Kuopio, Dept . Chem . P .0 .0 .21, 

Kuopio, Finland 

MX is a strong bacterial mutagen found in drinking water . In this study the 

changes in the mutagenicity of synthesized MX were investigated after its preincubation 

with liver 59 (L-S9), human placental S-9 (P-S9), vitamin-C and their combination 

utilizing Ames" Salmonella test . The effect of serum albumin on the mutagenicity 

of MX was also studied . The etability of MX mutagenicity when diluted in 

water and buffer with and without vitamin C was followed during storage at room 

temperature . 

MX lost its mutagenicity in the presence of P-S9 or L-S9 . Preincubation with vitamin 

C (10 ug) enhanced MX mutagenicity whereas adding of P-S9 or L-59 into this mixture 

caused a drastic decrease in the mutagenicity . However, no inactivation of the mutogenicity 

occurred when P-S9 was incubated with vitamin C before adding MX into thre 

mixture . In all cases L-S9 was a very potent inactivator . Albumin decreased the 

mutagenicity of MX when added onto the plate but low concentrations of albumin in 

the preincubation mixture caused an increase in MX mutagenicity . MX was very stable 

when stored in buffered C-vitamine solution whereas it lost its mutagenicity in 

water and buffer during two weeks . 

These results suggest that inactivation of MX might be due to enzymatic systems but 

also to an unspecific binding to proteins . The antioxidents might protect the 

reactive genotoxic sites in MX-molecule . 

50869 3622


IMPROVERD METABOLIC CAPABILITY BY DRUG METABOLIZING GENE TRANSFECTION. Notes 

R . ~Lan~enbach', C . Crespi', R . Davies', P . Smith', and K . Rudo', 'National 

Tnstitu~Environment Health Sciences, Research Triangle Park, NC 27709 and 

'Genetest, Woburn, MA, 01801 . 

The technique of gene transfection was employed to introduce the cDNA coding 

for specific cytochrome P450 enzymes into transformable C3H/10TYe mouse embryo 

ce11s and mutable human AHH-1 lymphoblastoid cells . The long term objective of 

this work is to enhance the metabolic capability of these cells for exploring the 

role of cellular metabolism in genotoxic and nongenotoxic mechanisms of carcinogenesis 

. C3H/10TS4 cells containing transfected rat cytochrome P450IIB1 (P450b) 

were more sensitive to the cytotoxic effects of acetylaminofluorene and dimethylnitrosamine 

than parental cells . Transfection of either human cytochrome P450IA1 

(methyl cholanthrene inducible form) or cytochrome P450IIA2 increased the mutagenic 

response of AHH-1 cells to aflatoxin B1 and dimethyl-/diethylnitrosamine, 

respectively . The manipulation of drug metabolizing genes provides an experimental 

strategy for delineating initial stages of carcinogenesis and/or mutagenesis by 

toxic agents in mammalian cells . 

319 

RECENT DEVELOPMENTS IN THE GLYCOPHORIN A ASSAY FOR SOMATIC CELL 

MUTATIONS IN HUMANS . Richard G . Langlois, William L . Bigbee, and Ronald H. Jensen, 

Biomedical Sciences Div., Lawrence Livermore National Laboratory, Livermore, CA (USA) . 

The glycophorin A (GPA) assay utilizes Immunofluorescent labeling and flow cytometry to 

identify and enumerate rare erythrocytes which lack the expression of one allelic form of 

GPA presumably due to mutations in erythroid precursor cells . Variant cells with both 

hemizygous and homozygous phenotypes are observed in normal individuals at a 

background frequency of about 10 per million normal cells . Transient Increases in variant 

frequency (VF) were observed in cancer patients during chemotherapy, but VFs returned to 

near normal values six months after therapy was completed . Significant dose-depehdent 

increases in VF were observed in A-bomb survivors suggesting persistent stem cell effects 

from radiation damage . Elevated spontaneous VFs have also been observed In individuats 

with some cancer-prone syndromes . Hemizygous VFs were elevated about 10-fold in 

individuals with ataxia telangiectasia, while approximately 60-fold elevations in both 

hemizygous and homozygous VFs were observed In Individuals with Bloom's syndrome . 

VFs for individuals with xeroderma pigmentosum, In contrast, consistently fell within the 

range of normal individuals . A new cell labeling method has been developed to allow the 

assay to be performed on a single-beam flow cytometer .Work performed under the auspices 

of the U .S . Dept . of Energy by the Lawrence Livermore Nat . Lab. under Contract No. W- 

7405-ENG-48 with support from the U .S. E .P .A. Grant R-811819-02-0 and the U .S . 

N .I .E .H .S . Interaqemcv Agreement NIH-ES•83-14 . 

320 

ONCOCENE CHANCES IN CHEMICALLY-TRANSFORHED RODENT RESPIRATORY EPITHELIAL CELLS . 

J .A . Lasleyl, S .J . Austinl, N .S . Schorschinskyl, D .K . Beeman2, and M .J . Mass2, 

lEnvironmental Health Research and Testing, Inc ., Research Triangle Park, NC 27709 

and 2Carcinogenesis and Metabolism Branch, MD-68, U .S .E .P .A ., Research Triangle 

Park, NC 27711 

Much evidence has accumulated linking alterations in oncogenes and the development 

of cancer . However, most of these studies have utilized tumors, which are 

late stages in the neoplastic process . We utilized the rat tracheal epithelial 

(RTE) cell transformation system to observe the participation of oncogenes in comparatively 

early stages of cell transformation in vitro . Eight cell lines and 

normal cells were studied for alterations in K-ras . H-ras, and c-mvc . These lines 

were transformed in culture with the carcinogens benzo(a)pyrene, 

7,12-dimethylbenz(a)anthracene (DMBA), and/or the tumor promoter 12-0tetradecanoylphorbol-l3-acetate 

. Southern blot hybridizations indicated no 

amplification of H-ras, K-ras, or c-wc in the linas studied . We did not find 

alterations in H-ras codon 61 that have been found in DMBA-induced tumors in vivo . 

H-ras probing of Hpa II digests could distinguish normal from transformed cells . 

One cell line had 2 types of o-wc RFLPs when digested with Bam HI or Hind III, and 

also exhibited methylation changes in CCGG sequences compared with normal cells . 

Northern analysis showed elevated H-ras and c-vic expression in some transformed 

cells . We conclude that transformed RTE cells can demonstrate a number of oncogene 

alterations, however, the relationship of each to the transformed state requires 

further study . This is an abstract of a proposed presentation and does not 

constitute EPA policy . 


A 

r


Notes ENHANCED MORPHOLOGICAL AND MALIGNANT TRANSFORMATION OF SYRIAN HAMSTER EMBRYO CELLS 

CULTURED AT pH 6 .70. R .A . LeBoeuf and G .A . Kerckaert, The Procter & Gamble Company, 

Miami Valley Laboratories, P .O . Box 398707, Cincinnati, OH 45239 . 

Studies conducted in our laboratory have focused on the development of a cell 

transformation model for investigation of mechanisms of carcinogenesis and the assessmnt 

of carcinogenic potential of chemicals . Our initial studies indicated that proliferation 

of SHE cells is optimal at pR 6 .70 compared to pH 7 .35 which has been used 

historically for Syrian hamster embryo (SHE) cell culture . Subsequent inter-laboratory 

studies demonstrated that the frequency of morphological transformation (MT) 

induced by several carcinogens (both DNA reactive and non-DNA reactive) from different 

chemical classes is greater at pH 6 .70 compared to pH 7 .35 . All carcinogens tested 

have induced a statistically significant increase in MT compared to concurrent pH 6 .70 

controls whereas non-carcinogens do not cause a statistically significant increase in 

MT frequency . In order to determine the relevance of the MT phenotype, we have characterized 

the malignant potential of SHE cells derived from colonies of different 

morphological phenotypes transformed at pH 6 .70 . Fifteen to 30% of the MT colonies 

induced by the carcinogens 3-methylcholanthrence and benzo(a)pyrene give rise to 

established cell lines . 85% of these lines progress to the malignant phenotype in an 

apparent step-wise manner . In contrast, no morphologically normal colonies from 

control cultures and less than 3X from carcinogen treated cultures escape senescence 

upon cell isolation . These results will be discussed in terms of the use of early 

passage SHE cells cultured at pH 6 .70 for both carcinogen screening and mechanistic 

studies . 


322 

COMPARATIVE GENOTOXICITY TESTING OF MAINSTREAM WHOLE SMOKE FROM CIGARETTES WHICH 

BURN OR ONLY HEAT TOBACCO . C . K . Lee, D . J . Doolittle, C . T . Burger and A . W . 

Hayes, R . J . Reynolds Tobacco Company, Bowman Gray Technical Center, Winston-Salem, 

NC 27102 

Mainstream whole smoke (MWS) from test cigarettes which heat but do not burn 

tobacco was compared for its genotoxio potential to that from a reference cigarette 

which burns tobacco . MWS was collected from lR4F, a Kentucky reference cigarette, 

and two test cigarettes, one with regular flavor and the other with menthol flavor . 

The cigarettes were smoked on a smoking machine under standard conditions and the 

particulate phase was collected on the Cambridge filter pad and the vapor phase 

passing through the pad was bubbled into a DMSO trap . The filter pad and DMSO in 

the trap was combined and extracted with additional DMSO to obtain MWS . Identical 

concentrations of MWS were tested with all samples to make a direct comparison on 

their genotoxic properties . MWS of 1R4F registered positive results in the Ames 

assay in the presence of metabolic activation but negative results in the absence of 

activation . The frequencies of both sister chromatid exchanges and chromosome 

aberrations were significantly increased in Chinese hamster ovary cells exposed to 

1R4F with and without metabolic activation . The MWS from the test cigarettes, with 

either regular or menthol flavor, did not induce a positive result in any of these 

in vitro assays in the concentration range tested . The MWS from 1R4F was cytotoxic 

at higher concentration range, while MWS from test cigarettes was not . In summary, 

the MWS from test cigarettes was neither genotoxic nor eytotoxic under conditions 

where MWS of 1R4F was genotoxic or cytotoxic in a concentration-dependent manner . 

INITIATION OF DNA SYNTHFSIS WITH ALTERED PHOSPHATIDYL INOSITOL METABOLISM 

Myung Ae Lee, Hyeong Ok Lee and Cheol . Joe 

Department of Biology, Korea Institute of Technology 

Taejon 302-338, Korea 

323 

Intracellular inositol lipid hydrolysis involved in the initiation of DNA synthesis 

was examined using chromatographic analysis . NIH 3T3 cells metabolically labeled with 

3H-inositol were growth-arrested by serum starvation for 14 hours followed by 3mM hydroxyurea 

treatment for 6 hours and were allowed to initaiate DNA synthesis in the normal 

media . The increased breakdown of phosphatidyl inositides into inositol 1,4,5-triphosphate 

and di~cylglycerol was observed with the maximal DNA synthesis rate which was 

measured by the H-thymidine incorporatio~ for 30 minutes during the progression of cell 

cycle into S phase . The incorpJoration of H into phosphatidyl inositol-4-phosphate and 

inositol-1,4-bisphosphate in H-myo-inositol labeled cells was markedly increased with 

the initiation of DNA synthesis . These data suggest that increased phosphatidyl inositol 

turnover is associated with the initiation of DNA synthesis, and this process is in 

close relationship with the phosphorylation and hydrolysis of phosphatidyl inositides . 

50869 3624

324 

DEVELOPMENT OF A PROCEDURE FOR QUANTITATION OF MUTATIONS AT THE NGPRT LOCUS IN 

CHINESE HAMSTER OVARY (CHO) CELLS WITH EXPHESSION AND SELECTION IN SEMISOLID CULTURE 

NEDIUM . P .S . Lee, K .Y . Henry, and C .J . Rudd, SRI International, Menlo Park, CA (USA) . 

We are developing a modified procedure for quantitation of mutations at the HGPRT 

locus to reduce the cost and potential errors associated with the suhculturing of 

cells during the expression period . In established procedures, CH0 cells usually 

require subculturing every 2 to 3 days for 7 days prior to treatment with the selective 

agent, 6-thioguanine (TG) . Colonies of TG-resistant CH0 cells can be selected 

either attached to tissue culture dishes or suspended in semisolid medium . In our 

laboratory, the cloning efficiencies of unselected cells and the numbers of mutant 

colonies were comparable under the two conditions . Using a modified procedure, we 

eliminated the need for subculturing during the expression period by growing lower 

densities of cells in semisolid medium after the induction of mutations . Mutant 

colonies were selected after 4 to 7 days by adding TG (final concentration 30 uM) as 

an overlay with additional medium . The colonies were counted after a total of 14 or 

more days incubation . The recovery of HGPRT- cells in reoonstruetion experiments was 

equivalent in the presence or absence of wild-type (HGPRT+) cells under these conditions 

. Small HGPRT- colonies could be distinguished from non-viable microcolonies of 

HGPRT• cells by staining with Thiozolyl Blue (MTT, 2 .5 mg/dish) . By growing the 

cells in semisolid medium for the duration of the experiment, olonal populations were 

maintained . Therefore, with the modified procedure, the number of TG-reslstant colonies 

should more closely represent the number of cells with a mutation at the HGPRT 

locus at the beginning of the expression period . This inereased accuracy, and the 

savings in labor and materials costs, are clear advantages of the modified protocol . 

325 THE ELECTROPHORETIC SPECIFIC LOCUS TEST IN THE MOUSE AND ANIMAL NOOELS 

OF HUMAN DISEASE 

Susan E . Lewis 

Research Triangle Institute . P . 0 . Box 12194, Research Triangle Park . NC 27709 

The mutagenicity of x-rays and a variety of chemicals have now been studied 

in the electrophoretic specific locus system . Comparisons of mutation frequencies 

in the treated groups in the electrophoretic specifiE locus tests . with those in 

the visible specific locus test . will be made . Electrophoretic screeniny has 

identified a number of electrophoretically detectable mutations, both spontaneous 

and induced by various agents . The underlying molecular basis for, and the physiological 

impact of selected mutations are discussed . Although animals homoZyflous 

for null alleles at many of the loci under study appear to be perfectly 

healthy . certain others are either not viable or impaired . Mouse models of human 

diseases have been recovered in the course of this and other biochemical screening 

programs . These are discussed as to their potential utility in experimental therapeutics 

and the relative susceptibility of the relevant loci to mutagenic change . 

(Supported in part by Contract No . N01-ES-2-5012 . NIEHS .) 

326 

Hypothesis : Modification of Oncogene Expression as a Major 

Mechanism of Action of "Nongenotoxic" Carcinogens 

A . P . Li, Monsanto Company Environmental Health Laboratory, 645 

ewstead, St . Louis, MO 63110 . 

Recent findings in cancer research have consistently associated 

mutations in oncogenes with carcinogenesis, therefore supporting 

the relevance of genetic damage in this process . However, 

findings in genetic toxicology now point out that a large variety 

of carcinogens, especially hepatocarcinogens, do not have 

genotoxicity readily measured using conventional assays (e .g . 

Ames test) . Examination of the biological effects of these 

"nongenotoxic" carcinogens include the following : a . Cell 

proliferation, either via a mitogenic effect (e .g . phenobarbital) 

or cytotoxicity-induced compensatory cell division (e .g . CC14) ; 

b . Enzyme induction, such as P450 (e .g . PCBs) or peroxisome 

induction (e .g . clofibrate) ; c . Alterations in DNA methylation 

(e .g . choline-deficient diet) . I hypothesize that these events 

share one common phenomenon which is key to carcinogenesis : 

Induction of oncogene expression . Cell proliferation is known to 

lead to sequential expression of several oncogenes which are not 



Notes


Notes expzessed in quiescent calls . Enzyme induction may be a 

pleiotropic lnduetion effect on gene expraasions of the inducers . 

oNA methylation has been linked to alterations of gene 

expression . Induction of oncogene expression leads to the 

expression of aberrant oncogene products in "preinitiated" cells 

which are essential for their ultimate development into tumors . 


327 

1+iUTA(iENETIC EFFECT OF 15 C0IPOUNDS IN DROJOPHILA Al1BIIPI+OIDY '1S8TIACt . 

Li Hua1y1, Ca1 0on®min, Huang Jintang and Nan$ Zinmin, Department of 

Tozicoiogy, Second Military Medical Ooilege, 200433 8hanghai, P .R . CHINA 

In order to determine the sensitivity of Drosophila aneuploidy detecting 

syatem, the females (yw/yw) and the malea (yB/Yy) are used in our 

tests . The treatment sex male and/or lemale . The treatment stage lsrva 

and/or adult . The treatment route feeding method . The brood schedule of 

2-3-3 day broods ia carried out in all teats . Tested chemicals inoludes 

(1) Colcemid, (2) Colohioine, (3) Catieine, (4) Aotinomyoin D!( 5)Mett~yl 

methaneaullonate, (6) 5-Flurodeoxyuridine, (7) Proflavine, (8)0y0lophosphamide, 

(9) Mitomyoin-C, (10) Meprobamate (11) 8orbitor, (12)Urethane, 

(13) N-Methyl-N=nitroN-nitroaoguanidine, ~14) Sodium lluoride, (15) 5- 

Bromodeox}ruridine . Normal test described by Margolin et al (1983) are 

used in examine data for statistical aignifioanoe . The compounds which 

aignilicant difference at the 5% level between the treated and oontrol 

lrequencies of chromosome gain in treated d'inolude (1,2,5,7,9) and (3, 

4) in treated Q . The compounds which significant difference at the 5% 

level of chromosome loss in treated d inolude (4,6,7,8,9,11,12,13,14,15) 

and (1) in treated Q . U! 15 compounds tested for non-diajtimotion and 

loas, only six (4,5,8,9 .12,13) have definitive oaroinogeneais olaaaif ication 

(a11 positive) . F ve (4 8,g,12,13) of these were significant di!lere 

oe for loas, three ~4 5 .9) o3 these rrere aignilioant di!lerence !or 

non-disjunction . Senaitivi~y ia 835: and 50>+, respectively. 

328 

MECHANISM OF ARSENITE INHIBITION OF REPAIR OF N-METHYL-N-NITROSOUREA-INDUCED DNA DAMAGE 

J .-H . Li and T.G . Rossman 

Institute of Environmental Medicine 

NYU Medical Center, NY, NY 10016 

Although inorganic arsenic compounds are known human carcinogens, they are not 

mutagens . The lack of arsenic mutagenicity has led us to study its comutagenicity . 

We have found that sodium arsenite at relatively nontoxic concentrations (5 uM for 

24 hours or 10 aM for 3 hours) is comutagenic with N-methyl-N-nitrosourea (MNU) at 

the hprt locus in Chinese hamster V79 cells . A nick translation assay, which measures 

DNA strand breaks in permeabilized cells, was utilized to show that strand breaks 

resulting from MNU or its repair accumulate in the presence of arsenite . MNU-induced 

poly (ADP-ribose) synthesis, measured by the incorporation of [3H]-NAD in the permeabilized 

cells, was also increased by post-treatment of the cells with araenite . This 

supports the hypothesis that arsenite inhibits the completion of DNA repair . The 

accumulated strand breaks in the presence of arsenite are probably not due to direct 

inhibition of DNA polymerase alpha, the presumed repair polymerase of MNU-induced DNA 

lesions, since very high concentrations of arsenite (about 7 .5 mM for 50% inhibition) 

are needed to inhibit this enzyme . We thus suggest that the repair of MNU-induced DNA 

lesions may be inhibited by arsenite by interfering with the ligation step . 

Supported by grant CA29258 from National Cancer Institute . 

GiNOlOIICMY Of ORCANIC gXIRAClS Q f3ggT foILS IN fICBI DIfZRIClS 

IN NgBo NANIN , pNILIppIlt3 

. .!s~ liaaoo . V . 1 . Isotiasds aad N . V . gotuysa , Iwtitute of 

stsy, o sge of Saiewoe, Uaiwrsity of tka pkilippias ., Dilioaa , 

Quesoa City , pkilippiass 

Seventy-five per eeat of air pollutiee !a lMtro Naaila is ooatributed by 

emaissieas LrN sre tha 500,000 motor velioles operstiag daily . A awbsr of 

th.se vehicles are disssel powered vbiek oaa expose tlr urksa populatiea to 

diesel exlrusts wbiek eaatsia polyoyelie aresrtie Wreearkooa which posseas 

kvmaa eareimejaale poteatial . Tbase pelyeyelie aremetie Iqdreesr6elM esa 

aeeu .,late in street oils . orpaie extracts trem sieved soil Nmples trom 

lodustrial, oasrroial sad reaidsatisl areas ia eiakt distriets around Nstro 

Maaila - Nakati, Narikias, pasia, !sa Juaa, IMadaluyoaa, Csiata, Wleaswla 

and parsasque - were studied wlag tlr Rsa assay, bost-msdiated assay aad 

50g69 3626 

329


.feronucl.us tast. Mo direct DNA darsiai capacity aas observed of tAs Organic Notes 

axtracts of strsst soil sa.plss from all eight dist:iets . AowesY . Mutagsoieiq 

aftsr rtatiolic acti .atien and ckro .osesn brsakia= effects wrs exhibited by 

orpnic oztraeta of street soil sasplos fro . industrial and oe.mreial areas 

from four districts . Q"otoxicity wws correlated with ttr prsssncs of 

bsnso(a)pyr .as . 

330 

ASBES'T06 PSSOCIATID III.IS=NP HESOTF>ELICNGi : GCNSISTLNP CFROMOG0t9iL QiF,NM IN 1S2Ctt 

CELL LIAES FROtd SIX PATIENIS . 

Lirviairiaa, K .I ., PalirrShlun3, K . Tanmilehto, L ., 1Mattson, K ., Jantunen, K ., Husgafvel- 

Pursiainen, K . Insti . of Occupational Health and 1 Univ . Hospital, Helsinki, FICIIAND 

Exposure to asbestos fibers is lanwri to be associated with malignant mesotheliema in 

at least 80% of the tumor cases . Karyotypic studies of m3sothelioma tissues have 

revealed a variety of changes involving different duomosares . A consistent kazyotypic 

abnormality oa :mon to niesotheliomas has not been reported previously . We have chracterized 

cytogenetically seven mesothelio:na cell lines and a short-term eulture whic3i have 

been established in our laboratory from ttsnor tissue or pleural fluid sanples fram six 

patients with pleural malignant mesothelioma . All the cases of our study have a heavy 

asbestos exposure history . Complex structural and ntmerical abnannalities were observed 

in all the cultures studied. Excess chromatid material of the short atm of diranosams 5 

was consistently seen in the cells of five patients . In four of the cases, monoea :y of 

c:lrcc,nsane 13 was observed . Additionally, double minute chrarosaees or harogeniously 

staining regions were frequently noted . The observed specific absormalities eottman to 

several patients, e .g . the excess of the p-ana of chrarosans 5 and monosony of chratrosare 

13 suggest the importanee of these karyotypic changes in the development of malignant 

nesotheliana . We have shawm that asbestos and glass fibers are able to induce ctu :anosanal 

damage in human primary mesothelial cell cultures in vitro . The possible association of 

these findings with the kaYyotypic changes in turor cells remains to be solved . 

331 

A STUDY ON THE WORKERS OF COKING PLANT WITH MICRONUCLEUS (MN) FREQUENCY . 

FRAGILE SITES (FRR .) AND SISTER CHROMATID EXCHRNGE (SCE) 

Liu Yongchang . Shanxi Cancer InstitutQ,Taiyuan .Shanxi (PRC) 

In this study, thrQa experiments had been done in coking workers and 

in normal control .ThQy were divided into 5 groups according to different 

testing spots : 1 . Workers of coking workshop ; 2 . Other workars . 3 .Staffs 

who were not in the workshop . 4 . The individuals of the steel institute . 

S .Normal control :ThQ results were as follow : 1 .Ths :data of MN frQquancw 

in sequence were 3 .13. 3 .25, 2 .21 . 2 .10, 0 .44 : 2 . The sequence of 

chromosome aberration (CR) were 4 .13, 5 .05, 1 .50 . 2 .?2 . 0 .20 . 3 . The 

sequence of SCE were 9 .60. . 8 .58 . 8 .88' . 6 .32 . 6 .40 .ThQra were significant 

differences among all these data . The concentration of Bap detected at 

this 5 spots list as logarithmic valua* 2 .38, 1 .78 . 1 .00 . 0 .59 . E .41, 

respectivnlu . Compared these data with MN . CA and SCE by means of 

correlation tast- the results showed that all were positive correlations 

TharQ were 251 fra. at all . 60% in the chromosome group A and 22X in 

group B . Of the 113 identified fra . . 34 .5X were inheritable (3p14) and 

47 .78% were corresponding to the nonrandom carcinoma fra . . 

332 

CHROMATIN ALTERATIONS DURING EXCISION REPAIR 

M. Ljungman and G . Ahnstrbm, Department of Radiobiology, Arrheniua Laboratories for Natural Sdeneea, 

University of Stockholm, S-10961 Stockbolm,Sweden . 

DNA excision repair in mammalian cells scema to be associated with chromatin modification events . Cells 

exposed to ultraviolet light appear to undergo a general type of chromatin modification while methylmethane 

sulphonate treatment induce a more localized modification . It has been our Intention to further investigate the 

mechanismc of these chromatinmodityingeventsinmammalian ceDa .Inapreviousttudy, kinedaofrepairinduced 

DNA strand breaks were followed in Y79 hamster cell cultures treated with methylmethana aulphonate (Id1vIS) . 

The shapes of the DNA strand break curves when adding the drug 3-aminobenramide suggest that poly(ADPriboso)polymerase 

was involved in locally modifjdog the chromatin prior to the rejoining step . Thia modification 

might be required in order for the ligase to gain access to the repair site. To study ehromatin alterations associated 

with nucleotide excision repair in human fibroblasts, we used the DNA-damagiog agents bleomycin, gamma 

radiation and8-methoxypsoralen .Ithasbeenshownthatthese agents react preferentiallywith DNA in open regions 

of the chromatin. Thus, they are suitable for talnng'snapahots' of the chromatin, revealing how open its structure 

is at any particular time . Results obtained on cells undergoing repair after UV-'uradution indicate that the 


115


Notes chromatin ia becoming altered by a time-dependent process. After 10-20 min of poet-UV incubation at 37T~ the 

relative DNA-damaging effects (measured as DNA strand breaks or DNA cross-links) of bleomycin, gamma 

radiation and 8-methoxypsoralen was increased 50% . However, after 60 mm the eHectswere back to control levels . 

These alterations might reflect events preparing the chromatin for the repair of W-induoed DNA damages . 


333 

LACK OP INDUCTION OF SCEs IN HUMAN LYMPHOCYTES EXPOSED TO MONOENERGETIC NEUTRONS 

L .G . Littlsfield, E .L . Proae*I, E .E. Joiner, apd S .P . Colysrt, Oak Ridge Associated 

Universities and Oak Ridge National Laboratoryl, Oak Ridge, TN (USA) 

Low-LET X- or gamaa rays induce few (if any) DNA lesions in boasn lymphocytes that 

are axpressed as SCEs . Recently, however, increased SCEs have bsen observed in 

lymphocytes exposed to 42 MsV d-Ee neutrons pro .pting the suggestion that the REE for 

SCE induction by higb-LET radiation suy be effectively infinite (J . Savage, M . 

Holloway, Sr . J . Radiol . 61 :231, 1988) . We conducted studiss to determine whether 

aonoenergetic neutrons induce DNA lesions that lead to SCE formation, and if so, 

whether such damage is modifiable by radioprotsctive cheaicals that selectively 

scavenge OH radicals . Busan lymphocytes suspended in culture sedium with or without 

1M DMSO were exposed to 5 doses each of 6 .0 and .44 M.V neutrons at RARAP . Chromosome 

aberrations and SCEs were evaluated in 1at or 2nd division staphases from 48br 

cultures . As expected, the two high-LET radiations ware considerably more efficient 

in inducing aberrations than X-rays, with maxisum calculated RBEs of 6 and 16 for 

dieantric induction by the 6 .0 and .44 NeV neutrons . Eased on results of curve 

fitting data, we estimated that OH radical aisdiated lesions in DNA contribute 402 and 

15% . respectively, of the dose dependency for aberrations resulting from exposures to 

the 6 .0 and .44 MsV neutrons . In contrast, SCE frequencies showed no correlation with 

neutron energy, radiation dose, nor presence or absence of radioprotector during 

exposure . We conclude that while high LET radiations are extremely efficient in 

inducing DNA lesions that lead to chroaoso .e aberrations, sonoensrgetic 6 .0 or .44 M .V 

neutrons do not induce DNA lesions that lead to SCEs . Supported by DOE contract 

DOE-AC05-760R00033 . 

334 

MUTAGP.NICITY SLREENING OF WATER EXTRACT8 PROM 102 XIKIIS OF CRUDE DRUGR IK CHIKF.SE RF.DICINC9 

Liu Dexiana , Yin Xuojun 

Laboratory of Medical Gonetics of Kestern Region Hospital,Orweehi,Xinjit-ig , 

- People's Republic of China 

In an a!tenpt to explore the autagonicity of the Chinese Medicinal herbs,102 crudo drx s :.n nu-.ber, 

eeployed comm,~only as traditional Chineso medicines,vore chosen and boiled,and then uaod tlieir obtained 

water extracts as the sample of aasay .Each extract vas assayed by Ames bioaa.ay system with TA 38 and 

TA 100 strains .At aame time,tho intreperitoneal injection in mice vaa aivon with the ditferent doses . 

and obaerved their ehromoaomal aberration (CA) and micronucleir (PC3KF) .Aaes test revealed thut,p 

extracts of the crude drugs (for instsnos,Astr .galus Meaberaneous Ege,,3ophora Japonica L.and xuceh~+la 

Ulnoidea Oliv .) vore poaitivs (3/102 ;2 .97t) .Aatragalua Kenberancous Ege in the presence of E9 mix vith 

strain TA 98 could induce tho number of revertant colonies to increase threo tieos (40mr,/platc) enre 

than spontaneous revertant colonies ;Sopho{~a Japonica L .in the presence or in tha abscnce ol £9 aix vith 

strain TA 98 could induce the number of revertant colonies to increase nine to ton point two tiscc 

(40mg/ylste') more than spontaneous ones (in those samples' probably eontainin~ certain frnaer .lift r.utegena) 

;Eucommia Ulaoidss Oliv .in the presence or in the absence of 59 aix with strain TA 100 could 

induce the number of revertant colonies to increase three to four timee (40ag/plati) morro than sppntaneua 

ones (in these samples' probably containing mutagenie factor indueir,g base chantoa in DNA)a ihe 

increasement of the number of revertant colonies inducing by these 3 hinds of erude drugs van eort'olated 

to incoparation into four dosea 

.Yith CA and PCEKN asaays in mice,the postive results voro riven to 

13 water extracts of the crude drugs (13/102;12 .7¢),suoh as,Astragalus 1(embranaceus BE,Euconnia 

tRaoides Oliv.,Sophora Japonica L.,Datura Metal L .,Artemisia Capillaris Thunb.,Rehmennia Olutinosa 

(Oaertn .)Liboach,Carthsaue Tinctoriu L .,Forsythia Suspensa (Thunb .) Yahl,Paeonia Suffruticosa Andr ., 

Platycodon Orandiflorum (Jacq .)A .D.C .,Cinnanomum Cassia Presl,Kotopterygiua Incisum Ting .and Sophora 

Flavescens Ait .The CA and PCEMN induced by these drugs rere positive correlation to injecting medicines 

into intraperitoneal cavity in nice . 

The results have shown that the Aaes assay and the miee test (CA and PCE?4f) were positive results 

(23%;3/13) .The false negative rate of Ames aasqy was higher than the CA and PCEMK if the aberration of 

sucaryotio cells was taken .as assay eriteria . 

A QUANTITATIVE ASSAY OF DNA POLYMERASE-a IN SITU AT SINGLE CELLS USING FLUORESCENCE 

PSEUDO-COLOR IMAGE AND ACAS 470 . 

P .K . Liu, B . Goudreau, and G .S . Hsu, Case Western Reserve University, Cleveland, OH 

(USA), and Chia-Cheng Chang, Michigan State University, E . Lansing, MI . (USA) 

Aphidicolin is a speoifio inhibitor of DNA polymerase-o and 8 in eukaryotio 

oells . Because of the specificity of this inhibitor, it is potentially a useful 

probe for the detailed studies of the function of these polymerases . A DNA 

polys,erase-o mutant isolated on the basis of resistance to aphidioolin has been 

described (Proo . Natl . Aoad . Soi .,USA 80 :797-801, 1983) . We and others have 

isolated 4 variants which exhibit hypersensitivities to aphidicolin (Aphho) from 

Chinese hamster V79/743X fibroblasts (In : DNA Replication and Mutagenesis, ASM . Pp . 

163-172, 1988) . These variants are designated aphhs-1, aphhs-2, aphhs-3 and aphna- 

4 . We reported here results of studies involving i®unoohemieal characterization . 

The Aphhs phenotype in all mutants was stable for at least 30 days in the absence 

50869 3628 

335


of selection pressure . The dCTP pools in the 743X and Aphhs cell lines were not Notes 

significantly different . The level of total DNA polymerase activity in crude 

extract from aphhs-2 clone was 30% of that observed in the parental clone . We 

developed a method to quantitate DNA polymerase-a antigen at single oells in situ 

using monoclonal antibody SJK 132-20 and fluorescence pseudocolor image . We found 

that the antigen of DNA polymerase-a in aphhs-2 was 40 - 50% of that in the 

parental 743X cells . The underproduction of the antigen of DNA polymerase-o 

provides a basis for the observed Aphhs phenotype . A possible mechanism for the 

underproduction of DNA polymerase-a in aphhs-2 clone is presented . (Supported by 

grants from NSF : DCB 8600659 and from CWRU : RIF-Liu to PKL) . 

336 

4ICR0NU,C(LEI INDUC!? BY FORMALDEHYI~E IN ERYTHROCYTES OF MICE, F . P . Loaroal, G . G . 

Arreola , A . Perez and T . H . Ma , Centro de Eatudios Aoalemicoe sobre Contaminacion 

Anbiental, Universidad Autonoma de Queretaro, QRO Mexico, Institute for Environmental 

Mamagement and Department of Biological Sciences, Western Illinois University, Macomb, 

IL 61455 (USA) 

Subchronic doses of formaldehyde were tested for the clastogenioity using Mouse- 

Peripheral Erythrocyte-Micronucleus bioassay . Young (6 weeks old) female white aioe 

(CD-7) were divided into groups of 5, and administered biweekly with 5 mg/&g, 10 

mg/Kg, and 15 mg/Kg of formaldehyde (diluted with saline solution) through intraperitoneal 

injection for a period of 3 months . Peripheral erythrocytes were collected 

repeatedly from the tail monthly from the beginning of the experiment . Blood smears 

were double stained with hematoxylin and Giemsa for micronuclei in the peripheral 

erythrocytes . Micronuclei frequencies were scored (10,000 cell per slide) from each 

of the treated and control (saline solution) groups . Significantly higher frequencies 

of micronuclei in all the treated groups (around 0 .42) than that in the control 

(around 0 .2%) groups were noted in the first month blood samples . The differences of 

micronuclei frequencies in the blood samples of the second and third months were 

reduced to the insignificant levels (around 0 .1% and 0 .2%) . Whether this was due to 

the aging effect or adaptation to the chronic exposure or sexual specificity requires 

further investigation . Results of similar studies conducted earlier in .male mice 

showed no decline of MCN frequencies at the end of the third month . 

337 

The Role of Carcinogen DNA Adduct Structure in the Induation of Nutations . 

L .L . Loechler,1 M . Benaautti,2 A .K . Basu,2 C .L . Green,2 and J .M . Lssigmann2 ; 

1Boston University, Boston, MA 02215 ; 

2Massachueetts Institute of Technology, Cambridge, MA 02139 . 

Carcinogens induce cancer by reacting with DNA to form DNA adducts, which are 

processed by cells to yield mutations ; particular mutations in proto-oncogenes can 

lead to their activation to oncogenes . One of the key questions in earoinogenesis 

is : what are the mechanism by which carcinogen DNA adducts induce mutations? To 

answer this question, the mutations induced by specific DNA adducts in vivo must be 

known, and rationale for the mutations that each induces must be der vd. Using a 

combination of chemical synthetic and recombinant DNA techniques, individual DNA 

adducts have been built into several vectors in vitro, these vectors placed into 

bacterial cells, and the mutations induced in v vo n progeny vectors by each 

individual adduct determined . Adducts to be considered include 06-Methylguanine 

(o6MeGua), which is produced when carcinogenic methylating agents react with DNA, and 

Thymine Glycol (TG), which is produced both by ionizing radiation and through 

oxidative pathways . O6MeGua induces G to A mutations, and TG induces T to C 

mutations . Proposed mechanisms of mutagenesie for each adduct will be discussed, 

including the use of molecular modeling techniques to interpret the results with TG . 

Preliminary data on the mutations induced by the major adduct formed in DNA from 

activated benro(a)pyrene (i .e ., BP-N2-Gua) will also be discussed . 

338 

COMMON AND UNCOMMON INDOOR SOURCES OF MUTAGENIC AEROSOL PARTICULATE MATTER . 

G6ran Ldfroth and Charlotte Stensman, Nordic School of Public Health, 

S-402 42 Gothenburg (Sweden) 

A number of pyrolysis precesses, which are performed indoors, have been investigated 

with respect to the emission of aerosol particulate matter and its mutagenic activity 

in the Ames Salmonella assay with the plate incorporation and microsuspension 

methods . The emission of the gaseous pollutants carbon monoxide, isoprene and benzene 

was also determined . Processes studied include smoking (sidestream) of tobacco and 

herbal cigarettes, mosquito coil and incense burning and frying of minced, lean pork . 

Expressed in the unit of mg per 9 material, the emission of aerosol particulate matter 


a


Notes was about 0 .06 for frying, 20-30 for smoking and 50-60 for mosquito coil and incense 

burning . The mutagenic activity was highest in the presence of S9 and the response, 

expressed as TA98 revertants per ug particulate matter, was in the microsuspension 

assay 1-2 .5 for mosquito coil and incense smoke and 6-8 for sidestream cigarette smoke 

and frying fumes . With the exception of frying, all the other processes, which burn 

vegetable materials, emitted the gaseous pollutants . Benzene emission was about the 

same, 0 .4-0 .5 mg per g material, for all processes . Isoprene and carbon monoxide 

emission varied depending on process with tobacco cigarette smoking giving the highest 

isoprene emission and incense burning the highest carbon monoxide emission . It is 

apparent that both common activities, as smoking and frying, as well as uncommon 

activities, as incense and mosquito coil burning, can cause indoor air pollution . 


339 

MUTAGENIC EFFECTS OF SODIUM AZIDE ON THREE VARIETIES OF PEPPER (CAPSICUM ANUUM L . 

C .S . Longid, Institute of Biology, University of the Philippines, i iman Phil .) 

The objectives of this study were to determine the mutagenic effects of sodlum azide 

on the M1 generation of three varieties of pepper and to study the frequency of chlorophyll 

muti .tions in the M2 . Saeds of California wonder (CW), Long slim (L8) and Chinese 

(C) were soaked in water for two hours befose treatment in 0 .12, 0 .25, 0 .50 and 

0 .75 mM sodium azide at pH 3 for two hours at 30 C . The criteria used to assess the 

mutaganic effects of azide in the !1~ included germination percentage, seedling and plant 

height, saad set, number of leaves ~ar plant and presence of chimeras . The frequency 

of chlorophyll mutations was determined in the M2 . Sodium asida induced a dose-effact 

relationship in the indices used for mutagenicity except in the number of leaves par 

plant, which did iiot show any relationship between the number of leaves per plant and 

the dosage of the mutagen . The most frequent chlorophyll deficient mutations were the 

viridisr, followed by chlorina, xantha and albina . Very few xantha and albina were 

observed . Sodium azide was observed to be mitagenic in the three varieties of pepper 

studied . 

340 

RESPONSES OF PEPPER VARIETIES AND THEIR FI HYBRIDS TO ETHYL METHANESULFONATE 

C .S . Longid and J .D . Soriano, Institute of Biology, University of the Philippines, 

Diliman (Philippines) 

Seeds of California wonder (CW), Long Slim (LS) and Chinese (C) varieties of pepper 

(~Ca e~icum Anuum L .) were treated with 0 .3% and 0 .6% ethyl methanesulfonate (EMS) at pH 

9 tol at3U°C . The objectives of this study were to determine the effects of EMS on 

three pepper varieties and to study the chemo-sansitivity to EMS of the F crosses of 

chemo-reeistant and the chemo-sensitive varieties . Data obtained in the Mi showed decrease 

in germination percentage, shoot length, plant height, seed set and survival 

percentage . Somatic chimeras were obtained . In the M2 seedlings, chlorophyll-deficiant 

mutants induced in decreasing frequency were viridis, xantha and albina . The shoot 

length response of a species is considered an important measure of its reaction to mutagenic 

treatment, since it is based on the degree of biological damage . Hence, in this 

study, C whose shoot length at 30 days was reduced by 49 .68% is considered chemo-sensitive 

while LS and CW which had a 27 .48% and 29 .20% reduction in shoot length respectively 

are considered chemo-resistant . Crosses were made batween the LS and C varieties . 

Their F1 hybrids were treated with 0 .6% EMS . Same biological effects as in the .Ml were 

obtained in the F2 seedlings . The three varieties of pepper showed varying responsas 

to EMS, with only one variety (C) which was very sensitive to its effects . Their F1 

hybrids were not very sensitive to EMS . (This study is a portion of the senior author's 

dissertation presented for a Ph . D . in Botany, University of the Philippines in Diliman, 

Philippines .) 

341 

•iTUDIES ON TEE EFFECTS OF GAMMA RADIATION ON KALANCHOE PINNATA Ys:hS . 

C .S . Longid, Institute of Biology, University of the Philippines, Diliman,(Phil .) 

Kalanchoe innata Pers ., Kataka-taka (Pil .), life plant (~g .) is a vegetatively 

propagatscf species, which gives rise to plantlets at the leaf wargins . the objectives 

of this study were to determine the effects of gamma radiation on the regeneration o : 

leaves, plant height, general morphology and the frequency of chlorophyll mutation@ ir 

the VM2 generation of Kalanchoe . Leaves from plants propagated from a single plant war : . 

washed, dabbed dry and were subjected to different doses of radiation . One set was 

exposed to 1000r per six minutes and the rest at doses of 2000r to 6000r per hour . 

The biological effects of gamma radiation on this plant were studied using different 

criteria such as percentage of reganuration per leaf, plant height, types and fraquenc7• 

of chlorophyll mutations . Percentage of regeneration was markedly reduced at 2000r . 

Plnntlet growth was also decreased . Reduction increased linearly with increasing radiation 

dose . Chlorophyll mutant plantlets obtained were dark green (viridis),yallow-green 

50869 3630


(chlorina), yellow (xantha), and white (albina) . Changes in number of shoots and ir Notes 

ieaf features such as abnormal serratione and cordate apices were seen . Somatic an, 

Renetic abnormallties were induced by gamma irradiation of Kalanchoe leaves at doser 

of 1000 per six minutes and 2000r to 6000r per hour . The greatest frequency of chlo 

rophyll mutations was recorded at moderate doses of 3000r and 4000r . Kalanchoe is sen 

3ltive to radiation and it is seen in the reduction of regenaration decreased plan' . 

qrowth, presence of morphological abnormalities which all increased linearly with in• 

creasing dose and the presense of chlorophyll mutati .one . 

342 

TERATOTOGENIC AND GENOTOXIC EFFECTS OF ETHYLENE GLYCOL (EG) 

Yin Longzhan, Liu Zheng, Shi Lihua, Bo Kemin . 

Shenyang Res . Institute of Industrial Hygiene and Occupational Disease, 

Shenyang, P .R CHINA 

Ethylene glycol was administered to pregnant Wistar rats during 10 days 

from the 6th to 15th day orally by a stomach tube at dose levels of 253, 

638, 858, 1073, and 1595mg/Kg . The fetuses' mean body weight and crownrump 

length in the 858-1595mg/Kg groups were significantly less than the 

control group (p 0 .01) . 1 .8-43 .6% of fetuses among these groups presented 

gastroschisis, exencephalia, meningoencephalocele, harelip and rib malformation 

. Malformation frequencies (mainly bastroschisis) showed a doseresponse 

relationship . 638mg/Kg was non-teratogenetic . 858mg/Kg was 

threshold teratogenetic and 1073mg/Kg was distinctly teratogenetic . 

We employed subcutaneous implantation of Agar-coated BrdU tablets in ICR/ 

JCL mouse for SCE analysis . The results showed no significant difference 

in bone marrow cells SCE between control and EG groups treated with 

1/50 LD50- 1/5 LD50 of EG (EC LD50m7 .26g/KG . No significant difference 

in chromosome aberation in mouse bone-marrow cells was found between 

control and EG groups, when doses of 253-1595mg/Kg were used . The frequencies 

of micronuclei in "Kun-Ming" mouse palychromatic erythrocytes 

showed no significant difference between control and EG groups, with 

253 - 1595mg/Kg doses . At 5-500 ug/plate doses with salmonella typhirium 

TA98 and TA 100, no significant mutation increase was found . 

343 

EVIDENCE FOR A PCN-P450 ENZYME IN CHICKENS AND COMPARISON OF ITS DEVELOPMENf TO THAT 

OF OTHER PB-INDUCIBLE FORMS . N .A . Lorr, S .E . Bloom, S .S . Park, H .V . Gelboin . H . 

Miller, and F .K . Friedman . Cornell University . Ithaca, NY (USA) and National Cancer 

Institute, Bethesda, MD 20205 (USA) 

The sensitivity of a developing organism to mutagens is dependent in part on the 

state of development of the cytochrome P450 enzyme system . In previous studies, we 

have shown that the chicken embryo is capable of activating a wide spectrum of xenobiotics 

to DNA damaging metabolites . In order to compare the chicken P450 system to 

that of the rat, 4 monoclonal antibodies (MAbs) to purified rat liver P450s, including 

those from 3-methylcholanthrene, phenobarbital (PB), ethanol, and pregnenolone- 

16-a-carbonitrile (PCN) treated rats, were used to lmmunodetect proteins in chicken 

liver microsomes after blotting from SDS-PAGE . Only the MAb against PCN-inducible 

P450 reacted with chicken liver microsomes . The immunodetected protein was most 

predominant at 1 day posthatch (DPH) while such lower levels were observed in the 

embryo and at 36 DPH . PB and dexamethasone were both effective inducers of this 

protein . Chicken liver microsomal erythromycin demethylase, a characteristic activity 

of rat PCN-inducible P450, had a similar developmental profile and inducibility 

to that of the immunodetected protein with a high degree of augmentation at 1 DPH 

compared to that in the embryo end at 36 DPH while aldrin epoxldase, and benzphetamine, 

ethylmorphine, and eminopyrine demethylase were more similar to each other in 

development and induction and were less well correlated with the immunodetected 

protein . This evidence suggests the presence in chicken liver of at least two types 

of P450, one a form related to the PCN-inducible P450 family . This result agrees with 

sequence information suggesting the early evolution of this form . (NIEHS ES03499) 

344 

SYNTHESIS OF [2-14C]3-CHLORO-4-(DICHLOROMETHYL)-5-HYDROXY-2(5H)- 

FURANONE (MX) . 

S . Ltltjtlnen, A . Eakelinen, P. Kauranen, T . Vartiainen , Dept . of 

Chemistry, Univ . of Kuopio, P .O .B . 6, SF-70211 Kuopio, Finland and 

*National Public Health Institute, Dept . of Environ. Hygiene and 

Toxicology, P .O .B . 95, SF-70701 Kuopio, Finland . 

It is well documented that MX is a significant contributor to the 

mutagenity of drinking water . To evaluate the potential health risks 


, 

Ln 

m 

OD 

~ 

10 

W 

41 

W 

Ir 

119


Notes associated with the drinking water consumption, investigations of 

the toxicological properties of MX are necessary . We now report a 

convenient method for the preparation of 14C labelled MX needed in 

those investigations . Bromo[1-14 C] aoetic acid (Ameraham) was first 

converted to its methyl ester by general procedure with diazomethane 

. Reaction of methyl bromoacetate with triphenylphosphine and 

treatment of the adduct with base gave 14C labelled carbomethoxymethylenetriphenylphosFhorane 

. Starting from this compound and 

tetrachloroacetone [2-1 C] MX was then obtained in five steps using 

the method of Padmapriya et al .l . The total yield was about 10 ~ . 

The method is also suitable for the synthesis of (2-13C] MX . 

REFERENCE : 1 . A .A : Padmapriya, G . Just and N .G . Lewis . Can . J . Chem . 

63 (1985) 828 . 


345 

TRADESCANTIA-MICRONUCLEUS TESTS ON CLASTOGENS AND IN SITU MONITORING, Te-Esiu Ma, 

Institute for Environmental Management and Department of Biological Sciences, Western 

Illinois University, Macomb, IL 61455 USA 

Tradesoantia-Mioronucleus (Trad-MCN) bioassay is a highly sensitive test for 

chemical clastogens of gaseous or liquid forms and nuclear radiatlon . Its high 

sensitivity is attributed to the high degree of synchrony and fragility of the meiotio 

chromosomes which are the targets of exposure . Quantitative data of chromosome damage 

in terms of micronuclei frequency can be obtained from the synohronised tetrads (4cell 

stage of the meiotic pollen mother cells) 36-48 hr after the exposure . The 

microslides were prepared using the aoeto-carmine squash method . Since the 

establishment of this bioassay, about 130 chemicals were tested . These include 

carcinogene and mutagens, common beverages, common toxic compounds, non-prescription 

drugs, house-hold chemicals and radioisotopee . Among these agents, 50% of them gave 

positive responses at relatively low dosages (uM - mM) . Trad-MCN test is an unique 

monitoring bioassay which can detect olastogens in the air on the site of pollution 

without using the air condensates, and olaetogens in the polluted water directly 

without using the sediments or concentrates . More than 30 different air pllution 

sites and 20 sites of polluted water sources were ∎onitored in the US, Canada, Mexico 

and People's Republic of China . Both surface water and ground water sources yielded 

seasonal positive responses when tested through the year, Brackish water and diluted 

eeawater samples from the polluted sites were all positive as compared with the 

oontrol group . 

346 

PROFICIENCY OF TRE T~tAD-NCN 1MAGE ANI~SSIS BYST~1 FOR SCZORING MCN FfjEQ>~ENCY AND DATA 

PROCESSING, T . H . Ma , J . Zu , W . Iia , I . Jong , W . Sun and G . Lin-, Institute for 

Environmental Management and Depe~tment of Biological Sciences, Western Illinois 

Oniveraitu, Macomb, IL 61455 USA, 'Department of Computer Science, Fudan University, 

Shanghai, PRC, and 3Institute of Oceanology, Academia Sinioa, Qingdao, PRC . 

Tradeecantia-Micronucleus (Trad-MCN) bioassay is an efficient short term test for 

genotoxioity of pollutants . In order to increase the efficiency and to standardize the 

MCN scoring process, an automated scoring system was developed using the principle of 

image analysis in computer science . This assemblage is called the Tradescantia- 

Micronucleus Image Analysis (Trad-MCNIA) System ." Results scored by Trad-MCNIA system 

was compared with that scored by human observer for its proficiency, i .e . the accuracy 

and speed . A set of low MCN frequency (lese than 10 MCN/100 tetrads) slides prepared 

from EMS treated materials and a set of high MCN frequency (more than 50 MCN/100 

tetrads) slide prepared from X-ray treated materials were used for this study . In low 

frequency slides, The Trad-MCNIA system scored about the same value ae human 

observers . In high frequency slides, MON frequencies scored by the System was lower 

than that scored by human observers . This desorepanoy was corrected by increasing the 

power of the objective of the microscope in the Trad-MCNIA System from 10 X to 20 I . 

The MCN frequencies scored by the System was about 90x of that scored by human 

observer at the present time . The scoring speed of the System was about 4 times ae 

fast as that of the human observer, and the data can be processed and statistically 

analyzed immediately after scores were recorded . Further improvement can be made by 

upgrading the video camera and the computer speed .

347 

CLASTOGENICITY OF HEPTACRLOR ON ERYTHROBLASTS OF THE MOUSE, T. $z l,in, J . J . Temenak, 

$ . C . Oh, E . Zhou and T . D . Chen, Institute for Environmental Management and 

Department of Biological Sciences, Western Illinois IIniveraity, Macomb, IL 61455 

(USA) 

Neptachlor, an agent commonly found in the uncontrolled industrial waste site, was 

tested for its clastogenicity using Mouse Peripheral Erythrooyte-MSoronucleus 

bioassay . The chemical was first dissolve in ethanol and diluted with tapwater to the 

final concentrations of 0 .5 and 5 .0 uM to feed the mice . Fifteen Balb white mioe were 

divided into 3 groups and two groups were fed with the heptachlor solution and one 

group with tapwater as the control . Animals were treated with these chronic low doses 

of heptachlor for 3 weeks, with a weekly change of fresh aolutions . Two mice were 

died during the second week in the 0 .5 uM treated group, and 1 died during the third 

week in the 5 .0 uM treated group . Peripheral blood was collected from their tails 7 

days after the end of treatment . Two blood smear slides were made from each the the 

mice and stained with hematoxylin and Giemea to reveal mioronuolei in both 

polychromatic and normochromatio erythrocytes . Mioronuclei (MCN) frequencies were 

scored from 10,000 cells in each elide . Preliminary results show that the means and 

the standard errors of the control group , 0 .5 aM and 5 .0 uld treated groups were around 

0 .7 (+0 .3) ; 1 .7 (+0 .4) ; and 1 .4 (+0 .1) MCM/1000 cells respectively. This indicates 

that heptachlor is toxic and also exhibits clastogenicity to the ohromoeomes of 

erythroblasts of the mouse . 

348 

MICRONUCLEATED ERYTHROCYTES AS A MARKER OF CYTOGENETIC DAMAGE IN MAN . J .T . MacGreaor, 

Department of Biochemistry, University of California, Berkeley, CA (USA) 

Splenectomized individuals constitute a unique population for studies of chromosomal 

damage in man . Because micronucleated erythrocytes derived from damaged erythroblasts 

remain in peripheral blood with a lifespan approximately equal to that of normal 

erythrocytes, the incidence of micronucleated cells in peripheral blood can be used as an 

index of chromosomal damage in nucleated precursor cells . The target cell population is 

rapidly dividing in vivo, and therefore is sensitive to agents and conditiohs which act 

during cell division . This is a significant advantage for studies of conditions such as 

transient nutritional imbalances, to which nondividing cells (e .g .,lymphocytes) are 

relatively insensitive . Two distinct age populations of erythrocytes can be monitored : 1) 

newly-formed erythrocytes, identified by their RNA-positive staining characteristic, which 

reflect damage occurring 4-6 days prior to sampling, and 2) RNA-negative erythrocytes, 

which reflect the average damage over the 4-month period prior to sampling (corresponding 

to the turnover time of the mature erythrocyte pool) . Studies to date have established that 

micronucleated cell frequencies increase dramatically following treatment with clastogenic 

drugs, and that the kinetics of the observed increases in erythrocyte subclasses parallel 

the kinetics of erythrocyte formation and turnover . Studies of environmental factors 

associated with modification of the spontaneous frequency of micronucleated cells have 

established significant associations between observed frequencies and caffeinated beverage 

consumption, folate status, calcium supplement consumption in women, and consumption of 

antioxidant vitamin supplements . Age was strongly associated with the micronucleated cell 

frequency when other factors were not considered, but not when adjusted for the above 

factors . 

349 

AMPLIFICATION OF mRNA OF THE HPRT GENE FROM LYSATES OF MUTANT HUMAN CELLS AND DIRECT 

DNA SEQUENCING TO DETERMINE THE SPECTRA OF MUTATIONS INDUCED BY CARCINOGENS . V .M. 

Maher, J .-L . Yang, and J .J . McCormick, Carcinogenesis Laboratory, Michigan State 

University, East Lansing, MI 48824 (USA) 

Strong evidence points to mutation induction as one mechanism by which changes 

are introduced into normal cells to convert them into cancer cells . To understand 

the mechanisms by which mutations are induced in human cells by carcinogens, we 

are determining the kinds and spectra of mutations induced in the coding region 

of the hypoxanthine(guanine)phosphoribosyltransferase (HPRT) gene . This region, 

composed of 654 bp, represents nine exons from a 44 kbp gene . To be able to analyze 

a large number of independent mutants rapidly and economically, we have optimized 

the conditions for copying mRNA directly from lysates of a small number of cells 

(e .g ., 200) from a thioguanine resistant clone using reverse transcriptase and 

oligo(dT)1P_18 primers . Then two 20-mer primers, specific for the cDNA of the 



Notes


Notes HPRT gene, are used to amplify the first and second strand cDNA 5 x 107 fold during 

30 cycles of polymerase chain reaction (PCR) . The product (2 to 10 ng) is then 

purified by ultrafiltration, diluted 1 :1000, and subjected to an additional 30 

cycles of PCR, using two 20-mer primers located just interior to the first set . 

The product (-10 ug) is then sequenced directly using three end-labeled sequencing 

primers and Sequenase . With this system, we have sequenced 26 BPDE-induced mutants 

and found that 24/26 involved base substitutions . 97% of these involved G•C, 

predominantly G-C--*T•A, distributed throughout the 9 exons but with many located 

in exon 3 . (Supported by NCI Grant CA21253 .) 


I 

350 

ANEUPLOIDY FREQUENCIES IN MOUSE METAPHASE II OOCYTES AND FIRST-CLEAVAGE ZYGOTES 

FOLLOWING ORAL DOSAGES OF COLCHICINE . John B . Mailheal, Zhi Ping Yuanl, and M .J . 

Aardema2 . lDepartment of Obstetrics and Gynecology, L .S .U . Medical Center, 

Shreveport, LA 71130 ; 2The Proctor and Gamble Company, Cincinnati, OH 45247 . 

Certain chemicals can interact with cellular organelles responsible for orderly 

chromosome segregation and enhance the frequency of aneuploidy . Our objective was to 

determine the proportion of colchicine-induced aneuploid metaphase II (MII) oocytes 

that were transmitted to first-cleavage (lCl) zygotes . Female, CD-1 mice received 7 .5 

I .U . PMS and 5 .0 I .U . HCG was given 48 h later . Colchicine (2, 3, or 4 mg/kg) or 

sterile distilled water (solvent) was given by oral intubation iamnediately following 

HCG . MII oocytes were collected 16 h post HCG, whereas 1C1 zygotes were collected 16 

h post mating . The proportions (and percentages) of hyperploid MII oocytes were 3/216 

(1 .4), 81/512 (15 .8), 71/411 (17 .3), and 98/413 (23 .7) for control, 2 .0 mg/kg, 3 .0 

mg/kg, and 4 .0 mg/kg, respectively . Whereas, the proportions of hyperploid 1C1 

zygotes were 2/320 (0 .6), 23/364 (6 .3), 68/372 (18 .3), and 98/337 (29 .1) for control, 

2 .0 mg/kg, 3 .0 mg/kg, and 4 .0 mg/kg, respectively . The proportions of hyperploid 

cells differed (P0 .05) between MII oocytes and lCl zygotes in controls, and the 3 .0 mg/kg and 4 .0 

mg/kg groups . •the level of hyperploidy was greater (P


nucleation, micronucleation, chromosome structure or number . To further define the Notes 

action of chrysotile asbestos upon human cells, asbestos effects were examined in 

cultures of the human lung tumor cell lines A549, Calu-1, KNS62, and A1188 . Asbesto3 

induced 50% and 100% cytostasis of HBE cells at treatment concentrations of til ug/cm 

and 4 yg/cm , respectively . In contrast, three to thirty-fold higher concentrations 

of asbestos were required to inhibit tumor cell growth . Maximal growth inhibition was 

cell line dependent and ranged from only 20% to 50% . Asbestos produced only two-fold 

elevations of binucleation (spontaneous binucleation rate '% .2X) in HBE, KNS62, and 

A1188 cells . In contrast, dose-dependent elevations of binucleation (up to 15-fold) 

were observed in cultures of A549 and Calu-1 . These results are consistent with observations 

that asbestos amplifies the effects of human lung carcinogens such as 

cigarette smoke and suggest that this synergy may in part be mediated by 1) asbestosinduced 

inhibition of HBE cell growth that permits clonal expansion of pre-existing 

carcinogen-induced lesions and/or 2) enhanced susceptibility of some of these lesions 

to asbestos-induced cellular events that facilitate neoplastic progression . 

353 

ISSUES IN THE EVALUATION OF SHORT-TERM TEST PERFORMANCE AND TESTING STRATEGIES . 

Barry H . Margolin, University of North Carolina at Chapel Hill, Chapel Hill, NC . 

The formal definitions of statistical independence and dependence are reviewed 

within the context of construction of batteries of genetic toxicity assays for 

prediction of carcinogenicity . The existing empirical evidence in support of independence 

or dependence among assays is examined and the impact of statistical dependence 

on carcinogenicity prediction systems is explored in depth . Finally, carcinogenicity 

screening policies based upon short-term tests are reviewed and studied for their 

sensitivity to assumptions or inferences regarding statistical independence or dependence 

among tests . 

354 

HUMAN SPERM CHROMOSOME COMPLEMENTS, EFFECTS OF DONOR AGE, FREEZING AND SEGREGATION 

IN TRANSLOCATION AND INVERSION CARRIERS . Renee H . Martin, Division of Medical 

Genetics, Department of Pediatrics, University of Calgary and Medical Genetics 

Clinic, Alberta Children's Hospital, Calgary, Alberta, Cpnada T2T 5C7 

We have studied the effect of age on the frequency of sperm chromosomal 

abnormalities in 30 normal donors, stratified by age into 6 age groups (20-24, 

25-29, 30-34, 35-39, 40-44, 45+) . Data from newborns had suggested a possible 

increased risk of Down syndrome with paternal age but we found no inereased risk 

of disomic sperm with advanced patornal age . In contrast, the frequency of 

hyperhaploid (n+l) sperm decreased while the frequency of sperm with structural 

chromosomal abnormalities increased with age . To assess the effects of sperm 

cryopreservation, ejaculates from 10 normal men were split and studied pre- and 

post-freezing . A minimum of 100 sperm karyotypes were studied for each donor . 

There was no significant difference in the frequency of numerical chromosomal 

abnormalities (using a conservative estimate of aneuploidy, 2 x hyperhaploid sperm) 

or structural chromosomal abnormalities before and after freezing . There was no 

evidence for donor heterogeneity . The sex ratios were also not, affected by 

cryopreservation and did not differ significantly from 50% . Studies on 23 men 

with constitutional chromosomal abnormalities have demonstrated dramatic variations 

in the frequency of chromosomally unbalanced sperm from 0% to 77% . This information 

is useful in elucidating basic principles of how rearranged chromosomes segregate 

during meiosis and also in providing more accurate genetic counselling for these 

men . 

355 

MUTAGENICITY OF NITROARENES BY NEW TESTER STRAINS, TA100/PY0216, TA100/PY0219, TA98/PYO 

216 AND TA98/PYG219 . H . Matsushita, 0 . Endo, H . Katsushita,Jr ., M . Kochizuki, M . 

vatanabe, and M . Ishidate, Jr ., National Institute of Public Health, Kinato-ku, Tokyo, 

(Japan), Eyoriteu College of Pharmacy, Minato-ku, Tokyo (Japan), and National Institute 

of Hygienic Sciences, Setagaya-ku, Tokyo (Japan) . Mutagenicity of nitroarenes, potent 

environmental mutagene, was tested by new tester strains, Salmonella typhimurium TA 

100/PYG216, TA100/PYG219, TA98/PYG216 and TA98/PY0219 as well as the parent strains, 

where TA100/PYG216 and TA98/PYG216 have nitroreductase activity and TA100/PY0219 and TA 

98/PYG219 have acetyltraneferase activity . Mutagenicity test was carried out by the 

pre-incubation method in the presence and absence of S9 mix . Nitroarenes tested were 

21 nitro-derivatives of benzene, biphenyl, naphthalene, anthracene, fluorene and pyrene, 

in which 8 dinitropyrenes were included . The mutagenic activity was generally higher 

in the absence of S9 mix than in the presence of S9 mix for all ∎trains tested . Mutagenic 

activity of each nitroarene was generally in the order of TA100/PYG219, TA100/PYG 



Notes 216 and TA100 irrespective of presence and absence of 89 ∎ix . The activity for TA300/ 

PY0219 was about 10 to 600 times higher than TA100 in the abeence of S9 mix . In the 

case of TA98 strains, the order of mutagenic activity was complicated, but TA98/PYG216 

or 219 gave generally higher mutagenic activity than TA98 . For example, ratio of mutagenic 

activity for TA98/PYG219 to that for TA98 ranged from 245 to 630 (mean :430) for 

3 kinds of mono-nitropyrenes, from 6 to 370 (mean : 78) for 8 dinitropyrenes in the 

absence of S9 mix . The ratio between TA98/PY0216 and TA98 ranged from 130 to 590 (mean : 

350) for 3 nitropyrenes and from 0 .1 to 89 (mean : 18) for 8 dinitropyrenes in the absence 

of S9 mix . These results demonstrate clearly the usefulness of theme strains for 

the detection of nitroarenes in the environment . 


MODIFICATION OP SALMONELLA MUTATION TEST AND ITS APPLICATION TO ALKYL HYDRAZINES 

H . Natsushita,Jr ., K . Yamamoto, M . Mochizuki, O .Endo, and H . Matsushita, IKyoritsu 

College of Pharmacy, Minato-ku, Tokyo (Japan), and National Institute of Public 

Health, Minato-ku, Tokyo (Japan) . 

356 

Many environmental hydrazines are carcinogenic . However, information on their mutagenicity 

is few and the mutagenic activity reported 1s generally very low in contrast 

of their carcinogenicity . We modified the mutagenicity test procedure mainly on the 

pre-culture conditions using Salmonella typhimurium strains TA100 and TA102, and 

applied the modified method to the survey of mutagenicity of 12 alkylhydrazines : 

four 1,1-dialkylhydrazinea, four 1,2-dlalkylhydrazines and four monoalkylhydrazines 

with alkyl group from methyl to butyl . In this method, 10 out of 12 alkylhydrazines 

were detected as positive in the strain TA100, and all the 12 hydrazines were positive 

in the strain TA102 . Mutagenic activity obtained here were generally stronger 

than those reported hitherto . The mutagenic activity was stronger in the absence of 

metabolic activation, and the presence of 59 mix reduced remarkably the mutagenic 

activity . The inhibition by S9 mix was proved to be due to the capturing of mutagens 

by protein of S9, since bovine serum albumin also inhibited the mutagenicity of alkylhydrazines 

tested . The procedure developed here is useful in detection of many kinds 

of environmental mutagens, and also in elucidating mechamisms of mutagenesis and 

carcinogenesis of alkylhydrazines . 

COMPARISON OF TWO DIFFERENT PROTOCOLS FOR DETECTION OF CHEMICAL-INDUCED 

TRANSFORMATION OF BALB/c-3T3 CELLS . E .J . Matthews, Hazleton Laboratories America, 

Inc ., Kensington, Maryland . 

In 1983, the NTP furnished this laboratory 55 coded chemicals for testing in a standard 

transformation protocol using the A-31, 1-13 BALB/c-3T3 cells . This investiyation 

revealed that the standard assay was insensitive : 4 chemicals were active, 6 had 

limited evidence of activity, and 45 were inactive . Recently 61 of these chemicals 

were retested in a modified protocol : 26 chemicals were active, 6 had limited evidence 

of activity, and 20 were inactive . Neither the standard nor the modified protocol 

used an exogenous activation system . The enhanced sensitivity of the second 

protocol was attributed to several procedural changes . First, the initiat seeding 

density was increased from j to 3 .2 x 104 celts/60 mm dish . Second, the treatment 

time was reduced from U to 48-hours and treatments were begun dav-2 versus O,av-1 

after seeding . Third, the method of measuring chemical cytotoxicity changed from a 

standard clonal survival assay employing M wild type (wt) cells to a co-culture 

assay using 3 .2 x 104 wt and 100 ouabain-resistant ce11s . Additional assay improvements 

will be discussed, including : changing the positive control [3-methylcholanthrene 

to benzo(a)pyrene], and an alteration of the method of dosing . Finally, many 

NTP chemicals had limited solubility in water . This problem was diminished for many 

chemicals by dissolving them in an organic solvent at high concentrations and 

diluting them 100-fold into medium supplemented with a SX concentration of Pluronic 

F68 (1 .25k w/v) . Investigations were supported by NIEHS Contract N01-ES-65150 . 

TRANSFORMATION WITH BALB/c-3T3 CELLS . fv J . Matthews, Hazleton Laboratories America, 

Inc ., 5516 Nicholson Lane, Kensington, Maryland . 

Chemical-induced transformation of A-31,I-13 BALB/c-3T3 cells was investipated using 

a modified procedure . Chemical-induced cytotoxicity was measured using a clonal survival 

assay employing co-cultures of 200 ouabain-resistant and 3 .2 x 104 wild type 

(wt) cells . The transformation assay used vessels seeded with 3 .2 x 104 wt eells 

(DAY 0) and 48 hour chemical treatments were started on Dav-2 . Chemicals with solubility 

problems in water were dissolved at high concentrations in an organic solvent 

and diluted 100-fold into culture medium supplemented with Pluronic F68 (1 .25k w/v) 

50869 3636 

357 

358


and then 5-fold into culture dishes . Cultures were incubated 28-days and evaluated Notes 

for the presence of Types I-III foci . All experiments were conducted in the sene 

of any exogenous activation system . Seventy carcinogens and 58 noncareinoaens were 

tested in two or more transformation experiments . Carcinogens included 43 chemicals 

that were relatively cytotoxic to the BALB/c-3T3 cells (LD < 2 .0mM), and 27 noncvtotoxic 

chemicals (LOse > 2 .0mM) . Similarly, noncarcinogens included 27 cytotoxic 

chemicals and 31 noncytotoxic chemicals . All chemicals were evaluated at cytotoxic 

treatment doses . Transforming activities of the 128 chemicals will be compared to 

their reported structural alerts and flenotoxic activities in four in vi r assays : 

including Salmonella, mouse lymphoma (TK+/-), and Chinese hamster ovary sister chromatid 

exchanges and chromosomal aberrations . Chemieal-induced activities detected 

in the presence and absence of an S9 activation system will be discussed separately . 

Experimental investigations were supported by NIEHS Contract N01-ES-65150 . 

359 

TISSUE SPECIFICITY OF THE MUTAGENICITY OF 1,8-DINITROPYRENE IN A MOUSE-MEDIATED 

ASSAY . M .A . McCartney, S . Knasmfiller, E .C . McCoy and H .S . Rosenkranz, Department of 

Environmental Health Sciences, School of Medicine, Case Western Reserve University, 

Cleveland, Ohio 44106 . 

1,8-Dinitropyrene (1,8-DNP) is a highly mutagenio environmental contaminant which 

is also carcinogenic to rodents . DNA adduct formation, mutagenioity and presumably 

carcinogenicity are dependent upon nitroreduction to the corresponding 

arylhydroxylamine followed by 0-esterification by a transacetylase . Bacteria 

(Salmonella typhimuriurn TA98/1,8-DNPs) deficient in this transacetylase do not 

exhibit mutagenicity when exposed to 1,8-DNP . In a mouse-mediated assay (MMA) 1,8-DNP 

induced mutations in S . typhimurium TA98 recovered from liver and spleen of treated 

animals . However, when the MMA was performed using S . typhimuriurn TA98/1,8-DNPa 1,8- 

DNP-induced mutants could be recovered only from the spleen but not from the liver . 

The relevance of these findings to the tissue-specificity of 1,8-DNP will be 

addressed . 

360 

MALIGNANT TRANSFORMATION OF HUMAN FIBROBLASTS BY• ONCOGENE TRANSFECTION OR 

CARCINOGENS. J .J . McCormick and V .M . Maher, Carcinogenesis Laboratory, Michigan 

State University, East Lansing, MI 48824 (USA) 

Data indicate that carcinogen exposure is the cause of most human tumors, but 

human cells in culture have not been successfully transformed to malignancy by 

exposure to chemical carcinogens or radiation . One possible explanation for this 

failure is inability to recognize the phenotypes of carcinogen-treated cells that 

have undergone intermediate changes, so they can be expanded and exposed a second 

time to cause further changes . To Identify possible intermediates, we transfected 

diploid human fibroblasts with oncogenes known to be active in cells derived from 

fibrosarcomas and determined the phenotypes they produced . H- or N-ras oncogenes 

flanked by suitable enhancer and promoter sequences caused the celfs to acquire 

many characteristics of malignant cells, but not to acquire an infinite life span 

or form tumors . When we transfected these ras oncogenes in the same constructions, 

or a viral K-ras oncogene, into an infinite -Me span, near-diploid, non-tumori genic 

cell strain developed in this laboratory (MSU-l .l cells), distinct foci of 

morphologically-altered, anchorage independent, and growth factor independent cells 

were found which formed progressively-growing, invasive malignant sarcomas in athymic 

mice . These cells expressed the p2ls of the transfected ras genes . Transfection 

of two other infinite life span human cell lines with the~T-ras oncogene in the 

same construction also yielded malignant cells . We are currenETy using carcinogen 

treatment to activate cellular proto-oncogenes of the MSU-1 .1 cells and have 

succeeded in malignantly transforming cells . (Supported by DOE Grant 60524, NCI 

Grant CA21289 and NIEHS Contract ES65152 .) 

361 

HEPATIC AND LUNG MICROSOMAL METABOLISM OF ENVIRONMENTAL POLLUTANTS : EFFECTS OF !!1 

INDUCER PRETREATMENT ON THE METABOLISM OF 1-NITROPYRENE, 3-NITROFLUORANTENE AND m 

NICOTINE . 00 

G .D . McCoy , D . R . Koop and P . C . Howard, Case Western Reserve University, School of 

Medicine, Cleveland, OH 44106 tp 

The ability of microsomes isolated from adult male and female New Zealand 

rabbits to metabolize 1-nitropyrene (i-NP) , 3-nitrofluranthene (3-NF) and nicotine W 

(N) has been studied . Hepatic and lung microsomes were isolated from animals 01 

pretreated with either 1-nitropyrene, P-napthoflavone (J)-nf) or phenobarbital (Pb). W 

J 

The C-oxidation of 1-NP, 3-NF and N were significantly increased only in mlcrosomes 

from phenobarbital pretreated animals . In contrast, both Pb and 0-nf pretreatment 

significantly decreased the rates of nicotine N'-oxidation . Our previous studies 


125



Notes have shown that reconstitution experiments utilizing purified rabbit cytochrome P- 

450 isozymea that all three xenobiotics are preferentially metabolized by isozyme 

3b (IIC3) a constitutive form thus far found only in rabbits . The ability of Pb to 

induce the metabolism of 1-NP, 3-NF and N was also anticipated since Ssozyme 2(II 

81) one of the major forms induoed by phenobarbital also exhibited significant 

activity with these compounds in reconstitution studies . Signifioant metabolism of 

all three xenobiotios occurred in pulmonary miorosomes . Since environmental 

exposure to these three xenoblotlos will most likely be via inhalatlon and that 

common pathways of C-oxidation are involved in their metabolism, significant 

interactions between the three can be expected when present concurrently in complex 

mixtures . Supported in part by grants ES 03648 and AA 07219 . 

362 

COMPARISON OF-MUTAGENICITY OF TEN CHEMICAL MUTAGENS WITH THE SALMONELLA 

(AMES) ASSAY, UMU TEST, AND SOS CHROMOTEST . Audrey E . McDaniels, 

Antolin L . Reyes, and Larry J . Wymer, U .S . Environmental Protection 

Agency, Environmental Monitoring Systems Laboratory, Microbiology 

Research Division, Cincinnati, Ohio 45268 . 

With the greater number of environmental samples examined each year 

for mutagens, there is an increased need for methods that are less time 

consuming yet do not sacrifice sensitivity or reproducibility . Two alternative 

methods to the well established Ames test are the Umu test and 

SOS chromotest . These two colorimetric assays are based on production 

of B-galactosidase in response to DNA damage . The three methods were compared 

in 5 independent experiments . Dose response curves were obtained 

using 10 chemical mutagens representing 6 different classes of mutagens . 

Sensitivities, expressed as minimum significant doses (MSD), and precisions 

were also measured . Ames test MSDa ranged from 0 .002 pg to 2 .65 pg 

per plate for Salmonella ~ty himuri~um strains TA100 and TA98 . SOS chromotest 

MSDs rangedom ~0 2 p~ g to 6 . .9 pg per assay, and Umu test MSDa 

from 0 .005 pg to 0 .39 pg per assay . The range of precisions among the 

assays for all mutagens extended from 0 .07 to 0 .19 . The results of this 

study indicated the Umu test was either equivalent to or significantly 

better than the Ames test in sensitivity and precision for all of the 

chemicals examined . The SOS chromotest presented toxicity and solubility 

problems not observed with the Ames and Umu tests . 

363 

CYTOGENETIC EVALUATION OF THE IN VIVO GENOTOEICITY OF THREE QUINOLINE COMPOUNDS 

A .F . McFea and K .W . Lowe, Oak Ridge Associated Universities, Oak Ridge, TN (USA) 

Quinoline-derived compounds are widely used in medicine and industry as antiseptics, 

solvents, dyes, and components of various chemical processes . In vitro assays of their 

genotoxicity have yielded varying indications of potency, and few studies of their in 

vivo activity have been reported . Ye implanted male 86C3F1 aice with BrdU tablets and 

1 hr later gave injections of quinoline or 8-bydroxyquinoline in corn oil solution, or 

4-nitroquinoline-l-oxide (4NQ0)dissolved in DMSO . Each was tested at its maximum 

tolerated dose (MTD) and at 0 .5 and 0 .25 MTD . Chromosome aberrations were scored in 50 

first-division metaphases from each of 8 mice per dose level at 17 hr post-treatment, 

and SCEs in 25 second-division metaphases from 4 mice per level at 23 br . Compounds 

showing no effect on an endpoint were further tested for aberrations at 36 hr, and for 

SCEs at 42 hr . Unusually low control values resulted in a significant p-value for 

aberration induction by quinoline at 17 hr ; however, a repeat study showed no effect . 

Quinoline also showed no effect on aberration levels at 36 hr, no elevation of SCEs at 

either 23 or 42 br, and no indication of an effect on the rate of csll proliferation . 

8-bydroxyquinoline was also without effect on either aberrations or SCEs at any 

post-treatment time, although a modest depression of call proliferation rates occurred 

at the higher dose levels . 4NQO was a very potent inducer of both aberrations and 

SCEs, causing a significant increase in aberration rate at 30mg/kg, and in SCE at 

15mg/kg ; a depression of cell proliferation was also evident . Both quinoline and 

8-hydroxyquinoline seea to be without in vivo genotoxic activity, whereas 4NQO retains 

a significant potency under in vivo conditions . Supported by NIEHS Interagency 

Agreement Y01-ES-20100 and DOE/ORAU Contract DE-ACG5-760R00033 . 

IN DITRO MAMMALIAN CELL CENOTOXICITY ASSAYS : THEIR USE AND INTERPRETATION . 

Douglas McGregor, Dept . of Toxicology and Safety Assessment, Soehringer 

Ingelheim Pharmaceuticals, Inc ., Ridgefield, CT 06877, USA . 

To address the interest in identifying all aspects of genetic toxicity and its 

relevance to Man, more than 100 different test methods have been developed . 

50869 3638 

364

Few are commonly used . Mammalian cell assays hold a special position in this 

Irategy because of the hroad spectrum of damage that is theoretically 

deteccable with them, but which is not acceasable to prokaryotic assays . 

Reasons for this privilege include the higher order structure of mammalian 

chromosomes, the different repair mechanisms and the different chemical and 

metabolic reactions possible in mammalian as compared with prokaryotic cells . 

Fundamental to the acceptability or otherwise of mammalian cell assays is 

their success in rodent carcinogen prediction, although they also have value 

in biochemistry . Their primary function will be discussed and it will be 

concluded that certain assays have reasonable sensitivity (eg ., mouse lymphoma 

cell tk locus and sister-chromatid exchange assays), while others have higher 

specificity (eg ., hprt locus and primary hepatocyte unscheduled DNA synthesis 

assays) . Chromosomal abberition assays show moderate sensitivity and 

specificity . These properties of the assays can lead to proposals that some 

of them should be discontinued or that, if retained, their use shoul~ be 

-d bv scientific and ethical objectives . 

365 

UV-INDUCED CYTOGENETIC DAMAGE IN WILD-TYPE AND THYMIDINE KINASE DEFICIENT FRIEND MOUSE 

ERYTHROLEUKAEMIA CELLS . V .J .McKelvey and P .G .McKenna, Biomedical Sciencee Research 

Centre, University of Ulater, Coleraine BT52 1SA, N .Ireland . 

Deficiency of the salvage pathway enzyme thymidine kinase (TK) in Friend mouse 

erythroleukaemia cells results in increased sensitivity to cell killing and mutagenesis 

following UV-irradiation (McKenna,P .G . and Hickey .I . (1981) Cell Biol .Int .Reps . 5 :555) . 

Work with other malignant cell lines indicates that TK deficiency only confers eZevated 

sensitivity in those cell lines which are normally proficient in DNA excision repair as 

evidenced by their ability to undergo unscheduled DNA synthesis (UDS) (McKenna,P .G ., 

Yasseen,A,A . and McKelvey,V .J . (1985) Somat .Cell Mo1 .Genet . 11 :239) . Further work has 

shown that TK deficiency in Friend cells does not inhibit UDS dccurring (McKenna,P .G . 

and McKelvey,V .J . (1986) Somat .Cell Mol .Genet . 12 :325) . In this study vild-type clone 

707 Friend cells and two TK deficient eubclonea 707BUE and 707BUF, having TK activities 

of 1 .4% and 0 .7% that of wild-type cells respectively, were examined for cytogenetic 

aberrations following UV-irradiation . Three doses of UY light were used, namely 2 .4, 

4 .8 and 7 .2 J/m and UV-irradiated cultures were harvested for chromosome spreads at 15 

hours following treatment . Fifty randomly selected chromosome spreads per treated and 

control culture were scored for thirteen types of cytogenetic aberrations . The 

frequency of very severely damaged (pulverised) cells was observed to be considerably 

greater in each of the two TK deficient aubclones,707BUE and 7078UF, relative to wildtype 

clone 707 cells, for each UV treatment examined . Increased UV-aensitivity in the 

TK deficient subclones was also reflected in the total aberration frequencies exhibited 

by the 3 cell types . The imp rtance of thymidine kinase for accurate DNA repair of UVinduced 

damage ia indicated .(The support of the Ulster Cancer Foundation is acknowledge40 

366 

METABOLISM OF FOOD MUTAGENS WITH PURIFIED AND cDNAl EXPRESSED CSCfOCMROMES P-450 . M+ 

McManus, W .M . Byrgess, M .E . Ver3nese, J .S . Felton , M .G . Rnize , E .G . Snyderwine , 

L .C . Quattrochi and R .Y . Tukey . Department of Clinical Pharmacology, 2Flinders 

University, Australia, Lawre3ce Livermore National Laboratory U .S .A ., National 

Institute of Health, U .S .A ., & University of California, San Diego, U .S .A . 

We have investigated the specificity of six purified forms of rabbit liver cytochrome 

P-450 to activate the food derived haterocyclic amines . IQ and PhIP, to mutagens 

in the Ames test . The polcyclic hydrocarbon inducible isozymes Forms 4 and 6 

were efficient activators of both these compounds whereas, Forms 2,3b,3c and 5 were 

inactive in metabolizing IQ and PhIP to mutagens . The number of revertants produced 

in the Ames test per 10 pmol of Form 4 with IQ and PhIP as substrates were 136, 160 

and 1,521, respectively . In the presence of 10 pmol of Form 6 and IQ as substrate 

16,000 revertants were obtained, whereas PhIP gave 4,577 revertants . When MeIQ, MeIQx 

and DiMeIQx were used as substrates Form 4 was at least 3-fold more efficient than 

Form 6 in activating these compounds . The gene of the human equivalent of the rabbit 

cytochrome P-450 Form 6(P450IA1) and a human cDNA equivalent to Form 4(P450IA2) have 

been expressed in Coa-1 cells and their capacity to activate the heterocyclic amines 

IQ, PhIP,MeIQ,MeIQx and DiMeIQx was determined . Both the human P4SOIA1 and P450IA2 

were capable of activating these amines to mutagens . The order of mutagenicity using 

either P450IA1 or P450IA2 expressed cell lysates as the activation source were MeIQx > 

IQ > DiMeIQx > MeIQx > PhIP, respectively . The IC50's for a-naphthoflavone inhibition 

of the expressed human8P450IA1 and P450IA2 activities with IQ as a substrate in the 

Ames test were 2 x 10 and 2 x 10- N, respectively . These data indicate that the 

human P4501A subfamily is functionally similar to its rabbit counterpart . 



Notes


Notes 


367 

DHYDRO7CY-2(5H)~F~gT~Ip ONE (MX ~FJ I~R ~Na ier`, A .ID . CDsAna 1o13-FH B .RDaniel1C K~~Schenck1, 

M . F . Ske~ly2 and S . L . HuangZ lU .S . Environmental Protection Agency, Cincinnati, 

OH 45268, Environmental Health Research and Testing, Cincinnati, OH 45245 NC 27709 . 

MX is a potent bacterial mutagen and mammalian cell clastogen that forms in drinking 

water during water chlorination . Concern over potential health hazards stems from the 

finding that !DC is a major contributor to the mutagenic activity of drinking water samples . 

The present work was done to obtain preliminary information on the nature of the DNA 

damage which accounts for the potent genotoxic activity of MX . DNA adduct formation 

was examined in Salmonella tyohimurium TA100 cells, primary rat hepatocytes, nd in a 

rat liver embryonic cell line (Clone 9) . DNA adducts were anal~zed by the 3~P-postlabeling 

method of Randerath and Gupta . Mutation frequency (his revertants) was also 

determined for the TA100 cells . The Salmonella cells were exposed to !D( concentrations 

of 0, 1 and 3 pg/ml for 30 min . at 37°C whereas the mammalian cells were exposed for 6 

hr to concentrations of 0, 1, 5, 10 and 50 pg/ml . Mutation induction was linear over 

this dose range in the Salmonelia cells, whereas higher concentrations were toxic . The 

mutation frequency was 1 x 10- per pg/al . A dose-dependent increase in DNA adduct 

formation was observed for all three cell types . In each case a single major adduct 

appeared to be formed . The levels of #dducts at equivalent doses were similar in the 

two mammalian cell types (ca . 2 per 10 DNA bases at the 10 pg/al dose) . A comparable 

adduct level was observed at 1 yg/ml in the Salmonella calls . Further work to characterize 

the DNA adduct formed by !IX is needed to elucidate the role of this lesion in 

the genotoxic action of this compound . (This abstract does not necessarily reflect EPA 

policy) . 

368 

MUTAGENS IN CHLORINATED WATER . J .R . Meier, Health Effects Research Laboratory, 

U .S . Environmental Protection Agency, Cincinnati, OH 45268 

Over the past decade, substantial evidence has accumulated to show the widespread 

presence of genotoxins in drinking water . The sources of genotoxic contaminants can 

be generally classified into three groups ; contaminants of the raw water, chemicals 

added or formed during water treatment, and chemicals formed or unintentionally added 

during distribution . In many cases, the genotoxic activity can be directly attributed 

to the chlorination stage of water treatment . The genotoxic activity appears to originate 

primarily from reactions of chlorine with humic substances in the source waters . 

Cenotoxic activity in drinking water concentrates has been most frequently demonstrated 

using bacterial mutagenicity tests but results with mammalian cell assays are generally 

consistent with the findings from bacterial assays . There is currently no evidence 

for genotoxic damage following in vivo exposure, although little work has been done in 

this area . Organic acids appear to account for most of the bacterial mutagenicity and 

recovery of these compounds from water requires a sample acidification step prior to 

extraction . Recently, one class of acid cospounds, the chlorinated hydroxyfuranones, 

was found to be responsible for a major part of the mutagenic activity . Approaches 

for drinking water treatment aimed at reduction of genotoxins in drinking water include 

granular activated carbon (GAC) filtration, chemical destruction, and the use of alternative 

means of disinfection (i .e ., ozone, chlorine dioxide, and monochloroamine) . 

The question of how best to minimize exposure to genotoxins in drinking water while 

maintaining a microbiologically safe water remains to be resolved . (This abstract 

does not necessarily reflect EPA policy) . 

369 

HERITABLE VARIATION IN THE RESPONSE OF A CLINICALLY NORIIAL, AU!tAN POPULATION TO ION- 

IZING RADIATION . T . Merz, D .Y . Harrison, L .A . Corey, Medical College of Virginia, 

Virginia Commonwealth University, Richmond, VA (USA) 

This is a study of the inheritance of variability in the response of clinically 

normal individuals to ionizing radiation . The micronucleus assay is used to measure 

response and since micronuclei frequencies are dependent on cell proliferation, cell 

growth kinetics are also considered . Then twin method is used to determine whether 

there is a heritable component of variation in the response of cells from clinically 

normal individuals . Ten pairs of monozygotic twins were examined for their responses 

to radiation . The variation of the response of twins within a pair is compared 

to the variation between pairs of twins . An analysis of variance does indlcate 

that there is considerable variation in observed micronuclei frequency . Nost 

of the variability can be accounted for by the differences between twin pairs . The 

large interpair variation compared to the intrapair variation demonstrates that 

twin micronuclei production is more alike (correlation of 0 .92) than non-twins . It 

is suggestive of a genetic influence on mlcronuclel production .

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MOLECULAR PATTERNS OF APRT GENE REARRANGEMENTS . M . MEUTH, G . SARGENT, C. MILES, AND Notes 

G . PHEAR, IMPERIAL CANCER RESEARCH FUND, Clare Hall Laboratories, South Mimms, Herts . 

EN6 3LD, U .K . 

Deletions and other gene rearrangements appear to be an important step in the 

process of oncogenesis and are a result of many forms of DNA damage, but very little 

is known of the mechanisms responsible for these mutations . We have been studying 

gene rearrangements at the hamster adenine vhosnhoribosvl transferase . (anrt)locus with 

the intention of identifying sequence featurea and functions involved in such 

alterations . A striking feature of deletions at aprt is the directionality of the 

mutations . Deletion breakpoints frequently occur within aprt and often upstream of 

the locus but rarely downstream . This suggests that an essential function or structure 

lies downstream from the locus and limits the mutations recoverable . On the other hand 

this directionality aids molecular analysis by providing a "tag" for deletion junction 

fragments allowing their cloning or recovery by the polymerase chain reaction . Many 

types of rearrangements (both small and very large deletions as well as several 

insertion mutants) have now been characterized at base sequence level . These 

alterations have a number of distinctive properties which will be discussed in detail . 

371 

INSTABILITY OF CHROMOSOMES CONTAINING AMPLIFIED REGIONS IN CHINESE BAMSTER CELLS . 

M . Miele, S . Bonatti, G . Fronza, L . Ottaggio, S . Yiaggi,and A . Abbondandolo, National In 

stitute for Research on Cancer, Genova (Italy), University-of Genova (Italy), and UO of 

CNR, Pisa (Italy) 

With the aim to study the effect of gross morphological modifications on chromosome 

stability, the behaviour of chromosomes carrying amplified CAD (carbamyl phosphate synthetase-aspartate 

transcarbamylase-dihydroorotase) or DH7R (dihydrofolate reductase) genas 

was studied in V79 Chinese hamster cell derivatives resistant to PALA (N-phospohacetyl- 

-L-aspartate) and MTX (methotrexate), respectively . In both metaphase chromosomes and in_ 

terphase nuclei, amplified regions were localized by in s1 u hybridisation . In MTX-resis_ 

tant cells, the amplification bearing chromosomes was lagging behind at anaphase and gaw 

rise in interphase nuclei to bud-shaped formations . Apparently, these buds could eventually 

separate as micronuclei . In both MTX- and PALA- resistant cells, micronuelei in in_ 

terphase and displaced chromosomes in metaphase, both containing amplified DNA, were ob 

served . The presence of chromosomes in micronuclei was confirmed by fluorescent staining 

with antikinetochore antibodies . Finally, amplification,bearing dicentric chromosomes 

were found at high frequencies in both drug-resistant call lines . All together, these ob 

servations indicate that the presence of an amplified region makes chromosomes unstabla, 

since : (i) they tend to be excluded from cells, and (ii) they rearrange sare frequently 

than normal chromosomes . 

372 

SPONTANEOUS AND IN VITRO RADIATION-INDUCED CHROMOSOME ABERRATIONS IN HUMAN SPERMATOZOA : 

APPLICATION OF A NEW METHOD 

Mikamo, K., Kamiguchi, Y. and Tateno, H . : Department of Biological Sciences, Asahikawa 

Medical College, Asahikawa 078, JAPAN 

Chromosomes of the spermatozoon can be analyzed only after they replicate and become 

condensed in the ootid as male pronuclear chromosomes . Therefore, difficulty of using 

human oocytes had long been a severe limitation for the human sperm chromosome study . 

Fortunately, however, development of the interspecific in vitro fertilization system 

using zona-free hamster oocytes made It possible to carry out a large scale study of 

human sperm chromosomes without relying upon human oocytes . In the present talk, we 

describe briefly the procedure of our lmproved method and the results thereby obtained 

in the in vitro experiments . (1) Spontaneous incidences of human sperm chromosome 

aberrations in a total of 9280 spermatozoa from 87 samples of 26 men. Incidences of 

aneuploidy and structural anomaly were 1 .35 Z(hyperhaploidy, 0.68 x; hypohaploidy, 

0.67 x) and 13.9 x, respectively. The latter incidence varied considerably among the 

donors, ranging from 3.6 x to 21 .5 2. (2) Radiation (X-, y- and 8-rays)-induced human 

sperm chromosome aberrations in a total of 6974 spermatozoa from 57 samples of 12 men . 

Incidences of spermatozoa with structural chromosome aberrations increased linearly 

with increase of radiation dosage . The slope of the dose-effect equation was nearly 

the same between the three kinds of radiation . The incidence of breakage-type 

aberrations was far higher than that of exchange-type aberrations, both of them showing 

linear dose-dependent increases . 


129



373 

Notes 'itD; AOIE OF C1.T2, PRALiFFItATZCN IN CfmCAL CARCIIvmw7s . Jan C. Mirsalis . SRT 

International, Menlo Park, c1. 

Most agents that irxh :oe increases in celi proliferaticn have been st :oan to be 

effective tumor praootoss in liver and othar tissuasf indeea, the cnrcim9enicity of 

~r ~ be entu+rx:ed by partial hepatectony followirg c~ioal exposAme . 

grvwirg evidsnoe ~ t oall proliferation alone, in the abmenoe of 

e:mger:ous initiation, may also irduoe an incn .vased incidsnoa of liver t:moors in 

rodents, Ixutioularly mice . We have investigated the role of chaai~ 

1 mechanians includirq pxtaocticn of spantnneasly initiated tumors, altered 

gena zegulation, la~.ion of RA, ar activatacn of 

fhis mec3ianism of h~ycancinogenesis will enable batter estima~ of t~ ris~k .~ 

374 

MUTATIONAL SPECIFICITY OF CIS-PLATIN IN YEAST . J .R .A . His and g .A . Kunz, Microbiology 

Department, The University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2 

The chemotherapeutic agent cisplatin Jois-diamminedichloroplatinum(II)) produces 

DNA monoadducts and crosslinks and is mutagenic but the DNA sequence changes caused 

by this agent in eukaryotic cells have not been characterized . We are using DNA 

sequence analysis of mutations induced in the yeast suppressor tRNA gene SUP4-o to 

assay the mutational specificity of cisplatin . =-o mutants were selected following 

cisplatin treatment that reduced survival by 701 and increased the mutation frequency 

five-fold . 100 independent cisplatin-induced mutants have been characterized to date . 

Although the frequency of induction was relatively low, the spectrum of induced mutations 

differed from the spontaneous spectrum . Single base-pair substitutions constituted 

a smaller fraction (62%) of the total mutations and G-C -> A-T, C-C -> C-C and 

C•C -> T•A events each accounted for approximately 301 of the base-pair changes identified 

. The substitutions occured predominantly at dipurines and where changes can be 

assigned to specific dipurines, 90% (36/40) were found at 5'-GG-3' or 5'-CA-3' sites . 

The fraction of single base-pair deletions induced by cisplatin was 10-fold greater 

(30% of the total mutations) than observed spontaneously and the majority (75%) of 

these events are found in a run of 5 C-C pairs . In addition, a small fraction (3/100) 

of non-tandem double events involving base-pair substitutions and/or deletions were 

recovered . Taken collectively, our results suggest that both monoadducts and 

crosslinks may play roles in determining the mutational specificity of cisplatin . 

Currently, we are analyzing additional induced mutants . (Supported by NSERC Canada) 

375 

THE RELATIVE ROLES OF PHARMACOKINETICS AND ORGAN SPECIFIC ME'fABOLISK IN THE 

SELECTIVITY OF CYCLOPHOSPHAMIDE-INDUCED IIQIUNE CELL DAMAGE IN VIVO . R .R . Misra and 

S .E . Bloom, Cornell University, Ithaca, NY (USA) 

In avian embryos bursectomy is achieved after subchronic administration of cyclophosphamide 

(CP) . However, the mechanism(s) by which organ-directed toxicity is 

achieved has not yet been elucidated . To this end, studies of xenobiotic metabolism in 

bursal and thymic cell fractions were undertaken . Three assays of mixed-function 

oxidase activity, as well as an assay of alkylation potential, were employed to detect 

differences between the abilities of bursal versus thymic micrososes to activate CP . 

Additionally, an aldehyde dehydrogenase (AD) assay was used to monitor differences in 

cytosolic detoxification activity . Compared to the liver, constitutive levels of P450 

activity were quite low in the bursa and thymus, and of the two lymphoid organs 

tested, the thymus exhibited higher levels of P450 activity . Alkylating activity was 

clearly demonstrated in hepatic microsomes, but fell below our limit of detection for 

bursal and thymic fractions . Similarly, iemune-organ AD levels were approximately 

one-tenth as high as those of the liver, and between lymphoid tissues, no significant 

difference in AD activity was apparent . The toxicokinetics of systemically ad .inistered 

CP as well as in vitro binding of the activated compound to lymphoid cells, were 

also examined . Results from these latter experiments indicate that in the intact 

animal, higher concentrations of CP and/or activated metabolite reach the bursa as 

compared to the thymus but that in vitro, no significant differences in binding occur . 

Our'findings suggest that while drug distribution patterns may be involved, differences 

in xenobiotic metabolism are probably not a major determinant in the selectivity 

of CP-induced immune-organ damage . (Supported by NIEHS ES03499 .) 

50869 3644


CLONING OF DNA REPAIR GENES IN HAEMOPHILUS INFLUENZAE RD Notes 

R. Mody, V.P. Joshi and N.K. Notani, ion e ica 3roua BFi-fiFa AtQn ic Research Centre, 

B3nbay 400085, India. 

We have reported a recanbinational DNA repair systan which Is more clearly manifest 

in strains like Uvr1 in which excision repair is deficient . With another UV-sensitive strain 

Mbo2 also, we now observe much more of recQnbinational repair than is noted in a wild 

type strain. We have earlier reported cloning of uvrl gene on an 11 .3 kb insert. The 

plasrid pKuvrl fully cQnplanents Uvrl strain but not a Uvr2 strain . We now report cloning 

of mbo2 gene on a 17 .7 kb insert which fully canplanents the UV-sensitivity of Mbo2 

strain but not of Uvrl strain indicating that uvrl and mbo2 are not allelic . EcoRl cutting 

produces two fragm ents from the 17 .7 kb insert, the Terger one of 13 kb anChe sn aller 

one of 4.7 kb . Both these fragn ents have been subctoned 13 kb fragm ent subclone does 

not conpianent the UV-sensitiwty of Mbo2 strain but 4 .7 kb subclone gives a partial camplem 

entation. It is inferred that atleast san e of the genetic infotm ation for expressing 

UV-resistance is carried on 4 .7 kb fragm ent. 

377 

ETHENO-ADDUCT PRODUCTION BY CARCINOGENIC AND ENDOGENOUS AGENTS 

Ruth A . Modzelewski, Mary K . Conner, Noriko Kawatani, Departments of Biostatistics and 

Industrial Environmental Health Sciences, Graduate School of Public Health, University 

of Pittsburgh, PA ., 15162 (USA) 

Ethyl carbamate (EC) is a carcinogen which produces highly fluorescent etheno-adenine 

(c-A) adducts in RNA . Its active metabolite is presumed to be an analogue of 

chloroacetaldehyde . The abundant adenylate pool is an obvious target for c-A adduct 

formation . Several e-A derivatives are potent inducers of SCEs and multiple complex 

chromosomal aberrations . 

Choline is an essential dietary element, cell membrane component, and a biochemical 

substrate . Arsenocholine (AsCh) is a arsenic analogue of choline found in seafood, including 

shrimp, crab, and some fish . Simple oxidation of the hydroxyl, moiety of choline 

or its arsenic analogue produces an analogue of chloroacetaldehyde . 

EC, Choline, and AsCh yielded incredibly similar fluorescent chromatogramS in extracts 

of murine red blood cells exposed in vivo . HPLC elution times of several fluorescent 

peaks correspond to those of standard c-adenine derivatives . The significance 

and long term consequences of c-A adduct production by carcinogens and endogenous 

agents remains to be elucidated . Supported by : BRSG 2 S07 RR05451-27, Biomedical Research 

Support Grant Program, NIH, and Center for Environmental Epidemiology, University 

of Pittsburgh, EPA CR 812761 . 

378 

METHODS FOR SCREENING FOR GERMINAL MUTATIONS: DETECTION OF "NON-POINT" 

MUTATIONS USING A MODIFICATION OF THE "RFLP" ANALYSIS STRATEGY . H . W . 

Mohrenweiser and B . A . Perry, Biomedical Sciences Division, Lawrence Livermore National Laboratory, 

Livermore CA 94550 

Insertions, deletions and rearrangements (I/D/R) of the DNA of the human genome have been identified 

during molecular analysis of both de novo germinal mutations and inherited variants causing genetic diseases . 

This class of molecular alteration in DNA structure is expected to predominate among radiation induced 

mutations. A modified restriction enzyme site (RFLP) mapping strategy, using only a single restriction 

enzyme digestion and then repetitively analyzing each sample with a series of probes for different loci, has 

the potential to screen a significant portion of the genome in each proband for heritable, "non-point" 

mutations . Results from screening 130 unrelated Caucasian individuals for variation at 40 independent loci, 

using this RFLP mapping strategy, indicate the frequency of rare, inherited I/D/R variants is 3 .1 variants/ 

1000 loci screened. A prototype mutation screening experiment that involves screening DNA from 50 

independent, clonally derived human lymphoblastoid cell ltnes, established in the absence of selective criteria 

following exposure of cell cultures to a mutagenic agent, has been initiated . Two ap nt mutations have 

been identified during the initial scteening of the DNA from these cell lines with sevet~NA probes, one in 

the progeny of ENU treated cells and one in a cell line established following exposure of the parental cells to 

X-ray. Confirmation of these apparent mutations and analysis of the molecular alteration in DNA structure is 

in progress, as is continued screening of the DNA from these cells with probes for additional loci . It should 

be possible with only minor enhancements of this RFLP mapping strategy to generate a significant data base 

regarding the frequency of germinal I/D/R mutations in an exposed population . Workperfotmmed under 

auspices of the US DOE by the Lawrence Livermore National Laboratory ; contract No.W-7d05-ENO-48 

379 

METABOLISM AND GENOTOXIC EFFECTS, IN VIVO . OF A MARKER FOR NfIRO-PAH . 2-NlIRO- 

FLUORENE . COMMONLY FOUND IN THE ENVIRON1vIENf . 

L . MBllert, J . Raftert . S . Tbmqulstt . B . BeIJe9, R ToRgArdt . T. Mldtvedt2 . M . Cortie2 and J-A . 

Gustafssont, Departments of Medical Nutritlonl and Medical Microbial Ecology2. ISarolinaka 

Institute and Department of Genetic and Cellular Toxicology3 . University of Stockholm . 

Stockholm. Sweden 


131



Notes Durtng incomplete combustion of organic matter there is a formation of po)ycyclic aromatic 

hydrocarbons IPAH) which can react with nitrogenoxides, with the formation of nttro-PAH'a 

as a result, a reaction which Is catalyzed by a low ptl . 2-Nitrotluorene (ÌF), a marker for nftro- 

PAH, is in vivo metabolized via two different routes . After inhalation there ts a formation of 

potent mutagenic metabolites . OH-NF's, which are distributed In the body. After oral administration. 

NF is reduced to the amine, a reaction mediated by the intestinal mtcrollora . and 

further acetylated to 2-acetylaminoAuorene (AAF), a potent carcinogen . Further ringhydroxyladon 

of AAF leads to detoxification and excretion . 

Induction of cytochrome P450 c .d affects the metabolism tn that more OH-NF'a are formed . As 

a consequence, more mutagenic metabolites are found In the circulation . The liver excretes OH- 

NF's as . In terms of mutagenictty. , totally harmless glucuronide conjugates . When these 

conjugates are excreted via the bUe . Intestinal beta-glucuronidase can liberate direct-acting 

mutagens tn the intestine . Thus, inhalation of NF can lead to formation of potent mutagens in 

the mtesttne. 

NF induces DNA-repair. In vivo, and is an tnttlator and a weak promotor, measured as formation 

of preneoplastic lesions in the liver. Risk estlmates, by two different methods . Indicate 

that nltro-PAH's, extrapolated from the marker NF. can expose humans to a cancer risk . 

380 

POINT hlJTATION AND CYTOGENETIC A'MLYSIS OA' L1TiPFDCY7ES FROM NH .RtOCYSTICFtCOTIC PATIFATS 

TRFATID WITH PRAZIWANTFL, R . Montero , D . Valencia , F . Moreno *, *f . Sandoval * and 

P . Ostrosky-Wegman . Instituto de Investivaciones Riomddicas, U .N.A .N ., Hosnital de 

Psnecialidades, Centro MBdico la Raza . Ando . Postal 70228, C .P . 04510, Wxico, n .F . 

Evaluation of genotoxic effects of praziouantel on neurocysticercotic piv. lyrtmhocytes 

showed an increased frequency of poliploids with res±+ect to non-infected animals and 

also a greater suscentibility to clastogenic damaqe . When same study was intented on 

humans we met a major problem on choosing adequate controls for neurocysticercotic 

patients since they are exnosed to several mutagenic agents before the correct diagnose 

is done ; these include com?wtarized axial tanopranhy, anticonvulsants, anesthetics, 

antiinflamnatory druqs and antibiotics . Our controls, therefore, include non-infected 

persons in one hand and neurocysticercotic natients exoosed to different combinations 

of treatments, all of them before they received the nraziauantel treatment, on the 

other hand . The terminal noints studied were : structural and numerical chronosomal 

aberrations, sister chromatid exchanges, cell cycle kinetics and 6-thiopuanine resistant 

lymhocyte frequency . Results show that neurocysticercotic status, which involves 

exposure to the agents mentioned, causes retardation on cell cycle kinetics ; 

besides, hprt assay results show that in some aatients there is also an increase on 

point mutations . After praziquantel treatment it was found that cell cycle kinetics 

return to normal values . 

THE COMPARISON OF MUTAGEN-INDUCED THYMIDINE KINASE (TK) MUTANT FREQUENCIES IN HUMAN 

AND MOUSE LYMPHOMA TESTING CELLS . M .M . Moorel, K . Harrington-Brock2 L . Parker2 . 

1U .S . Environmental Protection Agency, Research Triangle Park, NC 27711 USA ; 

2Environmental Health Research and Testing, Inc ., Research Triangle Park, NC 27709 

USA . 

The TK6 line of human lymphoblastoid cells can be used to detect mutants at the 

heterozygous thymidine kinas* (1k) locus . Little et al . (1987, Banbury Report 28 : 

Mammalian Cell Mutagenesis, p . 225) have reported that a class of slow-growing TK 

mutants can be recovered and that at least some of these mutants may result from 

mitotic recombination . These results are similar ta our findings for small-colony 

TK-deficient mutants of mouse lymphoma cells . We wished to make quantitative 

comparisons between the induced mutant frequency in the human and mouse lymphoma 

cells . Slow-growing mutants are difficult to recover and count using the procedures 

standardly used with the TK6 cell line . We are itnrestigating modifications which 

might optimize growth and quantitation of slow-6roving mutants . Modifications 

include using 24 well plates rather than 96 well plates and plating cells at 1x103 

to 5x103 rather than 4x104 cells per well . Using these procedures, we are comparing 

the ICR-170-, EMS-, and MMS-induced TK mutant frequencies in human and mouse 

lymphoma cells . (This 1s an abstract of a proposed presentation and does not 

necessarily reflect U .S . EPA policy .) 

381 

382 

IN VIVO REPAIR DURING G~ OF GA!!IA RAYS INDUCED LESIONS ELICITING SISTER CHRQIATID E% 

CHANGES (SCEs) IN MURINE SALIVARY GLAND CELLS . 

Pedro Morales-Ramfrez, Teresita Vallarino-Kelly and Re ina Rodr2 uez-Reyes . 

Instituto Nacional de Investigaciones Nucleares, Mgxico, D .F ., I~XICO) . 

The in vivo ability of mouse cells to repair gamma radiation induced lesions capable 

of eliciting SCEs was examined . The experimental protocol was done in mouse salivary 

gland cells induced to parasynchronical division by isoproterenol (1 umole/gm bd 

wt) . Two groups of mice were irradiated with 0 .38 Gy in a 6OCo source (Vick Rad) at a 

50869 3646

dose rate of 6 .9 Gy/min either at early or late GI, followed of a round of division in 

presence of bromodeoxyuridine (BrdU) dose of 0 .5 mg/gm bd wt . There was a significant 

decrease in SCE frequency at early Gj with respect to those irradiated at late G1 . The 

se data suggest that BrdU substituted cells are able to repair about fifty percent of 

the lesions induced by gamma radiation during G1 . Also the adequeate BrdU dose to obtain 

a clear differentiation of sister chromatids and the capacity of ga- a radiation 

to induce SCEs in salivary gland cells were determined . The BrdU dose which permits a 

good differentiation of sister chromatids (0 .3 mg/g bd wt) was one third of that required 

in bone marrow cells . The gaama radiation induced a significant increase of -- 

SCEs with a similar efficiency to that obtained in bone marrow . 

Acknowledgments : We wish to thank Jorge Mercader N ., Angel Reyes P ., Perfecto Aguilar 

V ., Felipe Beltran B . and Enrique FernEndez V., for their excellent technical assistance 

. 

383 

RESTRICI7ON ENZYMES AND CYTOGENETIC DAMAGE . W.F. Morgan, H .W. Chung. J .W. Phillips and RA 

Winegar, Laboratory of Radiobiology and Environmental Health, University of California, San Fraacisco, CA (USA) 

Bacterial restriction endonucleases recognize specific, rather short sequences of DNA as binding sites and produce 

either blunt-ended or cohesive-ended DNA double-strand breaks . Eleetroporatioe Is a rapid and extremely efficient 

method for pcrmeabilning Chinese hamster ovary (CHO) cells, permitting the introduction of restrictioa enzymes into 

exponentially growing cells. We have used restriction enzymes with different recognition sequences and different 

cutting frequencies to generate double-strand breaks in CHO cells and have examined the role of these breaks in 

cytogenetic damage. Restriction enzymes electroporated into cells readily induced chromosome aberratioos at all 

stages of the cell cycle. Enzymes generating blunt-ended DNA double-strand breaks induced more chromosome 

damage compared with enzymes generating coheaive-ended breaks . When restriction enzymes were eledroporated 

into exponentially growing cells during the second replication cycle in bromodeoxyuridise, and sister chromatid 

exchanges (SCEs) analyzed at the subsequent mitosis, we found no inaease in SCE frequency . Many enzymes 

induced aberrant metaphase chromosomes, but SCE frequency was not increased in those cells. These results indicate 

that in our hands, restriction enzyme-induced DNA double-strand breaks give rise to chromosome abenaNons, but do 

not lead to SCE formation . Work supported by the Office of Health and Environmental Research of the US . Dept . of 

Energy under contract DE-AC03-76-SF01012 . 

384 . 

GENOTOXICITY IN RODENT HEPATOCYTES AND CARCINOGENICITY OF ANTHRAQUINONES . 

H . Mori, N . Yoshimi, S . Sugie, H . Iwata, T . Tanaka, and K . Kawai, Gifu University School 

of Medicine, Gifu (Japan), and Chukyo Women's University, Ohbu, Aichi (Japan) 

A large number of anthraquinones and their derivatives have been isolated from higher 

plants and fungi, and some of them have been widely used as colorants in food, cosmetics, 

hair dyes and textiles . Luteoskyrin and rugulosin isolated from Penicillium islandicum 

are known as hepatocarcinogenic anthraquinoids . Emodin from the samerun-gus 

has been shown to be mutagenic in bacterial mutagenicity assay . We have demonstrated 

genotoxicity of some anthraquinones such as 1,8-dihydroxyanthraquinone (chrysazin) 

which has been used as a popular laxative, in the hepatocyte/DNA repair assay . In the 

genotoxicity assay, 1,2-, 1,4-, and 1,5-dihydroxyanthraquinones were negative, although 

1-hydroxyanthraquinone and 1,8-dihydroxyanthraquinone generated clearly positive res- 

ponse of DNA repair, suggesting a mutual relationship between the genotoxicity and structures 

of anthraquinones . Carcinogenicity testing of chryeazin was done using rats 

and mice . In rats, chrysazin was tumorigenic to cecum and colon . In mice, the chemical 

showed carcinogenic potentials in the large bowel and liver . Carcinogenicity of 1-hydroxyanthraquinone 

was also examined in rats . Carcinogenicity of this naturally occurring 

anthraquinone was manifested in cecum, colon and liver . The carcinogenic effect of 

1-hydroxyanthraquinone appeared to be stronger than chrysazin . The result was agreement 

with that of the genotoxicity assay with hepatocytes . It appears that genotoxicity in 

rodent hepatocytes is more consistent with mammalian carcinogenesis than bacterial 

mutagenicity for these anthraquinone chemicals . 

385 

EVALUATION OF CLASTOGENICITY OF NON-PHYSIOLOGICAL PHs IN CULTURED MAMiALIAN CELLS . 

MORITA, T ., TAKEDA, K ., and OKUMURA, R ., Tokyo Research Laboratories, 

NIPPON GLAXO LTD ., Nerima-ku, Tokyo, Japan . 

Using Chinese hamster ovary K1 cells, chromosomal aberration tests were carried 

out of formic acid, acetic acid, lactic acid and a mixture of S9 and NaOH, and the 

relationship between pHs of the media and their clastogenic activity was examined . 

The medium used was Ham"s F12 supplemented with 17 mM NaHC03 and lOx FCS . All of 

these acids induced chromosomal aberrations at the initial pH of ca . 6 .0 or below 

(10-14 mM of each acid) either in the presence or absence of S9 mix . Exposures of 

cells to pH 5 .7 or below (12-16 mM of each acid) were found toxic . In the culture 

media which were first acidified with each of these acids and then neutralized to 



Notes


Notes pH 6 .4 or pH 7 .2'with NaOH, no clastogenic activity was observed . Using F12 medium 

supplemented with 34 mM NaHC03, or 30 mM HEPES as buffer, we observed metaphases 

at doses up to 25 or 30 mM of these acids and no clastogenic activity at 20-25 mM 

or below . A mixture of S9 (final 10X) and NaOH induced chromosomal aberrations at 

initial pH of ca . 10 .8 . When the mixture was incubated at 37°C for 1 hr and then 

neutralized with HC1 to pH 9 .3 or pH 7 .4 . it showed no clastogenic activity . The 

above results show that these acids and base are non-clastogenic and thus the 

pseud-positive reactions attributable to non-physiological pH conditions could be 

precluded by either neutralization of the treatment medium or enhancement of the 

buffering ability . 


386 

HUMAN SOMATIC MUTATIONS AT THE AUTOSOMAL HLA-A LOCUS 

A .A . Morley and D .R . Turner, Haematology Department, Flinders Medical Centre, 

Adelaide, Australia . 

Human lymphocytes mutated at the HLA-A locus were isolated by immunoselection 

using monoclonal anti-HLA-A2 or -A3 antibody and complement followed by cloning in 

micropslates . Mutant frequency in 21 normal individuals, with mean age 35, was 3 .0 

x 10- and in 10 elderly individuals with mean age 78 it was significantly higher 

at 7 .2 x 10-s . Analysis of the T-lymphocyte receptor gene in 98 mutants showed 

that ~


at the later time points . Relative cloning ability was determined by exposing P3 Notes 

cells to concentrations of 0 .1 to 1 .0 VM BRdU or CLdU for 7 days . Each toxicant 

reduced cloning ability from 100% to less than 1%, although CLdU was more efficient on 

a concentration basis than BRdU . Not only were the toxic effects of CLdU greater than 

those of BRdU, but also its ability to perturb the cell-cycle . 

389 

INSECTICIDES AND CAF2CZNOGQI METAHOLIZING ENZYMES 

M .H . MU6'i'AFA AND A .H . Etrlt(%aEIDY . Institue of Graduate Studies and Research, 

Alria University, alexandria, Egypt . 

The effect of various insecticides on the activities of sane carcirtgertntt:tabolizing 

enzymes and the eytocliratr` P-450 of rat liver microsomes were studied . Organoc.hlorine 

insecticides, Lindane, DDT and Endrin significantly increased the activity 

of arylhydrocarbon hydroxylase (AHH) . Pretreatment of rats with the synthetic pyrethroids, 

fenvalerate insignificantly increased the enzyme activity . Pretreatment with 

carbaryl, a carbamate derivative and Ditnethoate, an orgartophosphrotls oatQottrd did not 

affect the AHH activity . Our data showed that all the tested insecticides which altered 

the AHH activity have similar effect on tht: cy''.,ochrane P-450 oontent . The data revw 

ealed the ability of various insecticides to increase the atsotlrtt of cytochrattta P-450 

and the AHH activity which ttssy increase the nutagenic and carcinogenic activity of 

benzo-a-pyrene, since such an activity is dependent an the induction of cytocYurome 

P-450 dependent AHH . A second enzyme was also investigated, dintethylnitrosamine (DM4) 

demethylase I and II . Pretreatntutt of rats with Lindane, DDT and Endrin decreased the 

activity of Dm-demethylase I and increased the activity of DMN-danethylase II . 

Fenvalerate and carbamate pretreatment groduoed insignificant change an both enzymes . 

Pretreatment with Dimethoate inhibited the oxidative N-darethylaticn of DMD1 . Since 

the metabolism of carcinogens by cytochrrnie P-450 dependent mx0aager>ares often 

facilitates their elimination or activiation, alteration of norntal tuetabolicpn t2xaays 

of these carcinogens by various insecticides may alter the intensity and duratiort or 

action of these environmental carcinogens . 

390 

GE\OTOXIC POTENTIALITIES OF ::-NITRU-METIOXYNAP T8O ('2,1-b) FURAN (R 7000) 

IN RELATION TO Ti{E GENERAL PROBLEM . 

J . Moutschen, J . Gilot-Delhalle, M . Moutschen-Dahmen, and F . London, 

University of Liige, Sart-Tilman 1322, D-4000 Liege (BELGIUM) 

Mutagenic activity of R'7000 was tested on two strains of Sehizoe? ccharomyces 

pombe ade 7 and ade 6 (at doses ranging from 0 .0Gto 32 . .0 M) . 

It was different in both strains . R 7000 showed clastogeniciSy in Nigella 

damascena chromosomes at doses ranging from 0 . 12 to 32 .t0-5M for seeds 

and 0 . 12 to 2 .10-5M for root tips . Treatments of dry seeds revealed the 

sensitivity of GO whereas treatments of presoaked seeds showed the aensivity 

of Gl, S and G': . These results were confirmed by treatments of 

growing root tips with a much higher sensitivity of the later phases of 

the mitotic cycle . These treatments yielded a high amount of gaps (G'and 

G") . The results are in agreement with those obtained after treatments 

of animal cells . Additionally, some treatments induced strong stathmokinetic 

effects analysed in details . The spectrum of induced chromosome 

damage shows similarities with the spectrum of lesions induced by cytosine-arabinoside 

and other related substances, wich suggests some mechanisms 

of action . In conclusion, R 7000 belongs to the class of substan-, 

ces with multiple genotoxic effects, and its position in the important 

class of furans has to be revaluated . 

391 

AN SAR STUDY OF THE MUTAGENICITY OF PAH COMPOUNDS IN SALMONELLA 

TYPHIMURIUM . Susan R . Moyer and Peter C . Jurs, Chemistry Department, Penn State University, 

University Park PA 16802 

Many Polycyclic Aromatic Hydrocarbons and their derivatives have been shown to be potent 

mutagens in Salmonella ryphirnuritun via the Ames test . A group of 110 PAHs (48 active and 64 

inactive) were obtained from the literature and were used to carry out a structure-activity relationship 

study. All of the compounds were tested by LaVoie and coworkers using S . ryphimtvitun TA100 with 

metabolic activation . The molecular structures were entered into the ADAPT software system by 

sketching them on a computer terminal . Three-dimensional molecular models were constructed using 

molecular mechanics . Then, topological, geometrical, and electronic molecular structure descriptors 

were calculated for each compound . Specific structural features were encoded by using substructure- 


135


Notes based descriptors that focus on details of the molecular structures . The descriptors were analyzed 

statistically in order to develop a minimal set to characterize the compounds . Pattern recognition 

techniques were used to classify the compounds as active or inactive based on the structural 

descriptors. A training set of 80 compounds (40 active and 40 inactive) was classified almost perfectly 

using 15 descriptors, and the remaining compounds formed a prediction set of unknowns . Studies in 

progress are aimed at refining our SAR model for PAH mutagenicity . 


CLONING ILLID PARTIAL C11WCP6RIZATION OF RftE H(A7)1N IXCISION RLPAIR GENB ERCC-5 . 

J .S . Mudgett, G .F . Strniste, and ri .A. MacInnes, Genetics Group, LS-3, M886, Los 

A,lamos National Laboratory, Los Alamos, t39 87545 (iJSA) . 

The human excision repair gene DtCC-5 was identified and isolated by complementation 

of the W-sensitive Chiness hamster ovary cell mtutant UV-135 and 

subsequent cosmid library construction and analysis . Genomic DtA from repair 

proficient human skin fibroblasts was ligated to pSV2gpt and transferred into 

W-sensitive Ctq cells of complementation group 5(W-135) . DNA from a primary 

MAX (aycophenolic acid, adenine, xanthine) resistant, W-resistant cell line was 

transferred into W-135 cells to isolate a secondary Mhe, W cell line, from 

which tertiary MaX`, i)V' cell lines were isolated by gene transfer . Cotuid 

clones which contained inserted human sequences wsre obtained from a 20X genumic 

library of the secondary transformant . None of thse individual (101) cosmid 

clones isolated and tested were able to ca .plement the repair defect, but 

specific overlapping pairs of cosmids co-transfected into UV-135 cells yielded 

transformnnts at high frequency which exhibited wild-type W resistance, suggestit~g 

that the repair gene is as large or greater than the cosmid insert size 

(-40 kb) . The overlap between the appropriate cosmid clones, and the coaplete 

contiguous human genomic insert, will be napped to identify the sise and functional 

boundaries of the human nNA repair gene . Unique huswt sequence probes 

derived from these specific cosmid clones were used to : 1) demonstrate a positive 

hybridization correlation with genomic tx4A from independent UV-resistant 

transformants and human cell hybrids ; 2) to confirm the previous assignment of 

ERCC-5 on chromosome 13 ; and 3) to screen c0Nuh libraries . (Supported by the 

U .S . Department of Snerqy under c^ntract W-7405-lN".-36) 

392 

393 

IN VIVO GENOTOXICITY OF TERTIARY BUTYL HYDROQUINONE IN GERM CELLS OP MALB 

NICE . A . Mukher,)ee and A . Sharma, Centre for Advanced Study in Cell and Chromosome 

Research, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, 

Calcutta 700 019 (INDIA) 

Effect of tertiary butyl hydroQuinone (doses of 10, 20 and 100 mg/kg/day during 

14 days) on germ cells of male mice were investigated . The mode of application was 

stomach intubation . The germ cell stages analysed were spermatids (for the heritable 

effects) and differentiating and stem-cell spermatogonia (for direct effects) . A lack 

of heritable translocations, sperm abnormalities was demonstrated in F males originating 

from treated P males . Significant effects in treated males were found with respect 

to . (1) sex-chromosomal univalency in the diakinesis-metaphase I stage after the 

treatment of stem spermatogonia, (2) sperm-head abnormalities after treatment of 

differentiating spermatogonia and (3) fertility after treatment of spermatids . 

394 

MICRONUCLEI INDUCED BY TERTIARY BUTYL HYDROQUINONE (AN ANTIOXIDANT) IN BONE MARROW 

CELLS OF MICE . Anita Mukherjee . Human Genetics Unit, Centre for Advanced Study 

in Cell and Chromosome Research, Department of Botany, University of Calcutta, 

35 Ballygunge Circular Road, Calcutta 700 019 . India . 

Tertiary butyl hydroquinone (TBHQ) a phenolic antioxidant was administered to 

Swiss albino male mice of 20, 50, 100 and 200 mg/kg as a single (i .p .) injection . 

Corn oil and cyclophosphamide were used as the vehicle (negative) and reference 

(positive) control respectively . Samples of bone marrow were examined for the 

incidence of micronuclei in the poiychromatie erythrocytes at 24 hours after dosing . 

A positive dose response effect in the micronuclei freQuency was observed following 

the Cochran Armitage Trend Test . 50, 100 and 200 mg/kg of TBHQ caused an increase 

in the incidence of micronuclei compared to the negative vehicle control . Cycl,ophoephamide 

caused a significant increase in the incidence of micronuclei . 

50869 3650


THE ROLE OF NUCLEAR MATRIX IN REPAIR Notes 

L.H .F. Mullenders', J . Venema', A.•van Hoffen', L. Mayne', A .T . Natarajan' and AA. van Zeeland") Department 

of Radiation Genetics and Chemical Mutagenesis, University of Leiden, The Netherlands ; p) Centre of Medical 

Research, University of Sussex, U.K . 

The organization of the eukaryotic genome into a series of loops anchored to the nuclear matrix bas been 

related to functional compartimentalization of the nucleus in order to facilitate processes such as replication and 

transcription. We have investigated the role of the nuclear matrix in processing UV-induced damage in human 

fibroblasts . Pulse-labelling of normal fibroblasta followed by enzymatic digestion of DNA-nuclear matrix complexes 

revealed a time and dose dependent preferential repair of nuclear matrix associated DNA . Pulse-ehase experiments 

showed no evidence for compartimentalization of excision repair at the nuclear matrix Le, lesions do not require prior 

attachment in order to be repaired . The results favour a model of preferential repair of DNA sequences permanently 

associated to the nuclear matrix . Pronounced differences in distribution pattern of repaired sites in DNA-nuclear 

matrix complexes were found among normal and UV-sensitive cells exposed to UV-'vradiation . Xeroderma 

pigmentosum group C (XP-C) efficiently repaired nuclear matrix associated DNA, but not regions of the genome 

further extended into thc DNA loops . In Cockayne's syndrome (CS) cells the reversed situation was found : efficient 

repair of loop DNA and inefficient repair of nuclear matrix-associated DNA . These differences in distribution oan 

be correlated to efficiencies in repair of UV-damage in transcriptionally active genes . Despite the low overall repair 

XP-C cells were found to be as proficient in repair of pyrimidine dimera from the ADA and DHFR gene as normal 

fibroblasts. The efficient removal of pyrimidine dimers from active genes was found to be absent in CS cells . The 

results suggest the existence of two independent repair pathways directed towards repair of pyrimidine dimers in 

either active or inactive DNA . 

396 

SENTINEL AND OTHER MUTATIONAL EFFECTS IN OFFSPRING OF CANCER SURVIVORS . John J . 

Mulvihill, Clinical Epidemiology Branch, National Cancer Institute, Bethesda, MD, USA 

To date, no agent has been documented to cause germ cell mutation in human beings, 

with the possible exception of radiation causing abnormal meiotic chromosomes in 

testes . For studies in humans, mutation epidemiologists prefer the cohort approach, 

start ing with an exposed population and looking for mutations that may be expressed 

in offspring as variants in health, chromosomes, proteins, or nucleic acids . Currently 

patients with cancer are the cohort exposed to the largest doses of potential 

mutagens, i .e ., radiotherapy and drugs . In 12 large studies with over 825 p,atients 

and 1573 pregnancies, 46 (4%) of 1240 liveborns had a major birth defect, a'rate comparable 

to that in the general population . One of these was a classic sentinel phenotype, 

i .e ., a new sporadic case of a dominant mendelian syndrome . In collaboration 

with 5 U .S . cancer registries, we interviewed a retrospRctive cohort of 2383 patients 

diagnosed with cancer under age 20 years, from 1945 thrbugh 1975 . Records were sought 

to verify major genetic disease, defined as a cytogenetic or single gene disorder or 

1 of 15 isolated birth defects . In 2308 offspring of survivors, 5 had a chromosomal 

syndrome, 11 had a single gene disorder, and 62 had at least one major malformation . 

Among 4722 offspring of sibling controls, the respective numbers were 7, 12, and 127, 

nonsignificant differences . 7% of the parents of the offspring with possibly new 

mutations received potentially mutagenic therapy, compared with 12% of parents of 

normal children . Since pregnancy in or by cancer survivors is still a rare event, 

future efforts to document germ cell mutation may be best studied through international 

cooperation coupled with diverse laboratory measures of mutation . 

397 

MMC INDUCED SCE IN DIVIDING AND NONDIVIDING HUMAN PURIFIED LYMPHOCYTES AND 

CHINESE HAMSTER OVARY CELLS . H . Murli, Department of Molecular and Cellular 

Services, Hazleton Labs, Kensington, MD . 

Repair of MMC induced lesions that result in SCE was studied in purified human 

lymphocytes (PHL) and in Chinese hamster ovary cells (CHO) . PHL at 0~ and 

confluent cultures of CHO cells were treated with MMC for two hours and held 

in G, or confluent for 0, 18, 25, or 48 hours . PHL received PHA and BrdUrd 

after the appropriate interval and were recultured for -72 hours . CHO cells 

were split after the appropriate interval and recultured for -26 hours with 

BrdUrd . SCE frequencies were similar for all the cultures with both cell 

types . Thus PHL and CHO cells were incapable of repair of MMC induced SCE 

damage under nonreplicating conditions . CHO cells were cultured in log phase 

for -14 hours after MMC exposure before Brdurd was added or confluent cultures 

exposed to MMC were split after 0 or 24 hours, cultured for 14 hours before 

BrdUrd was added and recultured for -25 hours . SCE frequencies were near 

control levels for all exposure conditions indicating repair of MMC induced 

lesions . PHL were treated with MMC, PHA was added 0 or 24 hours later, and 

BrdUrd was added 48 or 72 hours later, respectively . Again SCE frequencies in 

all these cultures were near control levels . MMC induced SCE in PHL were 

tested under differing exposure conditions to PHA and BrdUrd . These experiments 

indicate that MMC induced lesions are repaired only after one round of 

DNA replication . 


137 

W 

01 

fr 

N


Notes ETHICS AND THE ALLOCATION OF RISK : SCIENCE AND VALUES 

Thomas H. Murray, Ph .D . 

Center for Biomedical Ethics 

School of Nedicine 

Case Western Reserve University 

Cleveland OH 44106 


A large number of ethical questions posed by research on 

environmental mutagenesis need to be sorted out. They include 

research on human subjects, the responsible use of information, 

privacy and others . This paper will focus on an ethical question 

with considerable social importance : How should scientific findings 

and moral values be used in making public policy decisions about 

the control of mutagenic substances in the environment and in the 

workplace? The values that guide judgments about the relative 

certainty of scientific claims differ from the standards that are 

and should be used in making public policies that ought to be 

informed by scientific findings. The struggle over regulation of 

asbestos will form a case study of the interplay between science 

and values in the making of public policy on toxic substances . 

Such social decisions pose unavoidable questions about the 

allocation of risk, which are also ethical questions, specifically 

questions about justice . 

398 

399 

APPLICATION OF THE 32P-TECHNIQUL+ TO DETECT DNA-ADDUCTS BY CISPLATI N 

R . Mustonen and K . Hemminki . Institute of Occupational Health . 

Helsinki . Finland 

The main reaction products of Sjg-diamminedichloroplatinum (II), 

cisplatin. with DNA are intrastrand cross-links with dGpdG and . to a 

lesser extent, dApdG . Platinum can be conveniently measured by atomic 

absorption spectroscopy but with patient white blood cell DNA this 

technique is presently at the limit of its sensitivity . We have 

therefore applied the 32P-postlabelling technique to the DNA adducts 

of cisplatin . The standard compound, intramolecular crosslink Pt-dGpdG 

is labelled with T4 polynucleotide kinase and Y-32P-ATP, and the 

products are separated by PEI-TLC and visualized by autoradiography . 

standards containing a 5'-phosphate group are used as internal 

standards . The phosphorylation of the cisplatin-adducts is a slow 

process as compared to that of polycyclic adducts . but the levels of 

adducts detected with the standard compounds are presently belo w 

100 fmol . Adducts are also detected in DNA platinated vitro and 

digested enzymatically to nucledtides . 

CATECHIN AS AN ANTIMUTAGEN AND ANTICARCINOGEN . 

M. Nagabhushan, Northwestern Universit , Department of Pathology, 303 

E. Chicago Ave., Chicago, IL 60611, U~A . 

Betelquid chewing with or without tobacco is correlated with high incidence of oral 

cancer in India. Betelquconsists of betelnut, betelleaf, catechu, slaked lime and spices . 

Catechu is a nonawtagenic constituent of betelquid contain catechin as the principal 

compound. Catechu extract and catechin inhibits the muugeniciry of tobacco, masheri, bidi 

and cigarette smoke condensates, smoked meat charred and noncharred extracts, benzo(a) 

pyrene (BP) and dimethylbenz(a) anthracene (DMBA) in a dose dependent manner in 

salmonella/microsome test. Catechin inhibits the activity of Cyt P 450 and has no effect 

on GST, but increased (1SH content in rat liver. Simultaneous treatment of catechin 

prevents the mutagenicity of BP and DMBA metabolitis but pre or post-tteatment of 

bacteria with catechin has no effect on the mutagenicity. Catechin also inhibited the in vitro 

binding of 3HBP metabolitis to calf thymus DNA . Oral administration of catechin inhibits 

the incidence of BP-induced forestomach tumors in female swiss mice . This study 

indicates that catechu in betelquid may act as an antimutagen and anHcarcinogen and may 

suppress the mutageniclcarcinogenic potential of other betelquid ingnedients . 

400


CARCINOGEN INHIBITION OF HAMSTER BUCCAL POUCH EPITHELIAL CELL Notes 

REPLICATION jj`[ VITRO M . Nagabhushan, P. Polverini and D . Solt . Northwestern 

University Department of Pathology, 303 E . Chicago Ave ., Chicago, Il 60611 

Precancerous lesions induced in rodent liver with chemical carcinogens are 

resistant to the inhibitory effect which these agents exert on liver cell replication . 

This functional property of "resistance to cytotoxicity" has been used to detect 

very early precancerous liver cell populations . We wish to apply this same strategy 

to detect and characterize precancerous epithelial cell populations in hamster 

buccal pouch(HBP) -- a tissue which is structurally and functionally similar to human 

oral mucosa . As a first step toward this goal we have developed an approach 

to monitor HBP epithelial replication, and its inhibition, upon exposure to carcinogens 

L vitro . In a representative experiment, fragments of HBP were incubated 

in supplemented media for 24 hrs in the presence and absence of 7,12-dimethylbenzanthracene(DMBA) 

or methylbenzylnitrosamine(MBN). [3H]Tdr was added to 

the media for the final 2 hrs of incubation and acid insoluble counts were determined 

. At concentrations of 0 .15, 0.30, and 0 .60 mM, DMBA and MBN inhibited 

[3H]Tdr incorporation by 35%, 76%, 87% and 91%, 93%, and 98% respectively . We 

conclude that this approach, combined with autoradiography, may be useful for 

in vi detection of precancerous HBP lesions resistant to chemical carcinogens . 

402 

INHIBITORS OF NITROSATION IN VITRO . M . Nagabhushan, 

Northwestern University, Department of Pathology, 303 E . Chicago Ave., 

Chicago, IL 60611 . 

Nitroso-compounds are mutagenic/carcinogenic compounds formed by the 

interaction (nitrosation) of amine/arnide and nitrites . Human stomach maintains acid pH, is 

the most probable site of formation of nitroso-compounds . Fish, preserved meat, 

vegetables and drinking water are rich sources of amines/amides and nitrites . Several 

inhibitors of nitrosadon are known to be present in human diet . In the present study some 

of the inhibitors of nitrosation of tnethylur ea and fish extract by sodium nitrite at pH 3 .6 

and 2.0 are reported. Salmonella strain TAIOO and TA1535 are used to monitor the 

formation of mutagenic nitroso-compounds . The extracts (principles) of turmeric 

(curcumins), betel leaf (eugenol, hydroxychavicol), tea (Tannic acid) and catechu (catechin) 

exhibit dose dependent inhibition of nitrosation . The simultaneous treatment of inhibitor 

with precursors is essential for the inhibition . Pre or post-treatment of inhibitor does not 

modify the mutagenicity of nitroso - compounds. In case of phenolics pm - hydroxy 

group is essential for nitrosation inhibition . The nitrosation inhibition by phenolics in 

through the scavenging of nitrite ions from the media, thus making it non-available for 

nitrosation. 

403 

TRENDS IN ENVIRONtWNTAL CARCINOGENESIS 

B. Nagarajan, Cancer Institute, Madras - 600 020 INDIA 

rlost cancer is caused or promoted by life style and environmental 

factors . Carcinogenesis is a multiple process involving initiation, promotion 

and progression . Identification of several risk factors helps 

evolve effective approaches to control and contain the disease . In our 

laboratory, we carried out in-depth studies on hexachlorocyclohexane 

(ttCH), a commonly used pesticide . It is a hepatotoxic compound with 

potential tumorigenecity that induced gamma glutamyl transpeptidase 

positive foci, mostly in periportal hepatocytes . A complete carcinogen 

such as dimethylaminoazobenzene hits selected target cells and only that 

clone's cells proliferate to malignancy . In HCH treated cells, marker 

analysis both for differentiation and proliferation established an 

epigenetic promoter component . 

Clofibrate is an extensively used hypolipidemic drug . Cytogenic 

studies on drug-treated rat bone marrow cells and human lymphocytes in 

vitro showed minimal damage that repaired to normal on withdrawal of the 

drug . Biochemical studies also agreed with this observation . However, 

if the cells were pre-exposed to an initiator such as diethylnitrosamine 

and followed with clofibrate, they tend to exhibit tumorigenecity . A 

similar hazard risk assessment is warranted in the use of halogenated 

hydrocarbons . 


F 


Notes EFFECT OF GLUTATHIONE ON MITOMYCIN-C IN SWISS ALBINO MICE AND HUMAN LY1pNDCYTES 

RITA NARAM . D . GsethanJali and P .P. R@My 

Institute of Genetics Hospital for Genetic Disaases . owania Universitv . Baauaoet . Hvd.rabad-300 016 . 

A.P . India . 

AWIMOT 

Mito,yc in .C (MMC) is a potent antibiotic with autayenic and antitumor activity presusably through 

its covalent binding to DNA. MMC was tested for its autapanic potential in the presanca of plutethiona 

(GSH)in invivo and invitro systas . 

404 

Swiss albino .ice were fed orally with 2s0/Kp . b .w. M(C and 20.40 .80 and 160 ap/kR b .w . GSH slmultaneously 

to four groups of anLsals . The doses were administered at 0 hrs and 24 hrs . 6 hrs after the second 

dose the animals wra killed and aicronucleus test was dona as described by Schsid (1973) . Ll.phocyte 

cultures ware initiated fros healthy donors and 0 .2 uylUl MMC and 2 .5 . 5 .0 . 10 .0. 10 .0 and 20 .0 up/al 

GSH was added simultaneously to four se-ts of cultures . 72 hrs after treataant the cultures were tenli- 

neted and slides ware prepared as described by Moorhead et al ( 1960) . Thare was a significant decreese 

in the frequency of aicronuclei !n young erythrocytes and chroaosasal ebearrations in lyaphocytes 

treated with MMC in casbination with OSH when coapered to MMC alone . 

405 

EFFECT OF NICOTINAMIDE ON HEPATOTOXICITY ASSOCIATED WITH CARBON TETRACHLORIDE, 

BROMOBEN2ENE AND ALLYL ALCOHOL, L .M .Narurkar, J .P .Ramat, S .J .D'Souza, R .Krishnamoorthy 

& M .V .Narurkar, Biochemistry Division, Bhabha Atomic Research Centre, 

Bombay, India . 

Recurrent toxicity with cell necrosis resulting in regenerative stimuli has been 

proposed as one of the mechanisms of tumour promotion . Tumour promoters which are 

usually hepatotoxic agents may bring about selective inhibition of the cytochrome 

P-450 dependent microsomal drug metabolizing enzymes . It was observed that administration 

of tumour promoting and hepatotoxic agents such as carbon tetrachloride 

(0 .2 ml/kg .b .w .), bromobenzene (0 .2 ml/kg . b .w .) and allyl alcohol (30 mg/kg . b .w .)to 

rats brought about a significant inhibition of hepatic mixed function oxidase 

(MFO) system, while the conjugating UDP-glucuronosyl transferase activity was 

increased . The serum glutamate-oxaloacetate transaminase and serum glutamatepyruvate 

transaminase activities were also highly increased followin~ administration 

of these toxic agents . Further, when nicotinamide (250 mgs/kg . b .w. , an endobiotic 

shown in our laboratory to be an inducer of MFO, was administered simultaneously to 

rats along with hepatotoxic agents in independent experiments, the biochemical toxic 

manifestations were significantly modified . Besides the MF0 inducing ability, 

nicotinamide has been shown to bring about stabilization of polysomes as evidenced 

by marked reduction in monomers and dimers with simultaneous increase in the heavier 

aggregates, increase in total polysomal RNA accomganied by decreased alkaline RNase 

activity as well as enhanced incorporation of 1 C-leucine pulse in the ribosomal 

aggregates . 

406 

COMPARISON OF SISTER CHROMATID EXCHANGES IN SPLEEN AND THYMIC LYMPHOCYTES FROM AKR, 

B6D2F1 AND CBA MICE FOLLOWING IN VIVO EXPOSURE TO N-NITROSO-N-METHYLUREA . R .E . Neft, 

H .M . Schol and D .A . Casciano, National Center for Toxicological Research, Jefferson . 

AR 72079 

In order to evaluate the ability of the in vivo/in vitro murine lymphocyte sisterchromatid 

exchange (SCE) assay to predict carcnicity . SCE induction by N-nitro- 

N-methylurea (MNU) was studied in spleen and thymus lymphocytes from ARR mice which 

are highly susceptible to MNU-produced thymomas, CBA mice which are much less sensitive 

to induction of thymomas by MNU, and B6D2F1 mice . Following a single i .p . injection 

of 0 .033, 0 .066, 0 .131 mmol/kg MNU or PBS (vehicle control), clear dose-related 

increases in SCE were observed in LPS-stisulated spleen lymphocytes and Con A-stimulated 

spleen and thymus lymphocytes from ARR, CBA and B6D2F1 mice at 1 and 24 hours 

post-exposure . In general, MNU-induced SCE were higher in Con A-stimulated spleen 

lymphocytes compared to LPS-stimulated spleen lymphocytes and Con A-stimulated thymas 

lymphocytes from each mouse strain . On the whole, MNU-produced SCE were lower in 

AKR and CBA spleens than in B6D2F1 spleens . In addition, for the most part, l4iUinduced 

SCE levels in thymus lymphocytes from all three strains of mice were similar . 

In the present study, differences in MNU-induced genotoxicity in ARR, CBA and B6D2F1 

thymus lymphocytes could not be ascertained by use of the in vivo/in vitro SCE assay . 

( 

r 

F 50869 3654 



GENOTOXICITIES OF N-NITROSAMINES ON DROSOPHILA AND A SEARCH FOR THE INHIBITORS OF THE Notes 

TOXICITIES 

Tomoe Negishi, Teruko Shiotani and Hikoya Hayatsu 

Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700, Japan 

N-Nitrosamines are potent carcinogens for rodents, and their possible rr_levance to 

human cancer has been intensively studied . In spite of the strong carcinogenic activities, 

this class of compounds show only weak mutagenicity in the Ames bacterial tests . 

We have now found strong genotoxicities for several N-nitroso compounds using the Drosophila 

wing spot test . The test detects the activity of a given compound to cause gene 

mutations and recombinations in the larval somatic cells . N-Methyl-N-nitrosourea (MNU) 

and N-nitrosodimethylamine (NDMA) gave strong responses in this system, corresponding 

to their potent carcinogenicities . Although N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) 

is a strong mutagen against bacteria, its genotoxicity on Drosophila was moderate . The 

order of genotoxicities of these compounds in Drosophila was as follows ; MNU> NDMA 3-Nnitrosomorpholine 

> N-nitrosopiperidine Z N-nitrosodiethylamine > MNNG > N-nitrosopyrrolidine 

. Non-carcinogenic N-nitrosodiphenylamine and N-nitrosoproline were not genotoxic 

to Drosophila . Since this Drosophila system seems to be suitable for the detection of 

N-nitrosamine-mediated genotoxicities, it may be expected that this system is useful 

to search for modulators of the genotoxicity . We examined the inhibitory potential of 

several vitamins on the NDMA-mediated genotoxicity in Drosophila . We observed, however, 

no suppressive effect in 6-carotene, vitamin B1, vitamin C or vitamin D2 on the genotoxicity 

of NDMA . 

408 

EFFECT OF POLYUNSATURATED FATTY ACID ON DMH INDUCED COLON CANCER . 

Nehru, B ., Kaur, R ., and Bansal, M .P . 

Deptt . of Biophysics, Panjab University, Chandigarh-160014, India . 

The :e is a strong positive correlation between the dietary fat consurption and the 

prevalence of cancer of breast, colon, prostate and pancreas . The underlined mechanism 

are quite complex, multifactorial and different in different types of cancer . The 

present study was undertaken to study the role of unsaturated fatty acid in the initiation 

of colon cancer by a chemical carcinogen 1,2 Dimethyl-hydrazine administered 

in a dose of 20 mg/ g body weight / week subcutaneously . Three levels of dietary fat 

manipulation was used 10% fat, 20% fat, and 30% of the total calore . Marked alteration 

were seen in the drug metabolizing enzyme of colon follgwing a high fat dietary regime . 

The observations were similar when DMH was given along with the high fat diet . The 

cytochrone P 450 content showed a 4 fold increase in the high fat group . A decrease 

in phase II enzyme namely glutathione and glutathione-S-transferase was noted . 

409 

A MULTI-FACTOR RANKING SCHEME FOR COMPARING THE CARCINOGENIC ACTIVITY OF CHEMICALS . 

S . Nesnow, Carcinogenesis and Metabolism Branch, U .S . Environmental Protection Agency, 

Research Triangle Park, NC 27711 USA 

This activity scheme uses as its base, dose potency measured as TD50 (Gold et al ., 

Environ . Health Perspect ., 67, 161-200, 1986) The TD,r0 is converted into an inverse 

log scale, a decile scale, and then adjusted by weighting factors that describe other 

parameters of carcinogenic activity . These factors include positive or negative 

weightings for the induction of tumors at tissues or organs associated with high 

historical control tumor incidences ; the induction of tumors at multiple sites ; the 

induction of tumors in both sexes of the species ; and the induction of tumors in more 

than one species . In order to construct a measure to express the inactivity of 

chemicals as inducers of cancer, a measure analogous to the TD50 has been developed : 

the highest average daily dose or HADD . The HADD is the highest average daily dose in 

mg chemical/kg body weight administered in a chronic cancer study, and that did not 

induce a statistically significant increase in tumors . HADD values were converted to 

log decile units and adjusted by weighting factors relating to inactivity in both 

sexes of a species, and the inactivity in more than one species . Three activity 

ranking schemes were developed using a 142-chemical data set from NTP studies : the 

Carcinogen Activity-F344 Rat, an activity scheme based on cancer data obtained with 

the F344 rat ; the Carcinogen Activity-B6C3F1 Mouse, an activity scheme based on cancer 

data obtained with the B6C3F1 mouse, and the Carcinogen Activity-Combined, an activity 

scheme based on selecting data from both the F344 rat and the B6C3F1 mouse . 

This is an abstract of a propos .d pnfantation and does not nflact EPA policy . 


142 1989 EMS Abstracts _ 410 


NOteS- "" y'"RECOMMENDED 1TAMM PROTOCOLS BASED ON A SURVEY OF CURRENT PRACTICES IN GENOTOXICITY 

TESTING IABOR{RORIES . E .R . Nestmann(1), S .H .H . Swierenga, R .L . Brillinger(1), and 

J .P .W. Gilman ~.Directorate, Health Protection Branch, Ottawa, and (1) CanTox 

Inc ., 627 Lyons lane, Suite 200, Oakville, (Canada) 

The most commonly used genotoxicity assays for cultured mammalian cells are 

mammalian cell mutagenesis, chromosome aberrations/SCE, hepatocyte UDS, and cell 

transformation . Since their inception, protocols for these assays have been modified 

in various laboratories . It has been observed that minor but potentially significant 

method modifications frequently remain unpublished (Swierenga et al ., J . Tiss . Cult . 

Meth . 8 :7, 1983) but should be considered in the development of standard protocols . 

The present study was undertaken to determine the current "state of the art" for these 

tests . Detailed questionnaires on culture conditions and testing protocols for both 

stock and test cell populations were designed with the assistance of an international 

advisory committee and sent to all research and contract laboratories that could be 

identified in Canada, USA, and Europe . Responses from 425 completed questionnaires 

were analyzed to determine the most commonly used approach and modifications for each 

procedural step . As expected, the results show a large degree of interlaboratory 

variation . Detailed protocols for conducting each assay have been prepared and 

include : stepwise instructions, precautionary measures and practical solutions to 

common problems associated with each assay ; recipes for media and solutions ; formulas 

for quantifying genotoxic responses ; reference lists of related assays ; guidelines for 

interpretation ; and discussions of the applications, advantages and disadvantages of 

each test . 

411 

CRITERIA FOR ASSESSMENT OF GENOTOXICITY DATA WITH RESPECT TO IN VIVO MUTAGENICITY AND 

MECHANISM OF CARCINOGENICITY . E .R . Nestmann and L .D . Kier, CanTox Inc ., Oakville 

(Canada), and Monsanto Co ., St . Louis (U .S .A .) 

Evaluation of genotoxicity databases often is complicated by a mixture of positive 

and negative results . We have identified general criteria that can be used in a 

"weight of evidence" assessment . Evidence for Jja y1yQ genotoxicity is strenothened by 

any or all of the following : induction of genetically stable effects (i .e ., mutation) 

rather than indicator endpoints of DNA damage (e .g ., SCE, UDS) ; activity for multiple 

endpoints ; in vivo (rather than only in vitro) effects ; induction by appropriate route 

of exposure and at not overtly toxic doses ; structure and pattern of activity related 

to known mammalian mutagens ; strong responses . The case for j,p yjys genotoxicity is 

weakened by : negative in vivo results ; positive results only In vitro, especially 

activity that is reduced or eliminated in the presence of metabolic fractions ; activity 

that is only observed under conditions known to cause artifacts (e .g ., high 

cytotoxicity or osmolality, low pH) . Evidence for carcinogenesis through a genotoxic 

mechanism is strengthened by : consideration of the above criteria for in vivo 

genotoxicity ; correlation of in vivo genotoxicity and carcinogenicity findings (e .g, 

tissue or species specificity ; route) ; similar pattern of response as chemically 

related carcinogens . Evidence for a nongenotoxic mechanism is strengthened by : 

activity only in tests with low specificity ; other evidence for nongenotoxic or 

promoter mechanism of carcinogenesis . (Supported in part by the AIHC, CMA and 

ILSI/RSI ; contributions of the AIHC Mutagenicity Subcommittee are gratefully 

acknowledged .) 

412 

MOLECULAR ANALYSIS OF Zl= MUTATIONS ARISING jg VIVO IN HUMAN T-LYMPHOCYTES, J .A . Nicklas, 

T .C . Hunter, J .P . O'Neill, L . Recio, D . Simpson, T .R . Skopek, and R .J . Albertini, VRCC 

Genetics Lab, Univ . of Vermont, Burlington, VT and CIIT, Research Triangle Park, NC . 

hort and T-cell receptor (TCR) Southern blot analyses were performed on 96 wild type 

and 330 somatic h= mutant clones from three normal individuals . h= analysis showed 

that 16 .0, 16 .5 and 9 .6% of the mutations had visible b= structural alterations in the 

3 individuals, respectively . The breakpoints of the deletion mutations were spread across 

the gene in proportion to length (r- .94) with 0 .73 breaks/kb, allowing an estimate of the 

nearest flanking vital genes of 18kb 5' and 26 .5kb 3' . TCR analysis determined that 91, 

85 and 85% of the mutants were i .ndependent T celi clones (i .e . had different TCR gene 

rearrangements with TCRj8 or y gane probes) . Doublets, triplets, quadruplets and one 

nonamer ∎et of sibling clones were observed . The average persistence of a clone was 

2 .8,2 .9 and 1 month, respectively although the nonamer siblings persist after 2h years . 

We have previously described the extensive proliferation of a T call clone in another 

normal individual resulting in an elevated mutant frequency with greater than 90% sibling 

clones . One individual had 6 mutant clones with the saue apparent exon 2-3 deletion and 

new size axon 1 fragment ; by TCR analysis 4 are sibling clones while 2 share only TCRB 

gene rearrangements . This latter fact suggests an intrathymic mutation or extra-thymic 

TCRy gene rearrangement . This same apparent deletion was found in a mutant from another 

individual suggesting a"hotspot" for mutation . We found two cases of an apparent TCR 

clone (i .e . the same TCR gene rearrangements) containing 2 different b= mutations which 

is evidence for sensitivity of a dividing TCR clone to mutation . We are currently 

sequencing the mutants which did not show hp= gene alterations to define a spectrum of 

spontaneous" somatic mutation in man . Supported by NCI CA30688 . 

50869 3656

413 ~ 1989 EMS Abstracts 143 

DNA SEQUENCE CHARACTERIZATION OF ALKYLATION-INDUCED YERMILION Y{TPANTg IN DROSOPBILA. Notes 

t( .J .11 . Nivard, A .PastinkYand E .1f .Voge1, University of Leiden, Leiden (The Netherlands) . 

The aim of this study hsi been the characterization of base sequenoe changes induced 

by ethylnitrosourea (ENU), ethylmethanesulfonate (Et(8), and methylaetbanesulfonate 

(MMS) at the vezm{Zion locus of Drosophila, after treatment of postmeiotie male germ 

cells . The analysis included consideration of the position of the carcinogen on the 

potency scale for carcinogenicity (TU50 in relation to initial alkylation pattern ; 

collaboration with Drs .B .Bartsch and A .Barbin, IARC, Lyon), and alteration of the relative 

distribution of primary DNA lesions . Sequence analysis of mutants was performed 

using the recombinational screening method by 8eed in combination with dideoxy-sequencing, 

and the PCR method . All vermtZion mutatlons induced by ENU and EtE were due to 

base-pair changes, and all 22 mutations induced by EIIS represented OC : AT transitions . 

With ENU, 29 nucleotide substitutions were found in 26 mutants (3 mutants had 2 basepair 

changes) . Of these mutations, 23/29 (79%) were transitions and 6/29 (21%) transversions 

. In 18/29 (62%) of the mutations tiC : AT transitions were observed . Our results 

support the view that 08-ethylguanine is the premutagenic lesion in DrOsophila after 

treatment with EYS and ENU . In the latter case, other products of O-ethylation (02EtT, 

04EtT) seem to be involved . Characterization by DNA sequencing of 40 mutations induced 

by MMS revealed a decidedly different spectrum in relation to EMS and ENU : 21/40 

mutations (53%) were tranversions, 8/40 (20%) deletions and 10/40 (25%) transitions . 

Misrepair and indirect miscoding seem the predominant mechanisms of action of high S 

value type carcinogens . It is this type of genotoxin for which strongest hypermutabllity 

effects are obtained in an excision repair defective background . 

414 

NA REPAIR TESTS ON FOOD ADT)ITIVBS 

N'ichiko Non :,ka 

iaboratory of Food Chemistry, Faculty of Agriculture, 

Kyushu University, ?iunzoka, Japan . 

The liquid Bacillus subtilis rec-assays were carried out 09 25 food 

additives, all of which are eurrently used in Japan . Briefl .y, the overniBht 

culture of . subtilis strains, H17 eLnd ;d45, was mixed with test 

solutions and incubated for 30min at 37'C . After treatment, viable 

cells aere co•.i .Vltec2 and the ratio of 5M, .• stu'vi,va.l, coace:ltr,itions were 

calculated . 18 additives ((acetone, L-asoorbic acid, bettsoio acid, ethyl 

acetate, Food Red No .2(tanaranth), Food 3ed No .3(erythrosine), Food Red 

No .106(acid red), Food Yello•,v :To .h(tartrazine), glycerin, hexane, pota,ssium 

sorbate, propylene rlycol, nropyl gel3e.te, sacchexin sodium, 

socitvn benzonte, sodium dehydro3cetate, sodium nitrate, sodium sl-te .rtrste) 

ehovaed D :•', damaging potential e.lthough they had been negative in 

the ~ .~nes test . Seven additives (erythorbic acid, Food Green No .3(Fast 

Green FCP), hydrogen peroxide, potassium bromate, soflium nitrite, cacao 

;i&nent, cochineal) showed D'Ta damaging potential, which had been positive 

in the Ames test . These s+tr~-eRi ; ''s,+." ~; the ltj,tt' Bs subtilis 

rec-=- se : y 1-~ y be more sensitive than the Ames test . - 

415 

THE MICRONUCLEUS ASSAY IN LYMPHOCYTES. 

H . Norppa, M . Hayashi, and J . Miki-Paakkanen, Institute of Occupational Health, Topeliuksenkatu 41 aA, 

SF-00250 Helsinki, Finland . 

Cytogenetic monitoring of human populations exposed to genotoxic agents has almost exclusively 

relied on the use of peripheral lymphocytas . The sensitivity of the lymphocyte culture system In detecting 

in vivo exposure may not always be as good as desired, but it could be improved by more extensive 

analyses, method development and automation . In the human lymphocyte micronucleus assay, a major 

break-through has been the use of cytochalasin B, introduced by Fenecb and Morley, to idenNfy cells 

that have divided once in the culture . The recognition of these eells by the presence of two nuclei has 

abolished the earlier problems experienced with differences in cell growth and haa made the scoring of 

micronuclei in lymphocytes more accurate . Our experience on human monitoring studies, however, suggests 

that - in order to catch the cells after their first division - cytochalasin B treatment has to begin 

considerably earlier than originally suggested . As cytochalasin B appears to have some toxic effects on 

the lymphocytes, it would be good to have an alternative way to recognize the relevant eell population . 

At our laboratory, we have been developing a technique based on S-bromodeoxyuridine (BrdU) 

incorporation combined with a monoclonal antibody against BrdU-DNA, in order to produce a method that 

could be used as a basis for automated scoring of micronuclei by image analysis . BrdU can also be used 

without the antibody by applying a differential staining technique, but the antibody method is more 

specific and more suitable for automation, because only labelled nuclei are visible. For both of these 

approaches, the timing of BrdU treatment and cell harvest are ths critical points . It Is probable that 

micronucleus analysis in lymphocytes, with its rapid scoring and suitability for automation, will be an 

important alternative for the traditional analysis of metaphase chromosome aberrations in monitoring 

human exposure to genotoxins as well as in screening of elastogens in vitro . 


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144 1989 EMS Abstracts - • s -- 

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416 

NOteS ^ ENVIRONMEN?AL MaU AGENESIS RESEARCH IN DEVELOPING COUNTRIES 

N .K . Notani, Biorcal Group, Bhabha Atanic Research Centre, Borbay 400085, India 

Indian reseaTcFfers-~trave wide interests and work on problens ranging from DNA repair, 

gene cloning, mutagens and entinutagens, test-syste•ns to nature of heritable variation in 

man, etc. Environ•nental Mutagen Society of India affiliated to IAEMS provides a major 

annual forun within the country and has also periodically organized courses, both international 

and national, for teaching of new techniques. Besides, it has cosponsored m eetings in related 

areas with other societies. In our Biorrsedical Group which m ay be considered as representative 

of research activities in the country has projects on mutagenicity testing, modification of 

mutagenicity by antrnutagens, mutation surveillance in control and high background radiation 

areas, cloning of DNA repair genes, Restriction Fragnent Length Pol)morphisn analysis 

and ostrn eiotic expogure leading to transn issible genetic dan age . Two of the researches 

are ~escribed below : i) DNA repair and gene cloning : We have self-cloned two genes, uvrl 

and mbo2, in Haenophi(us Tnt uenzae, u g e s carried on an 11 .3kb insert. This OIm1 

clone fully canple•nents UV-sensitive strain Uvr1- . A directedtnutagenesis system was 

developed which then induces mutations only in the 11 .3kb insert (Kanade and Notani,1987) . 

Another DNA repair gene mbo2 has also been cloned which fully canplenents UV-sensitive 

Mbo2- strain but not UvrT~strain . Subcloning of two EcoRl fraqnents yield clones; only 

the snaller of which partially canple•nents UV-sensitivity of Mbo2- strain (Mody, Joshi 

and Notani, 1989). ii) Newborn surve of o ulations residi in control and hi h bac round 

radiation areas: Georg e et ai have exa~n ne new orns for wn syn ro•ne and 

'R>;L'h-06TSi1S88- date for Incidence related to m aternal age . Although, the data fror high 

background radiation areas are linited, these nevertheless see•n to be sinilar in incidence 

to the control area . 

CROSS ADAPTIVE RESPONSE BETVEEN HYDROGEN PEROXIDE AND ALDEHYDES IN E . croli. . 

T . NUNOSHIBA, M . HASHIMOTO, and H . NISHIOKA, Biochemistry . Doshisha University, Kyoto(Japan) 

417 

The phenomenon of the adaptive response that the bacterial cells acquire resistance to killing 

effect of a lethal dose of hydrogen peroxide(H202) . when they have been pretreated with 

its sublethal dose . has been noticed . Ve report here the existence of the adaptive response 

between H202 and aldehyde compounds . The phenomenon was na .ed by us as "Cross Adaptive 

Response" . The cells of E .2Qjj VP2 pretreated with a sublethal dose (30 or 80µM) of HzOz for 

30.in at 3TC acquired remarkable resistance to killing effect of for .aidehyde(FA) challenge 

at the concentration of B .M . The resistance acquirement due to pretreatment with a low concentration 

of H202 was similarly observed in the cases of challenge against the other aldehyde 

compounds such as chloroacetaldehyde . glutaraidehyde, glyoxal and methyl glyoxal . To elucidate 

the .echanism of the "Cross Adaptive Response", the effect of pretreatment with Hz02 on 

FA-sensitivity of ji . i strains with different DNA repair capacities was examined . The acquire.ent 

of the resistance was observed in VP7uvrA[V1f.l1] and ZA12[ug&] . but not in ZA80 

[recA] and CM561[IexA] . These results suggest that the "Cross Adaptive Response" Ray be attributed 

to induction of SOS genes coding some enzymes which are involved in reco .bination 

repair . the other unknown DNA repairs and aldehyde decomposition . 

418 

Cenotoxic effects of creosote oils : a relationship between ssutagenicity and the concentrations 

of A-S ring polycyclic aromatic hydrocarbons 

Yylund, L ., Heikkili, P ., Ji[rventaus, H ., Suhonen, S . . Hesso, A ., Himeill . !( . . 

Linnainsiaa, K . and Sorsa, tl . 

Institute of Occupational Health, Topeliuksenkatu •laA, Sy-00250 Helsinki, rinland 

Pour creosote oils iaported to Finland from different countries were tested using 

the Ames test, the SCS-test with CHg-c .lis and an automated version of the SOS chrosotest 

. All creosote oils were positive using $ . tvohimurius TA98tS9aix . With strain 

TA100aS9aix only two of the creosote oils were positive . The SCS-test showed results 

similar to the Ames test results with strain TA98i89aix . The two oils that were positive 

both with TA98 and TAlOO were also positive in the 808 chromotest .The taped plate 

assay for compounds evaporating at 37•C gave negative results for all the four creosote 

oils . The creosote oils wre fractionated by distillation and the fractions were 

tested using the Ames test . Mostly, the wtagenic activities were observed in fractions 

with high-boiling compounds . The cheaical compositions of the creosote oils were 

determined using OC/!tS-techniques . It was found that the wtagenic activities with 

TA98a89mix correlated well (r .0 .96, p

419 

, 

AN SVALUATION OF TBB C90/BGPRT MUTATION ASSAY INVOLVING SOSPSNSION CULTURES AND SOFT 

AGAR CLONING : RESULTS FOR 33 CHBKICALS . T . Oberly, M . Rexroat, s . Devsey, and K . 

Richardson . Lilly Reseatrh Laboratories, Rli Lilly & Co ., Creenfield, IN 46140 . 

The Chinese hamster ovary cell assay (C80) which measures forward mutation at 

the BGPRT locus is utilized in several laboratories for the detection of mutagens . A 

procedure involving treatment of CHO cells in suspension culture and mutant selection 

in soft agar cloning has been developed (Oberly et al ., Mut . Res ., 18St99, 1987) . In 

order to evaluate the effectiveness of these modHications, ff-chemMals representing 

six chemical classes were tested, and the results were compared to findings obtained 

with the additional assays "ployed in the battery of tests . A positive response was 

obtained with 20 chemicals, all of_vhich are recognized sutagens . Of the 14 

compounds that produced negative results, 4 were considered to be sutagens and/or 

carcinogens . For example, the rat hepatocarcinogen methapyrilene was negative in the 

CHO assay, and this finding agreed with all six test systems in the battery . In 

contrast, the suspect carcinogen, 4-nitrobiphenyl, was negative in the C80 assay but 

positive in 3 of 5 other test systems . Twelve of the compounds mentioned in this 

report have been previously tested in other laboratories, and as a comparison, the 

results reveal strong agreement between laboratories . These findings confirm that 

the use of suspension cultures and soft-agar cloning in the CBO assay maintains the 

sensitivity of the test in the discrimination of mutagens and is a viable alternative 

to the traditional monolayer procedure of O'Neill et al . (Hut . Res ., 59 :109, 1977) . 

In addition, the modified CHO assay has contributeT-to an overalrTsptovement in the 

cost effectiveness for the assay which will be discussed . 

420 

SISTER-CHROMATID EXCHANGES AND CHROMOSOME ABERRATIONS INDUCED BY 

CISPLATIN IN CULTURED HUMAN LYMPHOCYTES AND INHIBITORY EFFECTS BY 

SODIUM THIOSULFATE . 

T .Ohe, S .Tsuda*, Y .Sakata and T .Abe, Department of Hygiene and *Medicine, 

Kyoto Prefectural University of Medicine, Kyoto 602(Japan) 

Cisplatin(CDDP) is one of the most commonly used cytotoxic chemotherapeutic 

agents, but has serious side-effects including renal and 

haematological toxicities . On the other hand, it is known that CDDP 

therapy combined with STS brings effective relief from such severe sideeffects 

. In the present experiment, we examined the cytotoxicity, 

SCEs and chromosome aberrations induced by CDDP in cultured human 

lymphocytes, and in vitro interaction of CDDP4and STS on the frequencies 

of SCEs and chromosome aberrations . 

The mitotic index decreased ablyptly at 2 x_610-6 M and the yield of 

SCES was dose-related between 10- and 4 x 10 M . A dose-dependent 

increasg in frequency of chromosome aberra~ions w_is also found ug to 

4 x 10 M . Simultaneous treatment of 10 - 10 M STS and 10 M CDDP 

significantly reduced the number of CDDP-induced SCEs . Furthermore, 

treatment of cells with STS 3-hr later after CDDP administration 

significantly prevented toxicities as revealed by SCEs and chromosome 

aberrations . 

421 

RELATION OF LIVER C:.RCItiOt~.A TO ENVIRONME.NTAL FdC':ORS IN A TROPICAL RAIN-BELT CITY . 

:,.E . Ohwovoriole and G .C.E, Okeke, Dept . of Medicine, College of Medicine, University 

of Lagos, LAGOS, ATGERIA . 

Carcinoma of the liver is the commonest malignancy in many parts of tropical 

Africa . The reasons for its high prevalence in many countries are not certain but 

there are pieces of epidemiological evidence from other countries that environmental 

factors may play a dominant role in the aetiology of liver cancer in the tropics . 

Here we present an analysis of environmental factors in 104 patients with carcinoma 

of the liver seen in a large tropical hospital in the rain belt of Nigeria . There 

was a male predominance . Over 300 of the patients had historical or biochemical 

evidence of serum hepatitis (hepatitis B virus infections) . About 30 to 40 percent 

respectively gave a fairly strong history of alcohol and tobacco . A large rnanber of 

the patients admitted to ingestion of local herbs in the form of concoctions . The 

occurrence of ingestions of peanuts was no higher than in hospital controls . 

Environmental factors appear to contribute significantly to the occurrEnce of 

carcinoma in Lagos like in the rest of tropical Africa . However the sex ratio and 

peak age differ somewhat from that reported from the northern part of the country . 

The aetiology of liver carcinoma appears multifactorial . There is need to study 

further the role of co-carcinogens or other mutagens besides hepatitis B virus in the 

causation of liver carcinoma . 



Notes

146 1989 EMS Abstracts . 


- r~s - 

Notes - NOLECUIJ1It~YSIS Y OF N-HYDROXY-2-ACETYLMINOFUlONF1V~-INUCED MVfATIGNS IN 

CHIN~SE CELLS LnCRING DiA ERCISION REPAIR . R .T. Okinaka, S .L . Ansick, 

and G .F . StrQis~i..Genetics Group, Life Sciences Division, Los Alamos National 

Laboratory, LZSs-amos, M 87545 (USA) . 

It is generally believed that damage to DNA can be fixed as heritable mutations 

that are a conssquence of errors in repair or replication through the 

damaged sites . In order to better understand the role of DNA replication in the 

fixation of mutation, we have employed in our studies a DNA excision repairdeficient 

Chinese hamster cell line (CH0 W-5) . initial experiments were 

designed to define the magnitude of possible deletion mutations induced at the 

X-linked hypoxanthine-guanosine phosphoribosyl transferese (HPRT) locus 

N-hydroxy-2-acetylaminofluorene (N-OH-AlLLr) using restriction enzyme digesti~ 

Southern blotting and probing techniques (with a cloned hamster V79 cell HPRT 

cDNiA) . However, our observations indicate that none of the N-OH-AAF-induced 

UV-5 mutants analyzed to date contain large deletions or rearrangements at this 

genetic locus . In order to define the molecular nature of these mutations, we 

have recently employed a modification of the polymerase chain reaction (PCR) 

technique in conjunction with direct sequencing strategies . Preliminary results 

indicate that some of these mutants contain truncated HPRT s*As, possibly the 

consequence of small deletion events or processing errors in introNexon 

splicing . (This research is supported by the U .S . Department of Ehergy under 

contract W-7405-ENC--36) 

422 

423 

OVEP.VIEW OF IN VIVO MATQIALIAN TESTING SYSTEMS . F . B . Oleson, Bristol-Myers Company, 

Syracuse, New Yorc-(USA) . 

Both somatic and germ celle can be targets for genetic damage . In vivo mammalian 

assays for both types of damage will be described . Germ cell assays incfude spermatocyte 

cytogenetics or the new biochemical specific locus assay (currently under 

validation), specific locus and heritable translocation assays, and the rodent 

dominant lethal assay . Somatic cell assays have historically focused on the bone 

marrow, including cytogenetic studies for chromosomal aberration (CA) or, more 

recently, the erythrocyte micronucleus test . Other in vivo tissue-specific genotoxicity 

assays will briefly discussed, including 1) sistar c4rcomatid exchange (SCE) 

in bone marrow, 2) in vivo/in vitro hepatocyte DNA repair, 3) host mediated and 4) 

rodent lymphocyte CATSCE tests . Promising new test systems will also be presented 

such as the use of tranegenic mice for studies of oneogene activation and the single 

cell gel (SCG) DNA fragment electrophoresis assay . International regulatory 

guidelines for drug and chemical safety evaluation uniformly recommend one in vivo 

study be performed as part of a battery of mutagenicity tests . Bone marrow CK-or 

micronucleus assays are the recommended systems . Recent validation studies support 

the micronucleus test as the primary in vivo screen . The sensitivity and efficiency 

of these systems have been improved Sy moaffication of dosing/sampling time design, 

use of automated scoring techniques and the use of mouse peripheral blood for 

micronucleus testing . Finally, the rationale and scientific justification for the 

integration of multiple genetic toxicology endpoints into standard subacute (7- or 

30-day) rodent toxicology studies will be outlined . 

424 

AUTOMATION OF MOUSE PERIPHERAL BLOOD MICRONUCLEUS SCORING . F .B . Oleson . S .M . Getman, 

H . Mahran and G . Jenkinson, Bristol-Myers Company, Syracuse, NY (USA), and Cambridge 

Instruments, Inc ., Deerfield, IL (USA) and Cambridge (UK) . 

Procedures for the automated scoring of fluorescent stained mouse peripheral blood 

smears have been developed . . Peripheral blood smears stained with acridine orange are 

analyzed under high resolution microscopy in combination with a Cambridge Instrumants 

Quantimet 520 Image Analyzer . Orange-fluorescing polychromatic erythrocytes (PCEs) 

and yellow-fluorescing micronuclei are enumerated via the image analyzer . The total 

erythrocytes scanned in all fields analyzed is automatically estimated using a 

standard per field cell count . A motorized stage and autofocus allow for the rapid . 

analysis of individual fields for the total number of PCEs and micronucleated PCEs 

(MN-PCEs) . For each slide, the percent !Qi-PCEs and percent PCEs in total erythrocytes 

scanned are calculated and automatically tabulated by computer . Validation 

data comparing manual and automated micronucleus scoring will be presented including 

estimates of automated counting errors in the MN-PCE and PCE frequencies . Counting 

speed, limitations and problems with automated procedures will be discussed . Manual 

peripheral blood micronuclaus scoring for a standard study using 30 animals (1000 

PCEs/animal) and one evaluation time can be reduced from appror.imately 10 days to 2-3 

days using these automated procedures with no significant difference in results . 

These procedures should provide for improved assay sensitivity and reliability since 

appreciably more cells can be scored per animal than could be reasonably accomplished 

via manual scoring techniques . 

50869 3660

425 

r 

GENOTOXIC AND EPIGENETIC MECHANISMS OF CHEMICAL CARCINOGENESIS s IN VITRO AND IN VIVO 

STUDIES . L . Orfila, C . La ~ne, I . Chouroulinkov . Cancer Research Institute, C .N .R .S ., 

94802 Villejuif Cedex, France . 

Genotoxicity of chemicals ia generally considered as the only mechanism of carcinogenesis 

. However, it was found chemicals which are mutagenic and carcinogenic, or which 

are mutagenic but not carcinogenic, and finally which are not mutagenic but carcinogenic, 

such as TPA (PMA) . It is obvious that epigenetic mechanisms of carcinogenesis 

are clearly involved . The question is to know the'respective importance of each of 

those mechanisms in carcinogenesis . To give some answer,we have studied the effects of 

benzo(a)pyrene (BaP) 12-0-dimethylbenz(aYaT :thracene (DMBA), TPA (PMA),4-NQO, 3Me-4-NQO, 

testosterone and trembolone in the Syrian hamster embryo (SHE) cell transformation 

system, induction of ODC and epidermal hyperplasia as well as evaluation of BaP-DNA 

covalent binding in SHE and epidermal cells . Moreover, dexamethasone (D)040) was used 

as inhibitor of epigenetic effects in the different systems . The results showed that 

DXMO inhibits cell transformation,ODC and hyperplasia induction without inhibition of 

BaP-DNA covalent binding . From these results and data from long term skin tests we can 

classify the chemical carcinogens in 3 classes : with double activity,genotoxic and 

epigenetic activity (BaP,DMBA,4-NQO), only epigenetic activity (TPA,testosterone, 

trembolone) and only genotoxic activity (3Me-4-NQO) . The genotoxic effect appears to 

play a less important role in carcinogenesis than the epigenetic one . 

426 

IMMUNOHISTOCHEMICAL LOCALIZATION OF DNA-ADDUCTS FORMED DURING DNA R6PLICATION . 

Ofelia A . Olivero and Miriam Poirier . Laboratory of Cellular Carcinogenesis and Tumor 

Promotion, National Cancer Institute . Bethesda, MD 20892 . 

It has been previously reported that synchronized CHO cells exposed to H-acetoxyacetylaminoflurene(N-Ac-AAF)show 

a consistent pattern of preferential adduct formation 

in specific regions of metaphase chromosomes . Preliminary experiments suggested that 

chromosomal adduct localization correlated with the phase of the cell cycle in which 

the exposure took place . To further investigate this phenomenon, shake synchronized 

CHO cells were given simultaneous non toxic doses of 5-bromodeoxyuridine(BraU)and 

N-AC-AAF for 15 and 30 min periods respectively at times either early or late during 

the 8 hours of S phase . Cells were allowed to complete replication and were arrested 

in metaphase with colchicine . Metaphase chromosome spreads were processed for immunofluorescence 

using anti-BrdU antisera and a fluorescein :ltagged second antibody to localize 

sites of HrdU incorporation by DNA replicative synthesis . An antiserum specific 

for guanosin-(8-yl)-aminofluorene and a streptavidin-biotin second antibody system 

tagged with Texas red were used to localize DNA-adducts .The immunofluorescence staining 

showed evidence of adduct formation throughout the whole of each chromosome, but bright 

spots indicated regions of high adduct concentration . The BrdU incorporation sites were 

coincident with the areas of high adduct concentration, indicating a correlation of DNA 

damage and DNA replication . These results suggest that the most concentrated DNA-adduct 

formation is a cell cycle dependent phenomenon and occurs in those regions of the 

chromosome that were replicating during carcinogen exposure . 

427 

FORMATION OF MUTAGFNIC COMPOUNDS BY INTESTINAL BACTSRIA 

T .Osawa, M .Namiki, S .Kawakishi, K .Suzuki and T .Mitsuoka, Dept . of Food Science & 

Technology, Nagoya University, Chikusa, Nagoya 464-01(Japan), and The Institute of 

Physical and Chemical Research, Wako, Saitama 350-01(Japan) . 

In the past several years much attention has been given to the correlation between 

intestinal microflora and the formation of colonic cancer . One area of specific 

interest has been the correlation of microflora to cocarcinogens produced from 

dietary components through interference with intestinal bacteria, however, no clear 

evidence for this correlation has yet been obtained . 

Preliminary experiments showed that high mutagenic activity exists in the cellfree 

homogenates prepared from the six strains of intestinal bacteria in the fortyfive 

strains . High DNA damaging activity has been observed in the ethyl acetate 

extracts obtained from Streptococcus faecium IB 37 and Veillonella parvula ATCC 10790 

as shown by the differential growth inhibition between the Bacillus subtilis strains 

H17 rec+ and M45 rec- . After a large scale incubation of the strain S . faecium IB 

37, isolation and purification of the active principles have been carried out . 

Instrumental analyses confirmed the structures of two of DNA-damaging substances as 

indole and streptindle, respectively . Streptindole is the first genotoxic di-indole 

derivative found in the metabolites produced by intestinal bacteria . Streptindole 

was found to be negative by bacterial reversion assays with Salmonella typhimurium 

strains TA98, TA100 and TA104, however, positive in the colonic nuclear aberration 

assay using C57BL/6J mouse . 



Notes


- . 

Notes - 


428 

dv~ -- _ . _• 

.+ CENOTOXIC AC9MT#S06F MUCONDIALDEHYDE . Y . 0ehiro I C .E . Piper,l S .C . Gad,1 E . 

Rohrbacher,l 1r-S . Balwierz,l S .G . Soelter,l C . Nitzi and B .D . Goldstein .2 G .D . Searle 

& Co .,l Skokie, IL jMW77 and Dept . of Environmental and Community Medicine,2 

University of-4d nd Dentistry of New Jersey, Piscataway, NJ 08854 . 

Trans, trans-mucondialdehyde 1s a benzene metabolite that is speculated to cause 

leukemia in humans . We have examined its genotoxic activities using the Ames test, a 

CHO/HGPRT/micronucleus combined end-point assay and the rat primary hepatocyte UDS 

assay . In the first Ames experiment, TA1535, TA100, TA1538, TA98 and TA97 strains were 

treated with mucondialdehyde at six concentrations ranging from 0 .5 to 100 Ug/plate . 

The high dose of 100 yg/plate was toxic to the cells and there were indications of 

activity with TA100 and TA97 at 10 and 50 pg/plate . A second experiment using test 

concentrations from 5 to 70 Ug/plate showed a dose-related increase with TA100, but 

the response did not reach twice the value of the solvent control . However . TA97 

showed dose-related mutagenic activity that exceeded two times the solvent control at 

25 yg/plate and higher without S9, and at 60 ug/plate and higher with S9 . Mucondialdehyde 

was toxic at concentrations above 5 yg/ml in the rat primary hepatocyte UDS 

assay and there was no evidence of induction of DNA repair at lower test concentrations 

. Likewise, mucondialdehyde was toxic (17% survival at 0 .8 yg/ml), but negative 

for induction of HGPRT mutations in CHO cells, at concentrations from 0 .1 to 

0 .8 ug/ml without S9 . Cells from this experiment examined for micronucleus induction 

showed a dose-related effect, with significant clastogenic activity detected at 0 .4, 

0 .6 and 0 .8 ug/ml . Further studies with mucondialdehyde are ongoing, including in vivo 

micronucleus assays . The results to date indicate that mucondialdehyde is toxic, 

mutagenic, and clastogenic in vitro . 

429 

MODIFICATION OF AFLATOZIN B, INDUCED LYg00gNESIS IT ANTIBODIES . O .A . Osowole and A .0 . 

Uwaifo . Biochemistry Department, University of Ibadan, Ibadan, Nigeria . 

Lysogeneais, a consequence of induction of the SOS pathway, results from chromosomal 

DNA lesions persisting after constitutive repair . Lysogenic induction tests in 

prokaryotes are a good measure of the carcinogenicity and mutagenicity of a given 

carcinogen . The effect of antiserum against aflatoxin B1-bovine serum albumin complex on 

aflatoxin B1 induced lysogenesia was studied using Escherichia SAi Ku . Antibody against 

aflatoxin Bi-bovina serum albumin complex was obtained after a single intradermal multiple 

site injection of water in oil emulsion of the complex (66 .67yg/ml) into rabbits . The 

antiserum used was obtained in the seventh week after isseunization . The results obtained 

ahowed a marked reduction in the degree of lysogenesis induced by aflatoxin 81 after the 

addition of the antiserum to the reaction medium prior to microsomal enzyme activation 

of aflatoxin B1 . The result also showed that there was no detectable effects of the 

antibody when the antiserum was added after aflatoxin 31 activation . This suggests that 

the antibody in the aflatoxin B1-bovine serum albumin antiserum could interact with 

aflatoxin B1 prior to its activation . It seems probable therefore, that an immune 

protective effect may be exerted if the antibody intervenes before activation . Results 

from equilibrium dialysis study of the interactfon of purified immunoglobulin G from the 

antisera with aflatoxin 31 showed the average number of binding sites on the antibody 

molecule for aflatoxin B1 being 1 .65t0 .27 with a F1' of 5 .40 Kcal/mole while the average 

association constant was 1 .81f0 .19x10s 3 . These results indicate that the antibody has 

high affinity for aflatoxin B1 . A possible explanation for the reduction in aflatoxin B1 

induced lysogenesis is that antibody binding to aflatoxin B1 reduces epoxide formation . 

430 

X-RAY-INDUCED MULTILOCUS DELETIONS IN THE g4--3. REGION OF A TWO-COMPONENT HETEROKARYON 

OF Neurosoora rr Up COVERING THE 31174 AND hj;-l LOCI LACK THE WILD-TYPE hi;_.$ DNA IN 

RESTRICTION ENZYME ANALYSES . L .K . Overton . J .S . Dubins . R .R . Cobb and ~,Z ~g ~grres . 

Center for Life Sciences and Toxicology . Chemistry and Life Sciences . Research 

Triangle Institute, Research Triangle Park, NC 27709 (USA) 

4 mutant 1-226-565 results from a 1 .8 kb insertion in the hjgj- locus and as a 

consequence has an altered banding pattern upon Ps_% I digestion and hybridization to 

the hi,U- probe pNH6O (Oubins et al ., Mutations Res ., submitted) . After such 

treatment fragments of 1 .4 . 4 .9 . and 7 .9 kb were seen . Similar treatment of the DNA 

from the wild-type strain 74-0R23-1A showed fragments of 1 .4 . 4 .9 . and 5 .9 kb . A 

series of 30 X-ray-induced pA,1 mutants (genotype $cj:,3A jg=20) induced in a twocomponent 

heterokaryon (N-12) of " . Srassa have also been shown to cover the two 

proximal loci . IJ,i_;=1 and 1vs-4, (de Serres . Mutation Res . . in press) . These mutants 

have been subjected to restriction enzyme analysis to determine whether they resulted 

from interstitial deletion . The DNA from forced dikaryons between each of the 30 AQ- 

,3A ,gq=J@ mutants and mutant 1-226-565 was analyzed for the presence of "wild-type" 

DNA that should be missing if these 30 mutants resulted from deletions . The 

5 .9 kb fragment was missing in 27/30 forced dikaryons analyzed, indicating that the 

"wild-type" his-$ locus was ∎issing in these 27 mutants ; 2/30 mutants showed a partial 

50869 3662

; 

deletion of his-_3 . In the remaining forced dikaryon, the 5 .9 kb fragment was present 

in addition to a 2 .6 kb fpagment . Thus, the latter 3/30 Ad;_39 pdZR mutants probably 

resulted from multiple 4obus mutation . Thus, data from these studies demonstrated 

that the majority of X-ray-induced mutants classified as presumptive multilocus 

deletions by classical genetic tests are actual interstitial deletions . 

431 

Ai r"'UPIAIDY IN MOUSE 00CYTES AND 0NE-C°L .L ZYGOTf.'S AFfF:R ORAL DOSAGES OF GRIS. .OFULVIN 

F .?acchie :rotti,C .Tiveron,F .Marchetti,B .Bassani,Lab .Toxicology,FN'cA Casaccia,Roma,Italy 

Griseofulvin (GF) has been characterized as a mitoclastic agent in :namralian cell 

cultures probably causing spindle disrL4)tion by inhibition of cmrbination between .nicro 

tubules and microtubule associated proteins . Doses of 200, 666, 1332 or 2000 mg per kg 

of body weight dissolved in olive oil have been administered by gavage to young superovulated 

BC3F1 female mice either at the time of hunan chorionic gonadotrophin (ECG) 

injection or 2 h later . Metaphases of secondary oocytes or one-cell zygotes have been 

prepared 18 or 42 h (overnight cultivation in colchicine added mediun included) after 

HCG respectively . Cytogenetic analysis of secondary oocytes showed that when GF was 

given at the same time of ICG a dose-dependent induction of meiotic arrest was observed 

without any evidence of aneiploidy in the oocytes which had corrpleted the first meiotic 

division . When the same dose (2000 mg per kg) was given 2 h later a reduction by a 

factor of 3 .6 of the meiotic arrest was observed, while a preliminary analysis showed 

that 23 out of 40 (57 .5 %) analyzed metaphases were aneuploid . Observatims of one-cell 

zygotes showed that approximately all the oocytes which had been arrested at the first 

meiotic metaphase underwent irregular fertilization and gave rise to polyploid zygotes 

or zygotes with an MI or MII arrested female pronucleus . These data suggest the inportance 

of treatment time during meiotic progression as a flmction of the way of chemical 

adninistration and of the cellular target and mechanism .s of aneuploidy irxSuction . 

432 

PREDICTING CARCINOGENIC POTENCY : THE USE 0f A BATTERY 

OF GENOTOXIC AND ACUTE TOXICITY ASSAY RESULTS 

S . Richter Pack 

C .C . Travis 

Oak Ridge National Laboratory 

Recent studies focusing on the ability of short-term mutagenicity assays to 

predict the potency of positive carcinogens have found equivocal correlations 

between mutagenic potency and cancer potency . The squivocal correlation may be 

due to a failure of the short-term tests to account for the ability of some 

compounds to act as cancer promoters . It is difficult to assess the extent that 

a measure of promotional ability will increase the predictivity of short-term 

test batteries since an adequate short-term test for promotion is not available . 

We propose using acute toxicity data (LDSO) as a surrogate measure of promotional 

activity . The suggested biological basis for this relationship is that tissue 

damage and consequent cell proliferation may be a surrogate measure for tumor 

promotion . We have found that including an assay for cytotoxicity in a shortterm 

test battery results in an increase in cancer potency predictivity to 

roughly 0 .84, as opposed to 0 .40 for the Ames test alone . 

433 

MUTAGENICITY OP THE HUMAN CARCINOGEN TREOSULPBAN IN SALMONELLA . D . A . Pa ano 

and E . Zeiger, Cellular and Genetic Toxicology Branch, National Inst tute o 

Environmental Health Sciences, Research Triangle Park, NC 27709 (USA) 

Treosulphan (TREO) is an alkylating agent that is used for the treatment of 

ovarian cancer and has been shown to be a human carcinogen . As part of a 

study of the mutagenic effects of human carcinogens, TREO and its hydrolysis 

products, diepoxybutane (DEB) and methanesulfonic acid (NSA), vere tested for 

mutagenicity in Salmonella . TREO and DEB were direct acting mutagens in 

strain TA1535 . TREO induced 116 rev/plate at a dose of 1 .87 umols and DEB 

induced 50 rev/plate at 0 .88 umols . The use of a preincubation protocol or 

the addition of rat-liver S9 caused a slight enhancement of TREO mutagenicity, 

but a decrease in DEB mutagenicity . NSA was not mutagenic or toxic up to 128 



. 

Notes

150 1989 EMS Abstracts - -~~ ._ - . s~ 


Notes umols/plate in tha .presence or absence of S9 . TREO is knovn to hydrolyse to 

DES and MSA vf tlr~lsaasing pH . TREO mutagenicity vas tested at pH 6 .0, 7 .0, 

and 8 .0 and shovn to be p8-dependent, vith the highest autagenic response and 

the least toxicity seen at pH 6 .0 ; the lovest response and the greatest 

toxicity vas seen at pH 8 .0 . DEB mutagenicity vas unaffected by pH, and its 

autagenicity patterns vere similar to TREO at pH 8 .0 . At this pH, the 

hydrolysis of TREO to DEB is rapid . It vould be expected that the 

mutagenicity of DEB, rapidly produced from TREO (at pH 8 .0), vould be similar 

to DEB directly applied . The nonmutagenicity of NSA eliminates it from 

consideration as the active hydrolysis product . These results are consistent 

vith the conclusion that TREO is mutagenic through its nonenzyaatic hydrolysis 

to DEB . 

434 

BIOLOGICAL AND ANALYTICAL MONITORING OF RIVER WATER POLLUTION IN CAMPANIA, ITALY . 

G .Pagano, G .Melluso ; G .Corsale, A .Esposito, M .Ouida: and G .G .Giordano, Ystituto 

Nazionale Tumori, 1-80131 Naples, and * Dipartimento di Fisiologia Generale ed 

Ambientale, Facoltg di Scienze . Universitl di Napoli, 1-80134 Naples, Italy 

Several emissions of industrial, agricultural, and domestic sewage affect the Sarno 

river and the drainage system of the Volturno river-Regi Lagni in the Campania region, 

Italy . The possible consequences on marine biota near the mouths of the two rivers 

and the Regi Lagni have been investigated by biweekly sampling of river water from ten 

sites ; some experiments were performed by daily or circadian sampling from two selec- 

ted sitea . Biological monitoring was performed by testing river water samples diluted 

in seawater (10- to 10-') on sea urchin embryos and sperm, and scoring the outcomes 

in terms of : a) developmental toxicity ; b) fertilization success ; c) cytogenetic ab- 

normalities . Chemical analysis was carried out on water and sediment, and included 

several pollution parameters, as : i . COD and BOD, ; ii . NO=, N0„ and NH ; ; iii . some 

selected inorganice [Cd(II) ; Cr(III) and (VI) ; As(III) and (V)] ; iv . some selected 

organics (PAH's, PCB's, some pesticides) . The results showed : 1) the presence of re- 

markable pollution and toxicity at the mouths of the Sarno river and the Regi Lagni, 

whereas the Volturno river resulted to be less affected ; 2) daily and circadian 

changes of pollutant levels ; 3) a good agreement of biological vs . analytical data . 

(Partly supported by WHO, and by the Italian Ministry of Health) . 

435 

FANCONI'S ANEMIA (FA) LYMPHOBLASTS BELONGING TO COMPIEMENTATICN GROUPS 

"A" AND "B" ARE HYPOMUTABLE FOLLOWING TREATMENT WITH PHOTOACTIVATED 

PSORALENS . D . Pa ad oulo, B . Porfirio and E . Moustacchi . Institut Curie, 

Section de B o og e, UA 1292 CNRS, 26 rue d'Ulm, 75231 Paris cedex 05, 

France . 

Fanconi's anemia (FA), an eutoscmal•recessive disease, beldngs to the 

group of human genetic disorders cha;acterized by chranosaaal instability 

and a predisposition to malignancy . At the cellular level, FA cells 

are hypersensitive to DNA cross-linking agents, a feature likely to be 

related to a defect in the processing of lesions induced by these 

agents . Two genetic complementatien groups have been recently described 

"A" and "B" . We have previously demonstrated that FA group "A" cells 

are sensitive to cross-links and monoadducts (MA) induced by photoactivated 

bifunctional psoralens, whereas FA group "B" cells are eapecially 

sensitive to MA induced by these agents . In the present study, we measured 

in normal and FA lymphoblasts the frequency of 6TGr and ouar mutations 

induced by 8-methoxypsoralen or trimethyl,4,5',8,psoralen in the 

presence of 365 nm radiation . We observed that FA cell lines fraa both 

complementation groups are clearly hypaoutable for the two loci considered 

. The FA group "A" cells are almost immutable . The molecular analysis 

of the spectrum of HGPRT- mutants induced by photoactivated psoralens is 

in progress . 

436 

FISH CELL LINE DEB.IVEJ FROlf THE FIN OF TBE CEN(RAL MUDHINJOM/ (UMBRA L=) : A SUITABLE 

MC)DEL FOR CLASTOGENICITY ASSAY . E .-H . Park, J .-S . Lee, A .-K. Yi and H . Etch, 

Department of Biology, College of Sciences, Hanyang University, Seoul 133-791 (Korea), 

and Division of Biology, National Institute of Radiological Sciences, Chiba 260 (Japan) 

A cell line (ULF-23HU) from the fin of the central madminnow (UMbrA UML) was 

characterized and tested for its suitability to aseess cytogenetic damages induced by 

chemicals in fish . . Cells of this line exhibit a fibroblaat-like appearance and grew 

50869 3664

: 1989 EMS Abstracts 

optimally at 25 'C in ttSg TC-199 medium containing 10% fetal bovine serum, but slower Notes 

growth continued down ta 4°C, where they could be stored for prolonged periods . They 

had a 32-h cell cycle time laken up by a 20-h S period as determined by the 

autoradiographic analysis of the fraction of labeled mitosis . 'Itre cultures showed 

relatively high mitotic index (0 .84-2 .35%) during exponential growth phase lasting about 

7 d . Karyological analysis of the cells at the different subculture passages revealed 

constant chromosome modal number of 23 consisting of metacentric or submetacentric 

chromosomes which were primarily similar to those of in vivo cells except one additional 

chromosome . The spontaneous-sister chromatid exchange rate was 5 .3 per metaphase . When 

ULF-23HU cells were exposed to N-methyl-N'-mitro-N-nitoreoguanindine, mitomycin C, 

N-methyl-N-nitrosourea, 4-nitroquinolin-N-oxide and cie-platinum (II) diamine dichloride 

dose-dependent increases in sister chromatid exchanges were clearly detected . 

FLthermore, ULF-23HU cells responded to the indirectly acting mutagens (aflatoxin B1, 

cyclophosphamide and 4-aminoazobenzene) without adding a drug activation system . These 

rrsurts offered the high possibility of the use of this cell line as a suitable in vitro 

model for clastogenicity studies in fish . 

437 

CFUtOMOSOME CHANGES IN CELL TRANSFORMANTS INDUCED BY GENOTOXIC AND NON-GENOTOXIC AGENTS . 

E . M . Parry, J;M . Parry, T . Issa, M . Kadhim, R . Porter, D . Devi, School of Biological 

Sciences, University College of Swansea, Swansea SA2 8PP . 

Syrian hamster dermal (SRD) cultures exposed to carcinogens undergo progressive 

changes from primary isolation via the formation of immortal cultures to the production 

of transformed tumourigenic clones (Newbold et al 1982) . Thase culture rhanges may be 

induced by treatments with agents characterised as acting predominantly by mechanisms 

involving DNA interactions (genotoxic) and by a variety of non-genotoxins . We have been 

investigating the chromosomal (numerical and structural) and division characteristics 

(eg mitotic spindle fidelity) of SHD cultures exposed to both genotoxins and nongenotoxins 

. Immortal cultures which escape culture senescence (normally occurs after 15 

population doublings) generally show the presence of significant numbers of polyploid 

cells and high levels of spindle fidelity . Treatment of immortal cultures with 

carcinogens results in the progressive loss of chromosebes (predominantly from the 

polyploid population) resulting in the formation of hyperdiploids and the production of 

fully transformed colonies . Karyotype analysis of transformed SHD cultures has 

demonstrated a variety of aberrations with a transloc,ation of chromosome 11 being the 

most common event . This translocation is found in all the cells of clones transformed 

with alkylating agents and benzo(a)pyrene and initially at a low frequency in cultures 

transformed with diethylstilboestrol . With further subculturing, individual clones 

progress to become homozygous for the translocated chromosome 11 . Our data sugfest that 

the SHD system provides a suitable model for the study of the mechanism of action of 

both genotoxins and non-genotoxins . Newbold, R . F ., Overe11,R . W . and Connell, J . R . 

(1982), Nature ?Q9, 633-635 . 

438 

THE DETECTION OF ANEUGENS USING YEASTS AND CULTURED MA64fALIAN CELLS . J . M . Parry, E . M . 

Parry, T . Warr, A . Lynch, S . James . Biological Sciences, U .C . Swansea SA2 8PP . 

The EEC's aneuploidy project involves a study of a range of assays using a common 

series of chemicals . The core chemicals selected for their ranPe of cellular activities 

consists of colchicine (COL), econazole (EZ), Chloral hydrate (CR), hydroquinone (HQ), 

diazepam (DZ), thiabendazole (TB), cadmium chloride (CD), tbimerosal (TM), 

pyrimethamine (PY) and vinblastine (VB) . These chemicals are being tested in assays 

ranging from in vitro polymerisation of tubulin to the induction of numerical 

chromosome changes in the germ cells of intact rodents . We have been studying the 10 

chemicals to measure their ability to induce chroswsome loss in yeast D6, cell division 

aberrations, chromosome number changes and micronuclei formation in primary and 

immortal Chinese hamster cultures using a variety of protocols . In yeast D6, five'of 

the chemicals namely EZ, TM, TB, HQ and CH produced significant increases in chromosome 

loss, CD, PY and DZ produced only marginal increases in monosozd and COL and VB were 

inactive . These data suggest that fungi are at best only capable of detecting a 

fraction of those chemicals which are capable of inducing aneuploidy in mammalian cells 

(Parry and Parry (1988) . In our hands the most time and cost effective in vitro assay 

for detecting potential aneugens was the measurement of aberrations of aell division 

using procedures which differentially stain the spindle and chromosomes (Parry et al 

1985) . This assay was capable of detecting induced division abnormalities after 

exposure to all the test chemicals using relatively simple protocols and in a variety 

of cell types . Parry E . M . et al (1985) Mutation Res . 150, 369-381 . Parry E . M . and 

Parry, J . M . (1988) Aneuploidy, Part B : n uction and 3et Svstems, 169-1g8 . Alan R . 

Liss Inc . New York . 


151


Notes 


439 

THE HXPULSIQM OF MICRONUCLAI P~OM POLTCHROMATIC nTTHROClTAS OP MOUSB DONS MAAROp IN 

VIVO. J . Parto~ J . Deyers , and M . Carriott . Lilly Research Laboratories, sIT 

~flly and Co .'~y;'LTeenfield, IN 46140 . 

The mouse micronucleus test is a valuable tool for evaluating in vivo chromosome 

damage produced by test articles in polychromatic erythrocytes (PCTj o3-bone marrov . 

Compounds that are clastogens, such as cyclophosphamide (CP), induce ∎icronuclei 

(MN) that are smaller than MN induced by compounds that are spindle poisons, such as 

demecolcine (DE) (Hogstedt and Karlason, Mut . Aes . 1S6t229, 1985/ Tas~.moto and 

Kikuchi, Mut . Res . 71s127, 1980) . In vitro studies con ucted by Nito at al . (Mut . 

Res . 207 :5, T88)ihoved that siitomyc n and vincristine caused a dose-TepenNint 

rnluct3on of MN in mouse cell line L-929 but when the ∎icronucleated cells vere then 

treated with cytochalasin B, the rate of ∎icronucleated cells was reduced 31-39X due 

to MN extrusion . The eurrent study shows 1) MN are expelled from PCs in vivo and 2) 

expulsion is dependent upon ∎icronucleus sise . Male and female CD-1 ml-e : vere given 

0 .5, 1, 5, and 10 mg/kg of DE or 100 mg/kg of CP . The bone marrov was harvested 24 

and 48 hr after dosing and evaluated for micronucleated PCE . Expelled MN still 

attached to the PCE cell surface vere also counted . The clastogen, CP, produced MN 

that ranged in size from 0 .5 to 2 .8 ym' with a mean of 1 .5 vm' and no expelled MN 

were observed . The spindle poison, Dg, induced ∎icronuclei that ranged in sise from 

0 .8 to 7 .9 un' . MN retained within the PCE ranged from 0 .8 to 3 .4 ym' with a mean 

of 1 .9 um' while expelled MN ranged from 2 .1 to 7 .9 Ym' with a mean of 4 .3 um' . MN 

less than 2 .1 ym' are retained within the PCE and MN greater than 3 .4 ym' are 

expelled . MN ranging from 2 .1 to 3 .4 Ym' in size may or may not be extruded . 

440 

THE U .S . ENVIRONMENTAL PROTECTION AGENCY'S INTEGRATED RISK INFORMATION SYSTEM, J . Patterson, 

J . Swartout, R . Schoeny, R . Picardi, U . S . Environmental Protection Agency, 

Office of Health and Environmental Assessment, Cincinnati, Ohio and Washington, D .C . 

The Integrated Risk Information System (IRIS) is an electronic information system 

developed by the U .S . Environmental Protection Agency (EPA) containing data related to 

health risk assessment . IRIS is the primary vehicle for communication of chronic 

health hazard assessments representing EPA consensua after comprehensive review by 

intra-Agency work groups . This work group review includes an examination of the 

weight of evidence that an agent is likely to be a human carcinogen as well as a consideration 

of the validity of a quantitative risk estimate of carcinogenicity . Quantitative 

estimates of potential for noncancer health effects are reviewed by a separate 

group . The primary intent of IRIS is to provide guidance to EPA personnel in 

making risk management decisions . IRIS contains chemical-specific information in summary 

format for over 360 agents . The information is structured to provide a description 

of the basis for the hazard assessment and a discussion of the uncertainties in 

that assessment . An IRIS chemical file is initiated when consensus is reached on an 

assessment for carcinogenic or noncarcinogenic endpoints and contains the summary for 

that aseessment . Other information, such as Drinking Water Health Advisories, regulatory 

actions, acute toxicity, and physical-chemical properties, is added as available 

and as resources permit . IRIS is available to the general public by telecommunications 

link with a commercial carrier, and to Public Health Foundation members through 

the Public Health Network . Access through the National Library of Medicine's TOXNET 

is planned . 

441 

ANALYSIS OF ELECTROPHORETICALLY DETECTED MUTATIONS IN THE MOUSE, AND COMPARISON OF THE 

ELECTROPHORETIC AND SPECIFIC LOCUS MUTATION RESPONSE . 

J . Peters and S .T . 8a11, N .R .C . Radiobioloqy Unit, Chilton, Didcot, Oxon OXII 0RD (UK) 

Mutations occurring in mouse spermatogonial stem cells after exposure to 

ethylnitrosourea (ENU) or X-rays have been scored, both by the visible specific locus 

test and an electrophoretic test . One aim of the experiment was to compare mutation 

rates found by each method, and a second aim was to investigate the characteristics of 

the induced mutations . Significant differences in mutation rates per locus for 

recessive visible and electrophoretically detectable markers have not been found yet . 

Five independent null mutations of glucose phosphate iso .erase-1 were discovered 

among the offspring of mice treated with 250 mq/ky ENU . Heterosyyotes are fully 

viable and fertile, but homozygotes are not known . One homoayqote dies at about 

8 .5 days oost coitum (West et al ., in prep .) . Each mutation results in complete loss 

of gene product in all adult tissue~i t¢~ted, as judged by quantitative assay and 

electrophoresis, except one, Goi-lsD-mltl . This determines a polypeptide which can be 

found only in adult testis, and embryonic stages . Another mutant allele Gui-1sh:au 

also codes for a polypeptide which is only seen in embryos . Possibly the mutant 

polypeptides are unstable, and thus can be detected only in rapidly dividing tissues . 

Molecular analysis by Southern blotting has failed to show 4ifferences between DNA 

from the mutants and from the wild types goi-tsa and Goi-ts . These findings are in 

agreement with the suggestion that ENU induced mutations are mainly intragenic 

changes . 

50869 3666

442 = 1989 EMS Abstracts 153 

Notes 

, 

HUMAN DNA ADDUCTS DUE TO SMOKING AND OTHER CARCINOGEN EXPOSURES . 

D .H . Phillips, Institute oftancer Research, Chester Beatty Labs, London, UK . 

Human exposure to complex mixtures of potentially carcinogenic polycyclic 

aromatic hydrocarbons (PAH) has been monitored by 32P-postlabelling analysis of 

DNA isolated from human tissues . The presence of aromatic adducts in DNA from 

peripheral lung has been detected at levels proportional to the numbers of 

cigarettes smoked by the individuals . The complexity of the adduct patterns 

indicated that adducts were formed by-$ large number of different compounds . Lung 

DNA from former smokers of several years' abstinence generally showed a level of 

adducts similar to that in DNA from non-smokers . Analysis of bronchial DNA also 

revealed evidence of smoking-related adducts . Adduct levels in peripheral blood 

lymphocyte DNA did not differentiate between smokers and non-smokers . 

Occupational exposure to PAH amongst iron foundry and coke oven workers was, 

however, evident from the elevated levels of PAH-DNA adducts in their lymphocyte 

DNA compared to levels in unexposed control subjects . Exposure of human skin to 

PAH, monitored in psoriasis patients receiving topical applications of coal-tar and 

juniper tar, leads to the formation of adducts in this tissue . Corroborative studies 

on the formation of adducts in experimental animals provide further evidence of 

the potential carcinogenic risk to humans of exposure to these PAH mixtures . 

443 

CHROMOSOME-SPECIFIC PROBES IN CYfOGENETICS . D. Pinkel, J. Gray, R. Segraves, J. Lucas, W-L. 

Kuo, B . Trask, L C. Yu, D. Eastmond, M. Poggensee. Lawrence Livermore National Laboratory, 

Livermore Ca . 94550 . 

Cytogenetic analysis now relies primarily on staining procedures that produce patterns of bands on 

metaphase chromosomes. Analysis of these bands by skilled observers permits chromosome identification 

and recognition of structural and numerical aberrations. However the proce4ures are time 

consuming and limited to mitotic cells . Recent developoments in chromosome stainEng by in situ 

hybridization promise to extend cytogenetic analysis to new areas . Since staining is based on DNA 

sequence, the staining pattern can be tailored by the choice of probes, and information can be obtained 

from complex settings such as interphase nuclei . Two classes oj probes have proven useful . The first 

are probes for chromosome-specific repetitive sequences . These sequences occur in compact clusters, 

frequently near the centromeres . Specific probes for approximately 2/3 of the human chromosomes 

are known. Nuclei hybridized with these probes show a compact spot at the location of the target 

sequence, permitting identification of aneuplold cells . The second class of probes, composite probes, 

consist of collections of probes which produce a desired staining pattern on chromosomes . Composite 

probes capable of staining a specific chromosome type over its entire length are now available for all of 

the human chromosomes. They permit rapid detection of structural abnormalities such as translocations 

in metaphase spreads and interphase nuclei. In situ hybridization with probes of both types 

has allowed us to detect aneuploidy induced by in vitro chemical exposure, trisomy 21 and other 

important prenatal numerical abnormalities, and translocations induced by in vitro and in vivo 

radiation exposures . Work performed under the auspices of the USDOE by the LLNL under contract 

W-7405 ENG-48 and USPHS grants CA45919, GM25076, and HD17665 . 

444 

ENHANCED REACTIVATION AND NUTAGENESIS OF ADE 2 VIRUS IN CARCINOGEN - 

PRETREATED BELA CELLS 

Stelios N . Piperakis 

Institute'of Biology, N .R .C . "Democritos" 

Agia Paraskevi, Athens - Greece 

Enhanced reactivation of a UV - irradiated mammalian virus by pretreatment 

of susceptible host cells with a number of agents has now been 

found to exist in different systems . In many studies with mammaliancells 

an increased degree of viral mutagenesis has also been found with increasing 

damage to the virus,but there is still disagreement as to whether 

this mutagenesis is further enhanced by pretreatment of the cells . 

In the present study an investigation has been undertaken in the Ade 2- 

Hela system . Hela cells were treated with the agents sodium arsenite, 

EMS, and aflatoxin Bland the enhanced reactivation and enhanced mutagenesis 

of UV - irradiated Ade 2 was compared to the enhanced reactivation 

and enhanced mutagenesis obtained by UV, heat and MMS . 

The results show that the enhanced reactivation of UV-irradiated Ade 2 

observed in Hela cells pre-treated with these agents is not due (at 

least under the used experimental conditions) to the induction of an 

error - prone DNA repair mechanism . 




NOteS._ , .,; . ROLE OB Ai.TW4~D HEPATIC FOCI IN THE STAGES OF CARCINOGENESIS. n P't 

~ Yvonne Dt9t~an, Mark Pyron, Chris Laufer, and Tahir Rlzvi 

. 

. McArdle Laboratory Research, UmverWof Wisconsin, Madison, Wisconsin 53706 

Hepatoaltrcittogettrsis in the rat can be divided into the stages of initiation, promotion, and progression 

. Initiadon by a DNA-alkylating chemical results in the appcarance of individual hepatocytes 

and their progeny exhibiting altered gene expression of specific tnarker(s) . The stage of promotion is 

induced by exo~enous and/or endogenous chemicals, usually non-DNA-reactive, which stimulate 

both the re licatton rate and reversibly altered gene expression of clones of initiated cells . However, 

different chemical classes of promoting agents induce populations of altered hepatic foci (AHF) 

which, in general, exhibit different phenotypic characterls 'ttcs, unique to the class of chemical promoting 

agents . Furthetmore, when multiple markers are utilized to score the phenotypes of the 

clonally expanded inidated cell populations, all possible phenotypic combinations within the constraints 

of the action of the promoting agent itself are represented in the AHF populadons . The stage 

of progression, when not induced simultaneously with~ge of initiation, may be heralded by the 

appearance of phenotypic heterogeneity within individual AHF in contrast to the phenotyp ic homogeneity 

of most or all AHF during the stage of promotion . It is in the stage of progression that karyotypic 

changes, proto-oncogene activation, and malignant neoplasia occur either predominantly or 

exclusively. Progressor agents capable of inducing this stage of neoplastic development are characterized 

ubiquitously by their clastogenic effects . Based on this rtwdel, point mutations and clastogenic 

effects appear to play critical but different roles at the sta es of initiation and progression in the 

development of hepatic neoplasia respectively. (Supported ~y grants from the National Cancer 

Institute: CA-07175, -22484, -43700 and a contract from the National Toxicology Program : ES-3- 

5024.) 

446 

DOUBLE-STRAND GAP PLASMIDS DO NOT ALTER THE RATE OF MUTATION OR GENE 

CONVERSION IN Saccharornyres cerrvisiac . MJ . Plewa, S . 7>tylor, A. Kumar and R. Carr. Institute for 

Environmental Studies and the Department of Microbiology, University of Illinois, Urbana, 61801 USA . 

We are developing a system to locate and recover information from specific mutant sites in eukaryotic 

chromosomes . We are studying the mutational spectrum of forward mutation at CANl by the doublestrand 

gap repair of a series of yeast shuttle vectors . The advantage of this approach is that the 

information copied onto the gapped plasmid occurs after the mutational event on the chromosome . We 

constructed a progenitor shuttle vector - pMPS - which contains amp' . E. coli Ori, LEUZ GlNI, and 

the yeast 2µ Ori. Yeast that are resistant to canavanine (canl') become sensitive to canavanine after 

transformation with pMPS. However, it is important to determine if linear, double-strand gap plasmid can 

modify the rate of forward mutation at GlNl on chromosome V or gene conversion within the yeast 

genome. Linear pMHIS8 (a vector used in the construction of pMP5) at concentrations of 0, 1, 2 .5. 5 

and 10 µg were added to competent yeast cells (strain XY729) under transforming conditions . Forward 

mutation at CANI and cell toxicity were measured . There was no difference in the frequency of canl' 

cells among the groups with a mean value (t SE) of 39 t 4 .0 mutants per 3.5 x 10' competent cells 

plated . The same concentrations of linear pMH15S were added to competent D7 cells and gene conversion 

was measured between trpS-12 and apS-27 heteroalleles . Again there was no difference in the gene 

conversion frequency at dpS with a mean value of 750 s 20 .3 convertants per 3 .4 x 10' competent cells 

plated . These controls indicate that the use of double-strand gap shuttle vectors does not affect the 

processes of mutation or gene conversion in competent cells . 

447 

THE AC'ITVATION OF PROMUTAGENS BY PLANT CELL SYSTEMS. Michael J. Plewa . Institute 

for Environmental Studies, University of Illinois, Urbana, IL 61801 USA . 

Plant activation is the process by which promutagenic agents are activated into mutagens by plant 

systems. Many promutagens are activated by the familiar mammalian microsomal monooxygenase systems 

as well as by plants . However, several environmentally important agents are preferentially activated by 

plant cells. Plants have become a reservoir for the deposition and accumulation of environmental 

xenobiotics . With the widespread use of agricultural chemicals on crop plants and with the global exposure 

of plants to pollutants, the possibility that plant-activated agents may be introduced into the human food 

chain is a cause of concern . Due to these and other concerns, envitonmentaliy relevant agents should be 

evaluated with plant assays. The plant celUmicrobe coincubation assay uses cultured plant cell suspensions 

as the activating system and bacteria or yeast cells as the genetic indicator organism . After a treatment 

time, the microbes are plated on selective medium. In this way the activation system and the genetic 

system can be independently studied. In addition the viability of the plant cells and the microbial cells can 

be independently determined so that the toxicity of a test agent can be evaluated . We have employed 

cultured tobacco, cotton, carrot, maize and 7Yadescanlia cells to study the activation of test agents and 

complex environmental mixtures . In addition to screening, this assay is being used in basic research to 

elucidate the biochemical mechanisms of plant activation. Data using model monocyclic and polycyclic 

aromatic amines will be used to identify developmental and biochemical pathways involved in plant 

activation. This research supported by an OSWR grant and USAF grant N88-AF P-0511 . 

50869 3668

448 ' r 1989 EMS Abstracts 155 

VITAMLN C INTAKE DECRKAS .ES CHROMOSOME DAMACE IN GENETIC INSTABILITY ASSAY 

Pohl, H . and Reidv . J .1! 

Genetics Branch, Division of Environm,mtal Health Laboratory Sciences, Center for 

Environmental Health and Injury Control, Centers for Disease Control, Atlanta, CA 

30333, USA 

It has been suggested that some people are predisposed to cancer as a result 

of genetic instability and this instability can be detected by exposing their cells 

to a clastogen in vitro . We know very little, however, about person-specific 

factors other than genetic instability sbat might influence this assay . We report 

here that the amount of chromosome damage induced in human lymphocytes by an 

exposure to bleomycin during the last 5 hours of cell culture significantly 

decreased after the blood donors took 1 g of vitamin C per day for 2 weeks . 

Similar changes were not seen in lymphocytes from control individuals sampled at 

the same time but not taking vitamin C supplements . The initial study involved two 

persons who took vitamin C and two controls who did not take the vitamin . Results 

showed little variation among triplicate flasks and little variation over a month 

in the controls . Results were confirmed in a second experiment with eight people 

(p< 0 .01) . Each person was his own control . In addition, two laboratory controls 

(individuals not taking vitamin C) showed no differences during the experiment . 

From this limited study, we suggoet that it would be prudent to consider dietary 

and perhaps other lifestyle factors in the interpretation of results from this and 

related assays for genetic instability . 

449 

ORGAN SPECIFIC GENOTOXICITY AND CARCINOGENICITY B .L. Pool, P.Schmezer, A. 

Tompa`, S.Y. Brendler. Institute for Toxicology and Chemotherapy, German Cancer 

Research Center, INF 280, 6900 Heidelberg, FRG . 'National Institute of Occupational 

Health, Nagyvarad T€r 2, Budapest, Hungary . 

The factors governing the unique organ specific carcinogenic effects of Nnitrosamines 

are largely unknown . Feasible mechanisms include (a) organ specific 

metabolism (b) pharmacokinetics (c) peraistance of DNA damage. The role of each 

pathway is being investigated for nitrosamines carcinogenic in hepatic or extrahepatic 

tissues . Induced DNA single strand breaks were determined in primary rat cells of 

liver, lung, kidney, testes, thymus gland and blood . (a) Organ specific metabolism was 

not apparent in vitro using hepatocytes, since hepatotropic compounds were less 

genotoxic than extrahepatic carcinogens . (b) PharmAcokinetics were monitored by 

determining toxic and genotoxic effects in intact cells isolated from treated animals . 

This approach has the advantage that DNA damage arising from toxicity can be 

excluded, and that it is very sensitive . It was shown that < 1 mg/kg of the liver 

carcinogen N-Nitrosodimethylamine is genotoxic in the liver ; > 2 mg/kg are active in 

lung and kidney . No genotoxicity is detectable in cells of thymus gland or testes . (e) 

Persistance of DNA damage was assessed after 1, 4, and 16 h exposure, and was 

found to be greater in liver than in lung . Therefore, this compound's organ specificity 

in carcinogenicity and in vivo genotoxicity agree well . This sensitive, versatile (multiple 

organs), rapid (48 h) and efficient (few animals) in vivo method can identify tissues 

which may be susceptible to carcinogenesis. The value of this approach to study 

toxicokinetics of genotoxic compounds is stressed. Results with additional nitrosamines 

and new approaches to monitor remote target organs will be presented . 

450 

GENETIC TOXICOLOGY OF THIAARENES B .L . Pool, P .Klein, P. Schmezer, S .Y. Brendler, 

J.R. Schlehofer', and G. Grimmer", Institute for Toxicology and Chemotherapy, 

Ìnstitute for Virus Research, German Cancer Research Center, INF 280, 6900 Heidelberg, 

°Institute for Biochemistry of Environmental Carcinogens, 2070 Grosshansdorf, FRG . 

Three isomeric polycyclic aromatic sulfur heterocyclics (thiaarenes) have been 

assessed for genotoxicity. An evaluation of thiaarenes is important, since they are 

significant constituents of synthetic fuels . Compounds of this class may be structurally 

similar to several carcinogenic polycyclic aromatic hydrocarbons, but they have not 

been similarly well investigated for adverse biological effects . Benso(b)naphtho(2,1d)thiophene 

is a sulfur-containing analog to chrysene and the other two isomers are 

different by the position of their sulfur substitution (1,2-d and 2,3-d isomers) . 

Following results were obtained : (a) In S . t,vphimurium TA98, mutagenic activity was 

observed for 2 of the lsomers (2,1-d = 2,3-d; 1,2-d was negative) with standard 

metabolic activation by S9 from Aroclor pretreated rats . Stimulation of the system for 

glucuronyl acid conjugation did not reveal enhanced mutagenicity . (b) DNA Single 

strand breaks were not observed in metabolically competent primary hepatocytes nor in 

a Chinese hamster cell line. (c) Using the same cell line, integrated SV40-DNA was 

shown to be amplified by two compounds (1,2-d ∎ 2,3-d; 2,1-d was not tested) . Results 

of ongoing studies will be presented : (d) In a novel system, the amplification of Adeno- 

Associated Virus DNA is being measured in primary Syrian hamster embryo cells (SHE) 


Notes


_ ._--_-_ . 

NOteS - ~-F•infected -by

454 © 1989 EMS Abstracts 

CYTOGENETICS IN ENVIRONMENTAL MUTAGENESIS . R. Julian Preston . Biology Division, Oak 

Ridge National Laboratory, Qak Ridge, TN 37831 

It is 50 years since Karl Sax demonstrated that x rays could induce chromosome aberrations, and that the 

rc~pcrose was dose-dependent. In the period since•we have learned that a whole range of chemical agents 

c: n also induce similar alterations. It is also apparent that specific chromosome alterations arc as .cociated 

with many birth defects and with tumor initiation and/or progression . Important corollaries of these 

observations are can we determine which agents are mutagenic (or carcinogenic) and can we estimate the 

risk (genetic and somatic) from exposure to such agents? The answers are still equivocal . This stems from 

the fact that the mechanism of induction of chrotnDrame aberrations has not been determined, and the role 

of specific aberrations in the induction of birth defects and cancer are not generally known . Many new 

techniques have become available in the past few years to help in this endeavor . It is possible for example. 

to characterize chromosomes by using specific DNA probes, to identify and sequence chromosome breakpc 

ints, and to identify chromosomal regions by sophisticated banding techniques and in situ hybridization . 

These methods can be used to determine if agents present in the environment can induce specilic 

chromosome aberrations, not just aberrations in general . It is also of importance to determine if there is 

a range of sensitivities to aberration induction within the population and the bases for this, (e .g ., replication 

fidelity, repair competence, and chromosome fragility) . The incorporation of molecular techniques into shortterm 

assays (in vitro and in vivo) and population monitoring studies will enhance their utility, and hopefully 

allow truly predictive information to be obtained . Operated by Martin Marietta Energy Systems, Inc . under 

contract DE-ACA5-840R21400 with the U . S. Dept. of Energy . 

455 

RESTRICTION ENDONUCLEASE INDUCED CHROMOSOME DAMAGE : A MODEL FOR IONIZING 

RADIATION AND A PROBE OF INTERCHANGE HOTSPOTS . R.J . Preston, G .J. Hook, and G.J. Horesovsky, 

University of Tennessee Biomedical Graduate School, and Biology Division, Oak Ridge National Laboratory, Oak 

Ridge, TN 37831 . 

Type II restriction endonucleases (REs) are capable of recognizing specific DNA sequences and indbcing blunt 

or cohesive ended double strand cuts at these sites . Since only one type of DNA damage is induced by REs they 

represent a good model system for studying the importance of double strand breaks in the formation of CAs (CAs) . 

In addition since the cuts are persumabley at a specific location it should be possible to produce very specific types 

of CAs. Using cell electroporation we have designed experimental protocols by which the validity of RE induce 

damage as a model system, specifically for radiation damage, as well as the feasibility of looking at specific 

chromosome interactions can be tested. Cell electroporation has the advantage over other techniques used to 

introduce RE into cells in that very low doses of RE can be used, and .a greater percentage of the cells exhibit 

damage. Experiments have been carried out in CHO cells using the blunt end cutter Rsa I and the four base 

cohesive end cutter Sau 3A1 . The CHO cells were harvested 6 and 22 hrs after treatment so that cells treated in 

G= and G, would be sampled . The induction of chromosome abemations by concentrations of 1-g0 units for Rsa 

I and 5-30 units for Sau 3AI have been analyzed . As reported by others, REs induce a much higher percentage of 

exchange type aberrations than is found for X irradiation at similar levels of deletions . Both Rsa I and Sau 3AI are 

capable of inducing CAs In 01 and G= at all the concentrations tested. The shape of dose response curves are 

different from those reported elsewhere in that they are non-linear . For both Rsa I and Sau 3AI the dose response 

curves are biphasic ending in a plateau . No straightforward correlation can be made between double strand cuts 

induced by REs and double strand breaks induced by X irradiation . (Research sponsored by the Office of Health 

and Environmental Research, U . S. Department of Energy under contract DE-AO0S-840R21400 with the Martin 

Marietta Energy Systems, Inc. GJ Horesovsky is supported by NIH-CA 09104-13) 

456 

COMPARATIVE ANTIMUTAGENICITY OF Cff1 .OROPHYLLIN AND FIVE COMRtON ANTIMUTAGENS AGAINST 

FOUR LABORATORY I4UTAGENS IN Salmonella typhimurium STRAINS TA98 AND TA100 . 

K . Pupatwibul and N . E . Brockman, Illinois State University, Normal, IL (USA) 

Chlorophyllin (C1Q.) is a potent inhibitor of the mutagenlcity of 10 complex 

mixtures in Salmonella typhimurium strain TA98 (Ong, Whong, Stewart and Brockausn, 

1986) . Furthermore, CNL is more effective than 4 common antimutagens (B-carotene 

and vitamins A, C, and E) against the mutagenicity of 5 of these complex mixtures 

in TA98 (Ong, Whong, Stewart and Brockman, 1989) . Based on these results, we have 

initiated in S . typhimurium strains TA98 and TA100 a comparative study of the 

antimutagenic activity of CHL and commonly used antimutagens against the 

mutagenicity of laboratory mutagens with different mutagenic mechanisms ; we will 

extend this comparative study to other short-term tests for genetic toxicity . We 

report here our results on the antimutagenic activities of CHL . B-carotene, 

retinoic acid, and vitamins A, C, and E against the mutagenicity of N-methyl-N'nitro-N-nitrosoguanidine 

(MOING) and 6-N-hydroxylaminopurine in TA100 and of 

aflatoxin BI and the acridine mustard ICR-170 in TA98 . Vitamin A inhibited the 

mutagenicity of all 4 mutagens, and CHL inhibited the mutagenicity of all of the 

mutagens except PINNG : Retinoic acid and 8-caroteae inhibited the mutagenicity only 

of aflatoxin B . Dose-response curves of antimutagenic activity will be presented . 

(H .E .B . acknowledges the support of the U .S . EPA Distinguished Visiting Scientist 

Program .) 


Notes 

157 

I


Notes ~ -" IN VIVO IN 

'I 

TI~DS-TEST ON RAT HEPATOCYTFS EXPERIENCES IN TESTING XENOBiO- 


TICS AFTER SINGLE OR REPETITIVE TREATMENT . 

E .C . Puri, Th . _krtnowd D . MOlier, Ciba-Geigy Limited ., CH-4002 Basle (Switzerland) 

The in vivo-in vitro DNA-repair test gained significance during the last years and was also performed 

in our laboratories for testing of chemical products as well as of the following reference compounds : 4aminobiphenyl 

(ABP), benzo(a)pyrene (BaP), dimethylnitrosamine (DMN), methylmethanesulphonate 

(MMS) and nafenopin (NAF). To investigate the effects of an enzymatic induetion of the liver a repetitive 

treatment with two of these compounds was also carried out . Therefore, compounds were administered 

either once (all five compounds), or repetitively (BaP, NAP). The preparation of hepatocytes 

(perfusion technique) followed 2 hours after the single application of ABP (200 mg/kg), DMN (15 mg/kg) 

or MMS (100 mg/kg) and 4 hours after the single application of BaP (100 mg/kg) and either 2 or 12 

hours after the single application of NAF (200 mg/kg). For the repetitive treatment the animals was given 

either BaP at a dose of 20 mg/kg or NAF at a dose of $00 ppm administered in the food on seven consecutive 

days, followed at the eighth day by a dose of 100 mg/kg BaP 4 hours before isolation of hepatocytes 

or by a dose of 200 mg/kg NAF either 2 or 12 hours before Isolation of hepatocytes . NAF did 

not induce DNA-repair synthesis under any of the treatment conditions . Several experiments with ABP . 

DMN and MMS yielded all clear positive results . BaP yielded a clear positive result after repetitive treatment 

. These positive results obtained with BsP after enzymatic liver induction by the test substance itself 

are noteworthy, inasmuch as earlier in vivo-in vitro experiments with the same substance on rat 

hepatocytes (Mirsalis et al ., 1982, Environm . Mutagenesis 4, 553), rat pancreatic cells (Steinmetz and 

Mirsalis, 1984, Environm. Mutagenesis 6, 321) and rat tracheal epithelial cells (Doolittle and Butterworth, 

1984, Carcinogenesis S, 773), all performed without prior enzymatic induction, yielded negative results . 

458 

SOMiATIC NUTATIUN AND rtaCOMtlINA'TI0N TrST 0F FUnArYnINID0Nl ;, A Nr:W ANTIFILAaIAL AGEiVT, 

IN Dti0S0YHILA hh:LANOGASTan 

nei-Li Qian and A . F:iche 

Shanghai Institute of Pharmaceutical Industry, 1320 Beijing Xi rtoad, Shanghai, China 

Safety Assessment, Astra AS, S-15185, StidertRlje, Sweden 

The somatic mutation and recombination wing spot test in Drosophila melanogaater 

was used to evaluate the genotoxicity of Fluapyrimidone . Third inatar larvae, transhetero2ygous 

for recessive wing trichome mutationa were f6II food/test compound mixturea 

. A saturated solution or dilutions containing the test compound was added to 

Instant medium (cxp 1) and powdered Furapyrimidone was added to Yeast-Torula (&xp 2) 

at percentages of 0 .0j-S . F'urapyrimidone did not cause a significant change in the 

mutant spot frequency for any ty ;,e of spots in l:xp 1 . In Fxp 2, the frequency of large 

and twin spots was increased in flies treated with 0 .SdB Furapyrimidone . However 

in repeated experiment the differences were statistically insignificant . Fwrapyrimidone 

was positive on TA 100 and TA 98 at 0 .1 and 1 ntg/plate in Salmonella%microsome 

system for mutagenicity test but teat on Sex-linked recessive lethal test in Drosophila 

melanogaster Furapyrimidone showed no evidence of mutagenic potential . 

Acknowledgmente : This work was done at Safety Assessment, Astra AB . We wish to thank 

K . Sandelin and G . idexell for their technical assistance . 

459 

1SICROt1UCLEUS TEST IN VICIA PAnA ROOT TIPS : INITAOR1tICITT OP COAL TAR PITCH . Z . Qingfan, 

ChangFuju, and Z . Qingxia . Dept . of Pathology, Henan Medical University . Zhengzhou, 

Henan, P .R . China . 

The effect of three different processed (high, moderate, low temperature) coal tar 

pitches on the induction of micronuclei in yjglg ZAbg root tips was studied . The results 

indicated that CTP had potent mutagenicity on'the root tips of y1gjA I&a, but the 

mutagenicity may vary in different kinds of CTP . 

TABLE . Frequencies of Micronucleated cells in }(jsL j4g root tips induced by CTP 

1oa1 ~ T, 

ar Pitch Croilp - maan3SD (a) 

Control Grouo 

Concentration (mg/ml) j, OZa =s 

0 .324 14 .33t0 .88 36 .33±4 .44 16 .33t0 .88 

(16 13 14) (38 43 38) (16 18 15) 

6 .828 39 .67t1 .76 59 .00±3 .46 26 .33t2 .91 1 .67±0 .67 

(39 43 37) (53 65 59) (27 21 31) ( 3 1 1) 

10 .0 60 .33±8 .08 82 .00t9 .61 60 .67±6 .49 

(49 76 56) (63 94 89) (58 73 51) 

These results are consistent with the study on rat lung cancera induced by 

intratracheal instillations of CTP, which were made by us previoualy, Through the 

experiment, we consider that the micronucleus test in yicig ZAg can be used as a new 

alarm system to detect envirorueental pollution such as CTP, and that this test has the 

advantages of sensitivity, reliability, low cost and manageability . 

50869 3672

460 

_ 

DETECTION OP MICRONUCLSI IN PERIPHERAL BLOOD LYMPHOCYTES OF PATIENTS WITH ESOPHAGEAL 

CANCER AND IMPROVEMENT OF THE METHOD . 2 . Qingfan, F . Shuli, and Y . Bin . Dept . of 

Pathology, Henan Medicaltniversity, 2hengzhou, Henan, P .R . China . 

Thirty-four cases of esophageal cancer were chosen for this study . Their age ranges 

from 30 to 75 . The diagnosis of the patients were confirmed by cytological examination 

in our department . They had not received radiotherapy, chemotherapy or other 

immunosuppressive agents recently . Thirty-four healthy persons were taken as the control . 

The method consists of taking 2 to 3 drops of blood from the ear lobe of the examined 

person, treating the blood sample with Tris-ammonium chloride buffer solution to hemolyze 

the red cells and then using the simplifled cytocentrifuge developed by the author to 

prepare the smears, the white cells can be concentrated and spread monolayerly onto a 

small area of the microscope slide, and the morphological details of the cells can be well 

preserved .' The background of the smear is clear, which facilitates scoring the 

micronuclei in lymphocytes . The normal range of micronuclei in lymphocytes in our study 

was 0-3 0/00 . It shows that the results of our improved method are similar to those with 

the ordinary method in which blood was taken by venous puncture . The improved method is 

not only fast and reliable, but liable to be accepted by the persona examined . The mean 

frequency of the micronuclei in the peripheral blood lymphocytes of the patients with 

esophageal cancer was 2 .8 0/00 as compared with 0 .832 0/00 in the control . The difference 

is statistically significant (p < 0 .001), but there is no marked specificity for the 

diagnosis of esophageal cancer, because the frequency in half of the patients with 

esophageal cancer is below 2 0/00, which superimposes with the control . 

461 The SOS Chromotest : an analysLs from published data on 430 chemio3ls. 

P . Quillardet, E. Touati and M. Hofnung . Institut Pasteur, UPMTG - CNRS UA271 - INSERM 

U 163 - Paris France . 

We have made use of an E. coli strain carrying a fusion of gene lacZ to gene 4fu1, one of the 

SOS gene, to devise a rapid assay for genotoxins : the SOS Chromotest (Quillardet et al ., Proc. Natl . 

Acad. Sci ., 79 : 5971, 1982 ; Mutation Res . 147: 65, 1985). The assay is performed in few hours and 

involves simple enzymatic assays . It allows to classify compounds according to their SOS inducing 

potency (SOSIP), defined as their ability to induce the expression af the sfGl : aacZ fusion. To day, 

works from a number of laboratories using the SOS Chromotest have been published . We have 

reviewed data obtained with the SOS Chromotest on 430 chemicals issued from 40 publications arising 

from 20 different laboratories . This led us to evaluate further the potential of the SOS Chromotest to 

detect carcinogens and to compare its response to that of the Salmonella / microsome assay . The results 

confirm that in addition to its remarkable simplicity, the SOS Chromotest is a powerful method to 

detect and evaluate genotoxic agents 

462 

COOPERATIVE EFFECTS IN ASSAYS OF CHEMICAL MIXTURES OF ATMOSPHERIC POLLUTANTS . 

A .S . Raj and D .M . Logan . York University, North York, Ontario, Canada M3J 1P3 

Exposure to pollutants in natural atmospheres usually involves complex mixtures 

of chemicals rather than a single pollutant . In such cases risk assessment is 

difficult if not impossible because the chemicals may interact in several 

unpredictable ways . The object of this research is to measure chemical interactions 

in certain bioassaya and define quantitatively their cooperativity . Nine common 

atmospheric pollutants (4 promutagens, 1 direct acting mutagen and 4 non mutagens) 

have been tested alone and in combinations of two and three in the Ames and micronucleus 

assays . For each of the combinations a strictly additive dose response has 

been calculated and compared with the actual response . In the Ames assay most 

mixtures produce fewer revertants than predicted and this is not due to S9 

limitation . In cases where the predicted response is biphasic ag . BaP and DMBA due 

to different threshold dosages, the actual response is not biphasic but parallels 

that of the lower threshold chemical . In a few mixtures (three or more chemicals) 

inclusion of the direct acting mutagen 1-nitropyrene produces a highly synergietic 

response ie . more than 5 times the additive response . Weighting factors have been 

calculated for each chemical tested which can be used to predict the response of 

new mixtures of the chemicals . This approach should prove valuable in predicting 

the risk due to environmental pollutants . 

This research was supported by a grant from the Ontario Ministry of the Environment . 



Notes



463 

NULEES" "' °°''tYNERGISTIC-AL$qNReTOXICIIY OF ALCOH9L AND LB IN PRINTING PRESS 11ORKBRS . 

Talitha T Rajgh , NV Prasad , K Kashyap , YR AhuJeAq .1)Dept of Genetics, Osmania 

University . 2)C~,o_v~rlnting Press, 3)Bureau of Police . RBD, Hyderebad, INDIA . 

Lead (Pb) has been shown to have a mutagenic potential but reports on the 

mutagenicity of alcohol (A) are controversial . Since there are no reports available 

on the effect of Pb exposure in combination with A it was decided to study the 

cytogenetic effects of this combination . Air sampling was done to estimate the content 

of Pb In the working atmospherf~ The atmospheric Pb level was found to be elevited 

in the printing press (3 .144g/m ) as compared to the background value (0 .5A.g/m ) . 

Peripheral blood lymphocytes were cultured for 48 hrs from 20 adult males (10 

of whom were A consumers) working in a printing press . These individuals were 

exposed to Pb for more than 5 years . Twenty one healthy matched controls were 

also included . Chromosome aberrations (CA) were analysed from 100 metaphases 

per subject . A consumers did not show a significant increase in the frequency 

of CA (structual+numerical) as com pared to the control group (11 .35 vs 7 .54%) . 

But subjects exposed to Pb showed a significant increase In genotoxicity as compared 

to the controls (17 .60 vs 7 .54%) . When a comparison was made between alcohol 

consumers and nonconsumers within the Pb exposed group, there was a higher frequency 

of CA In the consumers than the nonconsumers (20 .72 vs 17 .60%) . This finding 

suggests that Pb and A interact In a synergistic manner In inducing genetic damage . 

Financial Assistance from ICMR Ss acknowledged . 

464 

MODULATION OF GENOTOXICITY, Claes Ramel, Department of Genetic and Cellular ToxicologK 

Wallenberg Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden . 

In the absence of any principle breakthrough in cancer therapy, cancer prevention 

must rely on hindrance of induction of malignant growth . Chemoprevention of cancer by 

means of anticarcinogenic agents is one way towards this goal . This approach is favoured 

by the multistage process of carcinogenicity, where each step can be subjected 

to modulation by various agents . The critical role of genetic alterations In carcinogenicity, 

revealed by activation of oncogenes and inactivation of antioncogenes, focuses 

the attention on genotoxicity in this context . Modulation of genotoxicity can be 

achieved at two levels - by preventing chemical induction of mutations and by affecting 

the expression and regulation of altered genes . It has been shown that chemical 

induction of mutations can be modified by a large variety of chemicals acting at 

different levels - from the initial exposure to genotoxic chemicals to interactions 

with DNA and the establishment of mutations . The development of neoplasm comprise 

alterations of cellular messengers and interaction of gene products . The elucidation 

of this cellular signal system opens new prospects for chemoprevention of cancer by 

interference with the expression and regulation of genes involved In this system . 

UV-INDUCED TNYMIDINE INOORPORATION IN A CLINICAL VARIANT OF XERJDER4A PIGMENTOSW 

A . Shobha Rani . A . Jyothy, C.Kususe Kuearl, M . SuJetha, P.P .Raddy, and O .S.Rsddi. 

Institute of Geiwtics . Hoapital for Ganetie Diseasae, Ossenle Universitv . Beuumpet,HVderabad-S00 016 . 

A . P. Ind ia . 

ABSTRACT 

Xerodeima piymentosus Ss a rare autosoaal reaassive disease In which patients develop abnorael pigmentation 

and salipnancies !n the areas of the skin ezposed to sunlipht . These patients are unable to repair 

normally a certain type of UV induced daaape !n their DNA . The present lnvestl9etion is carried out 

to sae Sf there is any Japsinssnt in the repair process In this clinical variant with only occul .r senlfastation 

without any cutaneous manifestation . 

For the scintillation eeasurewent of raoelr . we used leukocvte ooouletions contelnina 1Xio cells . The 

cells vsre suspended In 1∎1 culture media with 205 serus . The cells were lrradiated with UV and then 

HTdR was added and reincubated for 24 hrs ., at 37oC. The cells rsre washed In saline followed by Sf and 

10% TCA ( trichloroacatie acid) and finally In chilled es thenol . 0 .1 •1 of triton X-100 was added to 

dissolve the pellet and transferred to scintillation vials after which the activity was measured by 

B-counter . UV-induced thyaidlne incroporation rate 1s decreased to 20-37% In the affected individuels 

as compered to their unaffected sib (64%) parents ( 100% ) and controls (100f) lndicatin0 that ths 

rapair mechenis+. !n this clinical variant 1s Upaired . 

50869 3674 

465

466 

I)ISCRE:TE PRODADILITY MODELS IN MAMMALIAN MUTAGENESIS 

I~ .Ilamnanth fvra.~ DeparlrqpeFt of Genetics . Osmania University, 

Ilyderabad - 500 007 (A .P .) India . 

One of the objectives of genetic toxicology is to critically evaluate the 

mutagenicity of various industrial chemicals and environmental pollutants using 

dominant lethal system in mice . T he statistical problem arising out of such 

situations is to Identify the probability distribution of the variables underconsideration 

to assess the significance of the lethality . Preliminary distributions 

like binomial, and poissori have beeft' suggested by Haseman and Soares (1976) 

for the distribution of lethality in plants and mice and are currently in vogue . 

However, these distributions have been found to be inadequate . The complexity 

of the biological phenomenon envisages the possible role of the com pound 

probability distributions . Based on series of experiments conducted in our 

laboratory on dominant lethals in mice a new discrete compound probability model 

has been constructed . The study suggests that the model fits well for evaluating 

even weaker mutagens thus making it appropriate for wider application in the 

field of environmental mutagonesis . 

467 

PREVENTION OF GENETIC DANAGE INDUCED BY BENZO(a)PYRENE IN NICE BY PROSTAGLANDINS 

K .P .Rao, K .Sridevi, and K .V .Chandrika, Department of Genetics, Osmania University, 

Hyderabad-500007, INDIA . 

The present investigation is undertaken to see the possible antimutagenic action of 

Prostaglandin E1(PGE1) against Benzo(a)pyrene (BP) induced genetic damage in bone marrow 

cells of mice by micronucleus test . Recent investigations revealed that certain 

mutagens/carcinogens are known to influence the production of PGs and thus suggesting that 

an altered PG system could be responsible for mutation and cancer . Hence, two experiments 

using micronucleus test in bone marrow cells of mic$ were ~arried out . In the first 

experiment the effect of different doses of P~El (10- to 10- N) on BP induced genetic 

damage was studied . The effect of PGE1 (10- M) in relation to cell cycle against BP 

induced genetic damage was studied in the second experiment . The results obtained from the 

above studies substantiates the possible role of PGa in the pathogenesis of mutagenesis and 

carcinogenesis . 

468 

M1p1/LATIeN OF RADIATIGN-IMJ11C® GEMETIC DAMIIGE BY 2-0EO7fY-0-GLlIC06E IN TISSIR :S OF TRIGD)ELLA FOE 

K.V.S . RAO. G . Jayaraman and P .M . Gopinath . Department of Genetics . PGIBMS. llniwrsity of Madras, Tara .ani, 

Madras - 600 113 . India . Modulatory effect of 2-deoxy-D-Olucwe (2-OG) on radiation induced genetic 

damage has been assessed in the cells-of fenu0reek . Trioonells foenua-oraeoue . 2-DG . a Oluoose antioete- 

bolite and an inhibitor of glycolysis was reported to exert differential action in irradiated normal and 

neoplastic mammalian cells . Mhile I,t potentiates radiation induced genetic damage in tumor cells . !t 

reduces siailer damage in noraal cells . In t1a present study roots of fenu0reek were lrradiatW with 

ganee rays and post-treated with 2-nG . Cytological preparations obtained from the roots on recovery were 

scored to record aitotic indices . instances of ∎icronuclei and ∎itotic anomalies . Roots and calli were 

cultured in aediua containing 2-DG to record data on growth kinetics . DNA extracted from these tissues 

was irradiated both In the presence and absence of 2-DG and subjected to spectrophotoaetric characterization 

. For corroborative date . DNA from calf thyaus and Salaonella tvohieuriua was employed . 2-DG was 

found to reduce the incidence of radiation induced ∎icronuclei snd mitotic anoealies !n seedling snd 

cultured roots . Reduction of mitotic activity by 2-DG was significant . 2-DG Inhibited proliferation of 

cultured tissues . Spectrophotometric characterization of Irradisted DNA in the presence/absence of 2-DG 

reveals no direct interaction with DNA in v tr . DNA of calf thymus and ;; . tv6hiaurium also exhibited 

similar observations . From the observations It is concluded that 2-DG causes reduction !n radiation- 

induced genetic damage also in plant cells . 2-DG is found to be a eitotic inhibitor and reduces the growth 

of celli and cultured excised roots . 2-DG did not show direct Interaction with DNA,lrn vitro . 

A 


Notes 

469 hEOSEMOGENIC RI83C PREDICTION OF TFE CYTOBTATIC TREIT!'lEliT 

Tibor Raposa ?i .D ., and Judith Yirkonyi, n .D . Setttmelweis Univ . Ned . 

6chool,Ill . bept .of Internal riedicine, 1121 .~udapest,EBtv6s u. 12 .Eung . 

The genotoxicity of wide range of cytostatice, commonly used in the 

therapy of lymphonas and solid tumors wae measured in the " in vivo - 

in vitro " SCE assay . A comparison was them made to analyse the rela- r 


i; 

I



NOtES'- "-'"'fionship-bgt_t+e~he genotoxicity and carcinogenicity of the same cytotoxic 

agents . The model for this purpose has been the thorough literature 

surve' oLt herapy induced leukemias during the period from 1930 

to 1988 . Over 130_o secondary acute leukemias are included . It is established 

that the cytostatic compounds with strong BCE induction formed 

the basis off the chemotherapy of more than 9d1i of all the primary malignancies 

, which were later followed by an acute leukemia of the induced 

type . On the contrary, only a few secondary leukemias were repotked 

after the administration of an " 8CE negative " cytostatic treatment 

schedule . This observation supports the conclusion t at the BCE assay 

is a good indicator of the genotoxicity and carcinogenicity of different 

cytostatic agents, and as such it can reasonably be used in planing 

new therapeutical trials with low leukemogenic activity but also 

high clinical efficacy, in malignancies which have a high probability 

for cure . 

470 

NfIATIOYAL SPEY:IFIJITY OF 2-(:YANJE'D(YtZNE 07QDE IN HIlMN LV1PfOB[ .ASA)ID CELLS . L . Redo, D. Slspson, J . 

Cochrane, VLiber and T.R. Sknpak . Chmdcal IMustry Institute of Toxicology . Research Triangle Park, NC 

(USA) and `liatvatd School of Public Health, Boston, ?U (USA) 

The proposed sutagenic metabolite of the rat carcirogee acrylanitrile, 2-cyanethylene oxide (AND), is 

nutaganic at both the hunan tk and prt loci . To develop a better urdeistanding of its or:clanism of action 

in human cella, our grcup has determined the specific It(A sequsnce alterations irdueed by ANJ treataent . 

Analysis of several tk-/- and lrt- mutants by Southern blot liybridisaticn shawed that sost of the iMuced 

mutants had no detectable alterations . A collection of brt- mutaixs were further characterized by dideoery 

sequercing of cloned 1rt cINA . Point mutatiare in the lct codin6 region Wore obsetved in 9 of 17 lrtautants 

; 5 accua-ced at AT base pairs and 4 at OC base pairs . 8/17 ct- mutarxs displayed aberrant splicing 

of trt mRNA, resulting in the loss of single and maltiple ebxm, as tiiall as alternative splicing at a 

cryptic splice site vithin aeon 9 . Southern blot analysis of aitants wdth single axmn losses revealed no 

visible alterations . Analysis of one mutaix missing afmas 3-6 in its sRNA did reveal a visible deletion in 

its gecnodc 1NA. The intron/exon jurcticns of three aitanta (one sd,th somn 7 loss, one adth exan 8 loss, 

and one mutant exhibdting cryptic splicing in e:mn 9) tiere PCR aplified fram gemedc INA and analyzed by 

Southern blot using ema-specific probes . The amos missing ftcm the rt aRNA were present in the $enomdc 

rt aequence . The pertinent intton/exoa regions of l,xt genosd .c INA fram a nRant rdth axon 8 loss and a 

mrtant exhibiting cryptic splicing in a:mn 9 uare cloned into M13op19 and sequenced . Point sutations in the 

conceneus splice acceptor site of ewon 8(AT+fA) ard cmn 9(ATKiC) were observed . These observations 

indicate that AND icduces primarily point .utations in hmmn cells at both AT and OC base pairs, and that 

concensus splice acceptor sites are prone to ®rtageneeis . This wrk also suggests that there are at least 

two pramutagentc lesions irduced by AA1) . 

471 

MOLECULAR ANALYSIS OF HPRT- T-LYMPHOCYTES DERIVED FROM AN IN VIVO CLONAL 

AMPLIFNATION . L . Recio, D . Simpson, J . Cochrane, T . Skopek, J .A . Nicklaaa, J .P . 

O'Neill , and R .J . Albertinia) . Chemical Industry Institute of Toxicology, RTP . NC 

(USA) and the aUniversity of Vermont, Burlington, VT (USA) . 

T-lymphocytes rearrange their T-cell receptor (TCR) genes during differentiation 

in the thymus and then pass the unique TCR gene patterns to their clonal descendents . 

Analysis of TCR gene rearrangements has been used to establish the clonal relationship 

of hprt- T-lymphocytes isolated from the peripheral blood of humans (Mutagenesis 

2 :341) . A female subject has been identified with an extremely high and increasing 

frequency of 6-thioguanine-resistant T-lymphocytes . The majority (>92X) of these 

mutants display the same TCR pattern and therefore must be sibs (Environ . Mol . Hutat . 

12 :271) . To develop a better understanding of the genesis of these mutants we have 

isolated and sequenced hprt cDNA from 15 mutant clones displaying the same TCR 

pattern . All 15 mutants were missing exon 6 from the hcrt message, suggesting that 

this mutation was an early event during the clonal expansion process . One mutant was 

also missing axon 8 in addition to exon 6 . These results demonstrate that clonal 

expansion of mutants in vivo can seriously affect both the frequency and spectrum of 

hprt- mutants observed 1n vivo . The preliminary finding of one mutant possessing two 

separate alterations also suggests the possibility of extreme genetic instability in 

this population of dividing T-lymphocytes . 

POSITIVE CORRELATION BETWEEN SISTER CHROHATLD KXCHANCB AND COTININf! IN SMOKERS 

Reidy, J .A ., Chen . A .T .L ., Spiorto, F .W ., Waymack, P .P ., Jr ., and Smith, S .J . 

Division of Environmental Health Laboratory Sciences . Center for Rnvironmental 

Health and Injury Control, Centers for Disease Control, Atlanta, CA 30333, USA 

Resulta of numerous studies have shown that smokers have a higher 

incidence of sister chromatid exchange (SC6) in their lymphocytes and higher 

levels of cotinine (a metabolite of nicotine) in their serum than nonsmokers . 

50869 3676 

472

t 

SCe and serum cotinine were determined for 17 smokers . SCE for each person 

was determined by analyong 100 second-division lymphocytes from whole blood 

cultures . SCE per persoiC ranged from 4 .54 to 10 .16 exchanges per coll . 

Cotinine values were determined in duplicate for blood drawn at the same 

time . These values were between 13 and 500 ng/ml . In this study, we found a 

positive correlation (R .0 .53, p- 0 .03) between SCE and serum cotinine . Larger 

studies are needed to confirm this finding . 

473 

4Hk: lNTEGENICITY STUDY OF 69'NINGOCOCCAI: I'OLYSACCIIARIED-TETANUS TOXOIL CONJUGATE 

Ren Neiyue, Zhao Hong, tang Yaozhone, and Li genqing, Shanghai Institute of Biological 

Products, Shanghai, China 

Mer.ingococcal group A'polyaaccharide-tetanus toxoid conjugate was combined with po- 

lysaccharide and protein so as to increase the immunogenicity of both the polysaccheride 

and tetanus toxoid in the guinea-pigs and mice . It In hopeful that the conjugate 

will be used widely in human beings, eapecielly in very young children In Chine . Here 

we examined the mutagenicity of the conjugate for Ames assay, micronucleus test and 

chromosome aberration analysis in Chinese Han .ater Lung cells . The result indicated th- 

at the conjugate could not increase remarkarly either the revertants of four test strains 

( TA97, TA98 . TA100, and TA102 ) with of without 89 mix In Ames asaay at the con- 

centration range of 0 .2-125 ug/plate, or the rate of micronucleus after treatment for 

2t hours in the polyghromatic erthrocytes of tho bone mRrrow cells in mice at the con- 

centration range of 48-1200 ug/I(g body weight . The result also indicated that half of 

the cells was inhirited In vitro when the roriugete was et the concentration if 0 .92 

ug/ml and the conjugate could not increase remarkably the rate of chromosome aberrati- 

on after treFtmertt for u hours with S9 mix or for 24 or 48 hours without 59 mix at the 

concentration range of 0 .25-1 .0iug/ml in chromosorre aberration anelysis . It wss belie- 

ved that the conjugate hae no mutegenicity in our leb. f 

474 • 

THE LACK OF DNA HOMOLOGY IN PAIRS OF DNA DIVERGENT CHROMOSOMES SENSITIZES THEN TO 

LOSS BY DNA DAMAGE . M .A . Resnick and T .Nilsson-Tillgren . Yeast Genetics Group, 

National Inst . Environmen-EaT~eafth Sciences, Research Triangle Park, NC, USA ; 

Inst . of Genetics, U . Copenhagen, Denmark 

Chromosomal DNA is considered a priori to be a target for production of numerical 

(whole chromosome) aneuploidy and DNA repair would be expected to play a role . 

Using the yeast Saccharom ces cerevisiae, we have addressed the importance of 

recombinational repa r requ re or ouble-strand break [DSB] repair) in the 

maintenance of complete chromosomes . Specifically, aneuploidy induction by Ionizing 

radiation has been examined in diploids which had either one chromosome III 

or chromosome V replaced by a DNA divergent (homoeologous) chromosome from S . 

carlsber ensis . While they are functionally equivalent, the lack of precise DNA 

omo oof chromosome III or all of V was expected to prevent recombinational 

repair . The absence of recombinational repair (presumably of DSBs) in the 

divergent chromosomes results in aneuploidy levels of 5 to 15% at nonlethal doses 

and a low level of rearrangements . The induction appears to level off suggesting 

alternative ways of dealing with double-strand breaks . For homologous chromosomes, 

the aneuploidy frequency is 20 to 50 X lower . Based on genetic and physical analyses, 

the aneuploidy is due to chromosome loss, not chromosome deletions nor 

malsegregation . Thus, the absence of opportunity for homologous recombinational 

repair of double-strand damage results in chromosome loss . We suggest that nonhomologous 

regions of otherwise homologous chromosomes may be important targets for 

the induction of aneuploidy . The relevance of these observations to those in mammalian 

cells will be discussed . 

475 

BIOMOHITORIPG OF INDIVIDUALS OCCUPATIONALLY EIpOSED TO AROMATIC A11IwES . 

L .R .Ribeiro,D .M .F .Salvadori,E .M .M .Cerqueira,H .S .Barbosa,M .D .M .Oliveira and A .R .Silva . 

Universidade Federal da Bahia, Salvador, BA (BRASIL) . 

Several reports show high frequency of genetic damage and high incidence of urinary 

bladder cancer due to the presence of mutagenic and carcinogenic products in the urinary 

tract,apecially the aromatic amines .The genetic and carcinogenic risks of workers occupationally 

exposed to aromatic amines and the presumed antimutagenic and anticarcinogenic 

potential of provitamin a-carotene are being studied at the production plant at 

the Petrochemical Industrial Complex of CamaSari,BA,Brazi1 .30 male individuals exposed 



Notes


~ Notes 


~ 

to aromatic amines and 30 controls were exaeined .The clastogenic studies were carried ' 

out vith micronucleus test on exfoliated cells from the urinary bladder and the careinQ 

genicity was evaluated through the cytopathological analysis of these cells . Urinary 

bladder cells were recovered by centrifuging the urine and pipetting the asdiASnted eelis 

onto microscopic slides .Air-dried-alides were Giemsa-atained to micronuclei analysis and 

Papanicolau-stained to cytopathological analysis .Tbe possible correlation between preneoplasic 

cells and the frequency of micronuclei is being evaluated,whereas the possible 

effects of age,smoking habits,alcobol and coffee drinking are also discussed .The aim of 

the study presented here was to verify if a combined application of the micrawleus test 

and the cytopathological analysis in exfoliated urothelial cells could be used to identify 

population groups of high risk for urinary bladder cancer and to verify the antiinitagenic 

and anticarcinogenic 3-carotene supplementation effeet .The potential for this method 

to be used for noninvasive monitoring will be discussed .This work was supported by 

FINEP, CNPq and ROCHE . 

476 

CHROMOSOME CHANGES DURING NEOPLASTIC PROGRESSION IN RAT TRACHEAL EPITHELIAL (RTE) 

CELLS . K . Rithidech, D . G . Thomassen, and A . L . Brooks, Lovelace Inhalation 

Toxicology Research Institute, P .O . Box 5890, Albuquerque, New Mexico 87185 . 

Chromosome abnormalities are usually observed in tumor cells . Since it is not 

known if these abnormalities are the cause or consequence of tumor development, it is 

important to characterize chromosomal changes at all stages of progression . The 

purpose of this study was to analyze chromosomal changes in RTE cells at preneoplastic 

and neoplastic stages of tumorigenesis . RTE cells were exposed to 6 Gy of X-rays or 

N-methyl-N-nitro-N-nitrosoguanidine (MiNG) . Preneoplastic enhanced growth variants 

(EGVs), the first recognizable step in tumor progression of RTE cells to neoplasia, 

were isolated using a selective medium . Eighteen EGVs were isolated 35 days after 

exposure . All EGVa from passage 6 were evaluated for chromosomal changes and were 

injected into nude mice to test for their tumoriganicity . Five X-ray-induced ECVs 

have been analyzed . Four of the five cell lines were hyparploid and one was 

hypoploid . G-banding revealed unique chrosasomal alterations in each EGV . The 

specific type of changes were deletions (1q), isochromosomes (9q), translocations 

(5 . 12), marker chromosome/, and double minutes . Nost of the hyperploid EGVs possessed 

at least one extra copy of chromosome 1 . These results suggest that specific 

chromosomal changes can be detected in early stages of carcinogenesis . Cytogenetic 

studies of the remaining EGVs and of tumor cells produced in nude mice by these EGVs 

are underway . Results from these studies will help determine the role of chromosomal 

changet in tumor progression . (Research sponsored by the U .S . Department of Energy's 

Office of Health and Environmental Research under Contract No . DE-AC04-76EV01013 .) 

477 

CTTOGENETIC EVALUATION 0F THE IN VIVO GENOTOXICITT OF SUPERLBTNAL DOSES OF DIOXIN . 

S .D . Robertson and A .F . McFee, Oak Ridge Associated Universities, Oak Ridge, TN (USA) . 

The halogenated aromatic hydrocarbon, 2,3,7,8-tetrachlorodibanso-p-dioxin has 

received considerable attention as a highly toxic environmental pollutant . It !s a 

positive carcinogen in some test systems and a potent teratogen . Cytogenetic findings 

following various sub-lethal doses have ranged from no observable effect on either 

chromosome aberration or SCE induetion to modest but significant ineresses in the 

ineidence of aberrations . We administered single intraparitoneal lnjeetions of 250, 

500 and 1,000 ug/kg (lOx the acute lethal dose) to ule B6C3F1 mice and scored 

chromosome aberrations in marrow cells of 8 aice/treatment 17 hr later, and sister 

chromatid exchanges in 4 mice at 23 hr post-treataent . One-tail trend test analyses 

of the data indicated no significant change in the level of SCEs= the percent of calls 

containing aberrations was significantly incraased by the two lower doses but not the 

highest level . Sfnce dioxin is slowly excreted froa liver and fatty tissue storage 

and lethality of even very high dosas fs not expressed for about 2 weeks, !t was 

meaningful to study longer treatment-to-evaluation times . SCgs among 5 mice per group 

showed a significant lncruse (p< .05, t test) at 8 days after 1,000 vg/kg doses, but 

not at 2 or 4 days ; however, a repeat trial found no elevation of SCBs at 2, 4, 6, or 

8 days . The proportions of polycbro .atic erythrocytes baaring ∎icronucla! in the 

marrow of treated ∎ice were not different from controls at 1, 3, or 8 days, but 

micronuclei in peripheral blood erythroeytes were elevated at 3 days . We conclude 

that significant increases in cytogenetlc lesions are not consistently produced by 

superlethal doses of dioxin . Supported by NIRRS Interagency Agreement T01-ES-20100 

and DOE/ORAU Contract DE-AC05-760R00033 . 

~ 

m 

CO 

Oh 

%O 

W MJ 

CO

478 

_ 


CHLOROPHYLLIN IS AN ANTIMITAGEN IN DROSOPflIIA ItEIANOGASTRRf Notes 

Dra . R . Rodriguez-Arnaiz and S . Zimmering . Universidad Nacional Autonoma de Mexico, 

Coyoacan 04510, Mexico, D:f. MEXICO . 

In Drosoohila Melanogaster chlorophyllin was tested for its ability to inhibit or 

reduce SLRL using the $ggg method and CS males . A brooding scheme of 0-2 and 3-5 days 

was employed . Two groups of experiments were run . First, males were fed with DMN (75ppm 

for 48 hours) and then they were injected with a solution of 0 .5% chlorophyllin sodium 

salt in 5% sucrose solution . A second group of males was injected with the 0 .5% solution 

of chlorophyllin and the fed DMN at 75ppm for 48 hours . The negative control were only 

injected with chlorophyllin and the positive control only fed DMN . The SLRLT was run as 

usual . Results obtained show a decrease in the number and proportion of sex-linked 

recessive lethals in groups treated with DI4d+chlorophyllin and chlorophyllin+DMN . 

479 

Antomatic Evaluation and Comparison of Micronuclel Frequencies in Bone Marrow 

and in Peripheral Blood of Rats Treated with Various Model Clastogens . 

F. Romagna and A . Papapetropoulos . Drug Safety Assessment/fosieology, Sandoz 

Ltd., CH-4002 Basle, Switzerland. 

A new technology is presented which offers high quality slides suitable for fullyautomated 

micronuclei scoring by computerized image analysis and In addition, allows 

the enrichment of immature peripheral blood erythrocytes by using step•gradient 

centrifugation . Repeated dosing of rats for four consecutive days led to a steady state 

level of micronuclei Induction In bone marrow as well as in peripheral blood. In the 

latter, maximal response was delayed. In contrast, no elevated micronuclei frequencies 

could be seen in the mature erythrocyte population Indicating that micronuelelIn this 

cell compartiment were mainly removed by spleenic function . Theae results are In 

contrast to observations made in several mouse strains, where accumulation of 

micronuclei in mature erythrocytea does occur. Our data clearly demonstrated that the 

immature erythrocyte population of rat peripheral blood Is a highly sensitive system 

for micronucleus testing and that a steady state level with maximal drug response can 

be achieved by using a multiple dose regimen . It is hoped that this study may encourage 

further evaluation of the rat peripheral blood micronucleus test fdr routine purposes 

in genetic toxicology . 

480 

GENOTOXIC AND NON-GENOTOXIC CARCINOGENS CAN BE IDENTIFIED AND PREDICTED BY CASE, AN 

ARTIFICIAL INTELLIGENCE SYSTEM . Herbert S . Rosenkranz, Dept . of Environmental Health 

Sciences, School of Medicine, Case Western Reserve Univ ., Cleveland, Ohio 44106 

Analysis of short-term test and animal carcinogenicity results indicates that 

there are genotoxic carcinogens (GC) which are characterized by the ability to induce 

cancers in mice and rats, at multiple sites and in both genders, and there are nongenotoxic 

carcinogens (NGC) which are species- and site-specifio and may be 

restricted to a single gender . NGCs cannot be differentiated from non-genotoxic noncarcinogens 

by short-term teats or "structural alerts" . CASE, the Computer Automated 

Structure Evaluation system Was applied to this situation and a number of findings 

were made : 

1 . CASE was successful in handling non-congeneric SalmoneUamutagenieity data bases . 

The structural determinants (biophores) identified could be used to prediet 

mutagenicity and to study mechanisms of mutagenicity . In spite of many differences 

in the composition of the NTP and Gene-Tox data bases, the same major biophores were 

identified in both . 

2 . Analysis of the NTP carcinogenicity data base revealed that CASE was highly 

effective in classifying carcinogens and non-oaroinogens (sensitivity, 1,00 ; 

specificity, >0 .86) . Additionally, CASE allowed the recognition of NGCs based upon 

unique structural features . CASE identified three types of oaroirwgen-speoifio 

biophores : (a) those' specific for GCs (primarily eleotrophilio or potentially 

electrophilic biophores) ; (b) NGC-specifio biophorea and (a) non-eleotrophilio 

biophores shared by some GCs and NGCa . These findings reveal an unexpected structural 

commonality among NGCs and permits a systematic study of the basis of their action . 


r 

165

166 1989 EMS Abstracts _ • r -_ . . _. 481 

NOtE S 

~J•! 


--=iw- 

^ ~ PREDICTINC' lRtTAGENICITY AND MUTAGENIC NECHANISNS USING STRUCTURAL CONCEPTS AND 

ARTIFICIAL INTELLI E~NCE . H .S . Rosenkranz and G . Klopman, Departments of Environmental 

Health Soienoes,_ad1=hemistry, Case Western Reserve Univ ., Cleveland, Ohio 44106 . 

Heretofore structure activity relationships (SAR) have been confined to highly 

oongenerie data bases (i .e ., specific chemical classes) . Thus the study of individual 

molecules or small groups of molecules has not generally been possible due to the 

paucity of data . The CASE (Computer Automated Structure Evaluation) method developed 

by us is an expert system which is completely automatic and self-learning . We have 

demonstrated that it is further unique in that it can use non-congeneric data bases 

and still be highly predictive of mutagenioity . In the present study a subset of the 

Gene-Tox Sa/rnonella mutagenicity data base consisting of 808 chemicals was analyzed by 

CASE and the generated structural determinants were used to study the basis of the 

mutagenicity of a series of agents . Thus, it was shown that for phenylazoaniline 

dyes retention of the azo moiety is essential for mutagenicity . For 1-amino-2naphthol-derived 

azo dyes, on the other hand, reductive cleavage of the azo bond is 

required for activity . The basis of the inactivation of azo dyes and polyeyolie 

aromatic hydrocarbons by sulfonation was also elucidated and it was demonstrated that 

only certain sites are targets for inaotivation by sulfonation . 

It will be demonstrated that CASE can be used to design molecules retaining their 

beneficial properties but of greatly reduced mutagenioity . 

MODULATION OF GENOTOXIC EFFECTS IN HUMANS . M.P. Rosin and A .M Gilbert, 

Carcinogenesis Unit. School of Kinesiology, Simon Fraser University . Burnaby, B.C. and British 

Columbia Cancer Research Centre . Vancouver, B.C ., Canada 

482 

Human populations are constantly exposed to a multiplicity of environmental agents, the 

interactions of which can have a profound effect on cancer risk. This paper describes studies 

validating the micronucleus test on exfoliated cells (MEC test) as a technique for studying the 

interplay of carcinogens, cocarcinogens, genetics and dietln a biological response with relevance 

to cancer, chromosome breakage in target epithelial sites. The initial studies described in this 

paper involved carcinogen-exposed populations in which combinations of poor diet, alcohol, and 

tobacco chewing were shown to interact to increase MEC frequencies in the oral cavity . In these 

populations, oral supplementation with beta-carotene and/or vitamin A resulted in a reduction in 

MEC frequencies even when exposure to tobacco/alcohol remained unchanged . Recent studies 

are aimed at incorporating genetic factors (defective DNA repair processes, spontaneous 

chromosomal instability) into these models . The population currently being examined is 

comprised of patients with ataxia-telangiectasia (A-T), a cancer-predisposing syndrome 

characterized by a hypersensitivity of cultured cells to the action of free radical-producing 

chemicals and spontaneous chromosomal Instability . Our initial observations indicate that MEC 

frequencies are elevated in A-T patients and in some of the parents of such patients (at risk for 

breast cancer) . These studies suggest that the MEC test may be one approach by which the 

complex interactions of environmental and genetic factors can be delineated . 

483 

0°-METNYLGUANINE-DNA-HETHYLTRANSFERASE ACTIVITY IN SURGICAL SPECIMENS FROM HIGH GRADE 

HUMAN MALIGNANT GLIOMAS . 

0 . Rossi, G . Arena, G . Frosina, G . Fronza, A . Sobrero, S .L . Gentile, E . Bruzzone, N . Bal_ 

dini, C . Silvestro, and A . Abbondandolo, National Institute for Research on Cancer, Genova 

(Italy), University of Genova (Italy), and Neurosurgical Clinic, S . Martino Hospital, 

Genova (Italy) 

Chloroethylnitrosoureas (CENUs) are used, usually in combination with radiotherapy, 

in the clinical treatment of brain tumors . As pointed out by R .W . Kohn (1987), "It seeas 

likely that only tumors consisting predominantly of transferase-deficient cells would be 

potentially responsive to chloroethylnitrosoureas . Tumor tissues could be assayed for 

guanine-06-alkyltransferase, and treatment with these drugs could be confined to patients 

bearing transferase-deficinet tumors" . MT-deficiancy has been found to be relatively fre_ 

quent among cell lines derived from human brain tumors but has not been demonstrated so 

far, to our knowledge, in tumor tissues . We have started a study to measure the MT aetiv_ 

ity in surgical specimens from high grade human malignant glio®as, with the dual aim to 

(i), know whether lack of activity can be demonstrated in these tumors, and (ii), relate 

the measured levels of MT to the histology of the tumors and to the response of patients 

to chemotherapy with 1-(2-chloroethyl)-3-cyclohaxyl-l-nitrosourea (CCNU) . To date, 12 

gliomas have been assayed . In 11 tumors, MT activities ranging from 30 to 150 fmoles/mg 

protein have been measured . The only negative specimen derived from a patient who had re 

ceived radiotherapy before surgery . At the present stage of the study&therefore, we 

have no unequivocal evidence for the existence of MT-deficient gliomas . 

50869 3680

484 

_ 

IMUrID RE.VEFtSION OF A SPONfAt~OUS POINT NATPATICN WITHIN THE CHINESE HAFISTFR HPRT 

GENE~ Yrz~ ~~ 1 

B J F Rossiter ~DCMuzny , C T Caskey1 and r( ~, 2 Inst 1 for Molecular 

Genetics, Baylor College of Medicine, Houston :Texas 77030, USA, jDept of Biochem 

Genetics,Paterson Institute for Cancer Research, Manchester . 

Spontaneous HPRT mutants can revert spontaneously at different frequencies by 

anplification, point autation at the site of the original mutation or at a second 

site . HPRT mutants can also revert at widely different frequencies on exosure to 

alkane sulptnnates or alkyl nitrosaueas . The lowest frequencies (5 x 10 50pghnl 

1rIDtu1 were observed in mutants e .g . TG15_arith normal amounts of nozmal sized HPRT 

mRm . Two other mutants with no detectable HPRT s04A revert at a tenfold higher 

frequency . A cDNA library was made fzan 4G15 in gtlO and a full length clone 

isolated after screening with HPRT cDNA. Sequencing revealed an A -> G transition 

which results in the substitution of glycine for aspartic acid at position 134 . 

Allele specific screening of in vitro amplified DNA frem wild-type cells, TG15 and 

four independent M induced revertant clones showed that all the had regained the 

wild-type sequence . Thus the point mutation in the TG15 is responsible for 

inactivation of the HPAT enzyme . Amino acid 134 is thought to lie within the 

catalytic domain . These results and those in which a truncated form of the E coli 

ada gene transfected and expressed in TG15 (FCa and Margison, Mutagenesis 3, 409, 

1988) Eeduced the induced revertant frequency are consistent with the conclusion 

that 0 nmethylguanine is the promutagenic lesion and reversion is due to a point 

mutation at the site of the original lesion . 

485 

BETA-CA~~TENE IN AQUEOUS DISPERSION AS A SCAVENGER OF 

H~O2/CU /ASCORBATE GENERATED FREE RADICALS :EFFECTS OF PARTIAL 

PRESSURES OF OXYGEN . EJ .Rousseaus, AJ . Davison & M.P. Rosin . Bioenergetics Research 

Lab . Faculty of Applied Sciences, School of Kinesiology, Simon Fraser University, Burnaby, B.C . 

V5A 1S6 and BC Cancer Research Centre, 601 W 10th Ave ., Vancouver, B .C. VSZ 1L3 

We report on the antioxidant capacities of beta-carotene In aqueous dispersion as a 

scavenger of hydroxyl radicals under varying partial ressures of oxygen . Hydroxyl radicals 

(generated by hydrogen peroxide In the presence of oopper and asoorbate) pxid¢ed betacarotene 

resulting in a loss of absorbance at 460nm . A mixture of 0 .1mM Cu +, 0.5mM 

ascorbate, and 2.5mM H2050 iIn Hepes buffer at pH 7.4 and 25~, was used, oxidizing 2 .76ug 

beta-carotene/min . Each ofthe active ingredients was essential. If any were omitted, the rate of 

bleaching of beta-carotene decreased by more than 20% . This system was studied under high 

oxygen(62%), normoxic(21%), and low oxygen(5%), each with four concentrations of H 02 

(2 .5, 5, 7 .5, and 1omM) . Rates of bleaching of beta-carotene under 20% oxygen were a~f 

average of 15% and 30% higher than 62% and 5% pressures of oxygen . Thus beta-carotene is 

at its most efficient as an antioxidant at intermediate partial pressures of oxygen, In partial 

agreement with other workers who have shown enhanced protection at p02 below ambient 

levels. Beta-carotene is capable of scavenging hydroxyl radicals . The ability of beta-carotene to 

function as an antioxidant against free radicals could help define a mechanism by which betacarotene 

acts a chemopreventative agent . 

486 

TRADESCANTIA-MICRONUCLEUS BIOASSAY ON CI~STOGENICITY OB WASTEWATER AN{~ I~i SITU AIR 

MONITORING, E . F . Ruia1, E . R . Valtierra , S. U . Lecona , and T . H . Ma , 'Centro de 

Estudios Academ~cos sobre Contaminacion Ambientl, IInivereidad Autonoma de Queretaro, 

QRO, Mexico, Institute for Environmental Management and Department of Biological 

Sciences, Western Illinois University, Macomb, IL (USA) 

Tradescantia-Micronucleus (Trad-MCN) bioassay is known to be a highly sensitive short 

term test for clastogenioity of water directly without concentration and on site 

monitoring of clastogenicity of air pollutants without collecting air condensates . 

Results can be obtained within 36 - 48 hr . Trad-MCN bioassay was applied to 

wastewater samples collected from the canal at a point about 8 kilometers down stream 

of the industrial zone of Queretaro City , Mexico . Four liters of water samples 

were collected monthly for tests in the year of 1987 and part of the years of of 

1986 and 1988, for the clastogenicity of the water through the rainy and dry seasons . 

On site monitoring of the clastogenicity of the air with Trad-MCN test was done at 

three locations in the city . The clastogenicity which was reflected by the 

frequencies of micronuclei in the meiotic pollen mother cells was always higher in the 

wastewater treated groups than the negative control group group using tapwater . When 

the fluctuated MCN frequencies were compared with the monthly rainfall records, the 



Notes


Notes peak olaetog+nj&.tty-seems to be related to the runoff of the pollutants from the upper 

atream of:ftlte 0smF1: Five on site monitoring tripe were made at each of the three 

locatione, -U e . Conalep, Flores Magon (induetrial sone) Belles Aetee (Downtown) in May 

and June, 1988~Positive resulte were obtained in ∎ore than 60% of the tests 

conducted .. -~a--r- 


487 

PATTERNS OF MUTATIONAL SENSITIVITY IN POST-STEM-CELL STAGES OF MOUSE 

SPERMATOGENESIS AND IN ZYGOTES : 'I'HE GENERATION OF DELETION MUTANTS AND 

MOSAICS . Liane B . Russell, Oak Ridge National Laboratory . Oak Ridge, TN. 37831-8077. 

Because the majority of conclusive results in the mouse specific-locus test (SLT), are for to#onial 

stem cells, we are engaged in enlarging the SLT data base for post-stem-cell stages (from differentiating 

spermatogonia to mature spermatozoa), each of which spans a relatively brief time interval . Chemicals differ 

markedly in their relative effects on different post-stem-cell stages, both with regard to mutation rate and to 

productivity (determined by dominant-lethal incidence and germ-cell death) . Mutations induced by chemicals 

that have their greatest mutagenic effect in premeiodc (but post-stem-cell) stages appear to be smaller genetic 

lesions than mutations induced by chemicals that are most effective in postmeiottc stages . The correlation 

between patterns of germ-cell-stage sensitivity for specific-locus mutations and dominant lethals, which has 

been noted for some chemicals, does not hold for others . Of all post-stem-cell mutagens chlorambucil 

(CHL) is the mos~ effective : we have shown that exposure of early spermadds to only 10 mg CHIIkg 

induces 1 .3 x 10' mutations per locus . Almost all such mutations have proved to be deletions or other 

structural changes, which are highly valuable tools for molecular mapping studies . -- The zygote may be 

regarded as the final germ-cell stage, since maternal and paternal genomes are still separate . We have found 

that ethylnitrosourea (ENU) produces a very high frequency of pritttarily small genetic lesions in zygotes, 

most resulting mutants being mosaics. Mosaics can provide very useful biological tools for developmental 

studies . Thus . CHL administered to early spermatids, and ENU to zygotes, may be the mutagenic 

treatments of choice for generating maximum frequencies of, respectively, large-lesion whole-body mutants 

and smaU-lesion mosaic mutants . [Research 'p indy sponsored by the Office of Health and Environmental 

Research, U .S . DOE contract DE-AC05-84OR21400 with Martin Marietta Energy Systems, Inc ., and by the 

National Institute of Environmental Health Sciences under IAG No . 222Y01-ES-10067 .) 

488 

DOSE REPETITION GREATLY INCREASES THE MUTAGENIC EFFECTIVENESS OF ENU IN 

MOUSE SPERMATOGONIA : ADDITIONAL DATA . W. L . Russell, P. R. Hunsicker, and S . C. 

Maddux, Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-8077 . 

The maximum practicable single dose of ethylnitrosounea (ENU) that can be~g~ven intrapertioneaUy co 

mice in mutagenic studies is appro ximately 250 mg/kg . We reported earlier (Hitotsumachi et al ., 1985, 

Proc. Natl. Acad. Sci. USA, 82 : 6619-6621) that the effectiveness of ENU in inducing mutations at 

specific loci in stem-cell spermatogonia of mice could be increased above the level obtained with 250 

mg/kg by using 4 doses of 100 mg/kg spaced at weekly intervals . The increase obtained was 2.2 times, 

and was statistically significant (P, one-tailed - 0 .02), even when calculations were adjusted for the 

occurrence of clusters . However, since this adjustment is only a rough approxitnadon, and since the 

protocol of repeated doses is an attractive one for other investigators desiring a high mutation rate, we 

decided to increase our sample size by doing a teplicate experiment . A total of 37 mutations was obtained 

in 3428 offspring from treated stem-cell spetmatogonia. Clusters were again uent, but there were at 

least 20 independent mutations . The mutadon frequency is almost identical wI that obtained earlier . 

Furthermore, the significance of the difference of the combined data from the mutation frequency obtained 

with a single dose of 250 mg/kg is now more convincing (P, one-tailed - 0 .003), and the recommendation 

to other investigators of rhe value of the dose-repetition protocol can now be made with more confidence . 

As was emphasized in our earlier publication, the mutation frequency obtained is of a magnitude that 

seemed out of reach a few years ago, being 36 times the highest reported for pr .carbazine, the most 

effective chemical mutagen known for stem-cell spermatogonla before ENU had been tested . [Research 

jointly sponsored by the Office of Health and Environmental Research, U .S . DOE contract DE-AC05- 

840R21400 with Martin Marietta Energy Systems, Inc ., and by the National Institute of Environmental 

Health Science under IAG No. 222Y01-ES-10067 .] 

489 

CONCENTRATION OF MUTAGENIC COMPONENTS IN RIVER WATER BY USE OF THE BLUE RAYON METHOD 

Hiroshi Sakamotol, Katsuhiko Nakamuroz Yasuyoshi Sayato2 and Hikoya Hayatsut 

F~ulty of Pharmaceutlcal Sciences, Okayama University, Taushima, Okayama 700 

2Faculty of Pharmaceutical Sciences, Seteunan University, Hirakata, Osaka 537-01, Japan 

Activated carbon and %AD-resin are widely used for concentration of organic mutagens 

in river water . In these procedures, a large volume of water has to be taken out of 

the river and processed . Use of the blue rayon method gives facilty for concentrating 

mutagens with polycyclic structures from river waters . In this method, blue rayon is 

allowed to stand for a day in the river to make contact with the flowing water, and 

compounds adsorbed to the rayon 1s then eluted and assayed for mutagenicity . In this 

way, we have measured the mutagenicity of the water of River Yodo in Osaka, and that of 

the tributary rivers Katsura, Kisu and Uji in Kyoto . The mutagenicity is assayed with 

50869 3682


S . typhimurium TA98 and TA100, with and without metabolic activation . The samples from Notes 

the Katsura showed the highest mutagenic activity (3500 revertants with TA98, . +59 ; 87 

revertants with TA9B, -S9~ 141 revertants with TA100, +S9 ; negative with TA100, -S9, 

per 0 .1-g equivalent of'bIue rayon) . By further measurement at several different sites 

of this river, we found that the effluent from a waste-water treatment plant in Kyoto 

City is the possible source of the mutagenic pollution . When the samples collected 

from this river in the winter and summer of 1988 were fractionated by TLC, the mutagenic 

activity in TA98 with metabolic activation was found predominantly in single identical 

zones . It is suggested that River Katsura is constantly polluted with certain 

frameshift promutagens of polycyclic structures . 

490 

SALMONELLA MUTAGENICITY TEST ON VARIOUS KIND OF IRRADIATED SUGARS AND AMIlIO ACIDS 

K . Sakamoto, K . Kanazashi, S . Iwahara, K .Takatori, and R . Aibara, Hatano Research 

Institute, Food and Drug Safety Center, Hadano, [anagawa (Japan) 

Recently, various kind of irradiated foods were studied in mutagenicity tests . 

Every food comprises variety of components, but the volume of samples that can be 

used by these test systems is limited . Accordingly, even if certain components of 

the irradiated food should undergo mutagenic changes, it would be impossible to 

detect such changes if the volume of them are smaller than the detectable limit of 

the test systems . 

For this reason, it is necessary to study mutagenicity on each irradiated food 

components . And if the test results indicate the formation of mutagens in component 

by irradiation, we should evaluate the genetic toxicity of irradiated foods with the 

test results in due consideration of : 1) irradiation dose, 2) ratio of mutagenic 

products formed from the irradiated food components, 3) strength of mutagenic 

activity, 4) proportion of components in food changeable to mutagens and 5) possibility 

of elimination of the mutagenic products . 

We studied Salmonella/mammalian microsome mutagenicity test on various kinds ofsugars 

and amino acids irradiated in the dry condition and in liquid solution with 

gamma ray of maximum dosage upto 10 kGy . As a result, weak mutagenic activity was 

detected in the 5% solution of cystein and 10% solution of arabinose irradilted at 

the maximum dosage of 10 kGy, with metabolic activation system . The results indicate 

that the mutagenic products from each compound hardly forms in irradiated foods . 

491 

CLASTOGENIC EFFECT OF ELLIPTICINE ON DIFFERENT PHASES OF THE CELL CYCLE OF 

CULTURED HUMAN LYMPHOCYTES AND ITS SYNERGISTIC ACTION WITH INHIBITORSOF 

DNA REPAIR AT G2 . Elza T . Sakamoto-Hojo and Catarina S . Takahashi'(IBILCE- 

S . Jose Rio PreEo-UNESP and FFCL de Ribeirào Preto-USP, BRASIL) . 

Ellipticine, a pyridocarbazole alkaloid, has shown an antitumor effect 

on several types of experimental and human tumors, being an intercalating 

substance whose mutagenicity has been demonstrated in several systems . 

To characterize the mechanism of action of this compound at the cell cycle 

level, human lymphocyte cultures from 2 healthy donors were treated with 

3 pg/ml ellipticine in 30-minute pulses during different phases of the 

cycle and analyzed for chromosome aberrations and sister chromatid exchanges 

. The G2 phase was more sensitive in terms of induction of aberrations, 

followed by S and G1 . The 24-h treatment (S phase) induced a four 

fold increase compared to th8 control . SCE induction was significant only 

at G1, when SCE frequency doubled_3The effect of lymphocytg post-treatment 

with inhibitors of DNA repair (10 M caffeine and 5 x 10 M 1-B-D-arabino 

furanosylcytosine) was also tested by adding 3 yg/ml ellipticine at G in 

30-minute pulses and the inhibitors immediately afterward during the2last 

3 h before harvesting the cultures . In the two experiments performed on 

blood from the 2 donors there was a moderate effect of chromosome aberration 

potentiation (about 2-3 times), i .e ., ellipticine bad a synergistic 

effect with both inhibitors in terms of chromosome aberration induction . 

492 

THE ABSENCE OF PAH HYDROXYLATION ACTIVITY IN TOBACCO CALLUS S9 . M . F . Salamone', 

S . Richard', C . J . Gentile', J . M . Gentiles, and D . A . Rokosh', 'Ministry of the 

Environment, Toronto, Canada and *Hope College, Holland, Michigan . 

Microsomal enzymes (S9) from four week old photosynthetic and nonphotosynthetic 

tobacco (Nicotiana tobacum) callus cultures were prepared by two separate laboratories . 

The ability of each callus S9 to activate 2-aminofluorene (2AF) and benzo(a)pyrene 

(BaP) was then tested with the Ames fluctuation and plate incorporation assays . With 

each assay the callus S9 preparations from both laboratories increased the mutagenic 


. 

169 

Y


NotQs__ . w_ . _ 

,.+ .,tct)vity of:~Af, ~not BaP . Each S9 was then tested for 2,5-diphenyloxazole (PPO) 


" hydroxylationtsictivity in a fluorometric mixed function oxidase (MFO) assay modified 

from Niebert and G oin . No PPO hydroxylation activity was observed with any of the 

callus S9 prep_%ra In parallel experiments, rat liver S9 activated both 2AF and 

BaP, and exhibited strong HFO activity . Subsequently, nonphotosynthetic tobacco 

callus was grown on Aroclor 1254 and 3-methylcholanthrene enriched callus media to 

determine whether the hydroxylation enzymes could be induced . S9 preparations from 

these callus cultures also were negative for BaP activation and for MFO activity . An 

electron capture-gas chromatographic analysis of the callus grown on the Aroclor 

enriched media, indicated that Aroclor 1254 was being incorporated into the growing 

callus . These results suggest that callus from Nicotiana tobacum may not possess 

sufficient quantities of the enzymes necessary to ydroxylate PANs (PPO and BaP) and 

that these enzymes, if present, are not readily induced . 

493 

CYTOGENETIC EFFECT 0F PLANT USED IN CATTLE FOOD AND IN POPULAR MEDICINE . 

D .M .F .Salvadori*,A .R .Silva*,M .D .M .Oliveira*,A .R .P .L .Bautista**,and L .R .Ribeiro* . 

*Univeraidade Federal da Bahia, Salvador,BA (BRASIL),**Empresa de Pesquisa Agropecuaria 

da Bahia, Salvador, BA (BRASIL) . 

Indi¢ofera suffruticosa is one of the plants widely used as animal food,ard its ecanmic 

importance is due to the fact that the greater part of the cattle are fed with it .It is 

also used as a medicinal plant in Brazil .The main compounds present in this plant are the 

pyrolizidine alkaliods, A mutagenic activity of these alkaloids and their capacity to 

induce liver tumor in man and in experimental animals have been observed .In this present 

study,teste were made on the ability of aqueous and hexanic leaf extract of Indiaofera 

suffruticosa to induce chromosome aberrations in mouse bone marrow ce1ls .Groups of male 

mice were injected intraperitoneally with 3 different concentrations of aqueous leaf 

extract, 0 .312, 0 .625 and 1 .250mg/kg body wt . which correspond,respectively to 6 .25,12 .5 

and 25 .0% of the lethal dose (LD100)and with 3 different concentrations of hexanic leaf 

extract (0 .625, 1 .250 and 2 .500mg/kg body wt .),respectively 12 .5, 25 .0 and 50 .0% of the 

LD100• 24h after the treatment the animals were killed and bone marrow metaphase cells 

were prepared .No significant increase in the frequency of cells with chremoaare aberrations 

was observed between negative control (4 .7%) and the groups treated with aqueous leaf 

extract (3 .0, 5 .0 and 5 .0% respectively) . For hexanic leaf extract the results obtained 

were 5 .7, 3 .3 and 4 .0% . respectively . A,light increase (5 .7%) was observed only in the 

lower concentration as compared to negative control (4 .8x) .Tbeae results showed that the 

response decrease with increasing dose . Further experiments are in progress to examine 

the clastogenic effects of the other plants in Brazil . FINEP, CNPq end COMCITEC . 

494 

MUTAGENICITY ACTIVITY OF AIRBORNE PARTICULATE EXTRACTS FROM SAO PAULO, BRAZIL . 

PRELIMINARY RESULTS . P .S . Sanchez, G .U . Valent, M .C .L .S . Coelho, C .A . Coimbrao, C . Alon 

so and M .I .2 . Sato . Companhia de Tecnologia de Saneamento Ambiental (CETESB) . Sao Paulo, 

Brazil, CEP 05489 . 

It is well recognized that airborne particulate extractable organic matter may 

contain compounds which have both mutagenic and carcinogenic activity . The objetive of 

this study was to evaluate the mutagenicity of airborne particulate from Sao Paulo, in 

urban and industrial Segions, where the incidence of respiratory diseases is high . 

Air volumes of 2000 m were collected in glass-fiber filters using a high - volume 

(Hi-vol) sampler in a 24 h period . Filters were extracted twice by ultrasonication 

with 100 mL of 1 :1 mixture of methanol and dichloromethane . The extracts were filtered 

in AP20 membrane and evaporated to dryness under vaccum and dry nitrogen . After 

evaluation of dry weight, the extracts were dissolved in DMSO and assayed for 

mutagenicity by Ames Test, using S. typJiimu4tua strains TA98 and TAJ00, in the presence 

and absence of S-9 fraction . Results were expressed in revertents/m and revertents/yg 

of extractable material . Preliminary results showed the presence of mutagenic compounds, 

capable of inducing such frameshift mutation as base pair substitution, in several urban 

extracts sampled . The results suggest the necessity of carrying out more detailed 

studies about mutagenicity of particulate air pollutants in this area, which will be 

useful in evaluating future changes in air quality in Sao Paulo, as well as provide 

subsidies in the establishment of goals and criteria to an efficient action in air 

pollution control . 

~ 

m 

Cfl 

Ot 

W 

Oh 

CD 

~P

495 - 1989 EMS Abstracts 

IONIZING RADIATION AND GENETIC RISKS : CURRENT STATUS NO èS 

K . Sankaranarayanan, Daf'artment of Radiation Genetics and Chemical Hutagenesis, 

Sylvius Laboratories, State University of Leiden, Leiden, The Netherlands 

Estimates of the risk of genetic disease in the progeny of parents or populations 

exposed to ionizing radiai>on are made on the basis of mutation data from animal 

experiments and using a number of assumptions to bridge the gap Tetween induced 

mutation and genetic disease in man . The natural prevalence of genetic and multifactorial 

diseases in the population provides not only a framework for risk perception, 

but also is used in the act¢ffl calculattde of risks . 

Current estimates of genetic risks relate primarily to expected increases in the 

frequencies of those diseases with Mendelian patterns of inheritance ; their natural 

prevalence is of the order of 1 .25% . However, for multifactorial diseases, whose 

natural life-time prevalence is well over 50%, no reliable estimates of risk can be 

made in view of the fact that the relationship between mutation and disease is not 

well-understood . Animal studies using multifactorial traits as indicators of genetic 

damage and the genetic studies of the Hiroshima and Nagasaki populations (in the latter 

two of the indicators used are multifactorial) have failed to demonstrate significant 

adverse effects as a result of radiation exposures . However, as is well-known, 

mutation studies with experimental mammals have provided good evidence for the induction 

of mutations . The possible reasons for these discrepancies will be briefly 

discussed . Additionally, the relevance of knowledge on the nature of spontaneous and 

radiation-induced mutations in the context of risk estimation will be considered . 

496 

IMMUNOLOGIC METHONS FOR THE DETECTION OF CARCINOGEN ADDUCTS IN HUMANS 

R . M . Santella, Columbia University New York, NY 

Monoclonal ant,ibodies have been developed which recognize a number of 

carcinogen-DNA and protein adducts . These antibodies can be used in highly 

sensitive competitive enzyme linked immunosorbent assays (ELISA) to detect femtomole 

levels of adducts in human samples . With the most sensitive antibodies, DNK adducts 

in the range of 1/l0s nucleotides can be measured . In addition, antibodies to DNA 

adducts can be used to investigate localization of adducts in specific cell types . 

We have used antibodies recognizing the major edduct of benzo(a)pyrene (BP) to 

monitor adducts in lymphocyte DNA of foundry workers and smokers end nonsmokers . 

Adducts in lung tissue of cancer patients and placental tissue of smokers and 

nonsmokers have also be analyzed . Because of antibody crossreactivity with 

structurally related adducts of other polycyclic aromatic hydrocarbons, this assay 

is not specific for BP adducts . Monoclonal antibodies against 8-methoxypsorelen-•DNA 

adducts have been used to monitor adducts in psoriasis patients treated with this 

chemotherapeutic agent . Immunofluoreacence staining of skin biopsies from patients 

demonstrated adduct localization to epidermal cells . Studies with antibodies to 

aflatoxin-Ri-DNA adducts were used to detect elevated levels of adducts in liver 

tissue from Taiwanese hopatocellular cancer patients . Adduct detection in humans is 

now established as a viable method for determination of exposure to certain chemical 

carcinogens . The relationship of adduct. measurements to risk requires further 

investigation . 

497 

CYrOGEMETIC RISK ASSES9Etfr OF OPERATI011 T/EATRE PERSOI~EL 

S .T . SANIHIYA . V. Padsrani and A. Raassh. Departsant of Genetics . University of Madras, Madras - 600 113 

(India) . Occupational health risk to personnel working in operation theatres have been extensively 

studied . Although results are controversial . many agree that they constitute a potentially risk group and 

needs to be periodically aonitored . This is particularly relevant, to countries,Yhere safety NasurN 

at work places receive less priority than desired . Therefore, the present work is contemplated to assess 

cytoyenetic risk on anaesthetists and supportive staff aeployed in a major referral hospital of Madras . 

The study group consisted of 16 snaasthetists and 4 theatre assistants serving for 1-33 years (13 .50 s 

7 .6) in several surgical theatres, which do not have any scavenging device . N40, ether and halothane are 

being used aither singly or in combination . Control group (n - 20) consisted of persons with different 

occupational set up . satched for possible confounding variablas . Cytopenetic damage !s assessad in terms 

of chroaososal aberrations (CA) and sister chromatid exchanges (SCE) observed !n 48 and 72h lysphocyte 

cultures of peripheral blood . Stp-rise regression analysis was performed taking duration of service (xl), 

ege (x )u sex (x3) and saokinp status (x4) as indepandent variables and risk aeasures (f setaphuas with 

and vi~hout gaps - CA (G .) and CA (G -) and SCE/cell) as dependent variables using SPSS . Only xl !s the 

significant determinant of the variation in CA . This accounted for 609 upvards of the variation . For 

SCE . both x) and x3 are significant detersinants. xl alone explained eef of the variation and both together 

accounted for about 915 of the variation !n SCE . 8ased on these findings, it is concluded that persons 

working in theatres devoid of scavenging measures are at potential risk of genetic damage which appears 

to increase with duration of service . 

analysis . 

Thanks to our Prof .P .M.Gopinath for encouragement and Dr .M .Laks)ranan . CMC. Vellore for Computer 


171


Notes '" rULTRA-VIOLET-.1NDUCED MUTATION SPECTRA ON SIMIAN VIRUS 40 GENES AFTER DNA 

TRANSFECTION OR VIRUS INFECTION . 


A. SARASIN,. Az■LtOftENBEROER and F. BOURRE. 

498 

Laboratory of Molecular Genetics, Institut de Recherches Scientifiques our Is Cancer, B .P. n° 8, 94802 - 

VILLEJUIF (France) . 

The analysis of induced mutations In mammalian cells has become a major goal for the understanding of 

the carcinogenesis Initiation . Due to the complexity of the cNlular genome, the use of small and easily 

manipulatable DNA probes, depending on cellular enzymic machinery, is highly desirable . Two such systems 

have been developed : shuttle vectors and animal virus mutants . We have used the latter one with either natural 

SV40 or modified SV40. 

Simian virus 40 (SV40) has been used after being treated In vitro with ultraviolet light (UV) . Our 

mutation assay is based upon the reversion of a temperature-sensitive (ts) growth at 411C to a wild-type 

growth phenotype. UV Irradiation of is 8V40 DNA leads to the Induction of temperaturalndependent phenotypic 

revenants which are due to singie-base substitutions located opposite potential UV-Induced DNA lesions . This 

mutagenesis appears therefore to be targeted opposite lesions . Treatment of UV-irradiated DNA with the E . coli 

photolyase increased virus survival and strongly decreased virus mutagenesis . The mutation spectrum is 

different after photoreactivatfon implying that pyrimidine dimers and Py(8-4)Py photoproducts are both 

premutagenic lesions . 

UV-irradiated or unirradiated SV40 virions or SV40 naked DNA have been used with the same 

phenotypic reversion assay . It appeared that the transfection step, used with naked DNA, and the Infection lead 

to different results for mutagenesis . Indeed the UV-tnduced mutation spectra were similar but not Identical 

while the spontaneous mutation spectra were different regarding the location of mutation hot spots . 

GERMINAL IMPRINTING IN CHROMOSOME MUTATION 

M. S. Sasakl, Radiation Biology Center, Kyoto University, Kyoto (Japan ) 

499 

There is now a growing body of evidence indicating that the non-Robertsonlan de vo 

chromosome mutations have a strong bias toward paternal origin . Such non-randomness has 

been implicated as a reflection of differential susceptibility to errors in meiotic process 

between males and females. However, we meet some difficulties with this idea . Deletion 

or loss-of-function mutation of retinoblastoma susceptibility (RB) gene has a critical role in 

the development of retinoblastoma and also presumably Dsteosarcoma . Investigation on the 

parental origin of de novo chromosome mutations involving- RB gene In retinoblastoma 

patients showed that the germinal mutations were predominantly paternal origin, providing 

an additional evidence for the paternal melotic errors. In the non-hereditary sporadic 

osteosarcoma, the Initial mutation is presumed to be somatic origin . The mutation of 

tumor auppressing gene is often expressed by a subsequent loss of a large piece, or all, of 

the chromosome which harbors the normal allele. When the parental origin of chromosomes 

was identified with polymorphic loci, 12 out of 13 tumors showed loss of maternally 

derived chromosome 13 alleles, indicating that the Inltial somatic mutations were also 

non-random and occurred predominantly on paternally derived chromosome 13 . The 

preferential Involvement of paternal chromosomes both in germinal and somatic mutations 

rather suggests the involvement of germinal imprinting In the mutational susceptibility . 

500 

CYMIAGICAL ACfIVTTY OF NATURALLY OOCURRING 1K)GS --- S7UDIFS CN PFRILIA EICIRACPS 

Mieko Sasaki, 4bkyo Metr.Res.Iab .P,H.,3-24-1 Hyakunin-cho,Shinjuku-ku,7bkyo 169,Japan 

Naturally occurring substances have been widely used as drugs and food additives . 

Toxicology of these substances, however, is not studied enough because aLnost all of 

them are carplex chemicals and their activities are difficult to be examined . On the 

other hand, anti-mutagenicity or anti-wrcinogenicity has been found in some of these 

materials. Recently, Chinese medicine (naturally occurring drugs) has been interested 

and considered again in Japan, as a therapy for canoer or chronic diseases . The 

effects of these drugs have not been established enough yet, and the qualitative and 

quantitative studies bacome necessary now . 

Perillae (leaf of Perilla frutescens var.acuta) is one of popular herbs in Japan, 

and has been used as a food for expecting a good smell and anti-spoiled effect, and 

it is mixed into the Chinese medicine for armron cold concerning In its anti-allergic 

effect. The main active earnponent of perillae is known as perillaldehyde, and 

another toxic derivative, perilla ketone, is a potent puLronary edematogenic agent 

in farm animals. Biological activity of the ether-, ethanol-, water-extracts from 

leaves of perilla and purified 1-perillaldehyde was investigated in cultured CAD-K1 

cells. Chemical analysis of these samples was practiced by capillary OC or HPLC . 

Aberratiens of chratnsone were increased in cells treated with the ether-extract , 

and weak mutagenicity was also observed. The correlation of cytological activities 

to chemical analysis has been exadlLned, and the other biological effect, such as an 

effect on inaraua-earpetent cells, has been studied to estimate the Chinese medicine . 

50869 3686

501 

ANTIMUTAGENIC FACTORS IN VARIOUS AQUATIC PLANTS . 

T . Sato, Y . Ose, H . Nagase and H . Kito, Gifu Pharmaceutical University, 

Gifu City, Gifu 502 (•dlrpan) 

Various aquatic plants were collected from Nagara River system and 

the inhibitory effect of water extracts of the plants on mutagen-induced 

mutations were investigated by the Ames test (Salmonella tYChimurium 

TA100 and 98) . Smartweed, curled pondweed, Tuiopean cul- -grass an'a 

grass-wrack pondweed showed strong antimutagenic effect on benzo(a]pyrene 

(B[a]P), but had not for AF-2 . For 2-nitrofluorene (2-NF), 

curled pondweed, Europea.n cut-graju and grass-wrack pondweed had the 

antimutagenic effect, but smartweed had not . These antimutagenic factors 

were heat-resistant . The molecular weight of antimutagenic factor in 

curled pondweed and grass-wrack pondweed was above 300,000 . Four 

extracts did not show bioantimutagenicity . The active factors of curled 

pondweed, smartweed and grass-wrack pondweed acted as desmutagens . 

However, the active factor of European cut-grass did not show activity 

by desmutagen test, and not inhibit spontaneous mutation and S9 mix . 

The contribution of chlorophyll on antimutagenesis was little . B[a]P 

was treated by various amounts of the extracts and extracted by ethyl 

acetate . The extracted B[a]P decreased with the amount of plant 

extract . Almost all of adsorbed B[a]P was released with twice 20 min 

ultrasonication . By these results, it became clear that different 

antimutagenic factors exist in various aquatic plants . 

502 

APPROACHES TO DNA METHODS FOR THE DETECTION OF HERITABLE MUTATIONS IN HUMANS . 

C . Satoh, K . Hiyama, N . Takahashi and M . Kodaira . 

Radiation Effects Research Foundation . Hiroshima 732, Japan 

. 

We have examined feasibility of ribonuclease A cleavage at mismatches in RNA :DNA 

duplexes (RNase A method) and denaturing gradient gel electrophoresis (DGGE) of 

RNA :DNA duplexes for a study determining ns~leotide mutation rates . Identical 

RNA :DNA duplexes made by hybridization of P-labeled RNA probes and target DNAs 

were used in both techniques . Employing the RNase A method, 10 types of mismatches 

were examined in duplexes less than 771 bp made from cloned human $-globin genes and 

8 of them were cleaved . Deletion and insertion of 1, 4, 5 and 10 bp were detected . 

A polymorphic substitution of T to C at position 666 of IVS2 of P-globin gene was 

detected in genomic DNAs of 59 Japanese after amplification by polymerase chain 

reaction (PCR). Using DGGE, 8 types of mismatches were examined and all of them 

were detected . The deletions and insertions detected by the RNase A cleavage method 

were also detected. Three different polymorphic substitutions and a variable-length 

polymorphism [(ATTTT)n, n - 4 . 5, 6, 7] in the globin genes of 59 Japanese were 

detected in the genomic DNAs without amplifica~ion . Any large program screening for 

mutations requires examining of the order of 10 person .probe determinations. We 

are studying ways to optimize efficiency and cost effectiveness. These 139lude the 

use of multiple probes on a single DGGE gel and eliminating the need for P-labeled 

probes by the PCR technique . 

503 

MECHANISMS OF CHROMOSObIE ABERRATION 

John R .K . Savage, HtC Radiobioloqy Unit, Chilton, Didcot, Oxon . OX11 ORD . U .K . 

The subject of mechanisms has to be considered at several levels and not confined 

just to the initial molecular lesions and their repair/misrepair . Although there are 

many kinds of lesions which can be introduced by a wide variety of agents and there 

are many proposed pathways by which a cell can deal with them, there is only a 

limited number of ways in which "rejoining" can occur to produce structural changes 

visible in chromosomes at metaphase . This makes chromosomal aberrations a somewhat 

non-specific end point . Irrespective of the lesion types and their products, at some 

stage in time, some of them must come into "physical contact" if exchange is to 

occur . Is such contact predisposed? Are all regions vulnerable? Nhat is the 

relationship to and influence of chromatin structure and intranuclear architecture to 

such events? The many orders of magnitude difference between the molecular events 

and the observed result must not be forgotten, for the complex process of chromosome 

condensation and packing which intervenes, leads to aberration modification and 

disguise . Some mechanism must also exist to cope with entanglement, for even in the 

most complicated of,aberrations, this is a very rare phenomenon . Comments and 

observations will be made on these and other topics within the context of 

"mechanisms" . 



Notes


Notes - .+•FNTERLABOR/kTGRyoldMPARISON OF THE 32P-POSTLABELLING ASSAY FOR 

AROMATIC DNIFADDUCTS IN WHITE BLOOD CELLS OF IRON FOUNDRY WORKERS . 


K .Savela 1 . Heme~kil D .H .Phillips2 . A .Hewer2 K .L .Putman3 . 

K .Randerath3-'-f""-Tn titute of Occupational Health . Topeliuksenkatu 

41aA . SF-00250 Helsinki Finland . 2 . Chester Beatty Laboratories . 

Institute of Cancer Research . Fulham Road . London SW3 6JB . UK . 3 . 

Department of Pharmacology . Baylor College of Medicine . Houston . TX 

77030 USA 

The 32P-postlabelling method was compared independently in three 

laboratories in the analysis of human DNA samples, isolated from white 

blood cells of iron foundry workers . who were occupationally exposed 

to polycyclic aromatic hydrocarbons . The levels of aromatic DNA adducts 

from the exposed foundry workers in the three laboratories varied 

between 9 .2 +23 and 26+43 and from the controls between 1 .7t0 .7 and 

3 .1±1 .7 adducts per log nucleotides . No effect of smoking was 

observed in the present study . Each laboratory found large interindividual 

variations in the level of adducts . Good correlations were 

found between the results of the 32P-postlabelling assays carried 

out in the three laboratories ; the correlation coefficients between 

laboratories 1 and 2 . 1 and 3 and 2 and 3 were 0 .61 0 .62 . and 0 .45 

respectively, all being statistically highly significant . 

504 

505 

THE EFFECT OF CHWRINE ON THE MUTAGENICITY OF 3-CHLORO-4-(DICHLOROMER'HYW-5-HYDROXY-2(SH)- 

FURANONE (MX) AND ITS RECOVERY FROM NATER SAMPLES . K. M . Schenck, J . R . Meier, H . P . 

Ringhand and F C . Kopfler, U .S . Environmental Protection Agency, Cincinnati, OH 45268 . 

MX has been shown to contribute significantly to the mutagenic activity of a number 

of chlorinated drinking water samples . Preliminary studies on the recovery of MX by XAD 

resin concentration showed substantially lower recoveries fran tap water samples than 

from granular activated carbon (GAC)-filtered distilled water . These results prcmpted 

us to examine the effects of residual chlorine on the recovery of MK . GACrfiltered 

distilled water containing 0 to 3 mg/1 chlorine was spiked with MS at 50 rg/1 . The 

samples were concentrated 4000-fold on columns containing XAD-8 over XAD-2, and then 

tested for mutagenicity in the SaLnonellq/microsame assay using TA100 without S9 activation 

. The results showed that the percent of spiked MX recovered, as determined by the 

level of mutagenic activity present, decreased as the concentration of chlorine increased 

. This effect could be explained by the reaction of chlorine with MX . To examine 

this directly, the mutagenicity and UV absorbance of M8 in the presence of chlorine 

were monitored over time using high reactant concentrations (1-20 mg/1 for MXf 1-120 

mg/1 for chlorine) . At 1, 2, or 3 mg/1 chlorine, the decrease in the mutagenicity of 

Huc over time was significantly greater than that observed in the absence of chlorine . 

The concentration of MX . as determined by its absorbance at 250 nm, also decreased in 

the presence of chlorine . This decrease was both time and dose dependent . These results 

suggest that the level of MX present in tap water depends not only on the amount 

of MX produced by the chlorination of humic substances but also on the rate of degradation 

with residual chlorine. (Abstract does not necessarily reflect EPA policy) . 

506 

SYSTEMIC GENOTOXIC AND TOXIC COMBINATION EFFECTS OF N-NITROSODIMETHYLAMINE 

AND SO2 IN THE LIVER FOLLOWING INHALATION EXPOSURE . Schmezer,P. Pool,B .L . 

Liegibel,U . Zeller,W.J. Klein,R.G . Institute for Toxicology and Chemotherapy, German 

Cancer Research Center, INF 280, 6900 Heidelberg, FRO . 

A series of studies is being performed to determine which effects the environmental 

pollutant S02 may have on the rat liver carcinogen NDMA . Here, short-term in vivo data 

on systemic combination effects of SOz and NDMA are presented . Our previous studies 

had shown that pretreatment (inhalation) of Sprague-Dawle rats with 60ppm 802 for 

two weeks reduced the amount of DNA-single strand breaks ~SSB) observed in explanted 

hepatocytes incubated in vitro with NDMA. The strand breakage induced by 25-50 

pMoles/2x10s hepatocytes was reduced by apr. 20% . Accordingly, we have now 

investigated wether this decrease in genotoxicity caused by S0i is also apparent when 

both S02 and NDMA are administered simultaneously in viva We used a sensitive shortterm 

in vivo assay with which DNA-SSB are detected in intact cells isolated from treated 

animals . Inhalation exposure with NDMA alone revealed astonishingly low doses to be 

genotoxic in the liver. The doses were similar to those found to be effective after 

gavage . The inconstant respiratory frequency of the animals, however, made exact 

dosing of low NDMA levels difficult . Therefore, short-term oral NDMA application was 

preferred as exposure route for the combination experiments . We used lh in vivo 

exposure to NDMA at doses of 0 .5-1 mg/kg . This mode of application resulted in 

comparable amounts of strand breakage as the 25-50 pMoles NDMA induced in 2404 

hepatocytes in vltro. So far, the results of the combination treatment indicate that in 

contrast to the in vitro situation, the in vivo genotoxicity of NDMA was not affected by 

a two week SO2 treatment (10 and SOppm) . This observation as well as the possible 

impact of the formed sulfite (product of SOt + water) on the intracellular ATP level of 

the explanted hepatocytes will be discussed . 

50869 3688

507 

DIETHYLSTILBESTROL i(DES) INDUCES CHROMOSOME STICKINESS IN SYRIAN 

HAMSTER (SHE) FIBROBLASTS . 

R . Schnitzler, ' . Sch~ fmann and M . Metzler, Institute of Toxicology, 

University of Wtlrzburg, Versbacher Str . 9, W . Germany 

The genotoxic effects of estrogens are still a matter of debate . Existing 

data suggest that numerical chromosome changes rather than gene 

mutations are involved in estrogen induced neoplastic transformation . We 

have shown previously that DES induces micronuclei (MN) in a nonsynchronized 

cell population already after 1-2 hrs, whereas agents causing gene 

mutations (i .e . .4-NQO)_iriduce MN formation not before 12-18 hrs . This 

supports the hypothesis of a DES in3uced disturbance of the mitotic apparatus 

. - We report here that DES induces chromosome stickiness during 

early mitosis in SHE cells without measurable alterations of the mitotic 

index . After 7 hrs of DES treatment (40 }tM) mitotic cells showed a spheric 

aggregation of the chromosomes . The spindle apparatus was found to 

be morphologically altered in comparison to control cells . In many cases 

we observed single chromosomes separated from the chromosome aggregation 

None of them was connected to the mitotic spindle apparatus . In addition 

the centrioles were located on top of the aggregate and in some cells 

only one centriole could be detected . - These findings suggest that DES 

interacts with the protein matrix of mitotic chromosomes, presumably 

topoisomerase II . This interaction and the observed disturbance of . the 

spindle formation should result in numerical chromosomal aberrations and 

therefore could be the main reason for the formation of DES induced MN . 

508 

REPEATABILITY OF SPONTANEOUS AND CHEMICALLY-INDUCED SISTER CHROMATID 

EXCHANGE (SCE) ~4ND CHROMO$MAL BREAKAC (CB) IN UNRELATED INDIVI~UALS . S, 

Schwartz , L . Lasher , S.S. Wasserman , and M.M . Cohen ~ . Divi~on of Human Genetics . Division of 

Geographic Medicine, University of Maryland School of Medicine, Medical Biotechnology Center of the 

Maryland Biotechnology Institute, Baltimore, Maryland 21201 . 

It is well-established that considerable variability in the frequency of sister chromatid exchange (SCE) and 

chromosomal breakage (CB) exists within and among populations. Lymphocyte cultures from 27 individuals 

.e ere examined, on 2-3 occasions during a 6-month -2 year interval, for spontaneous and clast en-induced 

SCEs and CB following exposure to mitomycin C (MMC), bleomycin (BLM), streptonigrin~SN) and 4n 

itroquinoline- I -oxide (4NQO) . These experiments were performed to assess the reliability of single SCE and 

C B observations in evaluating an individual's response to environmental exposure . Data analysis utilizing a twoway 

nested analysis of covariance for each parameter (SCE or CB) yielded information concerning variability 

among the individuals as well as among the different cultures of a givenberson . These data clearly demonstrate 

that the interindividual variation of both spontaneous and induced SCEs and induced CB is significantly greater 

than among the cultures of the same person (pc0.01). A similar difference was not found for spontaneous 

chromosome breakage . For spontaneous SCEa. SN- and BLM-induced SCEa, spontaneous CB and 4NQOinduced 

CB, variation among sampling dates was not significantly greater than the variation within cultures ; 

Khile for MMC-and 4NQO-induced SCEs and MMC- . BLM-, and SN-induced CB, variation among sampling 

dates was significantly greater thin within sampling dates (p

176 1989 EMS Abstracts _ 510 


Notes --~~ gEQUENC'F.-iEtifALeR'M OF MUTATION INDUCED IN THE lac/ GENE OF Escherichla colr 

BY 2-AMINOPtJRINE . W .E . SCHY. AND B .W. GLICKMAN, Dept. of Biology, York 

University. 471N) Keelt*i, Toronto. Ontario, Canada M3J 1P3 

We have founzl`tl ~ r.t vses of mutational spectra to be a useful approach In 

elucidating mechanisms of inutation induction . To this end, we have characterized at the 

DNA sequence Ievel . '~-o-tminopurine (2-AP) induced mutations that lie within the first 180 

bp of the /acl gene (1-~ mutants) of Eschericlria cole. A saturated culture of NR 3835 

(wtld-t)•pe) cells was diluted in 25(1 Ed (if LB/2AP (600 q/ml) so that each well of a 96well 

mtcrotitrr dish contained less than 1(N) cells. Following overnight growth at 37oC 

and washing. appropriate dilutions were plated directly onto plates containing LB to 

determine survtval . and plates containing pbenyI .It .D-galactopyranoside (Pgal) as the sole 

carhim source tt) select Iacl' n~utants. One Incl- mutant was seleeted from each well . 

Mutants were mapped and I' mutants were selected for sequence analysis . Of the 42 

mutants at 14 sites that have been seyuenced thus far, all are transition mutations . 

Approximately 2-fold ax many mutants were G :C => A:T than A :T => G:C transitions ; . 

howevrr. when hotspot sitrs werr excluded from the analysis, transition mutation in 

eithcr direction apprars te~ hr ryually likely . With res(+ect to site specificity, we noted a 

pruminrnt hotspot at pc~sitiun e3 (G :C ==• A :T). This site Is surrounded by 5 neighboring 

G :C hase pairs . which may serve to stabilize a 2•AP :C mismatch . This regional sequence 

specificity is consistent with previous observations associating hotspot sites with adjacent 

Cr :C' huse pairs . 

511 

DEOXYNUCLEOTIDE TRIPHOSPHATE POOLS AND THE SPECIFICITY OF MUTATION FOR LACI IN E . COLI . 

W .D . Sedwick, M .L . Veigl, S . Schneiter, and S . Mollis, Dept . of Medicine,Case Western 

Reserve Univ . Cleveland, OH 44106 

The mutational specificity of trimethoprim (TMP) for the Lacl gene of E . coli was 

investigated . Mutants (500 each) were isolated for analysis from cultures which were 

treated 4 hours with 5 uM trimethoprim (TMP) or TMP + 100 uM thymidine (TdR), followed 

by a 4 hour recovery period which allowed resolution of the filanmentation seen only in 

the cultures treated with TMP alone . The frequency of mutation in lcI increased 10and 

2-fold under the two conditions, respectively . All isolated mutants were probed 

for occurrence of mutation at two of the most common sites represented in the spontaneous 

spectrum, the frameshift "hot spot" where the thrice repeated sequence, CTtk ;, is 

frequently duplicated or deleted, and the +6 position in the operator, the site of a 

recurrent transition of T-A -> C-G . Frame shift "hot spot" mutants represented only 20% 

with TMP and 43% with TMP-TdR vs >66% in the spontaneous background (Shaaper et al, JMB, 

189 :273, 1986) . The high frequency mutation in the operator represented 22% and 18% of 

the total mutants in the TMP and TMP-TdR sets, respectively, vs 3% in the spontaneous 

collection . About 60 mutants mapped to the first 200 base pairs of 1aal were carried on 

for DNA sequence analysis from both TMP and TMP-TdR treated cultures . An overall 

increase in single base nonsense mutations at mber_ and ochre sites was not observed, but 

the predominant isolates from TNP-treated cultures were T-A -> C-G transitions clustered 

at several different sites . A high frequency of A-T -> C-G and G-C -> C-G transversions 

was also observed . Preliminary analysis of the TMP-TdR mutants supports a similar 

pattern of base substitution mutations . The results of these experiments will be 

presented with correlative DNA triphosphate pool analyses . 

512 

CONTRASTING RESULTS IN HEPATOCYTE UDS ASSAYS USING AUTORADIOGRAPHY 0R LIQUID 

SCINTILLATION COUNTING e b r, C .Meli and R .Forster, Dept . Genetic Toxicology, 

Life Science Research Roma ox co ogy Centre, 00040 POMEZIA, Rome, Italy 

Contrasting findings in rat hepatocyte UDS assays performed using liquid 

scintillation counting after nuclear separatlon (LSC) or by autoradiographlc 

estimation of radiolabel uptake (ARG) have been observed with several products 

received for routine testing . Further experiments were with product X, which gave 

reproducible increases in UJS of up to 110% over control values in the LSC method . In 

the ARG method, negative results were obtained ; both cytoplasmic and nuclear grain 

counts remained similar to control values at all treatment-levels . Possible 

explanations for these results were examined : (1) The responses obtained with the 

positive control (2-acetylaminofluorene) suggested that the ARG method is the more 

sensitive method of detection . (ii) The percentage of cells in S-phase was determined 

in the autoradiographic preparations . There was no lncrease in S-phase cells which 

could explain the results obtained in the LSC method, but calculations show that the 

sensitivity of S-phase scoring may be insufficient to detect increases which will 

result in positive responses in LSC . (ii1) The experimental methods follow the same 

tine-plan and kinetic differences cannot explain the contrasting results . (iv) 

Parallel LSC assays were perfot7ned with or without hydroxyurea treatment ; product X 

produced increases in radiolabel uptake 1n the LSC method both in the absence and 

presence of HU, indicating that the observed UDS did not result from the swtagenic 

properties of HU . At present the basis of the differing results between the LSC and 

ARG methods cannot be satisfactorily explained . 

50869 3690

513 

PROTAMINES IN GERM ZELL MUTAGENESIS, Gary A . Sega, Biology Division, ORNL, Oak 

Ridge, TN 37831 . 

t 

A number of chemicals are'powerful mutagens in late spermatids and early spermatozoa stages of the 

mouse . Among these chemicals are ethyl methanesulfonate, methyl methanesulfonate, ethylene oxide, 

and acrylamide . We have found that all four of these chemicals bind at much higher levels in the late 

spermatids and early spermatozoa stages than in other stages . 11te increased binding in the sensitive 

stages has been shown not to be the result of increased alkylation of DNA but rather to alkylation of the 

protamine present in these stages . The sulfhydryl (-SH) groups present in the cysteine residues of 

immature protamine in the sensitive stages are susceptible to aUcylation by these chemicals . We have 

hypothesized that such alkylationVrevents nomsal disulfide bond formation in the protamine of the 

developing sperm and leads to stresses that break the sperm chromatin, with resulting genetic damage to 

the Fl progeny. Clearly, not all mutagenic chemicals act by the above mechanism, and other molecular 

targets may be important in other germ-cell staps. However, our observations of how some chemicals 

bind strongly to sperm protamine in mammals gives a new dimension to our understanding of mutational 

processes in mammalian germ cells . [Research jointly sponsored by the Office of Health and 

Environmental Research, U .S . DOE contract DE-ACOS-84OR21400 with Martin Marietta Energy 

Systems, Inc ., and by the National Institute of Environmental Health Science under IAG No . 222Y01- 

ES-10067 .] 

514 

POTENTIAL ROLE OF GENOTOXICITY INDUCED BY GANMA IRRADIATION OF SPODOPTERA LITURA IN 

ITS MANAGEMENT . S .S .Sehgal, and R .K .Seth, Department of Zoology, n ver y'8T-UEtihi, 

Delhi-110007 (INDIA) 

In accomplishing the laboratory evaluation of the biological effects of gamma irradiation 

of a polyphagous lepidopteran pest, Spodoptern litura, it was observed that 

as compared with other insects, Spodopteru required a very high dose (25,000 rad) 

of gamma irradiation to produce a complete sterility . However, a gamma dose of 13,000 

rad that induced a partial sterility (50%) in irradiated insects produced,progeny which 

not only exhibited an increased sterility (ca .80%) but also a higher degree of sexual 

competitiveness . The holokinetic nature of chromosomes could be a conceptual cytogenetic 

explanation for these results . The fragments of irradiated holokinetic chromosomes 

were probably represented in heterozyqous conditiorl in the offsprings, behaved as translocation 

heterozygotes during meiosls in F-1 generafion which gave rise to aneuploid 

gametes that caused dominant lethality in the next generation . The higher dose of gamma 

irradiation caused some physiological effects like female infecundity, inability to 

mate, reduced life span, malformations and sex-ratio skewed in favor of males . The 

excess of males was probably caused by meiotic drive of maleness locus in this moth 

and this phenomenon appears to have potential for the genetic control method for this 

pest . Indeed, it is conceivable to identify and 'engineer' the deleterious genetic 

mechanisms (or the genntoxicit .y)-induced by gamma irradiatinn, that might be transmitted 

by the released moths to regulate the field population of this major pest in India . 

515 

MECHANISNS OF CAFFEINE INHIBITION OF DNA REPAIR IN E . COLI . Christopher P. Selby and 

Aziz Sancar, Department of Biochemistry, University of North Carolina at Chapel Hill, 

Chapel Hill, NC 27599 . 

Caffeine inhibits excision repair and photoreactivation in E . coli in vivo . We 

used purified E . coli enzymes and DNasel footprinting to study the mechanism of 

inhibition . We do observe inhibition of ABC excinuclease and DNA photolyase by 

caffeine in vitro . ABC excinuclease catalyses early events of excision repair : 

recognition of damaged DNA and incision of the phosphodiester backbone on both sides 

of the damage . The UvrA subunit is involved in damage recognition . Using an 

oligonucleotide with a unique psoralen adduct, UvrA protects 33 base pairs 

surrounding the adduct from DNaseI digestion . In the presence of caffeine, the 

DNasel footprint of UvrA covers the entire oligonucleotide ; thus, caffeine promotes 

the binding of UvrA to undamaged DNA . UvrA subunits "trapped" by caffeine would be 

unable to catalyze repair . Photolyase binds to pyrimidine dimers, and upon 

irradiation of the enzyme-dimer complex reverses the dimer . Using an oligonucleotide 

with a unique thymine dimer, we found that caffeine binds specifically to the thymine 

dimer and interferes with binding of photolyase to its substrate . Thus caffeine 

inhibits the two repair systems in E . coli by entirely different mechanisms, by 

promoting the nonspecific binding of the nucleotide excision repair enzyme and 

interfering with specific binding of the photoreactivating enzyme . Supported by 

grants from the NIH (GN32833) and NCI (5 T32 CA09156) . 



Notes



_ 516 

Notes tes -- . .-DOMINANT SKBLB/Mtff MUTATION METHODS ARE BEING USED TO DETERMINE WHETHER 

DOMINANT MUTATIONS ACCOUNT FOR THE MANY ABNORMAL FETUSES FOUND 

FOLLOWING EXPOOF ZYGOTES TO ETHYLNITROSOUREA (ENU) . P. B . Selby, W. M. 

Generoso, G. D. Re , W. McKinley, Jr., and K. T. Cain, Biology Division, Oak Ridge National 

Laboratory. P.O. Box 2009, M.S . 8077, Oak Ridge, TN (USA) 

Generoso et al . (Mutat. Res. 176:269-274 and 199 :175-181) discovered that there are remarkable increases 

in the incidence of developmental abnormalities in fetuses after early rygotic stages are exposed to any one of 

several chemicals, including ENU . Russell et al . (PNAS 85 :9167-9170) found that a 50 mg/kg i .p. 

injection of ENU induces approximately 8 times more specific-locus mutations when It is administered 

2 .5-3 h after mating instead of 5-6 h after mating. We Injected female mice with 40 mi ;/hz; of ENU (i .p .) at 

either 2 .5 or 5 h after mating . Offspring are being examined for externally visible altered phenotypes and for 

skeletal damage. The frequencies of mice wtih significant external findtng;(e.g., a missing eye, shortened 

tail, misshapen head), among mice living 3 weeks or longer, were 8/207 (3 .9%) and 0/156 in the 2 .5-h and 

5-h groups, respectively . The frequency at 2 .5 h is statistically significantly higher, and this difference, in 

view of the spectfc-locus results, suggests that dominant gene mutations may be responsible for the 

externally visible altered phenotypes. Results of detailed skeletal analyses will be presented, as will 

preliminary results from breeding tests . Since dominant skeletal mutations are much more common than 

dominant visible mutations, it is expected that a high frequency of dominant skeletal mutations will be found 

if induced dominant mutations are responsible for the high frequency of abnormal fetuses . [Research jointly 

sponsored by NIEHS under Interagency Agreement Y01-ES-10067 and the Office of Health and 

Environmental Research, U .S . Deparanent of Energy under contract DE-ACOS-84OR21400 with Martin 

Marietta Energy Systems, Inc .] 

517 

SUGGESTIONS FOR IMPROVING THE DIRECT METHOD OF GENETIC RISK ESTIMATION. P. B . 

Selby . Biology Division, Oak Ridge National Laboratory, P .O. Box 2009, M .S. 8077, Oak Ridge, TN 

(USA) 

One of the methods used by committees for quantitative genetic risk estimation for radiation is based upon 

a measure of induced phenotypic damage (usually malformed skeletons or cataracts) in the offspring of 

irradiated mice . This method, which has been called the direct method, has several distinct advanta*es over 

the doubling-dose method of genetic risk estimation. Much of the emphasis to date has been on induced 

mutations that were proved to be transmitted to later penerations . However, If these are the only mutations 

that are included in mutation frequencies, as is sometimes recommended, the following important classes of 

mutations causing dominant damage are overlooked : (1) mutations causing sterility, (2) mutations causing 

death before breeding tests can be completed, and (3) mutations with such low penetrance that transmission is 

not confirmed in the relatively small numbers of offs'pring that can easily be raised in a breeding test . 

Accordingly, it seems that the ideal method for collecting data to be used for genetic risk estimation is to 

identify all of the first-generation offspring that exhibit any phenotypes that would be clinically important if 

they were to occur in people . The induced mutation frequency could be calculated by subtracting the 

frequency of offspring with clinically important phenotypes in the control group from that in the experimental 

group . Various other suggestions will also be presented for improving our ability to estimate genetic risk by 

the direct method . [Research sponsored by the Office of Health and Environmental Research, U .S . 

Department of Energy under contract DE-ACO5-84OR21400 with Martin Marietta Energy Systems, Inc .] 

518 

INFLUENCE OF OXIDANT STATE ON UV AND FINNO INDUCED MUTATION IN V79 CELLS- 

Sharmila Sengupta and 1 u a , Saha Institute of 

Nuclear Physics, I/AF, alt ce, a outta- 00 064, India . 

Present day idea that oxidant state could be involved in carcinogenesie 

makes it relevant to study the influence of sueh condition on the 

mutation induction by W light and Mt+110 (N-methyl-N'-nitro-N-nitroeoguanidine) 

. In our experiments, H20p aae been used to create the oxidant 

state in V79 cells . The dose of H2 02 was in the non-toxio region ; for 

tnis particular cell-line, it was 0 .9µg/ml . The end-point selected for 

our mutation analysis was resistance to the drug 6-thioguanine . Oxidant 

state itself did not create any mutation above the background level of 

0 .33±0 .08 per 105 viable cells . For UV-light, different fluences ranging 

frotn 4J/ms to 20J/m were usp~d to get the mutations wnieh varied from 

8 .0+12 to 25 .60+7 .40 per 10 viable cells . For MNNG, the doses used 

were in the region 0 .2µg/ml to 0 .6µg/ml and the correponding mutation 

frequencies varied between 5 .0+1 .0 to 32•5+4 .2 per 101 viable cells . The 

creation of oxidant state altered the value ; in case of MNNO, these 

raaged between 12 .8+2 .3 to 56 .5+3•6 per M1 viable cells and in case of 

UV light between 8 .7+1 .4 to 22 .3f8 .2 per 105 viable cells at the dose 

ranges mentioned . It-wae seen that such a condition enhanced mutation 

induction by NNN6 but had no effect on mutation by UV-light . Clearly, 

the oxidant state, if involved in oarcinogeneais does not produce any 

extra effect for the 254 nm UV-light but the situation is different in 

case of chemical agents like MNO . 

50869 3692

519 

XRS-5 CELLS ARE NOT 47PERSENSITIVE TO X-RAY-INDUCED MUTATION . 

J . D . Shadley, M . Toohill, J . Whitlock, J . Rotmensch and J . L . 

Schwartz, University-qpf Chicago Medical Center, Chicago, IL (USA) 

The Chinese hamster ovary (CHO) cell line xrs-5 has been shown 

to be hypersensitive to the cytotoxic effects of X-rays compared 

to the parental line K1 . In agreement with previous reports on 

this cell line, we find that xrs-5 cells are : 1) 3 fold more 

sensitive to the cytotoxic effects of X-rays (D10) than K1) 2) 

rejoin only 60% of their gamma-ray-induced double strand breaks 

compared to over 90% for Kl ; and 3) 7 fold more sensitive to Xray-induced 

G2 chromatid-aberratiotTS than Kl . Given its 

hypersensitivity to the above endpoints, we tested to see if xrs- 

5 cells are also hypersensistive to X-ray-induced HGPRT mutation 

compared to K1 . Xrs-5 cells gave similar 6-thioguanine resistant 

induced mutation frequencies per Gy dose (1 .9 x 10-5 for xrs-5 

vs 1 .8 x 10-5 for Kl) . When based on equal survival levels, 

either DO or D10, X-rays induce only one-half to one-third 

the frequency of 6-TG resistance in xrs-5 compared to K1 . Thus, 

xrs-5 cells are as mutable if not less mutable than K1 cells . If 

the deficiency in double-strand break rejoining is in part 

responsible for the hypersensitivity to X-ray-induced cytoxicity 

and chromosomal aberrations, then it does not appear to affect 

mutation induction . Research supported by Department of Energy 

DE-FG02-88ER60661 . 

520 

ExxAMCED ASSAYS DETECT INCREASED CNROMOSOME DAMAGE ARD SIST[R CNR0N0S0ME ExCNANG[S IN 

xER01N ADDICIS . D .A .Shaf .r, V .G .tlunbar, A .Falek, R .N .Don .hoe, J .J .Maddan . E~ery Univ . 

and GeorRis Mental Xeatth Inst ., Atlanta, GA 30306 

To refine previous studies of chromosome damage (CD) and sister chromatid exchanSes 

(SCE) in heroin addicts, we applled eethods re developed to enhance detection of the 

cytopenetic effects of lor-l w .l radlation expesure in hospital rerkers . For CD 

analysis, our TFC• .nhanced proeedure oonslsted of traatment at setup with 1x10•7N FdU 

and Lx10-SM dT to Inhibit thymidytate synthetase and to satisfy that induced 

require .ent, and treat .ent in G with oaffein . (2 .2 ∎M) to inhibit repair syfthesis . 

For SCE enhaneewent, only caffel ne was used but traat~ant ras extandod traek thru S 

phase (19 hrs before harvest) . UsinO s atandard and an enhaneed CD and fCE eulture per 

subject, blood samples rera evaluated from 20 street heroin addicts and 22 controls . 

Standard 2-day CD and 3-day SC[ assays showed insiGnificant sonotoxic Increases in 

addicts vhile the enhanced CD and SCE assays showed highly significant Increases . Most 

CD events were in the for∎ of ehromatid and ehromososa iruka . There rere no rings and 

a fev dicentrics Yare only observed in the TiC-enhanead eultures . AltheuGA 

quadrlradlals are usually rare, 10 vere found In addict TFC•cultures and 3 In control 

TFC-cultures . V(th the standard CD assay, the level of chromosome breaks per 100 e .lls 

~as .727 for controls and 1 .OS6 for addicts (not siGntf .) . Yith the TFC-enhancad assay, 

the same e usure shored t .i63 chrosose .e bruks for eentrols and 5 .163 fer .ddlet . 

(highly signif . p< .0001) . A hiGhly siGnifiaant dtfferenca ras also observed for 

chroeatid dasa9e with the TFC-anhancad assay although such damage ts not typloally 

considered indicatlve of J„a vlvo chro .oso .e daea0e (chro .atid breaks per 100 callst 

16 .793 for eontrols ; 4 l .191 for addicta) . The SCE data Gave sio ilar si0nif/eant 

differences . standard SCE cultures showed 10 .892 SCE/cell for eontrola and 11 .732 

SCE/cell for addicts . Vith CAF enhanca .ent thare rara 13 .0! SC[/oell for controls and 

17 .05 SCE/cell for addiets (p< .006) . Tha abeve flndlnGa lnd/eats that tAe deteetlen Of 

ICEWICD and SCE affeces can be siGnifleantly anAane .d by the use of tAass nar pree .durss . 

The findinG of increased chromatid damage also auGGU ts a ner interpretation of CO 

effects since chro .atld effects •ust have occurred poat S•phase . Perhaps exposure to 

cheoicals, druGS and environ .ental agents ∎ay not only leave a residue of DNA and/or 

chromosome damage but also an indueed sensitivity to further Genotoxic damage . 

521 

ACTIVITY OF DINITROPYRENES IN THE INTRASANGUINOUS HOST MEDIATED ASSAY . 

A .B .Shah, I .R .Rowland and R .D .Combes, School of Biological Sciences, 

Portsmouth Polytechnic, U .K ., B .I .B .R .A ., U .K ., and I .R .I . Ltd ., U .K . 

Dinitropyrenes, present in polluted air, are potent direct-acting 

mutagens in Salmonella tvnhimurium TA 98 . This mutagenicity is markedly 

reduced in the presence of mammalian hepatic S9 or microsomes . Since 

most in vitro test systems do not adequately simulate conditions 

encountered in the intact animal, we have investigated dinitropyrene 

mutagenicity to Salmonella in the host mediated assay . 1,8- 

Dinitropyrene (1,8-DNP) given 2.2. to BALB/c mice induced a weak 

mutagenic effect in S . typhimurium TA 98 recovered from the liver 1 

hour after J,y . administration ; over the entire dose range tested no 

toxicity to bacterial cells was detected . Mutation induction in vivo 

was dose related with maximum response (71t9 revertants/plate) at lmg 

DNP/kg body weight. This optimum dose however, was non-mutagenic to 



Notes 

I

180 1989 EMS Abstracts e . 

._J . _ _- -` 

. . -- 

.y . woo 


NOteS strains TA 9'BDNP (arylhydroxylamine esterase deficient) or TA 98NR/DNP 

(nitroreductasel,, and arylhydroxylamine esterase deficient) . 1,3- 

Dinitropyren8---EFRF-1,6-dinitropyrene wer weakly mutagenic to TA 98 at 

doses similar to 1,8-DNP . Studies with [14C] 1,8-DNP showed that 1 hour 

after oral dosing (lmg/kg), over 100nq of DNP were present in the liver 

(w 0 .73% dose) . However, only about 5 .5ng were present in the bacterial 

pellet, suggesting that hepatic components, in vivo as in vitro, bind 

to DNP, thus preventing its interaction with Salmonella . 

522 

1,8-DINITROPYRENE : DISTRIBUTION AND EXCRETION IN THE MOUSE . 

A .B .Shah, I .R.Rowland and R .D .Combes, School of Biological Sciences, 

Portsmouth Polytechnic, U .K ., B .I .B .R .A ., U .K ., and I .R .I . Ltd ., U .K . 

Despite considerable interest in the toxicology of 1,8-dinitropyrene 

(DNP), little is known of the toxicokinetics of this mutagen . We have, 

therefore, investigated the distribution nd excretion of DNP following 

oral administration of a single dose of C-DNP (0 .25mg/kg) to female 

BALB/c mice . At known times (2,4,6,24,48,72,96,168, and 216 hours) 

after dosing, mice were killed, various tissues removed and their 

radioactivity determined. The results indicate a slow uptake of DNP 

into the bloodstream and other tissues . Maximum amounts were present in 

the bloodstream, liver and kidneys after 6 hours, representing 0 .274, 

2 .9% and 0 .21% of the radioactive dose . The radioactivity in these 

tissues decreased after 24 hours to 0 .1%, 1 .6% and 0 .12% respectively, 

after which there was a gradual decrease amongst these and all the 

other tissues studied . Most of the excretion was within 48 hours of 

dosing. At 72 hours, radioactivity recovered from all the tissues 

studied and urine and faeces amounted to

_ 

presence of .c_,lyDhimuriu_.n TA 98, TA 98NR and TA 98/1,8DNP6 . Cytosol, (0.8 mg/plate) as the sole 

exogenous enzyme fraction, enlUnced the mutagenicity of 2- and 3-nitrofluoranthene (2-NP and 3-NF) and 

1,3 dinitrofluoranthene (1,3-I7NF) in the three strains 2- to 9-fold over that observed in its absence . However, 

the number of revertants with 1,2 dinitrofluoranthene (1,2-DNF) was decreased 70% by cytosol. The 

inclusion of microsomes alone (0.2 mg/plate) decreased the mutagenic response by 57-98% . The addition of 

both cytosol and microsomes, to reconstitute S9, resulted in a marked decrease In the mutagenicity of the 

compounds . In a related study, the mutagenicity of three dinitropyrenes, 1,3-, 1,6- and 1,8-dinitropyrene 

(1,3-, 1,6- and 1,8-DNP) was also rnarkedly enhanced by cytosol m TA 100. This was especialily noticeable 

when 1,3 DNP was the substrate . Amongst other possibilities, these findings suggest that cytosol may contain 

enzymes more potent than the constitutive bacterial .naroroductases and/or acetylases in the activation of 

nitroarenes to mutagens or that the bacterial enzymes are rate limiting components of the system . Microsomes 

contain deactivating enzymes and when combined with cytosol to reconstitute S9, the deactivation potential of 

the microsomes overshadows the activation potential of the cytosol and a decreased mutagenic response is 

expressed . 

525 

ALTEltATIONS IN DNA CONTENT AND CHRONOciOHE COMPOSITION AS 

RELATED TO AGEING IN MAMMALS 

A . Sharma, S,Sen, G . Talukder, Human Genetics Unit, Centre for Advanced $ 

Study in Cell & Chromosome Kesearch, Department of Botany, University 

of Calcutta,' Calcutta 700019, India 

In view of the hypothesis that DNA is involved in the process of 

ageing, the present investigation was undertaken to find out the 

relationship between DNA content and progressive ageing in ratss 

in vivo and human leucocyte cultures in ~itrQ Chromosome studies 

from bone marrow cells of Kattus norvea-Lcus of .twelve different age 

groups showed a significant increase in the frequency of hypodiploid 

cells with ageing . This increase was, however, not seen in the .gonadal 

cells . A similar enhancement was recorded in the number of hypodiploid 

cells in the cultured lymphocytes from sex-mrtched human subjects of 

ten different age groups . DNA estimated in situ in bone marrow nuclei 

did not change to a significant extent with ~ncieased age . The observations 

made in proliferating human lymphocytes supported those made on rats 

in vivo . DNA contents of differentiated tissues, including brain, lung, 

lve lr, heart and kidney from fetal to adult rats showed an organ-specific 

variation(within the limits of statistical significance) . It may be 

due to controlled DNA amplification during differentiation and 

maturation of the tissues . 

526 

GENOTOXIC EFFECTS OF INDU3TKIALISATION IN SELECTED POPULATIONS 

A .Sharma, A. Raychoudhury, A . Banerjee, M .De, T .Das, M.Joardar, 

K . Agarwal, A .K . itoy, S . Maity, G alukder, Human Genetics Unit . 

Centre for Advanced Study in Cel & Chromosome Research, Department 

of Botany, University of Calcutta, Calcutta 700019, India . 

A comparison was carried out between selected populations from the 

Eastern India, who had been exposed, directly and indirectly, to 

industrialisation for 10 to 40 years and non-exposed populations 

from totally rural areas . The endpoints observed were blood genetic 

markers and cytogenetic parameters . In 1648 blood samples from 

healthy male age-matched donors, the frequency of abnormal variants 

of lipoprotein and hemoglobin was significantly higher in the 

exposed populations . There was no appreciable difference, however . 

in the incidence of markers not related to life style or diet, like 

haptoglobin, transferrin, acid phosphatase, lactate dehydrogenase . 

ABO system and Rh factor . The frequencies of chromosomal aberrations, 

micronuclei and sister chromatid exchanges were significantly higher 

in leucocyte cultures set up from 437 individuals tested from the 

exposed group, as compared with corresponding cultures from persons 

belonging to the non-exposed group . The difference between persons 

who had been exposed directly and those who had been exposed 

indirectly was not statisticilly significant . 



Notes


NOteS 

. .. ... 


.. 

IN SITU DETECTION OF GENOTOXICITY - POSSIBILITIES AND MEASUREMENTS C .B .S .R . S harma 

Salim Ali School of~cology, Pondicherry University, Pondicherry 605 001, India 

±i- - 

Three approaches exist to monitor the genotoxicity in the polluted ecosystems - (a) 

to screen the plants and animals in the habitats for genotoxic symptoms in mitotic and/ 

or meiotic tissues, (b) to monitor impacts on specific endpoints in the standard test 

systems maintained in situ and (c) to test the in situ habitat samples like water, soil 

and sediments in the laboratory for genotoxicity i-n the experimental systems . While 

reviewing these possibilities, measurements are presented to suggest that some herbaceous 

terrestrial weeds, aquatic plants, ipsect populations and crop systems have 

potential to reveal the in situ genotoxicity of the habitats - (1) Of the 30 weeds 

screened from two habitaTs periodically inundated by polymer factory effluents and 

sewage Croton, Justicia, Cassia, Parthenium, Cleome and Solanum have shown significant 

meiotic karyotoxicity 0 .~3-9: an po len aterllity 1- ..) . (2) Eichornia, 

inhabiting ponds variously polluted by effluents from homesteads, dyestu f and polymer 

factories and washates from transport systems,has shown mitotic karyotoxicity in 

significant frequencies (1 .2-12 .6%) (3) _Chroto on~u_s inhabiting the ground cover 

around sewage ponds revealed highly signi ica~tic karyotoxicity (3 .49-10 .2%) . 

(4) Meristem Assays and progeny tests involving Allium and Hordeum irrigated by sewage 

and polluted groundwater or maintained in the sewage slud9e- Fiave all shown genotoxic 

symptoms to various degrees . Some of the weeds of terrestrial and aquatic ecosystems 

and local insects can therefore be sensitive indicators of the ubiquitious genotoxins 

in our ecosystems . 

USE OF NATIVE PLANTS AND INSECTS AS INDICATORS OF in situ CENOTOXICITY . Sharma, 

C .B .S .R ., Panneerselvam, N ., Arumikkili K ., Raiendran M ., Meenakahi N .D ., Gowri R ., 

Salim Ali School of Ecology, Pondirherry University, Pondicherry 605 001 . 

Plants and insects inhabiting the polluted areas in the cities of Vishakhapatnam, 

Madurai, Coimbatore and Mysore of Southern India are assayed for genotoxicity . The 

pollution was due to polymer factory effluents in Vishakhapatnam and sewage in 

others . The results are as follows . In Vishakhapatnam and Madurai 30 herbs are 

acreened for meiotic aberrations and Pollen fertility . Twelve of these have shown 

significant effects - The frequencies of abnormal cells ranged between 0 .87 to 23 .9A 

and sterility between 12% to 95% . Among the more sensitive plants ares Croton 

bon landianum, Juaticia sim lex, Cassia tore, Terosia ur urea, Cleome v scosa, 

ar hen um ystero o us a~n ?olanum ind~m . Tn t~Fe Eic orn a ro~aystem, !~ 

frequency of karyotoxicity atT~iahakhapatnsm ranged between ~3 end 12 .6, at 

Coimbatore between 3 .2 and 8 .5, at Madurai between 3 .0 and 8 .9 and at Mysore between 

2 .1 and 5 .8 . In situ analysis of the insect Chroto nus asusaurei inhabiting two 

sewage ponds in Combatore, revealed meiotic ary~ty ranging between 3 .49 and 

10 .20 at one pond and between 6 .99 and 9 .0 in the other . These studies suggest that 

in situ inhabitants can indicate habitat genotoxicity especlally the water hyacinth, 

rl-chernia and the grasshopper, Chrotogonus . 

WATER HYACINTH (Eichornia crasaipes) IS AN EXCELLENT in situ MONITOR OF AQUATIC 

GENOTOXINS . C .B .S .R . Sharma, Salim Ali School of Ecology, Pondicherry University, 

Pondicherry 605 001, India . 

The ubiquitous occurrence of water hyacinth (Eichornia crasaipes) prompted us 

to explore the possibility of its use as a monitor of aquatic genotoxins . Herewith 

reported are the results from five cities of India where Eichornia's responses are 

monitored from aquatic habitats with different histories . The cities are : Vishakhapatnam, 

Madurai, Coimbatore, Mysore and Pondicherry . The assay is restricted to 

root systems . The endpoints scored aree clastogeny (fragments, micronuclei and 

bridges) and turbagsay(vagrancy and micronuclei) . The highest karyotoxic frequency 

was obtained in the populations inhabiting waters contaminated by a polymer factory 

effluents at Vishakhapatnam (3 .5*0 .25 to 12 .6*0 .64) followed by automobile washates 

plus sewage at Coimbatore (3 .2*0 .16 to 8 .5*0 .78) ; dye stuff effluents plus sewage at 

Madurai (3 .0 :0 .22 to 8 .9s0 .56)g untreated sewage at Mysore (2 .1*0 .19 to 5 .8*1 .1) 

and roadside runoffs at Pondicherry (1 .2*0 .12 to 2 .6*0 .5) . The background frequency 

has ranged between 0 .89*0 .09 to 1 .02 :0 .15 . Thus the complex mixtures in the water 

bodies causing karyotoxicity have in them effluents from homesteads, dyestuff and 

polymer factories, washates from the traffic-busy roads and automobile workshops 

suggesting propensity of genotoxic exposure in the concerned human populations . 

These preliminary studies, suggest that Eichornis can serve aa an excellent in situ 

biomonitor of habitat genotoxins . 

50869 3696 

528 

529

530 © 

GENOTOXICITY EVALUATION OF CHLORTETRACYCLINE 

R .K . Sharma, K .A . Traul,-C . Caterson, J . Harbell, A . Thilagar, D . Putnam, American 

Cyanamid Co ., Princeton,~--NJ ; Sitek Research Labs ., Rockville, MD ; Microbiological 

Associates, Bethesda, MD . 

Chlortetracycline-HC1 (CTC-HC1), a broad-spectrum antibiotic, was tested in a 

battery of short term j.D vitro (microbial and mammalian point mutation, and 

unscheduled DNA synthesis) and JjI y.jy4 (rat bone marrow chromosome aberrations) 

tests . Microbial and mammalian point mutation testing was done with and without rat 

S9 metabolic activation ; all tests had concurrent positive and negative controls . The 

microbial mutagenesis assay .jn J . tyohi~yyrium and E . Sg]j was conducted up to a dose 

of 10 pg/plate, in the presence and absence of S9, limited by toxicity . No increase 

in revertants per plate was observed . In the CHO/HGPRT mammalian gene mutation assay, 

doses (based on solubility and cytotoxicity) of 100,80,60,40 and 20 pg/ml (+S9) and 

125,100,75,50, and 25 pg/ml (-S9) were found to be nonmutagenic . In the rat (Sprague 

Dawley) bone marrow cyto enetic test, CTC-HC1 was administered by oral gavage at 

5,000, 2,500, and 500 mgYkg in males and females, based on a range-finding study . 

Bone marrow was harvested at 12,24 and 36 hours after treatment . CTC-HCl induced no 

increases in chromosomal aberrations in either sex at any dose or time point . In the 

rat hepatocyte primary culture/DNA repair (UDS) test, doses tested ranged from 25 to 

75 pg/ml, limited by cytotoxicity . None of the doses produced a mean grain count of 

more than five compared to vehicle control . Positive controls in each test system 

produced significant positive responses . It is concluded that Chlortetracycline-HC1 

is not mutagenic in these test systems . 

531 

GENOTOXICITY EVALUATION OF SULFAMETHAZINE 

R .K . Sharma, K .A . Traul, C . Caterson, E . Johnson, R .R . Young, J .L . Ivett . American 

Cyanamid Co ., Princeton, NJ ; Hazleton Laboratories America, Inc ., Kensington, MD . 

Sulfamethazine (SULMET), a bacteriostatic antibiotic, was evaluated for genotoxic 

potential in a battery of short term j,n yitro (microbial and mammalian point 

mutation) and jIl yjys (rat bone marrow chromosome aberrations) tests . Microbial and 

mammalian point mutation testing was done in the absence and presence of rat S9 

metabolic activation ; all tests had concurrent positive and negative contro)s . 

The microbial mutagenesis assay in a . tvohimurium and F . & Q]j was conducted up to a 

dose of 10 µg/plate, in the presence and absence of S9, limited by toxicity . No 

increase in revertants per plate was observed . In the CHO/HGPRT mammalian gene 

mutation assay, doses (based on solubility and cyto~oxicity) ranging from 0 .5 to 

7 .0 mg/ml with and without metabolic activation, were found to be nonmutagenic . In 

the rat (Sprague Dawley) bone marrow cytogenetic test, SULMET was administered by 

oral gavage at 3,000, 1,500, and 750 mg/kg in males and females, based on a rangefinding 

study . Bone marrow was harvested at 6, 18 and 30 hours after treatment . 

SULMET induced no increases in percentages of chromosomally aberrant cells or in the 

frequency of aberrations per cell per animal in either sex at any dose or time point . 

Positive controls in each test system produced significant positive responses . It is 

concluded that Sulfamethazine is not mutagenic in these test systems . 

532 

MONITORING THYMIDINE CATABOLISM IN COMPLEX NATURAL SYSTEMS : A SIMPLE, NOVEL METHOD 

FOR ECOTOXICITY ASSESSMENT 

T . Shaw, D .G . MacPhee 6 R.H . Smillie*, Departments of Microbiology, La Trobe 

University and Biochemiatry, University of Melbourne* (Australia) 

Thymidine is one of the most important molecules in biology . Numerous bioassays 

depend on measurement of incorporation of radiolabelled thymidine into acid-insoluble 

macromolecules, usually presumed - often without justification - to be DNA . In the 

majority of steady-state biological systems, and in particular in natural ecosystems 

where cell growth and turnover are slow, thymidine catabolism and incorporation into 

non-DNA macromolecules is almost invariably quantitatively more important than its 

anabolism and incorporation into DNA . Consequently, we investigated the influence 

of toxic compounds on thymidine catabolism in various biological systems . Our work 

demonstrates that (1) meaeurement of thymidine catabolism is extremely simple even 

in complex systems, because at saturating concentrations of exogenous thymidine, its 

catabolites diffuse into the extracellular environment at a faster rate than they can 

be re-utilised, and (2) that in stable aquatic microenvironments, the rate of catabolism 

of exogenously supplied thymidine is influenced in a time- and dose-dependent 

fashion by an enormous variety of compounds including "classical" cytotoxins and 

genotoxins, as well as other compounds not usually considered to be in either of these 

categories . We conclude that monitoring thymidine catabolism yields useful information 

about the metabolic state of all systems investigated and seems to show great 

potential as an indicator of toxicity . 



Notes

184 1989 EMS Abstracts- 533 


Notes IWEVALUATION`bE'IIJM RODENT CYTOGENETIC TEST RESULTS COMPARED TO IN VITRO 

TESTS AND CARCIAbGENICITY . Michael 0 . Shelby, Cellular and Genetic Toxicology 

Branch, National I te of Environmental Health Sciences, Research Triangle 

Park, NC 27709 j~SA 

The role of in vivo short-term tests for genetic toxicity in testing schemes 

intended to detect potential chemical carcinogens has long been a matter of 

discussion . Whether in vivo tests should be part of an initial screening battery 

or should only be conducted on chemicals selected by in vitro tests has never been 

agreed . Over the past 7 years, a substantial data base of in vivo cytogenetic test 

results has been developed through NTP contract studies . Preliminary analyses 

have shown that in vivo tests for abearations and SCE perform as well or better 

than in vitro tests in identifying carcinogens and in discriminating between carcinogens 

and noncarcinogens . Published results and data acquired through NTP for 

the mouse micronucleus test are particularly encouraging . It appears that the 

micronucleus test, using either bone marrow or blood to score micronucleated 

erythrocytes, performs as well or better than tests for aberrations or SCE in bone 

marrow cells and at a considerable saving in resources . Use of the micronucleus 

test in place of other in vivo cytogenetic assays in initial testing efforts is 

under consideration . The results of chromosomal aberration, SCE and micronucleus 

tests on a variety of carcinogens and noncarcinogens and the relative performances 

of in vivo and in vitro tests will be presented . 

534 

BACKGROUND FREQUENCY OF HYPERPLOIDY IN BONE MARROW CELLS OF MALE CHINESE HAMSTERS 

CW Sheu, JK Lee, CA Arras . RL Jones and KS Lavappa, CFSAN/FDA, Washington D .C . (USA) 

Chinese hamsters, with 22 distinctive individual chromosomes, are an ideal species 

for aneuploid analysis . In a series of studies, the hyperploidy frequency in bone 

marrow cells of 154 male hamsters was determined . The animals were given a single ip 

injection of solvent or a potential aneuploidy-inducing chemical . Ten animals were 

used per dose and bone marrow was removed at intervals of 6-96 hr . Slides were coded 

and 50-100 metaphases were analyzed per animal . A metaphase with more than 22 chromosomes 

was classified as a hyperploid cell, and the data were evaluated using a onetailed 

Fisher's Exact test . In three experiments, the frequencies of hyperploid cells 

were 0 .43, 0 .91 and 1 .14% for the control groups . The combined controls had 17 hyperploid 

cells per 2,656 metaphases prepared from 32 hamsters, giving an average frequency 

of 0 .64% . The hyperploidy frequencies of treated groups ranged from 0 .50 to 1 .25%, 

and there was no significant increase in any treated group over its concurrent control 

with the exception of one group treated with vincristine at 0 .75 mg/kg . The observed 

increase was not significant when compared to the pooled controls . The treated groups 

when combined had 65 hyperploid cells per 7,355 metaphases from 90 hamsters, giving an 

average frequency of 0 .88% ; this value was not significantly different from that of 

the combined controls . In a fourth experiment, the hyperploidy frequencies based on 

800 metaphases from 8 animals per group were 3 .75% for the controls and 3 .13-4 .52% for 

the treated groups . These values were significantly higher than those given above . 

The discrepancy appeared to be related to the batch of animals and illustrates the 

importance of incorporating concurrent controls in assays for aneuploidy . 

535 

INDUCTION OF HICR01NCL6I IN RAT BOS6 MARROW BY S6LBCTION CHSHICALS . ! . Shi and T . OnB, 

Division of Respiratory Disease Studies, National Institute for ocoupational Safety 

and Health . HorBantown, WV (USA) 

A large number of chemicals have been tested for the induction of micronuclei in 

mouse bone marrow cells . Many of these studies were designed to determine proper 

sampling times and peak responses . Such studies and infors,ation, however, are rather 

limited for the rat bone marrow cells . Bfforts have been made in our laboratory to 

determine the dose and sampling time responses of micronuclei after Sprague Dawley 

rats were treated with triethylenem.lamine, mitomycin C, vincristin, and dimathylbensanthracene 

by a single intraperitoneal injection . Three concentrations were tested for 

each compound . Animals were sacrificed 24, 48 and 72 hrs after chemical treatment . 

The procedure of slide preparation and staining followed that of Schmid (Mutation Res . 

31 :9, 1975) . The number of micronucleated polychrom.tic erythrocytes among 2000 

polychromatic erythrocytes (PC6s) and the ratio of PCB to norsochroastic erythrocytes 

were determined for each animal . The results showed that all four compounds caused 

micronucleus formations in a dose related manner . The peak response sampling tisw is 

dependent on the chemical as well as the concentration of chemical . In all cases . 

however, an increase in the micronuclested PCis can be detected 24 hrs after chemical 

treatment . These results seem to indicate that two sampling times, 24 and 48 hrs, 

would be adequate for the micronucleus assay usin8 rat bone marrow cells . 

50869 3698

536 r 

THE COLLABORATIVE STUDY ON MOUSE SPOT TEST IN JAPAN . 

x .TUtikawa, T .Shibuya, S .}litotsumachi, T .Ohshima and A . Wakata, The Collaborative Study 

Group for the Mouse pot Test, Hadano, Kanagawa 257 (JAPAN) 

The collaborative study on mouse spot test (MST) was carried out in 11 different 

laboratory in Japan . Mouse strain used in this study were male PW (established by 

Tutikawa and Harada) and six different females ; C57BL/6 (B6), four 810 congenic lines 

(B10, B10A, B10BR and BlOD2) and KYG . The test campound employed was N-ethyl-N-nitrosourea 

(ENU) . ENU was dissolved in phosphate buffer (pH 6 .0) and intraperitoneally 

treated to female mice at dag_10 .5 of pregnancy . The observations on the F1 mice were 

done on the following characterst recessive color spots (RS), white midventral spots 

(WMVS), misdifferentiation spots (MDS) and external malformations at their weaning 

period . 

The incidence of RS lineally increased as dose of ENU in all matings . These results 

were clearly different from that of mouse specific locus test (Russell et al, 

1982) . These observations may suggest that repair capacity is deficient in melanoblast 

cells in mouse embryos . In the case of KYG, the incidence of RS induced by ENU 

was always higher than that in the other matings . 

The fertility rate in plug-proved females was extremely low in 86 females that the 

other females mated . 

From these results, we recommend to use male PW and female 810 on MST in Japan . 

537 

N-ETHYL-N-NITROSOUREA INDUCED RECESSIVE MUTATIONS IN MOUSE PRIMORDIAL GERM CELLS . 

T .Shibuya, N .Horiya, H .Matsuda, T .Hara, M .Ratoh and T .Murota, Hatano Research Institute 

Food and Drug Safety Center, Hadano, Kanagawa, and Mitsubishi Kasei Co, Yokohama(JAPAN) 

Previously we found that N-ethyl-N-nitrosourea (ENU) induced recessive mutation in 

somatic cells and killed primordial germ cells (PGC) in mouse embryos (Shibuga at al, 

1982) . We therefore carried out a specific locus test (SLT) on mouse PGC with ENU . 

Male and female C3H/He mice were mated and pregnant females obtained were treated 

with 30 or 50 mg/kg ENU at day 10 .5 of pregnancy . The F1 male mice obtained were mated 

with tester female PW mice after they were grown . The obtained mice of the next qeneration 

were checked for their coat•color and ear shape to evaluate whether or not PGC 

had been mutated by ENU . The fertility of male mice was 99% and 38% at 30 or 50 mg/kg 

ENU, respectively, much higher than the value in female mice . 

Three mutants were obtained in the 30 mg/kg group fram 5,823 offspring, and the two 

of them were recovered fram the same male, showing cluster mutations . Fifteen mutants 

were gained from 2,837 offspring in the 50 mg/kg group, and most of those had originated 

from cluster mutations . All mutants thus obtained are viable under hanozygous 

conditions . The high viability of mutant genes under homozygous condition is similar 

to that of an examination of the effect of ENU on stem-cell spermatogonia . 

SLT in mouse PGC at different developmental stages (8 .5 and 13 .5 day of development) 

by ENU are now in progress . Until now, mutants are obtained fram both experiments . 

It may be concluded that early stage of PGC is a sensitive stage to ENU in mice . 

538 

GENOTOXICITY OF BENZENE AND ITS METABOLITES 

H . Shimada, S . Itoh and S . Takayama 

Research Institute of Daiichi Seiyaku Co ., Ltd ., Tokyo (JAPAN) 

Benzene is known to have clastogenicity and myelotoxicity in animals, though which 

metabolites cause these damages remain unclear . We investigated the relationships 

between DNA single-strand breaks (SSBs) and ∎icronuclei formation induced benzene and 

its metabolites in vivo and in vitro . Male ddY strain ∎ice were singly administered po 

with benzene and killed at 12 to 72 h later . It was found that the SSBs in the bone 

marrow cells was markedly increased at 36 h, and the ∎icronuclei frequency was markedly 

increased at 42 h after the administration . In the ∎icronucleus test of the bensene 

metabolites, phenol and hydroquinone induced ∎icronuclei but other metabolites (muconic 

acid, hydroxyhydroquinone, 1 .4-benzoquinone, 2 .2'-biphenol . 4 .4'-biphenol and catechol) 

did not . In vitro studies, hydroquinone, 1 .4-benzoquinone, 2 .2'-biphenol and hydroxyhydroquinone 

induced SSBs in the bone marrow cells, while hydroquinone and 2,2'- 



Notes

186 1989 EMS Abstracts. - -- 

. ~ - .n„:-. 


Note s biphenol failed to induce SSBs in the Chinese hasster cells . The SSBs was detected in 

the -Chinesa _44nAfte-cells after the treatment of hydroquinone and peroxidase 

simultaneously . These results suggest that the Induction of ∎icronuclei by benzene 

follows after SSBs, and the several metabolites play an important role to Induce 

micronuclei and myelotoxicity through localized setaboliss within the bone marro . such 

as peroxidase-aediated metaboliss . 

SISTER CHROMATID EXCHANGES IN LYMPHOCYTES FRON INFANTS WITH DOWNS'SYNDROFg . 

E .K .Shubber, H .A .Hamamy, and B .M .A . AL-Allak, Dept . of Molecular biology, Nuclear 

Research Center, P .O .Box 765, Baghdad (IRAQ), and AL-Nustansiriya Medical College, 

Baghdad, (IRAQ) . 

This study investigated the relationship of sister chromatid exchanges (SCE) 

539 

frequency between normal and Downs syndrome infants ^nd their parents .•Blood .lymphocytes 

from 12 infants (3 females and 9 males) with Down's s'mdrome were cultured in RPHI-1640 

medium containing 10 ug/ml of brocodeoxyuridine for 63h . The mean frequency of SCE per 

metaphase for the patients (both sexes) was 9 .220 .8 which was significantly higher 

(P

earlier separation of the centromeres wes also noticed . The clestogenicity and 

cytotoxicity of the druge"was further substantiated by in vivo essays with mouse 

bone marrow cells where it caused dose dependent increase of chromosomal aberrations, 

sister chrometid exchanges and micronuclei . Hydracortisone, therefore, 

appears to be highly potent steroidal drug capable of directly attacking the 

genetic material inspite of its negative response in the Ames teat, 

542 

MUTAGENICITY IN SALMONELLA AND SISTER CHROMATID EXCHANGE IN MICE FOR BAY-REGION 

SYAL AND ANTI-DIOL EPOXIDES OF 1,4-DIMETHYLPHENANTHRENE 

J .E . Sinsheimerl . A .K . Gir11, E .A . Messerlyl, K-Y . Jung2 and M . Koreeda2, 

1College of Pharmacy and 2Department of Chemistry, University of Michigan . Ann 

Arbor, MI 48109, USA . 

Dose-response relationships for (±)-7a,8s-dihydroxy-5p .6p-epoxy-1 .4-dimethyl- 

5,6,7,8-tetrahydrophenanthrene and its Sa,Ba-epoxy diastereomer, the syn- and 

anti-diol epoxides of 1,4-dimethyl-phenanthrene respectively, have been tested 

for their mutagenicity In Salmonella strains TA98 and TA100 and for their In vivo 

sister chromatid exchange In the bone-marrow cells of mice . Both isomers were 

mutagenic In the nmole per plate range with the syn isomer being In the order of 

fifteen times more active with TA98 and five times more mutagenic In TA100 than 

its anti isomer . The anti lsomer was more genotoxic In the sister chromatid 

exchange assay than the syn isomer . Stetistically significant results were 

obtained as low as 1 .5 mg/kg body weight for the anti isomer and 3 mg/kg for the 

syn isomer . The present study supports the inclusion of methyl-substituted bayregion 

diol epoxides In the concept that the syn isomer, with quasi-diequatorial 

hydroxyl groups, contributes to the genotoxicity of the parent polycyclic 

aromatic hydrocarbons of those compounds which form hindered bay-region diol 

epoxides . Supported by grants R01ES0334S . NIEHS and R01CA25185, NCI . 

543 

THE ROLE OF PROTEIN ADDUCTS IN THE STUDY OF CHEMICAL CI4RCINOGENESIS . P . L . Skipper 

and S . R . Tannenbaum, Massachusetts Institute of Technology, Cambridge, MA 02139 . 

The reaction of carcinogens with proteins is an incidental but apparently inevitable 

outcome of the process of metabolic activation to ultimate carcinogens ; thus far no 

etiological role has been ascribed to these adducts . Nevertheless, they provide an 

excellent historical record of exposure and subsequent metabolic events which may be 

exploited to several ends . Firstly, they may be productively used in exposure 

assessment . In this category fall such applications as exposure monitoring, definition 

of exposed populations, and identification of cryptic exposures . Cigarette smoking, 

as well as exposure to environmental tobacco smoke, have been monitored by analysis 

of hemoglobin-bound 3- and 4-aminobiphenyl . These studies also demonstrated that 

cigarette smokers are a distinct group defined by five- to ten-fold higher adduct 

levels than nonsmokers . Similar evaluation of the contribution of cigarette smoking 

to benzo[a7pyrene exposure has been conducted . Secondly, protein adducts are useful 

in assessing the role of individual differences in biochemical response within a 

population exposed to relatively similar amounts of a carcinogen . Thus, hemoglobin 

adducts of 4-aminobiphenyl in smokers have been used to assess the significance of 

acetylator phenotype on the activation of this carcinogen through hydroxylamine 

formation . Finally, subject to many caveats, protein adducts have the potential for 

improving assessment of risk . Factors which affect the feasibility of realizing this 

potential will be discussed . This work was supported by the National Institutes of 

Health (grant nos . ESO0597, ES02109, ES01640) and the American Cancer Society (grant 

no . SIG 10-II) . 

544 

CORRELATION OF DNA ADDUCTS WITH SPECIFIC ALTERATIONS IN DNA SEQUENCEt IMPLICATIONS FOR 

CANCER RESEARCH . T .R . Skopek . Chemical Industry Institute of Toxicology, Research 

Triangle Park, NC (USA) 

A variety of DNA alterations have been observed in the activation of oncogenes and 

the inactivation of tumor suppressor genes . These include point mutations in protein 

coding regions, point mutations affecting aRNA splicing, gene rearrangements, gene 

amplification, and losa of hemisygousity . Molecular biology techniques are presently 

being used to learn how different chemical mutagens can effect these specific 

alterations in DNA . Knowledge of the DNA adducts and sequence alterations induced by 

chemical mutagens'permits the correlation of specific adducts with specific mutations ; 

this in turn can be used to infer a mutagen's ability to effect specific base changes 

commonly associated with oncogene/suppressor gene alterations . Once identified, 



Notes 

Ir


NOteS " : .. 

promutagenic adiucts can be studied as biologically relevant endpoints in tumor 

induction studies (~t e of adduct formation, persistence, repair), as well as in 

epidemiology-stddiei:3Md risk assessment calculations . Examples using model compounds 

(alkylating agents, acrylonitrile) will be presented to illustrate the importance of 

determining the types of mutations induced by chemicals and determining promutagenic 

adducts . 


545 

MOLECULAR ANALYSES OF A NOVEL LESCH-NYHAN SYNDROME MUTATION (hcstHon r ) 81 USE OP 

T-LYHPHOJYTE CULTU~ES . T .R~ Skogek, L . Recig, D . SimpsonD L . ba4f~ire , S .$ . 

Nelancon , H . Ogier , J .P . 0 Neill , H .T . Falta , J .A . Nicklas , and R .J . A1$ertini . 

Chemical Industry Institute of Toticology, Research Triangle Park, NC (USA) ; Hospital 

Saint Justine, Hontreal, Canada ; University of Vermont, Burlinton, VT (USA) . 

The frequency of hprt mutants in peripheral blood T-lymphocytes of two putative 

Lesch-Nyhan individuals and their parents was determined by a cell cloning assay to 

quantify the frequency of thioguanine resistant mutants . The results confirm the 

Lesch-Nyhan diagnosis and demonstrate that the mother has an elevated mutant frequency 

consistent with being heterozygous for an hprt mutation . Mass cultures of Tlymphocytes 

from both the children and their mother, as well as cultures of hDrt 

mutant clones from the mother, were employed as sources of aRNA for cDNA sequence 

analysis . These hprt mutants show a single base substitution (T + C transition) at 

position 269 (exon 3) . The predicted amino acid change is the substitution of 

threonine for methionineS7 . We term this new Leach-Nyhan mutation hertHontreal' 

546 

ROLE OF METABOLISM IN BENZENE-INDUCED MYELOTOXICITY AND LEUXEMOGENESIS 

Smith, M .T., Yager, J .W ., Robertson, M .L ., and Eastmond, D .A .' School of Public Health, 

University of California, Berkeley, CA 94720 and "Biomedical Sciences Division, 

Lawrence Livermore National Laboratory, Livermore, CA 94550 . 

The principal metabolitea of benzene include phenol, hydroquinone, catechol and 

trans, trans-muconic acid . The repeated co-administration of two of these metabolites, 

phenol and hydroquinone, produces bone marrow toxicity in mice similar to that caused 

by benzene . In parallel studies, phenol has been shown to stimulate the conversion of 

hydroquinone to the highly toxic 1,4-benzoquinone by myeloperoxidase, the major peroxidase 

enzyme in the bone marrow . A mechanism of benzene-induced myelotoxicity involving 

the accumulation of phenol and hydroquinone in the bone marrow and subsequent 

stimulation of peroxidase-dependent 1,4-bensoquinone formation has therefore been proposed 

. The same metabolites may also be responsible for benzene's genotoxic and leukemogenic 

effects . In order to study this further, we have determined the aneuploidyinducing 

and clastogenic properties of beniena's phenolic metabolites using a micronucleus 

assay in cytokinesis-blocked human lymphocytes . These studies revealed that 

hydroquinone was by far the most effective inducer of clastogenicity and the only 

phenolic metabolite which caused a consistent, dose-related increase in aneuploid 

cells . Interestingly, these effects were inhibited by the inclusion of ascorbic acid 

in the incubation medium . These results suggest that an oxidation product of hydroquinone 

is the metabolite responsible for the aneuploidy and clastogenicity observed 

following occupational benzene exposure and thus potentially benzene-induced leukemia . 

Supported by NIH grants P42 ES04705 and P30 9801896 . Work performed in part under the 

auspices of US DOE by the LLNL under contract no . W-7405-ENG48 . 

547 

PROTEIN CONTENT OF TOBACCO CELLS IN RELATION TO THE PLANT AC'TIVATION OF 

m-PHENYLENEDIAMINE AND 2-AMINOFLUORENE. S .R. Smith, M .M . Verdier, ED . Wagner and 

MJ. Plewa. Institute for Environmental Studies, University of Illinois, Urbana, IL 61801 USA. 

'Ibbacco cell suspension cultures activate the promutagens m-phenylenediamine and 2-aminoQuorene 

with his• reversion of Sa/mondla 7jphimurium strain TA98 as the genetic end point . 7b help identify the 

biochemical mechanism involved, a growth curve was established for these cells by first Inoculating several 

flasks with 3 g each from a 7-day culture and then measuring fresh weight at approodmately 12-h Intervals 

for 2 weeks. At the same time, protein content was analyzed using the Bio-Rad protein assay which is 

based on the absorbance of the dye Coomassic Brilliant Blue 0-250 that shifts to 595 nm upon binding 

to protein. Fresh tobacco cells were titered, sonicated, and centrifuged, leaving the extracted protein in 

the supernatant fluid. Frozen cells were also analyzed but gave iaconsistent results . A standard curve was 

determined using bovine gamma-globulin for each 24-h sample . After a lag phase of 3 to 4 days, the cells 

entered log phase ; stationary phase was reached by day 7 . Protein content, however, did not follow the 

50869 3702

growth curve, instead peaking just before the cells entered log phase . Data from preUminary activation 

studies using tobacco cells at diffeyent growth stages showed that lag-phase cells activated 2-aminofluorene 

better than those in log or stationary phase . Although the maximum 2-aminofluorene activation coincided 

with peak protein content, late log-phase cells were most competent in m-phenylenediamine activation, 

suggesting that tobacco cells activate 2-aminofluorene and m-phcnylenediamine through different metabolic 

pathways. Research funded in part by a WRC grant and USAF grant #88-AF-P-0511 . 

548 

THE POLY (ADP-RIBOSE) POLYMERASE GENE : DIRECT OR INDIRECT INVOLVSNIIirfP DJ DNA REPAIR AND 

MAllONANCYI 

Smulson, M .E .. Chemey, B ., Haque, J ., Huppi, C., Kang. V., Marr. K .. Mohrart . Z., Simpson. S ., and 8hatla, K . 

Dept . of Biochemistry, Georgetown University School of Medicine, Washington, D .C . 

Poly (ADP-ribose) polymerase Is a nuclear enzyme which appears to play a key role in modulating the 

repair of damaged DNA mammalian calls . The catalytic activity of the purified enzyme is stringently 

dependent upon the presence of strand breaks in DNA . The polymerase ls rapidly modulated In response to 

mutagen treatment, probably representing one of the very early responses to DNA damage . We have evaluated 

the role of endogenously and exogenously Induced DNA strand breaks on the transcriptional control of this 

enzyme and also studied the expression of this gane during the eeU cycle as well as during differentiation, 

both biological processes were endogenous discontinuous regions of DNA, and possibly strand breaks are 

operative. Experiments using the polymerase cDNA In an expression vector indieate an Increased rate of 

DNA repair In DNA damaged Cos cells, transiently transfeeted to overexpress the protein . 

We have also mapped the human ehromowmtl location(s) of this gane . It has often been suggested that 

cancer is associated with abnormal recombination events and mutations . We hypothesked that if any member 

of the potymerase gene family or polymerase-related genes were necessary for reducing inappropriate 

«combinationc, its loss might be associated with at least some human cancers. Tbe existence of a deletion 

polymorphism in one of the polymerase genes, located on chromosome 13q32-ter, allowed us to test whether 

deletion on both alleles on this gene correlates with inoreased tumor Incidence . There was a marked inereaso 

(4x) in the frequency of the deleted allele in tumor DNA ; allelic loss was also noted. These results suggest 

that deletions in the polymerase, or a gene linked close to it on chromosome 13 may operate to promote the 

development of some types of cancer 

. 'r 

549 A 

CHROMIUM(III) BINDS TO SINGLE-STRANDED DNA, INCREASES DNA POLYMERASE PROCESSIVITY, 

AND INDUCES MUTAGENESIS IN E . COLI . E .T . Snow, L .S . Xu, and M .D . Cohent Institute of 

Environmental Medicine, NYU Medical Center, P .O . Box 817, Tuxedo, NY (USA) . 

Chromium is a suspected human carcinogen, but the mechanism of chromium-induced 

carcinogenesis is unknown . Chromate ions are readily taken up by cells and are 

reduced intracellularly via unstable Cr(V) and Cr(IV) intermediates to stable Cr(II1) 

species . During this process DNA damage is produced and point mutations are induced 

in many target genes . However, it is not known how Cr produces mutagenic damage or 

if damage is produced indirectly by oxidative processes . Cr(III), the most stable 

form of Cr, has been implicated in chromium toxicityt but, most Cr(III) species do not 

cross cell membranes and are neither mutagenic nor carcinogenic in vivo . We have 

investigated the genotoxicity of Cr(III) in vitro using a DNA replication assay and 

in vivo by transfecting Cr(III)-treated DNA into competent E . coli and scoring for 

survival and mutagenesis . Single-stranded (as) M13 DNA was treated with 1 to 10 yM 

CrC13 (0 .5 - 24 hr, 370) and excess Cr removed by gel exclusion chromatography . The 

DNA was primed with a 5'-32P-sequencing primer and primer extension was carried out 

with DNA polymerase I(Klenow) or polymerase a-primase . Very low doses of Cr(III) 

(1 t0 5 uM) increased the processivity of both polymerases although 10 uM Cr(III) was 

inhibitory . When transfected into JM101 E . coli, Cr-treated es M13mp2 showed a dosedependent 

increase in mutation frequency at the lac2„ gene . These results suggest 

that Cr(III) alters the structure of the DNA template increasing the binding strength 

of DNA polymerases and decreasing the fidelity of DNA replication . These 

interactions may account, in part, for the mutagenicity of chromium ions in vivo . 

550 

A COMPARISON OF CHROMOSOME ABERRATION INDUCTION IN THE CHO AND CHL CELL 

SYSTEMS BY 25 CHEMICALS . T . Sofuni, A . Matsuoka, M . Sawada, M . Ishidate 

Jr ., and M . D . Shelby, National Institute of Hygienic Sciences, 

Setagaya-ku, Tokyo (Japan), and National Institute of Environmental 

Health Sciences, Research Triangle Park, NC (USA) 

The present study was conducted to compare the results of chromosome 

aberration tests using the CHL and CHO cell systems on 25 test chemi- 


1989 EMS Abstracts 189


_ _ .~-- __ 

Notes •`!'ca1s . In"~e HL system, cells were exposed to the test chemical for 

24h and 48h without S9 mix and for 6h with S9 mix . In the CHO system, 

cells were eo~or 10 .5-20 .5h without S9 mix and for 2h with S9 mix . 

In the absence of S9 mix, 6(24%) out of 25 test chemicals showed diffarent 

results : 4 were positive only in CHL, 2 were positive only in CHO . 

In the presence of S9 mix, 11(44%) chemicals showed different resultss 

8 were positive only in CHL, 3 were positive only in CHO . According 

to the combined results with and without S9 mix, 10(40%) showed qualitatively 

inconsistent results between two test systems . One of the 

reasons for these inconsistent results may be the differences in the 

experimental protocol used . However, a possibility that, even by the 

same experimental protocol, chromosome aberration induction by some 

chemicals miqht be qualitatively and/or quantitatively different 

between the CHL and CHO cell systems, still remains . 


551 

FURTHER CHARACTERIZATION OF A METHYL IigTRANESULFONATE-RESISTANT MUTANT FROM A CHINESE 

HAMSTER CELL LINE : ANALYSIS OF ITS HYPERSENSITIVITY TO UV 

A . Sono and K . Sakaguchi 

Division of Toxicology, Research Center, TOYO JOZO Co . Ltd .(Japan) and Department of 

Genetics, University of California, Davis(USA) 

A Chinese hamster mutant which was highly resistant to methyl mefhanesulfonate (MfS), 

referred to as lQ1Sr-1, was found to be hypersensitive to UV . 141S -1 was twice higher 

sensitivity to UV in cell killing than the parental line (Don D-6) . The increase was 

S-phase specific in the cell cycle . Ca€feine could enhance the UV-lethality of Don 

D-6 . However, the survival curve of NMS -1, irrespective of the presence of caffeine, 

was almost the same as that for Don D-6 with caffeine . The mutant might be deficient 

in post-replicational repair . It also accompanied with higher induction rate of 

sister-chromatid exchange (SCE) and chromosomal aberration . The UV-lethality of I41Sr- 

1 appeared to relate to the increase rate of lethal chromosomal aberrations, such as 

chromatid breaks and exchanges . Although inhibition of protein synthesis enhanced the 

hypersensitivity to UV and reduced the acquired resistancy to HHS, it could antagonize 

the induction of UV-induced SCE . Inhibition of DNA synthesis affected synergistically 

only on the UV-lethality . At the 502 lethal dose of tN, MMSr-1 was unexpectedly much 

less mutable than Don D-6 . This phenomenon makes it very difficult to explain only by 

a defect in the post-replicational repair system, but may be explanable by adding 

another system defect, so called "error-prone repair" which is known only in E . coli . 

From these facts, the possible defective feature for this mutant was discussed . 

PRCBIEbS Il1 CYTOGENE'PIC SCIIZVEIISd1PKE OF PECPLE ' 

Sorsa, M ., institute of Occupatiorlal Health, Helsinki, FINIAND 

552 

Sanatic chrmrosanle damage has been used for some deosdes already as indicator 

of exposure of specific grotlps of humans to clastogenic or genotoxic agents . The 

niain rationale in the approach is that dmmsge ebserved in the genetic material of 

human cells represents or reflects initial events in a process, which may eventually 

lead to ill health manifestations . lhus cytoyenetic surveillance has the potential 

to serve as an early indicator, enabling prevention of adverse effects . 

Several krowm and unoontrollable confoiuti3ers have been reoorded in population 

studies ; with proper preplanning most of these can be avoided . Since cytogenetic 

biamonitorin3 studies are always tedious and difficult, experimental identification 

of the ahrvnceane damaginy potential of the exposing agent(s) must be considered a 

prereyuisite to their performance . 

A11 stu3ies concerning humans directly also involve ethical aspects and the 

riyht-to-know of the persons giving the saaples . T41e planning stage of the study 

should also involve an action design for possible decrease of the exposures of the 

yroup, if necessary. Otherwise, the preventive nature of cytiogenetic surveillance 

is lost . 

553 

SUB-LETHAL ACIDIFICATION : ITS ROLE IN CHRONIC HEALTH EFFECTS . 

C .L . Soskolne ; 0 . Pagano, and 0 .0 . Oiordano, Istituto Nazionale Tumori, 80131 Naples . 

Italy, and •University of Alberta, Edmonton, Canada T60 203 . 

Mineral acids in the workplace, ae well aa in the general environment represent 

a recognized health concern as yet mainly focussed on lethal or acute outcomes . 

Over the past decade, long-term workplace exposure to high concentrations of sulfuric 

and other acid miste have been shown to be associated with respiratory tract cancers, 

50869 3704


~ 

especially laryngeal carcinomas (Soskolne et al ., 1984 ; 1989 ; Beaumont et al ., 1987). Notes 

Toxicologic evidence supgorts these human data and lncludes : a) in vivo and in vitro 

cytogenetic abnormaliti6s following a sub-lethal decrease of extra-cellular pH ; 

b) developmental toxicity as induced by acidic contaminants or drugs ; o) a crucial 

role for pH in cell cycle, mitosis, and differentiation . The underlying biologic 

mechanisms that explain adverse health outcomes include pH modulation of toxicity 

for a number of xenobiotics (including carcinogens, genotoxins, and teratogens), and 

low pH-induced changes of celle involving, for example, alterations in mitotic 

apparatus and enzyme regulation . A•multi-disciplinary effort is prompted in order to 

investigate the role(s) of environmental acids and acidic drugs in genotoxicity 

and carcinogenesis . (Supported by the Italian Ministry of Health) . 

554 

MITOMICIN C-INDUCED DOMINANT LETHAL MUTA't1VNS IN SYERMATOCYI'IiS ANS NOT DUE 1'0 CELL- 

KILLING . J .P . Soto, F P va ivia, M .M . Orellana, N .M . Lafuente and H . Katob . 

Facultad Odontologia, U . de Chile . STGO (CHILE) and Hatano Research Institute, 

F .D .S .C ., Kanagawa (JAPAN) . 

Mitosicin C(MC) is well known to induce doainant lethal autations in souse early spersatids and 

speraatocytes but has not effect in late spereatids and spersatozoa (Ehling, 1971) and Kratochvilova 

(1973) reported that MC cause high percent of unfertilized ova when MC-treatea anisals were sated at 

intervals corresponding to treated speraatocytes, suggesting a strong cell-killing effect of MC to 

speraatocytes . Histological studies and sperm head abnorsalities test ssre done to deteraine whether 

MC-induced failure of fertilization in speraatocytes is attributable to cell-killing effect . 9 weeks 

oid sale C('i aice were intraperitoneally (i .p) injected with 5 sg MC/kg . At 4 days intervals over a 

7-week period following treataent, cauaal epididiaal spera and testes of 3 aniaals were recovered . 

The nuaber of abnoraal spera head was 61 to 80 in the tiee points ranging froa 20 to 32 days post 

treataent and increased to -200 in the tiae points 36 to 49 days after treatsent . At ail others,earliers, 

tise points it was around the control nwber, i .e ., -10 abnorsal speras/1000 spera per anieal . The 

abnoreal sperm frequency to tiae after treataent parallel the unfertilized ova frequency curve for 

MC . In cross sections of testes tubules of these saae treated sice there was not evidences of cell-killing 

in sperwtocytes ; therefore, spersatocytes developed to spersatids but there was faulty diffirentiation 

of spersatozoa, rich appears to be a significant cause of failure of fertilization in sperutocytes . 

555 

ANTIMUTAGENIC EFFECT OF PROSTAGLANDINE IN `HUMAN LYMPHOCYTE CULTURES 

Sridevl,K . . K .P .Rao and K .V .Chandrika . Depariment of Genetics . Osmania University . 

Ilyderabad-S00 007 . INDIA . 

Prostaglandin E .(PGE) . is a derivative of -linolenic acid, an essential fatty 

acid . PGE was tested for its antimutagenic activity !n invitro human lymphocyte 

cultures . korbol myristate acetate (PMA) at 200ng/ml was used as the clastogen . 

Whole blood cu,;tures were set up with Tc 1919 medium . Both the clastogen (PMA) 

and PGE1 (10 M) were added at the synthesis phase of the culture (48 hrs) . 

Cultures were harvested at 72 hours by hypotonic treatment followed by fixation . 

Slides were prepared and 100 metaphases were scored for various chromosomal 

aberrations like breaks . translocations . dicentrics etc . Three individual samples 

were studied to minimize the sample variation . The frequency of chromosomal 

aberrations was reduced with PGEI in PMA treated cultures, suggesting the protective 

role of PGE3 . 

556 

DETECTION OF MAMMALIAN CELL MD3AGENESIS IN AS52 CELLS . L .F . Stankowski, Jr ., W .G . 

Tuman, M.J . Bieszczad and K .F . McLaren, Pharmakon Research International, Inc ., 

Waverly, pA 18471 

The AS52/XPRT assay is quite similar to the more familiar CHO/HPRT assay with 

respect to its low spbntaneous mutant frequency and cytotoxic response to a variety of 

agents . AS52 cells contain a single, functional, stably-integrated copy of the E . coli 

qpt gene in a CHO cell line cont=ining a partial deletion of hprt, and have been used 

to detect mutation of 5Wt (to TG ) under conditions identical to those for hprt in 

CHO-KI-BH4 cells . Based upon the demonstrated or presumed carcinogenicity of 

approximately 40 agents compared concurrently in the two assays, the AS52/XPRT and 

CHO/HPRT assays exhibit sensitivities of 100 and 80%, and specificities of 94 and 100%, 

respectively . Minor modifications further improve/simplify the AS52/XPRT assay . For 

example, recalculation of survival data in terms of relative cell viability [(relative 

cell density on Day) (relative cloning efficiency)1 provides a more accurate estimate 

of cytotoxicity . Transfer of AS52 cells from MPA to XAT medium for 24 hours prior to 

treatment prevents the poor growth occasionally observed after transfer to 

unsupplemented Ham's F12 . The 7-day phenotypic expression time can be reduced, due to 


A 

191



0 

.~ 

OteS- 'the F~gradual decii3se~n population doubling time observed over the past few years . 

(This decrease is likely responsible for the somewhat higher spontaneous mutant 

frequencies recent,}y„~ved .) In addition, the use of semi-attached, or other 

subculture conditions without trypsinization, contributes to a further reduction in 

phenotypic expression time . These data lend further support to the use of AS52 cells 

as a more sensitive alternative to the CHO/HPRT assay . 

557 

FLUORESCENT LIGHT NUTAGENESIS IN SALMONELIJ~T~ ~RINORIUM AND 8SCldERICNIA COLI AS A MODEL 

FOR SUNLIGNT-INDUCF.D GENOTOXICITY . L .F . Sta owsnk ki, Jr ., D .B . Say, N .J . Sieszcsad, 

W .G . Tuman, K .F . McLaren and D .J . Mecca, Pharmakon Research International, Inc ., 

Waverly, PA 18471 

During evaluation of a photoactivated therapeutic agent, it was serendipitously 

observed that fluorespnt light alone yas quite mutagenio to some tester strains used 

in the Salmonella his and E . coli tM reversion assays . The strains were exposed (in 

a plate incorporation assay, without petri dish lids) to eight unshielded/unfiltered 

40-watt cool white fluorescent bulbs in a laminar flow hood . Treatment 4der thes; 

conditions for up to three hours produced time-dependent increases in his and trp 

reversion frequencies in strains TA98 and WP2 urA (3- to 6-fold) and strains TA100 and 

WP2 uvrA pKM101 (10- to 15-fold), with a plateau generally observed after a 90- to 

120-minute exposure . Smaller/variable increases occurred in strains TA1S35 and TA1537, 

but none were observed in strains TA1538, TA102 or WP2 . Filtering the light through 

polystyrene lids did not completely abolish its mutagenicity observed in strains TA100 

and WP2 uvrA pKtt101 under standard plate incorporation conditions (without lids) . The 

presence of a photoactivated promutagen in the nutrient/top agars or overnight culture 

medium seems remote, since treatment in microtiter wells after washing and resuspension 

in phosphate buffer still elicits a mutagenic response . Thus, these cosmion tester 

strains may provide the basis for a simple, sensitive, inexpensive model to study 

genetic damage induced by natural sunlight, as indicated by the ability of consumer 

sunscreens to reduce the mutational response observed under standard plate 

incorporation conditions . 

MUTAGENESIS BY GLUTATHIONE AND OTHER THIOLS : MECHANISM AND RELEVANCE TO 

HEPATOCARCINOGENESIS . A .A . Stark, D .A . Pagano, and E . Zelger. Department of 

Biochemistry, Tel-Aviv n vOsTEy, Ramat-Aviv 69978, Tel-Aviv (Israel) ; 

Cellular and Genetic Toxicity Branch, N .I .E .H .S ., Research Triangle Park, NC 

27709 (USA) . 

The mechanism of glutathione (GSH) and other thiols' mutagenesis was 

Investigated (Stark at al ., Mut . Res . 177 :45-52, 1987 ; Carcinogenesis 

9 :771-777, 1988) . The activation of GSH to a bacterial mutagen 1s catalyzed 

by a single enzyme, y-glutamyltranspeptidase (GGT) . GGT cleaves the Yqlutamyl 

residue from GSH to form the reactive dipeptide cystetny191yctne 

(CG), which is mutagenic 1n the absence of GGT . The mutagenicity of GSH, CG, 

and other thiols depends on the pKa of the -SH group and the pH of the 

medtum . GSH and CG mutagenesis requires molecular oxygen, is enhanced by 

1ron, and Inhibited by lron chelators, radical scavengers, and 

H2O2-metabollztng enzymes . Autoxidation of the thiolate anion generates the 

(pen)ultimate mutagen H202 . GSH can also drive lipid paroxldatlon in vitro 

and tn cultured cells tn the presence of GGT . Elevated GGT levels are often 

found in hepatic preneoplastlc foci . The high probability of their 

progression to hepatocarcinomas may thus be due to the formation of oxygen 

radicals by the GSH-GGT system tn the proximity of these foci . 

558 

559 

DEVELOPMENT OF MONOCLONAL ANTIBODIES RECOGNIZING 4-AMINOBIPHENYL-(SUANOSINE 

M . Stefanidis, D . W . Roberts, F . F . Xadlubar and R .M . Santella, National Center for 

Toxicological Research, Jefferson, AR (USA) and Columbia University, New York, NY (USA) 

Several aromal.ic aminee, including 4-aminobiphenyl, (4-ABP), have been found in 

cigarette smoke and are recognized as potent human carcinogens . Exposure to aromatic 

animns is believed to be a contributing factor to the increased incidence of bladder 

cancer in cigarette smokers . Previously, a polyclonal antibody was developed against 

the major 4-ABP--DNA adduct (Cancer Res . 48 :6336,1988) and used to monitor adducts in 

lung and bladder epithelial DNA of smokers (IARC publications i89,p .166, 1988) . To 

develop monoclonal antibodies to this adduct, BALB/c mice were issiunized with 4-ABPguanosine 

coupled to keyhole limpet hemocyanin . The two most sensitive clones, 6D4 end 

10114, were characterized as to sensitivity and specificity by competitive enzyme 

linked immunoaorbent assay (ELISA) . For both antibodies, 50% inhibitor concentration 

is in the range of 1 pmol of adduct . Neither antibody crossreacts with unmodified 

50869 3706

guanosine at the highest lonr.entration tested (1 .50 nmol/well) . Antibody 1oB4 

re•rofnizod the 4-Al1}'-guHnosine adducl, with similar sensit .ivity when it is present. in 

d~rn atured DNA or as the moaoadduct . A rompetitive ELTSA with fluorescence endpoint 

d~_•tecl.ion wus developed lo increase assay svnsitivlty (SOt inhibition at . 400 fmol) . 

SPnsitivity can be further increased by enzymatic digestion of DNA and isolation of 

ndducte by immunoaffinit .y chromcd ottraphy or HPLC before analysis . These antibodies 

will be used to monitor adduct levels in biological samples from smokers and 

nonsmokers and r•valunted as a marker of evpusru-e• to aromat.ic aminos . This work wns 

supported by a grant from NCI-CA21111 . 

560 

BLEOMYCIN-INDUCED ABASIC SITES WITH CLOSELY OPPOSED STRAND BREAKS : STRUCTURE, 

SEQUENCE SPECIFICITY AND ROLE IN MUTAGENESIS R .J . Steighner and L .F . Povirk, Department 

of Pharmacology and Toxicology, Medical College of Virginia, Richmond, VA 23298 

Bleomycin-induced mutations in the lambda cI gene occur preferentially at hotspots 

which share the sequence C-G-C-C . When an end-labeled restriction fragment of _cI DNA 

was treated with bleomycin, and the resulting abasic (AP) sites were cleaved with 

putrescine, discrete shorter double-stranded fragments were produced, whose 

electrophoretic mobilities were consistent with putrescine-dependent double-strand 

cleavage at or near each of the mutational hotspots . The shorter double-stranded 

fragments were eluted, denatured and run on sequencing gels . Analysis of cleavage 

sites and termini in each strand indicated that bivalent lesions consisting of a 

strand break at the C residue in the C-G-C-C sequence, plus an AP site at the G 

residue directly opposite, were formed by bleomycin at each hotspot . In separate 

experiments, bleomycin-induced mutagenesis of repackaged lambda phage was reduced 2to 

4-fold by treatment of the DNA with putrescine prior to repackaging . This 

reduction in mutation frequency may be attributable to cleavage of AP sites with 

directly opposed breaks, since endonuclease IV (which cleaved only "nonopposed" AP 

sites) had no effect . Our results strongly suggest that AP sites with directly 

opposed breaks are intermediates in bleomycin-induced mutagenesis . The potent 

mutagenicity of these lesions is probably attributable to the simultaneous loss of 

coding information at the same position in both DNA strands . The sequence 

specificity for formation of these lesions was distinctly different from thsyt of 

bleomycin-induced single-strand breaks . 

561 

MUTAGENIC EFFECTS OF PHTHALATE ESTERS AND ASSESSMENT OF RISKS POSED 

TO THE ENVIRONMENT 

K . Strobel and T . Grummt, Research Institute for yyg iene and Microbiology, 

GDR - 9933 Bad Elster, H .-Heine-StraBe 12 (GDR) 

with an annual production of about one billion pounds phthalste esters 

are in wide use . In some plastic materials they make up to 50 % 

of volume . By now they are widely dispersed in the environment . Knowledge 

on mutagenic effects of phthalate esters is incomplete and partly 

controversial . Therefore, we investigated the mutagenic activity of 

i8 phthalate esters in the AMES-test and in cell cultures (FAF-celle 

of Chinese hamsters) . Mutagenic effects were detected in one or both 

test systems for a number of these substances . Their toxic, and partly 

mutagenic, end/or carcinogenic potential as well se the fact, that 

concentrations of e .g . DEHP and di-(n-butyl)phthelate in water samples 

and sediment were higher than those of DDT end PCBs, have to be considered 

in risk assessment for phthalate esters . The hazards which are 

posed by this group of substances to the environment give rise for the 

conclusion that guideline values for their presence in drinking water 

have to be elaborated, aspecielly in view of the revision of WHO Drinking 

water Guidelines which is being performed at present . 

562 

INHIBITORY EFFECT OF ASPARAGUS ON DMH-INDUCED MICRONUCLEI AND APOPTOSIS IN THE COLON 

CRYPT CELLS OF MICE . H . Sun, Q . Zhu, S . Fu, Y . An, G . Dou, B . Yang and Y . Wang . Henan 

Medical University, Zhengzhou, Henan 450052, People's Republic of China 

By assaying of micronuclei and apoptosis in the colon crypt cells of C57 mice, we 

studied the potential inhibitory effect of asparagus toward the intestinal carcinogen 

1,2-dimethylhydrazine (DMH) . The original liquid squeezed from fresh asparagus 

was applied as inhibitor in this study . Three groups (5 mice/group) were treated with 

asparagud liquid in the dosage of 0 .1 ml, 0 .5 ml and 1 .0 ml/ mouse respectively by 

stomach intubation, one hour before DMH were injected intraperitoneally . Mice injected 

with DMH only were positive control and mice injected with EDTA only were negative 

control . Twenty-four hours after infection with DMH, all the animals were sacrified . 


A 


Notes

194 1989 EMS-Abstracts~ '"-aF` 

Notes 


Sections of paraf%n-eiedded colo rolls were routinely made, and DNA damages were 

revealed by Feulgen"s'faMeg . Micronuclei and apoptosis were scored in 20 complete 

crypts for each sample with oil immersion microscope and the mean number of micronuclei 

and apoptosis of one crypt of each group was caculated . We found that the frequencies 

of micronuclei and apoptosis in the groups treated with asparagus liquid 

were obviously lower than in the group only in ected DMH . The number of micronuclei 

and apoptosis was 3 .99/crypt in only DMH treated group but was 2 .71-0 .76/crypt in 

groups treated both with DMH and asparagus . A clear dose-response relation was 

observed with different doses of asparagus liqutd . Our result indicated that asparagus 

might reduce the mutagenecity and carcinogenecity of DMH to colon crpt cells of 

mice . 

563 

EVALUATION OF GENOTOXIC POTENTIAL OF COMMONLY USED INSECTICIDES USING 

BARLEY (HORDEUM VULGARE) TEST SYSTEMS 

T .Suryakumari and K .Vaidyanath, Andhra University, Waltair (A .P) and Osmania 

University, Hyderabad-500 007 (A .P .) India . 

The modern agricultural practices entail intensive application of pesticides for 

plant protection, which are potential genotoxicants . To assess the mutagenic potential 

of Ekalux (00-diethyl 0-quinoxalin-2-yl phosphorothioate and Rogor (00-dimethyl 

S-methyl carbamoyl methyl phosphorodithiote) a study was made on barley using 

multiple end points . The cytogenetic end point consisted of screening for cytotoxic, 

chromotoxic and clastogenic effects on somatic chromosomes . The scoring for forward 

mutation at waxy locus (Wx -~ wx), chlorophyll deficient seedings !n M, and 

polygenic variability in M, generations, constituted germinal mutation assay . The 

frequency and spectrum of cytological abnormalities observed in the study indicated 

mitodepressive, clastogenic and chromotoxic effects of the insecticides . The high 

frequency of sterile pollen and chlorophyll mutations elicited suggests mutagenic 

potential . However, both the insecticides failed to induce mutations at the specific 

locus . The polygenic variability noted in M . generation also suggests that Ekalux 

and Rogor are capable of inducing mutations for quantitative traits . The positive 

correlation observed between effects on somatic and germinal mutation suggests that 

any one or a com bination of assays can be used profitably for m onitor)ng genotoxic 

effect of environmental pollutants . 

DNA Repatr Induced by Various MutaQens in Rat Xepatocyte Primary Cultures 

Measured tn the Presence of Hydroxyurea, OuonasoJe or Aphidicolin 

W . Suter and F . Romagna, WSP Toxicology, Sandoz Ltd ., CH 4002 Basel, 

Switzerland 

The goal of the present study was to find a selective inhibitor of DNA replication In rat 

hepatocyte primary cultures to replace hydroxyurea (HUI, which has been found to be 

genotoxic. Two agents were chosen for this study, ouanasole IoUI 18-amino-lH-1,2,4triazolel, 

which like hydroxyurea . is an inhibitor of ribonucleotide reductase, and 

aphidicolin (API, an Inhibitor of DNA polymerase a. Using the nuclei procedure developed 

by Althaua (Cancer Res . 42, 8010-3016, 19821, DNA repair induced by UV irradiation 

. MNNO. MNU, H,O,, 6-hydroxydopamine. 4-nitroquinolirro-N-oxide, 2acetylaminofluorene, 

benzo/alpyrene, cyclophosphamide and aflatoxin B, was 

measured in the presence of either 10 mM hydroxyurea, 15 mM tuanazole or 0 .015 

mM aphidicolin . The latter was found to inhibit DNA repair in addition to its effect on 

DNA replication . In the presence of guanazole and hydroxyurea the overall sensitivity 

of the detection of DNA repair was similar . Aflatoxin B, and MNU were slightly more 

active In the presence of hydroxyurea, while MNNO was more eaily detected in the 

presence of guanazole. 

Thus . guanazole can be used in hepatocyte primary cultures instead of hydroxyurea for 

selective suppression of DNA replication . 

564 

SCRUTINY OFPROTOCOI S r-OR Ti il's MICRONUCLEUS TEST WITH MICE ---ACI IIEVEMENTS 

IlY CSGMT/JEMS .MMS, CSGMT, prcccnters : S . Sutou (Itoham Foods Inc ., Tokyo), H . 

Shimada (Daiichi Seiyaku Co .. Ltd., Tokyo), A . Wakata (Yamanouchi Pharm . Co ., Ltd ., 

Tokyo), T. Awogi (Oisuka Iharm. Co ., Lld ., Tokushima), A . Ohuchida (Taiho Pharm . Co., 

Ltd., Tokushima), M . llayashi and 1' . Sofuni (Natl . Ins(. Ilygienic Sci ., Tokyo) 

The Mammalian Mutagenicity Study Group of the Environmontal Mulagcn Society of 

Japan (JEMS/MMS) has organized a collaborative study group for the micronucleus test 

50869 3708 

565

f 1989 EMS Abstracts 195 

Notes 

(CSGMT) to scrutinize protoc,Qts and to reach a standard protocol so that all participants 

can obtain data under optimal conditions, and thus results can be compared regardless 

of time, place, and person . Firstly, we examined sex-related difference in the test and 

suggested that the use of male mice is sufficient for general screening (Mutation Res ., 

172, 151, 1986) . Secondly, we examined strain-related difference and concluded that 

any strain could be used as a tester, though MS/Ae tended to show the highest 

response, especially at higher doses (op . cit ., 204, 307, 1988) . Thirdly, administration 

route (ip and po)-related difference was exaaained ; the results will be presented 

separately in the present meeting . Fourthly, dosing times and sampling timing are now 

under study . Some preliminary results are presented and discussed . 

566 

MATERNAL INHERITANCE OF ms GENE, S . Sutou and S . Sato 

(Central Research Institute, Itoham Foods Inc ., 1-6-21 Mita, Meguro, 

Tokyo, 153 Japan) 

MS/Ae mice, which are mutagen-sensitive in both the dominant lethal 

test and micronucleus test, and CD-1 mice, which are the parental strain of 

MS/Ae, were mated in all four possible combinations . Both male and 

female offspring were subjected to the micronucleus test using mitomycin 

C(MMC), colchicine (Col), and 6-mercaptopurine (6-MP). Col showed 

equivocal results . However, MMC and 6-MP clearly showed differential 

responses in that both male and female offspring from CD-1 dams had 

lower incidences of micronucleated polychromatic erythrocytes than those 

from MS/Ae dams regardless of sire strains . In addition, body weights of ' 

offspring from MS/Ae dams were lower than those from CD-1 dams 

regardless of sires . These results strongly suggest that 1he ms gene (or 

genes), which regulates the characteristics of MS/Ae, is maternally inherited . 

567 

IN VITRO tdICRONOCLEI IN THE MOUSE MAMMARY EPITSSLIAL C$LIS GROWN UNDER 

SERIIM-FREE CULTURE CONDITIONS 

Kunio ,Suzuki and Tomotari Mitsuoka, Animal and Cellular Systems 

Laboratory, and Frontier Research Programs, The Institute of Physical 

and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan . 

Although epidemiological studies suggest that dietary factors are 

associated with breast carcinogenesis, the specific carcinogen involved 

in cancer of the breast remains to be identified experimentally . In an 

attempt to establish an in vitro short-term test for breast carcinogens, 

we studied several known carcinogens tested by micronucleus assay in the 

primary cultures of mouse mammary epithelial cells . Mammary epithelial 

cells from 2 month old ICR virgin mice were cultured on collagen gel in 

serum-free Ham's F-12/Dulbecco's modified Eagle's medium supplemented 

with insulin, bovine serum albumin, epidermal growth factor, transferrin 

and cholera toxin . At day 6 of culture, known chemical carcinogens were 

added to the cultures, and the number of micronuclei per 1,000 cells was 

scored at 24 hr after treatment . Two breast carcinogens, g-methyl-gnitrosourea 

(MNU) and 7,12-dimethylbenz[a]anthracene (DMBA), increased 

the micronuclei incidence in the cultures . The cells grown in this 

serum-free medium metabolized DPIBAA to the activated form . These results 

suggest that the .quantitation of micronuclei in the mouse mammary 

epithelial cells cultured under serum-free conditions might be used as 

an in vitro short-term screen for breast carcinogens . 

568 

CANCER PROMOTERS AND THEIR EFFECT ON THE MUTAGENICITY OF MUTACARCINOGENS 

Dalisay Chionglo Sy, U .of Santo Tomas, Fac .of Medicine,Dept of Biochem . 

Studies have indicated the cancer-promoting activities of phenobarbital(pheno) 

and saccharin (sacl . The possibility that their effects occur 

thru enhancement of a pre-existing mutagenic condition was tested using 



~ . . .r -- . .. 

Notes- - 

.$c~ .-{~im id's Micromuel~eu.cs Test . Nine to 10 weeks old Strong A male mice were 

separately treated i,p with 2 mutacarcinogens : dimethylnitrosamine(DMN), 

10 mg per kg_$_V,_~mitomycin C (mito C),3 mg per kg BW . An hour after 

injection of the mutacarcinogens,pheno,90 *g per kg BW and sac,2 .S gm per 

kg BW were given to the animals treated with mutacarcinogens via the 

same route . Pheno was injected to the DMN-treated group and sac to the 

group receiving mito C . Bone marrow cells from femora of animals were 

isolated using fetal calf serum for suspension . Cells were collected by 

low speed centrifugation and smears prepared . Slides were stained using 

May Grunwald-Giemsa stain, Animals that received pheno and sac after injection 

with mutacarcinogens showed -inereased production of micronuclei . 

The mito C-sac combination showed peak micronuclei formation when sac was 

given 3h after injection of mito C . Statistical analysis showed significance 

at p= .05 . Results agree with earlier studies showing increased germ 

cell toxicity of DMN and mito C after treatment with pheno and sac . Both 

findings suggest that pheno and sac could exert their cancer-promoting 

actions by enhancing the mutagenicity of mutacarcinogens in somatic as 

well as germ cells . Whether all cancer promoters act in like manner 

remains to be seen, 

GENOTOXIC ACTIVITIES OF 3-CHLOROPROPIONIC ACID AND RELATED COMPOUNDS 

M . SZEGEDI 


Chemical Works of Gedeon Richter Ltd . Microbiological Research Laboratory 

H-1475 Budapest, 10 . P .O .B . 27 . Hungary 

The 3-cloropropionic acid (3CPA) has a strong genetoxic activity 

in Salmonella typhimurium TA 1535 nd TA 100 strains . We have tested 

a group of chemicals - withouth metabolic activation - in the E .coli 

SOS chromotest . They were similar to 3CPA different in the lenght of 

the carbon chain and the subtituens as follows : propionic acid, 

propionyl chloride, 2-chloropropionic acid, 3-chloropropionic acid, 

2-chloropropionyl chloride, 3-chloropropionyl chloride, chloroacetic 

acid, 2-chioropropane, 1-chlorobutane, 4-chloro-l-butanol, 1-chloropropane, 

1-chloro-2-propanol, 3-chloro-propionitrile, 

3-chloropropionitrile . The experiments were carried out in BIOSCREEN 

Analyzing System using BIDSOS program (LABSYSTEMS Ltd .) . Positive 

response was given only by 3CPA and 3-chlorpropionY1 chloride 

suggesting that C1-CH2-CH2C0- is resposible for activity . 

569 

570 

EVALUATION OF THE CLASTOGENIC ACTIVITY OF ROCBAGAN (BENZNIDAZOLE) IN 

MAMMALIAN SYSTEMS .C .S .Takahashi,S .C .Souaa and S .J .Santos, F .F .C .L .R .P ., 

USp and F .M .R .P .,USP,Ribeirao Preto,Sao Paulo,Brazil . 

Rochagan, a drug whose active compound is benznidazole(N-benzil-2 ni 

tro-l-imidazolacetamida), is used for the treatment of Chagas'disease 

and has been effective as a trypanosomicide . The drug was tested for 

clastogenic activity in Wistar rats treated by gavage at doses of 50 to 

1000 mg/kg body weight administered three times at 8 h intervals and in 

human lymphocyte cultures at doses of 250 pg/ml culture medium .Six rats 

were used in each treatment and sacrificed 6, 12 and 18 h after the 

last treatment . One hundred bone marrow metaphase cells per animal were 

analyzed for chromosome aberrations,showing frequencies similar to control 

values for all treatments (0 .30 to 1 .66Z) . For the in vitro test, 

blood was obtained from 5 healthy donors and benznidazole wasa ded at 

the beginning of culture .One hundred metapbases per individual were ana 

lyzed for chromosome aberrations and 50 metaphases for SCE . The frequen 

cies of chromosome aberrations and SCE were 3 .2x and 8 .01 SCE/cell in 

control cultures and of 62 and 13 .89 SCE/cell in treated cultures .A 

slight increase in SCE was observed in the treated group .The lack of 

positive results could be explained by the absence of reduction products 

. These reactive metabolites were observed under anaerobic conditions 

which does not occur in the systems used . 

50869 3710

571 

: 1989 EMS Abstracts 197 

Notes 

COMPARATIVE CLASrOGENIC EFFECTS OF ORGANIC AND INOROANIC TIN SALTS 

IN VITRO JP:. 

Geeta TALUKDER,Ban~ Bandana Ohosh,Archana Sharma 

Human Genet cs Unit,Centre for Advanced Study in Cell a Chromosome 

Research,Department of eotany,University of Calcutta,35 BallY9unj 

Circular Road,Calcutta 70001l .India . 

The clastogenic response of human lymphocytes ;S was studied 

following the administration of stannic chloriae and 2oiuq/ml)and 

trimethyl tin(0 .5 and 7juy/ml) .Blood was obtained from healthy donors 

of both sexes of six age groups .The end points screened after 72 hours 

were mitotic index(MI),chromosomal aberrations(CA)both with and 

without gaps and micronuclei(MN),Sister chromatid exchanges(SCE) and 

cell cycle kinetics(c:CK)were recorded after both 48 and 72 hours in 

culture . Both tin salts,analysed statistically,increased the frequency 

of CA,MN and SCE to a statistically significant level and depressed 

MI and CCK when compared to the untreated control . The effects were 

directly proportional to the concentrations used . The percentages of 

MN and CA were significantly higher in cultures from donors of older 

age groups(above 50 years) as compared to the lower ones . There was 

no difference between the relative effects of the two salts or 

between the cultures from the two sexes . SCE was significantly higher 

in teeated cases and in blood from persons who smoked . 

572 

Cytogenetic study of gossypol acetate 

Tan Yongbu, Zhang Zhongshu and Wang Renli 

Shanghai Institute of Planned Parenthood Research, 

2200-4 Xietu Road, Shanghai PRC, 200032 , 

r 

Gossypol, a yellowish phenolic compound isolated from seeds, stems and roots 

of cotten plant,has been advocated by the Chinese scientists as a antifertility 

agent for males . A 

Clinical trials indicated that the antifertility effect of gossypol was 

exellent, but occasional cases of hypokalemic paralysis might occur in the 

course of its administration . 

In the present work the effects of gossypol on the concentration of some of 

micronucleus, chromosomal abrration, SCE, gene mutation in somatic/reproductive 

cells and dominant lethal mutation, dose-response relationships are observed in 

rats, mice and men . 

The results indicate that gossypol of antifertility agent for males at lower 

dosages may be safe, but it should not be neglected that higher dosages might 

disturb or damage the DNA synthesis . 

573 

In situ detection of induced mutations with chemicals by Tradescantia 

TANO, S . Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, 

Tokyo 113, Japan 

Tradescantia stamen hair cells are highly sensitive to the induction 

of somatic mutations by chemical and physical mutagens . Five kinds of 

chemical mutagens (EMS, NMU, NEU, NDEA, and NDMA) were tested for the induction 

of somatic mutations with special regards to the dose-response 

relationships and minimum effective dose . Three different kinds of doseresponse 

relationships were obtained depending on the variety of chemicals 

. Linear dose-response was observed with EMS treatment . In the case 

of NDEA, the mutation frequency increased sharply at the low dose range, 

but it saturated at the high dose range . In the case of NDMA, there was 

a slow increase at the low dose range and a sharp increase at the high 

dose range . Similar differences in dose-response relationships between 

ethylating agent (NEU) and methylating agent (NMU) were also observed as 



_,_-- - .- 

Notes "ìÀ"'the case bgtw&WINDEA and NDMA . With regard to detectability of minimum 

dose foEr a chemical using Tradescantia stamen hair system, this depends 

upon the kigl'6of chemical and test clones . It may be stated that 

an effective-8ase`for inducing mutations is in the order of picograms of 

chemicals per flower . Therefore, Tradescantia may be possible to use as 

one of the indicators for risk evaluation of chemical mutagens . 


MUTAGENIC EFFECTS OF ENVIRONMENTAL RADON 

Tavera L ., BalcBzar M ., de la Rosa M . E ., 2immering S .* 

ININ . Sierra MoJada N447 . 2 y 3er . pisos . Col . Lomas Barrilaco C .P .11010 .MEXICO 

*Brown University, Providence U .S .A . 

574 

The internal radon progeny exposure exhibits a distinct maximum dose In the respiratory 

tract Inducing lung cancer . 

From epidemiological data available only a statistical significance Increase of cancer 

frequency with Increasing background radiation can be deduced . Because of these data 

are given for different ages and radiation environments a Radon Chamber was desloned to 

exposure bioassays for searching effects other than cancer induced by radon exposure . 

The radon atmosphere in the chamber reach 97% of saturation after 19 days, however it 

is possible to determine an equivalent average radon concentration by means of Solid 

State Track Detectors . The Radon daughters assessment Is also possible with these detectors 

. 

Samples of Drosophila exposured in this Radon Chamber have shown inter-and-Intraspecie 

differences in sensibility when fecundity and viability were measured . Additionally a 

very typical aberrant phenotype was induced in D . melanogaster wild-type . 

575 

GENOTOXICITY ASSESSMENT OF QUINAPRIL, A NEW ANTIHYPERTENSIVE DRUG . J .C . Theiss, N .L . 

Kropko, G . Krishna, and V . Ciaravino, Parke-Davis pharm . Res . Div ., Warner-Lambert Co ., 

Ann Arbor, MI(USA) 

Quinapril was assessed for genotoxicity in a variety of test systems . This drug was 

not mutagenic toward S . typhimurium (10,000 ug/plate highest dose tested) and did not 

increase the mutant or SCE frequency in V79 cells (1400 ug/ml highest concentration 

tested) . Quinapril did not induce micronuclei in mice (1430 mg/kg high dose-80% mouse 

LD50) nor did it induce structural chromosomal aberrations (SCAs) in rats (2130 mg/kg 

high dose-rat LD10) . Thus, quinapril was not clastogenic in vivo at doses far in excess 

of the maximum human dose of 1 mg/kg . There was a slight but statistically significant 

dose-related increase in SCAs in V79 cells treated with quinapril for 3 hours which 

occurred in the presence of S9 only and at 18 hours post-exposure only (12, 18, 24, and 

40 hour time points) . The maximun frequency of cells with SCAs detected (6t) was within 

the historical control range for this laboratory (0-6 .31 cells with SCAs, n-31) and the 

minimum concentration at which an increase in SCAs occurred (1400 ug/ml) was cytotoxic 

(

ackground peak respons ) . SCA frequency was increased by four quinolones, PD 117579 

(10 ug/ml minimal clasto enic concentration, 4 .5-fold background peak response) . PD 

117962 (45 ug/ml minimaclastogenic concentration, 9 .3-fold background peak response), 

ciprofloxacin (400 ug/ml minimal clastogenic concentration, 8 .9-fold background peak 

response), and ofloxacin (1800 ug/ml minimal clastogenic concentration, 6 .3-fold 

background peak response) . Norfloxacin and nalidixic acid were negative for all three 

endpoints . Thus, the SCA test is the moat sensitive of the three endpoints assessed . 

However, literature reports indicate that the two quinolones that increased SCA 

frequency in vitro only at high concentrations (ciprofloxacin, ofloxacin) failed to 

exhibit clastogenic activ-ity-in animals yr man, indicating that these high 

concentration in vitro effects may be of limited biological significance . Thus, an in 

vitro assessment of the clastogenic activity of quinolones should be coupled with an in 

vivo test to assess the biological significance of any in vitro activity . 

577 

Measurement of Mutational Spectra in Human Tissues 

William G . Thilly, Phouthone Keohavong, Neal Cariello, John Hanekamp 

Center for Environmental Health Sciences 

MIT E18-666 Cambridge, MA 02139 

Application of mutational spectra to diagnose the causes of genetic change in humans 

will be discussed . Recent advances in technology, including PCR improvements, 

designed to obtain such spectra from human cells and tissues will be reviewed . 

Topics to be covered are signal/noise requirements, multi-copy sequences and means 

to obtain spontaneous spectra from individuals . 

Sponsored by U .S . Department of Energy Office of Health and Environmental Research 

and the U .S . National Institute of Environmental Health Sciences. • 

578 

LACK OE' CYTOGENETLC EFE'E:CTS IN BONE MARROW OF TWO STRAINS OF MICE CHRONICALLY EXPOSfSD 

TO CLGARETTE SMOKE . M .A . Thomas*, D . Gulati**, J .P . Wojciechowski**, P . Sabharwal**, 

and C .G . Gairola* . *Graduate Center for Toxicology, and Tobacco and Health Research 

Institute, University of Kentucky, and **Rnvironmental Health Research and Testing, 

Inc ., Lexington, KY . 

This study was conducted to determine if chronic exposure to cigarette smoke induces 

cytogenetic damage in the bone marrow of aryl hydrocarbon hydroxylase- (AHH) inducible 

(C57BL/6J) and non-inducible (DBA) strains of mice . Using a nose only exposure system 

groups of female mice were chronically exposed (50 - 70 weeks) twice daily to mainstream 

(ttS) or sidestream (SS) cigarette smoke, from high-tar/high-nieotine University 

of Kentucky Reference cigarettes (2R1) . Room (RC) and sham control (SH) groups were 

also examined . Pulmonary AHH activity was increased 2-3 fold in C57BL but not DBA 

mice . Blood carboxyhemoglobin (%COHb) levels were increased approximately 11 and 22 

fold for MS and SS groups, respectively, in both strains . Average intake values for 

smoke total particulate matter were 17 .1 and 6 .9 mg/kg per exposure-session, for MSand 

SS- exposed groups, respectively . These dats confirmed effective inhalation of 

smoke by exposed animals . Two cytogenetlc endpoints, sister-chromatid exchange (8Cg) 

and micronucleus (MN) formation, were examined in isolated bone marrow cells . tlnder 

these exposure conditions neither MS nor SS cigarette smoke induced an increase in 

frequency of SCE or MN in either strain of mice . (Supported by KTRB 41031) . 

579 

GENETIC ANALYSIS OF HUMAN DNA REPAIR GENES, L. H . Thompson, C . A . Weber, 

K .W. Brookman, N .J . Jones, E .P . Salazar, and M .J. Sicilianot, Biomedical Sciences Division, Lawrence 

Livermore National Laboratory, P.O. Box 5507, Livermore, CA 94550 and tDepartment of Molecular 

Genetics, University of Texas System Cancer Center, Houston TX 77030 

Human genes that control DNA repair were identified by complementing repau-deficient hamster mutant 

lines with human chromasomes in hybrid cells or by transfecting with human DNA . Nucleodde excision 

repair (NER) genes, which control UV radiation sensidviry, are located on human chromasomes 2,13,16, 

and 19 . A total of eight complementadon groups of NER genes are now identified among rodent mutants . 

The human ERCC2 (Excision Repair Crass Complementing) gene, which corrects CHO mutant WS and 

was previously cloned in cosmids, was used to isolate homologous cDNA ciones from Okayama's pcD2 

expression library. Nucleotide sequencing of an incomplete cDNA clone and ERCC2 S' genomic 

sequences identified an open reading frame . The protein coding region of ERCC2 has a atrildng 5296 a a . 

identity with the RAD3 gene in yeast This similanty suggests conservadon of function and the possibiliry 


A 


Notes


Notes . ,,that.ERCC2 is requiiedAsreeU viability as well as repair, as in the case of RAD3 . The gross underrepresentation 

of CHO mutants recovered in the ERCCZ complementation group upon treatment with the 

frame shift agent ICR170rther suggests an essential function. Two other human genes, XRCCJ (X-ray 

Repair Cross Compl XRCC2, which help repair Ionizing radiation damage, were assigned to 

chromosomes 19 and 7, respectively . Analysis of cosmid clones of XRCCJ shows that this gene, which is 

33 kb in length efficiently corrects CHO mutant EM9 . The phenotype of EM9 is defective rejoinin~ of 

strand breaks and greatly elevated sister chromatid exchange (SCE) . The cDNA clone pXRI-30, obtauted 

from the pcD2 library, gave stable but incomplete correction of EM9 . Nucleodde sequencing showed that 

this clone lacked 26 nucleotides of protein coding region. The XRCCI protein, which appears to be 633 

a.a. in length, remains to be identified in terms of itsbiochemicai function in repair and SCE . This work 

was done under the auspices of the U.S . Dept. of Energy by 1.LN1, under contract No . W-7405-ENO-48 . 


580 

MOLECULAR APPROACHES TO CARCINOGENESIS : ONCOGENE INDUCED TRANSFORMATION AND LINEAGE 

SWITCHING OF RAT LIVER EPITHELIAL CELLS . Snorri S . Thorgeirsson, Laboratory of 

Experimental Carcinogenesis, Division of Cancer Etiology, National Cancer Institute, 

National Institutes of Health, Bethesda, MD 20892 

Tumors produced by a chemically transformed rat liver epithelial (RLE) cell line 

and its single cell-derived clonal subpopulations demonstrate wide-ranging morphological 

presentations including carcinomas, sarcomas, "mixed epithelial-mesenchymal" 

tumors, and undifferentiated tumors (Am . J . Pathol . 127 : 168-181, 1987) . To address 

the question of heterogeneity of tumors er ve rom transformed RLE cells, we have 

used recombinant retroviruses containing the following transforming oncogenes : 

v-raf (3611-MSV), v-raf/v-myc (J2), v- c(J5), and v-Ha-ras (pRNR16) . A11 of the 

oncogenes, with the exception of v-styc~J5), were efficient transforming agents in 

the RLE cells . Tumors derived from the v-raf- and, to a lesser extent, those from 

v-Ha-ras-transformed RLE cells showed mixed epithelial-mesenchymal morphology, 

whereas the combination of v-raf/v-myc (J2) consistently produced differentiated 

trabecular carcinomas . These data suggest that the lineage commitment of the RLE 

cells can be perturbed by a single transforming oncogene and that different tumor 

types derived from these cells may reflect the expression of a selective oncogene 

or a combination of oncogenes . 

581 

METABOLISM OF1-NITROPYRENE TO A MUTAGEN IN CAO CELLS BY NITROREDUCTION . Janice R . 

Thornton-Manning, Beverly A . Smith, Roberta A . Mittelstaedt, Frederick A . Beland and 

Robert H . Heflich, University of Utah, Salt Lake City, UT (USA), and National Center 

for Toxicological Research, Jefferson, AR (USA) 

Nitroreduction of 1-nitropyrene (1-NP), a tumorigenic environmental contaminant, to 

N-hydroxy-l-aminopyrene (N-hydroxy-l-AP) is an important pathway for DNA adduct formation 

in vivo . In previous studies, treating Chinese hamster ovary (CHO) cells with 

1-NP for up to 5 hr produced low levels of a DNA adduct indicative of nitroraduction, 

but no mutants were induced . In this study, CHO-KI-SH4 cells and repair-deficient CHO 

UV5 cells were incubated with 40 uM 1-NP for up to 24 hr . While none of the CHO-R1 

treatments induced mutants, treatment of UV5 cslls for 12 and 24 hr induced 5 and 12 

mutants/106 clonable cells (different from control at p

ultraviolet irradiation o'f DT resistance in cultures of the transformed, 

xeroderma pigmentosum complementation group-A cell line XP20S(SV40), and of 

the Chinese hamster ovary ~CHO) repair-defective mutant 43-3B . In both 

cultures, there was a do~e-dependent increase in the frequency of toxinresistant 

cells . On an equal-dose basis, higher frequencies were observed in 

the repair-deficient cell lines than in their respective repair-proficient 

controls, GM637(SV40) and CHO-9 . At equal levels of survival, however, the 

frequencies of DTs cells were similar in the repair- defi~ient and proficient 

cells . These result provide further evidence that the DT" cells detected by 

the autoradiographic assay are indeed mutants . They also indicate a simple 

method of studying various aspects of mutation in cells that, because of e .g . 

defective DNA-repair, are hard to clone and use in a colony assay . (Work 

supported by the Israel Cancer Association .) 

583 

SOFTWARE DEVELOPMENT FOR MICRONUCLEUS (MN) SCORING . R .R . Tice, J .T . MacGre9or*, C . 

Campfield, A . Pellom, L . Williams** and C .H . Nauman**, Integrated Laboratory Systems, 

PO Box 13501, Res . Tri . Park, NC 27709, *Toxicology Consulting Services . Inc ., 383 

Diablo Rd, Suite 100, Danville, CA 94526, and **EPA/EMSL, P0 Box 89193, Las Vegas, NV 

89193 . 

Based on input from a panel of experts in the field, a software program, suitable 

for an IBM compatible PC, has been developed for the collection and analysis of 

micronuclei data obtained from in vtvo test systems . Experimental design information 

and MN data obtained on a number of cell types are easily entered using a series of 

menus, statistically analyzed by a variety of statistical models and presented both 

in tabular and graphic form . In the statistical analysis, the data are evaluated for 

scorer, sex, treatment, sample time, and animal effects . The use of this software 

will help to standardize in vivo MN data analysis and presentation . Although the 

research described in this article has been supported by the U .S . EPA through 

contract number 68-C8-0068 to Integrated Laboratory Systems, it has not been 

subjected to Agency review and therefore does not necessarily reflect the views of 

the Agency and no official endorsement should be inferred . 

. 

584 • 

ANALYSIS OF MOUSE MICRONUCLEI BY HIGH SPEED FLOW CYTOMETRY . 

A. M . Tometsko, Litron Laboratories, Rochester, N. Y . (USA) 

The mouse micronucleus assay is widely used to evaluate the clastogenic activity of 

chemicals . The essay involves scoring the number of cells containing micronuclei (MNS) In 

either blood or bone marrow preparations using fluorescent or bright field microscopy . The 

conventional assay is labor intensive end tedlus, and requires many hours of microscopic 

examination to complete an assay. High speed flow cytometry provides the means for 

analyzing fluorescent cells as they pass through a laser beam at 2,000 cell per second and 

permits the analysis of 30-50 times more cells then manual screening tn e shorter time. For 

FCM analysis, the cells are stained with fluorescent dyes which effectively label cellular 

nucleic acids (e .g. MNs) . Experiments will be presented which highlight the distribution of 

micronucleated cells in peripheral blood of normal and ctasto0en treated mice. The location of 

MNs in different blood cell populations will be shown and will provide en indicetion of the 

feasibility of developing an automated FCM based analysis protocol• The distribution of MNcells 

will be presented using histograms and bivariate graphs. Correlations will be made 

between manual scoring and analysis by high speed flow cytometry . 

585 

COMPARISON OF IN VIVO SOMATIC CELL MUTATION, CHROlie/SOME ABERRATION, SISTER CHROMATID 

EXCHANGE, MICRONUCLEI FORMATION AND URINE MUTAGENICITY IN STEEL FOUNDRY WORKERS . D .J . 

Tomkins, D .R . McCalla, and E .S . Gibson, McMaster University and Dofasco Inc .,Hamilton, 

Ontario, Canada . 

Preliminary results of genetic toxicologic monitoring of 125 steel foundry workers 

with a higher rate of lung cancer have been reported (Environ . Mutag . 7(S .3) : 33,1985) . 

Chromosome aberration (CA), sister chromatid exchange (SCE), and morning and afternoon 

urine mutagenicity (MUTAM,MUTPM), but not micronuclei (HN), were all significantly 

elevated with smoking (p- .001, .004, .008, .005 respectively), but not with occupation . 

The somatic cell mutation test (SCMT) has now been completed for 37 workers and the 

statistical analysis did not show any significant effects of smoking or occupation . 

In the case of CA, there was a significant interaction between smoking and occupation, 

particularly when gaps were not included in the total number of aberrations per cell 

(p- .01) . When specific types of abnormalities were analyzed separately, breaks 

appeared to be the moat important contributors to the effect of the interaction . Two 



Notes


Notes important conf.unieta -were identified : age was positively correlated with CA, !IN and 

. . ° .-aelayed/proloapediEWl'division in 48 hr culturas, and drug exposure significantly 

affected IiUTPWand CA . The different genetic tests were often correlated with each 

other . In partic . ,HUTAH was positively correlated with CA (p- .02), MUT'PN with CA, 

SCE, and SCH1 , .04, .01 respectively) . On the other hand, SCE and SCMf may 

have been negatively correlated (p- .05), and there was no correlation between CA, SCE 

and ALN . This suggests that urine mutagenicity may measure the metabolic products of 

a genotoxic exposure, but that the types of genetic damage detected by the lymphocyte 

tests may vary from subject to subject . 


MAMMALIAN DNA LIGASE I : THE MOLECULAR DEFECT IN BLOOI,fS SYNDROME 

Alan E . Tomidnson, Dena D. Lasko, Ams E . WNia and Tarnas Lirqalt 

rnperml Caneer Research Fund, Clare Hall Laborabries, South t,6mms, Herts ., EN6 SLD, U.K. 

Mammalian cells contain two clistinct DNA i0ases. The major aclivity In proUteratkp oeNS and therefore prohabfy the enzyme 

involved in DNA replioation Is the NOh mdealar weipAt enzyme. DNA Npue I. A 1dOkd brm of frs enzyme has been purilkd b 

~ ope~ ~~ thymus and tunan oeos . Theae en:ymes tww been characwised and poytbrol antlbod~e ses talaed apakrot 

DNA Ipases from the bede:bphapea T4 and T7, yeasts.J.wevelas andand vaccM4a vkus tonlain conserved 

repions of amrw add sequence . Poydonai anbbodes have a t sidue peptlde representing one athese 

conserved raqons and these anobot

mutagenesis by DMN increasaaLwith S-9 level . 3-MCA muta9enesis reflected a 2AAF-like kinetic at low 

exposure, but gave results reminiscent of DMN at higher exposure levels . Mutation frequencies (X10s) 

varied as follows : 

~1 u~I/mIS-9 ~uI/m IS•9 

DMN (400 ug/ma) - 2i~ iT~ 

2AAF (40 uy7ml) S07 26S 

3MCA (10 ul/ml) 280 358 

(0 .625 ut/ml) 144 61 

Control (Average) 51 .54 

The data presented here support ou r choice of 20-40 ul S-9/ml for a 4 hr . treatment of L5178Vi cells in this 

assay. However, the response of 2 AAF and the biphasic response of 3•MCA is an indication of the care 

needed in choosing the optimum en zyme (S-9) concentration for routine or mechanistic investigations . 

589 

MUTAGENICITY AND COMUTAGENICITY STUDIES ON THREE NOOTROPII+St 

PIRACETAM, ANIRACETAM AND HUPERZIIQ A . Z .H . Tu, Y .Y . Wang and W .D . 

Tang . Institute of Materia Medica, Academia Sinica,Shanghai, China . 

The mutagenicity and comutagenicity of 3 nootropils, piracetam, 

aniracetam and hupersin A, were determined at vrious concentration 

using S t himurium as the tester stains . The plate-incorporation 

essay wea con uc e both in the absence and presence of an aroolorinduced 

rat-liver S9 mix . A 30-min preinoubation teat protocol waa 

used for experiments with Sq . None of them is mutagenic towara theae 

tester stains without or with S9 . All of three drugs did not increased 

the revertants incueed by p-nitroquinoline in TA98 and by MINS 

in TA100. Aniracetam at 2 .5 mg/plate decreased the mutagen-indueed 

revertans in TA98, it might be of the toxicity of aniraoetam . 

Aniracetam at bigher concentration exhibited an inhibition effect 

on S t himurium . The micronuoleus teata of 3 nootropile were 

con3uc~3n 3ux mice at the dosage from 1/8 to 1/2 LD50 . All of 

them did not effect on the frequency of micronucleated polychromatic 

erythrocytes . 

590 

KINETOCHORES IN MICRONUCLEI: DEVELOPMENT OF A.SIMPLE AND RAPID METHOD TO 

IDENTIFY EXPOSURE TO ANEUPLOIDY-INDUCING AGENTS . J .D. Tucker and D.A . Eastmond . 

Biomedical Sciences Division, Lawrence Livermore National Laboratory, Livermore, CA 94550 . 

The identification of agents causing aneuploidy in mammalian cells is currently limited due to 

the labor intensive nature of traditional cytogenetic analyses . We have developed a new and simple 

method to identify exposure to aneuploidy-inducing agents (aneuploidogens) . The assay involves the 

induction of micronuclei in cytokinesis-blocked cells and the use of an antibody with a specificity of 

> 99% for kinetochore regions . Micronuclei with one or more ceneromeres are indicat)ve of cells with 

a high potentia) for aneuploidy. Staining of the kinetochores is achleved with the use of a 

fluorescein-conjugated seeond antibody and the nuclei are atained with DAPL With the simultaneous 

use of phase contrast and DAPI excitation, it waa possible to ldentify both the cell membrane and the 

nuclei while scoring . Cella with mlcronuclei were txamined for the presence of fluorescein label to 

determine whether the m)cronuclens contained a kinetochore. Every agent tested produced a doserelated 

Increase in the frequency of micronucleattd cells . In human peripheral lymphocytes, the 

micronucleated cella indueed by the aneuplo)dogens eolehicine, vlncrlatine sulfate and 

diethylstilbestrol contafned kinetochore-positive m)cronuclef 92%, 87%, and 76% of the time, 

respectively. In contrast, the micronucleated celis induced by the potent clastogens Ionizing radiation 

and sodium arsenite contained kinetochore-positive micronucfe) only 3% and 19% of thq time, 

respectively. In Chinese hamster ovary cells, the micronncleated cells induced by the aneuploidogena 

benomyl and vinblastine sulfate contained kinetochore-positive miuonuclti 92% and 94% o£ the 

time, respectively . With methyl methanesulfonate, however, only 11% contained a kinetochore . 

These results indicate that this simple and ra id procedure can discriminate between aneuploidogens 

and clastogens, and may allow mort rap)d identillcation of exposure to aneuploidogens both in vitro 

and in vivo. Work performed under the auspices of the U.S . DOE by Lawrence Livermore National 

Laboratory under contract No . 7405-ENG-48, with additional support from the Alexander Hollaender 

Distinguished Postdoctoral Fellowship (D .A.E .) . 

591 

SMOKE EXPOSURE, AGE, SEX, RACE, AND POTENTIATION AS VARIABLES AFFECTING SISTER 

CHROMATID EXCHANGE INDUCTION IN NUMANS . D .A . Tulis, J .K . Smollinger, and W .H . 

McKenzie, North Carolina State University, Raleigh, NC (USA) . 

In vitro cytogenetic analysis was performed on peripheral lymphocytes of 49 

passively smoking children, ages 6 mo to 5 yrs . Mean SCE for non-smokers (7 .60 i 0 .46) 

was not significantly different (p > 0 .746) from mean SCE for passive smokers (7 .85 3 

0 .39) . However, passively smoking children demonstrated a highly significant 

(p < 0 .001) SCE increase to in vitro a-naphthoflavone (ANF) exposure, while the nonsmoking 

children showed a much lower but etill significant SCE increase to in vitro 

ANF exposure (p < 0 .03) . These results suggest that ANF has the potential to magnify 

and therefore detect an SCE insult experienced by passively smoking children . Age, 



Notes 

r


Notes , 


_~.~t-- - .- 

r sex, and race~nf3uences were also investigated for their effects on SCE . A 

significant (p < 0 .001) SCE age effect was observed, with SCE frequency increasing 

with age .- A a?l,gniftsnt (p < 0 .008) SCE sex effect was also observed, with average 

female SCE (8 .76 t 0 .26) higher than the average male SCE (7 .85 t 0 .23) . However, no 

significant (p > 0 .94) SCE race effect was detected, although the SCE race * smoking 

interaction was significant (p < 0 .01) . Urinary cotinine was determined to be highly 

correlated (r - 0 .70) with the number of cigarettes smoked by the children's parents, 

and was used as an estimator of the actual amount of smoke inhaled by the children . 

This continuing study is presently investigating the role of ANF as a potentiator of 

SCE induction in non-smoking, passively smoking, and actively smoking young adults, 

and is also using cotinine as an indicator of cumulative smoke exposure for each 

subject . 

592 

ACETYLATION AND ACTIVATION OF 2-AMINO-3,8-DRAUCHYLIIIMAZO[4,5-f]QU1NOXALINE 

(MeIQx) TO DNA REACTIVE SPECIES IN SALMONELLA BACTERIA . K . W . Turteltaub, B. E. 

Watkins, and J. S. Felton, Biomedical Sciences Division, Lawrence Livermore National Laboratory, P .O. 

Box 5507, Livermore, CA 94550 . 

The aminoimidoazaarenes (AIAs), a group of heterocyclic amines found in cooked meat, require 

metabolic activation to express genotoxicity in a number of in vitro assays, including the Arnes/SalmoneUa 

test. The AlAs are mutagenic to Salmonella strains TA98 and TA98NR (nitroreductase deficient) ; but not 

to TA98/1,8DNP6 (acetyltransferase deficient), suggesting a requirement for the O-acetyltransferase . 

Asan et al. (Carcino enesis, 1987, 8 :1589) have found DNA adducts In Salmonella strains exposed to 

the quinoline AIA, IQ . We examined DNA adducts generated in Salmonella following exposure to 2amino-3,8-dimethylimidazo[4,5-f)quinoxaline 

(MeIQx) by 32P-posalabeling to access the role of bacterial 

acetylation in the activation of one of the quinoxaline AlAs. DNA was isolated from Salmonella TA98, 

TA98NR, and TA98/1,8DNP6 foUowing exposure to MeIQx, in the presence of Aroclor-induced mouseliver 

microsomes, for 4 hr . Four adducts were found in Salmonella TA98 and TA98NR . The adducts 

formed in these strains were judged to be idendcal by eoo-chromatography . No significant difference was 

found in adduct frequencies between these two strains . The same 4 adducts were also detected in 

Salmonella strain TA98/1,gDNP6 but at significantly lower levels . Synthetic azido-MelQx was reacted 

with calf thymus DNA and produced identical adduct profiles to Salmonella DNA as did mice given 

MeIQx . These data support the belief that formation of an N-acetoxy AIA intermediate is required to 

generated a DNA-reactive species. These data also support the hypo thesis that a single pathway, 

independent of species, is responsible for the generation of an ekctrophilic (DNA-reacdve) AIA 

(Work preformed under the auspices of the U .S . DOE by the LLNL under contract No. W-7405-F~8 

and supported by EAG NIEHS 222Y01-ES-10063). 

593 

FSTABL•ISHED OR PRIMARY CELL LINES FOR CYTOGEHETIC TESTS? 

DJ Tweats and DG Gatehouse, Glaxo Group Research Ltd ., Ware, Herts, England . 

When choosing on a cell line for cytogenetic tssts, investigators take into 

account the degree of validation ; chromosome stability (in terms of amoku of 

chromosomes) ; low spontaneous aberration frequencyl donor variation for lymphocyta 

cultures etc . One parameter that is not often taken into account is that astablishei 

cell lines e .g . Chinese hamster V79, CH0 etc contain comprehensively re-arrangul 

karyotypes involving deletions, additions, inversions, translocations etc. It 

appears that many of these changes occurred at the time these cells became 

established, but it is clear that sublinea are progressively changing and diverging . 

In cells with such rearrangements genes concerned with DNA repair, cell division 

regulation, chromosome stability etc may well function abnormally due to 'position 

effects' or gene dosage effects, as for the activation of oncogenes . Such changes 

may influence the response of a particular subline to clastogens . There is a paucity 

of comparative tests on clastogens in different cell lines, or sublines . However 

discordant results have been obtained both due to differences in protocol and to 

inherent differences in the cells themselves . Primary cells e .g . human lymphocytes 

do not have the problem of tearranged and diverging karyotype . However, these cell 

are terminally differentiated and have to be induced to divide in culture, also donor 

variation can result in at least quantitative variations in response . However, ou 

oalance, the case for using primary lines appears to be stronger than the case fo• 

established lines . This poster will review the karyotypic changes that have occurred 

in the CHO and V79 sublines since their isolation and the results of testing with the 

same chemicals with both types of line . 

594 

USE Of INTERPHASE ANALYSYS TO DETECT CHEMICALLY INDUCED ANEUPLOIDY IN CULTURED CELLS . 

Vagnarelli P . . De Sario A . . Raismndi E.. Scariolo S. and De Csrli L . 

Dipartimento di Genetica e Microbiologia. Universita di Pavia . via S.Epitaaio 14 . Pavia . Italy. 

Ia rilu hybridization with cnromosome-specda DNA probes has Wen exploited for developmq an is ritro assay for 

chemically inducea aneuploidy . The following probes have been tested : T*7 which idenGfies a 100 fold repeated fraqment in 

50869 3718

'ne oencentromer c region of the Tchromosome : 011123. complementarv to a DNA sequence repeated 50 times in thee 

penrentromenc region of chromosome 9 : pNlt}it, honwiogous to the N-Myc oncogene and specific for chromosome 1 in the 

Neuroblastoma cell line LAN-1, wheWthe oncogene is amplified 50 times . /e situ hybridization experiments have been 

cerrred out on human lymphocytes with Y97 and Op23 and on Neuroblastoma cell hnes with oNb19-21 . Preliminary 

exper ments, performed on untreated cultures, to test the efficiency of the probes . showed an exceedingly high proportion of 

hvpodiplo,d nuclei due to technical artifacts . Therefore hyperdiploiov has been taken as a reliable index of induced sneuploidv . 

In order to check the validity of the assay we analyzed four known aneuploidy inducers, iumely fknomyl (8E), 6rieeofulvln 

!nF 1 . Chloral hydrate (CH), Diethvlstiibestroi (DES) and a putative carcinogenic agent NitrilTnacebcAcid INTAI . A significant 

increase in the percentage of hyperdiploid nuclei has been found with 8E . 6F, CH and DES ; a dose-related effect has been 

revealed w,ti` CH and DES . NTA did not show any effect in the induction of aneuplmdv . To improve the efficiency of the method 

we have compared the conventional aRO'f ddiographlc proCeDUre with that based on immunofluorescence . To this purpose 

parallel experiments heve been carried out with radioactive and biotmvlated probes using DES as a test compound . The nonrIclioaCtlve 

technique turned Out to be more sensitive and therefore more suitable for screening new compounds . The positive 

response obtained with agents that induce chromosome number variation by different mechanisms of action indicates that the 

rriethod we have devroloped may be of general application for testing aneuploidy Inducers is riua Moreover, the possibility of 

scoring large cell samples makes interphase analysis suitable for tne cytogenetic monitoring of exposed populations . 

595 

APPRAISAL OF GENOTOXIC EFFECTS OF AGROCHEMICALS IN HIGHER PLANTS USING IN VIVO AND 

IN VITRO END POINTS . 

K .Vaidyanath and T .Suryakumari, Department of Genetics, Osmania University, 

Hyderabad - 500 007 (A .P .) India . 

The use of different agrochemicals to control diseases, pests and weeds is 

an essential component of the modern agricultural technology . Though economic advantages 

of such practice is appreciated, it is belived that exposure to agrochemicals 

has many genetic consequences . To have a realistic appraisal of genotoxicity, three 

commonly used Chemicals viz ., Ethylene dibromide, Phenyl mercury acetate and Ekalux, 

have been studied using multiple end points like, somatic chromosomal aberrations, 

heritable germinal mutations at specific and non-specific locus, and in vitro growth 

of callus cultures . Significant frequency of chromosomal aberrations, -c-h1orophyll 

deficient seedlings in M. generation, specific locus mutations at waxy locus 

(Wx - wx), pollen sterility, polygenic variability in H3 generation and iphibition 

of callus growth have been observed . The overall results of the study suggests that 

any one or combination of genetic end points could be employed for the effective 

screening of environmental mutagens . 

596 

ACRYLAMIDE-1NDUCED CHROMOSONE-T1iPE ABBRRAT'IONS IN SPERMiOGENIC STAGES EVALUATED 

IN THE FIRST CLEAVAGE ME'1'AYHASES IN '1'Hh MOUSI : . N .Y . Vald>lvia, N .M . Lafuente and 

M . Katoh, Fac . Odontoloqia, U . de Chile, bTGO (CHILIi) and Hatano Research Instztute, 

F .D .S .C ., Kanagawa (JAPAN) . 

Because of the evidences reported by Seqa, et al . (19`" Annual Meeting BMS) that acr7laaide (AA) 

binding to protasine of speraioqenic stages in the souse appears to be correlated with the pattern 

of genetic dasage oroduced by this cneaical in late speraatids and early speraatoza stages (Shelby, 

et a1 ., 1986) cytogenatic evaluation of these sase sensitive stages was perforaed in sarlir cleavage 

of souse eabryos . Adult sale M sice were intraperitoneally (i .p.) injected with 150 sq AA/kq and 

aated to untreated fesales at intervals ranging fros 6 to 9 and 10 to 13 days after tratsent . The 

plugged fesales sere i .p . injected with colchicine and the fert:lized ova were collected to the first 

cleavage aitosis, at sich tiae the male chroaosou cosplesent su analyzed for strsctaral ohrosososal 

da.age . The chroaosose-t)

206 1989 EMS Abstracts . r -- - .- 

Notes ,~ ~ ~ g~ .•~ . .. 


of~-the adducts:Z in target tissues is ecarse . We visualized the DNA adducts Osmethylguanine 

and 7- ethylguanine in tissue sections of rats and hamsters six hours 

after treatment-asic3ff= .ag (i .v .) resp . 100 mg (s .c .) NNK/kg . Adducts were observed 

in all target tissues for tumor formation except rat liver and pancreas . Nuclear 

staining was strongest in the nasal cavity, particularly in cells of Bowman glands . 

Staining was weaker in sustentacular and basal cells of the olfactory epithelium, 

the respiratory epithelium and serous glands . DNA adducts were also present in the 

trachea (epithelial and glandular cells) and in the bronchiolar lining of the lung 

(Clara cells) . Accumulation of adducts was observed after twice weekly applications 

of NNK for 8 weeks (rats : 3 .5 mg/kg, i .v . ; hamsters : 10 mg/kg, s .c ., each) in Bowman 

glands, serous glands and respiratary epithelium of the nasal cavity and in Clarti" 

cells . These adducts disappeared within 4 weeks after the cessation of NNK 

treatment . The immunocytochemical approach permits to study the distributiun, 

accumulation, persistence and repair of DNA adducts at the level of the single cell . 

One of the potential applications is the detection of adducts in human tissues, e .g . 

in buccal or bronchiolar cells of tobacco users . 

METABOLISM OF DIETARY OENOTOXIN9 BY THE RUMAN COLONIC MICROFLORA R .L . Van Tassellt, 

D .G .I Kingeton2 and T .D . Yilkinst . Departments of Anaerobic Miorobiologyt and 

Chemistry2 . Virginia Polytechnic Institute and State University Blacksburg, VA . 24061 

598 

The microflora of the human colon is a complex ecosystem of anaerobic bacteria 

which have the capability of enaymatically transforming a variety of dietary (or 

biliary) compounds to genotoxic metabolites . In the past, most investigators studying 

the interplay between diet and colonic flora and its role in the etiology of cancers 

focused on the reductive and glycosidie potential of the bacterial enzymes - many of 

which reverse the oxidative and conjugative reactions performed by the liver . Recent 

work in our laboratory has focused on the metabolism of two relatively new classes of 

genotoxine, the heterocyclic amines (pyrolysis carcinogens) and the fecapentaenes . 

The heterocyclic amines, which originate from fried or broiled proteinaoeous foods, 

normally require activation by the liver before being potent mutagens or carcinogens . 

However, the "IQ" subclass (IQ . MeIQ and MeIQx) can be activated in the colon by 

Eubacterium and Clostridium spp . to a 7-hydroxy form which is directly mutagenic and 

thus may react directly with the adjaoent oolonic epithelium . The fecapentaenes 

(conjugated ether lipids) are produced in the colon by 8aoteroides epp . from 

polyunsaturated ether phospholipids (plasmalogens) whose origin is unknown . The 

fecapentaenes are potent direct-aoting genotoxins that are detected in the feces of 

over 75% of individuals on normal western diets . Although there is no direct evidence 

that the 7-hydroxy "IQ" compounds or the fecapentaenes influence risk for colon 

cancer, the potency and prevalence of these bacterial metabolites is cause for concern 

- they may be carcinogens or somehow affect risk levels for cancers, particularly 

colo-rectal cancer, by some other means . 

599 

INFLUENCE OF DNA EXCISION REPAIR ON UV-INDUCED MUTATION SPECTRA IN CHINESE 

HAMSTER CELLS. H . Vricfing, M .L. van Rooijen . J . Venema, M .Z. Zdaenicka, J.W.IM. Simons, , L.H .F. 

Mullenders, P .H .M . Lobman and AA. van Zeeland. Department of Radiation Genetics and Chemical Mutagenesis, 

State University of Leiden, P .O . Box 9503, 2300 RA Lciden, The Netherlands . 

The molecular nature of mutations induced by UV light was investigated in Aprt mutants from V79 Chinese 

hamster cells isolated after exposure to a high (12 J/ms) or a low (2 J/mY) dose . The nature of point mutations in 

hpn exon sequences was determined by sequence analysis of in virro amplified Apx CDNA (PCR method) . Among 

the mutants analyzed all possible classes of base pair changes were present, 70% being transversions . Since all 

mutations except one did occur at dipyrimidine sites, the assumption was made that they were caused by UVinduced 

photoproducts at these sites . At the low dose g0% of the mutations were caused by photoproducts in nontranscribed 

strand of the hpit gene . This was 64% at the high dose . We propose that the strand bias for mutation 

induction towards the non-transcribed strand is caused by preferential repair of photoproducts from the transcribed 

strand of the Apn gene . Removal of pyrimidino dimers from an ig kb EooRI fisgment of the hprt gene was 

determined after digestion with T4 endonuclease V and analysis on alkaline agarose gels. Pyrimidine dimer removal 

in the Aprt gene was 80% in 24 hours whereas this was 18% in the genome overall . UV-induced mutations were 

also analyzed in a repair deficient cell line V-Hi, which is a UV sensitive derivative of V79 . UV-induced mutation 

induction in V-Hl was 7 times higher per unit dase than in normal V79 cells. All UV-iaduced (2 J/ms) single base 

pair changes in V-Hl were GC to AT transitions . Furthermore, 90% of the base pair changes were caused by 

photoproducta in the transcribed strand of the Irpr gese . Removal of pyrimidine dimers from the hpt gene is 

completely deficient in V-HI cells. We propose that most UV-induced mutations in V-Hi are caused by pyria8dine 

duners and that in normal V79 cells a relatively large part of the mutations are caused by 6-4 photoproducts . The 

extreme strand specificity of mutation induction in V-Hl might be due to differences in fidelity of DNA replication 

of the leading and the lagging strand .

600 

MONOCLONAL ANTIBOSY IMMUNOASSAYS FOR COOKING-INDUCED MEAT 

MUTAGENS . 

Martin Vanderlaan, Bruce Watkiss ; Mona Hwang, Mark Knize and James Felton. Biomedical 

Sciences Division, Lawrence Livermore National Laboratory, Livermore, CA 94550 

Fifteen monoclonal antibodies have been produced that recognize five ne (AIA) 

mutagens formed in cooked meats (IQ, MeIQ, MeIQx, DiMeIQx, and Fhlp). These antibodies are 

being used to quatttifiy individual AIAs in cooked meats, to identify inunutachemically crosrteac6ve 

new AIAs, and to quan c'fy AIAs and their metabolites in human urine . Extracts of well-done cooked 

beef were separated on HPLC and competition enzyme linked ittununoaorbent assays (ELISAs) were 

conducted on each HPLC fraction using the panel of-antibodies . The plot of intmunochemical activity 

vs . HPLC retention time forms an "immunogram," similar to the mone familiar "mutagnm ' plot of 

mutagenic activity vs . retention time. Each antibody shows a unique immunogram . Peaks in 

inununochemica) activity can be associated with known AIA tnutagani, and withpteviously 

unidentified chemicals which immunochemically cross-neact with the antibodies . These findings 

suggest that the known AlAs are part of a larger family of immunochemically similar compounds 

produced by cooking . Immuno-affinity chromatography is being used to isoLte and identify these 

new AIA-like compounds . The antibodies provide a structure-based means of identifying new AIAs 

that complements the previously used bacterial mutageatc activity-based methods . Analysis of urine 

from people on vegetarian and well-done beef diets s6ows both mutagenic and intmunochemicallypositive 

material is associated with the diet high in AIAs . 

Research supported by NCIgrants ROl CA40811-03 and CA48446-02 and performed at LLNL under 

contract W-7405-ENG-48 with the Department of Energy . 

601 

MUTAGENICITY OF DRINKING WATERS IN FINLAND . T . Vartiainen, National 

Public Health Institute, Dept . Environm . Hyg . Tox . P .O .Box 95, 70701 

Kuopio, Finland 

Mutagenic activities of drinking and raw waters from the major 

waterworks, covering about 60% of all tap water distributed in Finland, 

were tested with the Ames Salmonella tvphimurium assay using tester 

strains TA100, TA98, and TA97 . The highest yield of organic mytagenic 

impurities from these waters was obtained from acidic water samples by 

continuous extraction with diethyl ether or by XAD adsorption . Mutagenic 

activity of drinking water was dependent on the amount of organic 

matter (and ammonia) present in water and thrA quality and quantity of 

disinfectant used ; mutagenic activity could be approximated by an equation 

of the type A(1-e-kO), where A and k are constants and c is a parameter 

that depends on total organic carbon, the chlorine dose, and the 

ammonia concentration . The main mutagens in humus-rich chlorinated 

drinking water were acidic polar compounds detectable without metabolic 

activation . The highly mutagenic compound 3-chloro-4-(dichloromethyl)- 

5-hydroxy-2(5H)-furanone (MX) and its geometric isomer E-2-chloro-3- 

(dichloromethyl)-4-oxo-butenoic acid (E-MX) were detected in all extracts 

that exhibited mutagenicity . The concentrations of MX in the mutagenic 

drinking water concentrates ranged from 5 to 67 ng/l and this 

compound was responsible for 15 - 57% (average 33%) of the observed mutagenicity 

. Linear correlations were observed between mutagenic activity 

in TA100 and concentrations of MX and E-MX . 

602 

ENHANCEMENT OF BLEOMYCIN TOXICITY AND MUTATION AT THE APRT LOCUS IN CHO CELLS 

M .L . Veigl, J . Bhatt and W .D . Sedwick, Dept . of Medicine, Case Western Reserve 

Univ . Cleveland, OH 44106 

In drug combinations causing greater than 95% toxicity for CHO cells, the 

calmodulin antagonist, N-(4-aminobutyl)-5-chloro-2-naphthalene sulfonamide 

(w13), is synergistic with bleomycin in clonogenic assays as analyzed by the 

method of Chou and Talalay (Adv . Enz . Reg ., 22, 27-55, 1984) . At concentrations 

causing less toxicity, however, this drug combination interacts in an 

antagonistic manner . It has been suggested that calmodulin antagonists may 

interfere with DNA repair processes responding to bleomycin-induced DNA damage 

. In these experiments, the effect of W13 on mutation caused by bleomycin 

was investigated by analyzing the impact of this drug combination on 

mutation in the adenine phosphoribosyl transferase gene (aprt) in cells that 



Notes 

l11 

m i= 

00 

Ya 

~A) 

J 

N 

I+ 

,, 

I 

i 

I



NOteS- 'were either hemfa`ygoSs or heterozygous at this gene locus . The calmodulin 

antagonist, W13, at concentrations between 20 and 40 uM increased mutation in 

the aprt gene of_tIffig,Epzygous CHO cell line, D423, several fold over that 

observed with bleomycin alone . The analysis of this drug interaction and preliminary 

characterization of the specificity of these mutants will be 

presented . 

603 - 

DNA REPAIR IN HIGHER PLANTS . J .Velemfnskjr, Institute of Experimental Botany, 

Czechoslovak Academy of Sciences, Vltavskd 17, 150 00 Praha 5,Czechoslovakia 

. 

Since 1972 several repair pathways analogous to that known in other 

pro- and eukaryotic models have been described also in organs and cells 

of a few mono- and dicotyledonous plant species . They include an excision 

DNA repair after the action of alkylating agents and UV light, repair of 

ionizing radiation-induced DNA single strand breaks and bleomycin-induced 

double strand breaks as well as recovery of short daughter DNA strands 

synthesized on templates damaged by alkylating agents . Several DNA-glycosylases, 

UV- and AP-endonucleases have been isolated and the role of DNApolymerases 

and DNA-ligases in the repair processes in plants has been 

investigated . Special topics in plants rpresent the repair of spontaneous 

DNA lesions accumulated in seeds during their ageing, repair capacity of 

pollen grains and chloroplasts and the clastogenic adaptation - an analogy 

of adaptive response at the chromosome level . At the absence of repair 

deficient mutants in plants, metabolic inhibitors and artificial conditions 

like storage of mutagen-treated seeds help to elucidate the role 

of repair pathways in the mutagenic efficiency . A new approach in determining 

the involvement of individual DNA lesions in the mutagenic processes 

in plants represents the transfer of an E .coli repair gene ada into 

tobacco that enhances resistance of Jobacco oe s against the a-cTion of 

alkylating agents interacting with 0 in DNA guanine . 

THE ROLE OF TRANSCRIPTION IN UV-INDUCED EXCISION REPAIR 

J . Venema, L .H .F . Mullendere, A . van Hoffen and A .A . van Zealand . Department of 

Radiation Genetics and Chemical Mutagenesis . State University of Leiden, The 

Netherlands . 

Preferential repair of active genes following exposure to UV-light (254 nm) is now 

a well established feature of mamaulian cells . We have shown that in normal human 

fibroblasts the transcriptionally active adenosine d .aminase (ADA) gene is faster and 

more efficiently repaired than the genoma overall . The inactive 754 locus is repaired 

to the saao extent as the genome overall . 

We have extended these studies by assessing the role of transcription in excision 

repair . First, we have analyzed repair in the ADA gene with strand specific probes . We 

show that in a fragment comprising the 3' end of the gene there is no difference in 

repair of both strands . A second approach was made by using a cell strain derived from 

a patient with severe combined immunodeficiency (SCID) which is homozygous for a 

deletion in the promoter region of the ADA gene . In primary fibroblasts no transcription 

of the ADA gene is detectable by Northern blotting' . Our results show that repair in 

two adjacent fragments of the ADA gane is similar to that in normal human cells . We are 

currently investigating strand specificity of repair in this cell strain . 

1 . Berkvens, Th .M . et al . (1987), Nucl . Acids Res . 15, 9365-9378 . 

OVERVIEW OF BACTERIAL TE8TIN0 FOR MUTA08NICITY 

S. VenNt, Institute of Csncsr Research: Royal Marsdsn Hosplul, 8udton, Sumy SM2 IfNO, United Kkqdom 

604 

605 

it Is dlHlcuM, N not Imposslble to say anything about bsdsrld gsnotoxdolty tats which has not bsw 

sak( already. The discovery of oncogsns activation by point mutation (a:9., bass-pak substitution) has phrsn 

a naw leass of life to bacterial mulagsnetiol.b. Point mutations are paRbulsrly easy to detact in the 

S.Imonslla/mkxosoms assay, which Is by far the most widely used mutation assay aver dsvbsd . A search on 

ToxNne using -Ames" as the key word resulted In 7,'00 oBatloia In February 1ssY. The Ama test has bssn Ln 

subjected to criticism (both Informed and uninformsd), natimwl and International ragdstlon (blind, or not 0 

bland), with easy, difficult or knpossibls chemksis . lt has nsisbd this global bsttsrkp with remarkable polas, Co 

and Is more or tas unchanged from the form 11 took In the wJd4wvonfts when M Mt emerged . Despite ths O% 

appearance of a vaMty of alternative tests (aE ., nvans•nwlatbn In straba of EsohMdda ood WP2; t0 

fluctuation tes(s ; forward mutation assays; methods, suoh as ths 808 CfKOmotsst, which employ Induction 

of the 808 DNA-prooastrp network as s signal of DNA danrgr dllbrsnllsl taodoKy In npat profbisnt and J 

N 

N

epalr-deflcient bacteria) this assay will continue to form the basis of all screening programmes for potential 

mutagenicity and carcinogenlcaPt . In addition to the role of the Ames test In screening pure chemicals, It Is 

used for InvestlgaUng the mutag7nlclty of complex mixtures, both of chemical and biological otipin . Virtually 

every kind of bodily secretion and excretion has been eub)eoted to scrutiny, the outstanding discovery being 

the detection of mutagenic activity In the urlne of smokers. Despite the vast quantities of bHormatlon on how 

to conduct the S.Imonella/mIcrosome test, purnab and regulatory authorities still ncelw data based on 

inadequate tests, or Inadequate data based on adequate tests, or various combinatlons of these defects . 

606 

STUDY OF TtE ACTION OF AN EXTRACT OF Stryphnodendron obovatim BENTH SEEDS ON DIFFERENT 

BIOLOGICAL SYSTEhS . 

V .E .P .Vicentini-Dias, C .S .Takahashi, Univ .Est .Maringa-PR, F.F .C .L .R .P .-Univ .SP (Brazil) . 

Naturall products of plant origin have been extensively used in human therapeutic . 

Stryphnodendron obovatun BENTH, popularly called 'Barbatim'eo" in Brazil, is used in folk 

medicine against hemorrhage, leucorrhea, diarrhea, hemia and chronic ulcer, but the 

lethal effects in cattle that ingest its pods are acccxtQanied by signs of photosensitization 

and liver damage . In view of these effects, we investigated the action of S . 

obovatun BFMH seeds on three test systems . An aqueous seed extraot at concentrations 

ranging frorn 5 .3 to 23 .8 mg/ml of water drastically inhibited cell division from 16 to 

1% after 24 h of treatment in Alliun cepa root tip cells, with no recovery of division 

after a period of 6 and 24 h in water. Cell spindle formation was also inhibited, leading 

to the appearance of colchicine metaphases (2 .5%) . In bone marrow cells of wistar rats 

treated "in vivo" for 24 h, the extract (1 .1 mg/g body weight) did not induce a statistically 

significant increase in the frequencies of chromqsome aberrations (2 to 4%) . 

In hianan peripheral blood lynphocytes treated in culture (0 .7 µg/ml culture mediun) there 

was no increase in the frequencies of chronasome aberrations or in mean nunber of sister 

chromatid exchanges (about 7 .5 SCEs/cell) . Even though a clastogenic effect was not observed 

in nwnalian cells, treatments with the highest concentrations showed a certain 

cytotoxic effect . 

r 

607 

GENOTOXIC ACTIVITY OF 1,3-BUTADIENE, NITROGEN DIOXIDE AND THEIR PHOTOCHEMICAL REACTION 

PRODUCTS . 

K . Victorina M . Stf,hlberga, L . Buskb H . Cederbergc and J . Ma~nussonc, aInstltute of 

Environmental Medicine, Box 60208, S-104 01 STOCKHOLM . Sweden . Svedlsh Food 

Administration, Box 622, S-751 26 UPPSALA, Sweden . cUniverstty of Stockholm, 

S-106 91 STOCKHOLM, Sweden . 

A small exposure system has been developed for mutagenicity studies of gases . UVirradiated 

mixtures of nitrogen dioxide and alkenes gave rise to mutagenic effects in 

Salmonella, strain TA100, in the order 1,3-butadtene > propene > ethene . A directacting 

mutagenic effect was evident after 40 min reaction time and 6 h exposure time at 

such low reactant concentrations as 0 .25 ppm each of butediene and nitrogen dioxide . 

Butadiene in itself was not mutagenic at 0 .5 - 20 % . Nitrogen dioxide was slightly 

mutagenic but also bacteriotoxic at 10 - 15 ppm . In vivo experiments were performed 

with the mouse bone-marrow micronucleus assay and the somatic mutation and recombination 

test in Drosophila (the wing spot test) . Butadiene alone was not mutagenic in 

Drosophila at 1 X, but induced micronuclei in mice at 10 ppm during 23 h . Nitrogen 

dioxide was not genotoxic tn either test system . The photochemical reaction products 

were toxic but not mutagenic at high doses tn Droso hila (1000 ppm butadiene + 50 ppm 

NO2 + UV), and not genotoxic in mouse bone-marrov highest non-toxic mixture 10 ppm 

butadiene + 10 pm N02 + UV) . The in vivo experiments thus did not confirm the strong 

direct-acting mutagenic activity of the photochemical products in Salmonella . 

608 

GENOTOXIC RESPONSE IN GROWING AND STATIONARY PHASE YEAST CELLS . 

Vierling, Th ., Boehringer Mannheim GmbH, Sandhofer StraBe 116, 

D-6800 Mannheim 31, Federal Republic of Germany 

Yeasts are known to provide suitable systems for the assessment of 

genotoxic potentials of chemicals . The diploid Saccharomyces 

cerevisiae strain MP1 allows for screening of intergenic and 

interallelic recombination and of forward mutation . In order to• 

investigate intrinsic differences in sensitivity of the Saccharomyces 

cerevisia,e MP1 testing system, the genetic responses which 

were obtained after treatment of proliferating or stationary phase 

cells with known genotoxic agents were compared . It was found that 


A 


Notes

210 1989 EMS Abstracts, 


Notes stationary phi[se cells were more severely affected by triethylene 

melamine, cyclophyWhamide and acridine orange treatment, whilst 

the qenotoxia :%f?8'dCY of sodium nitite and ethyl-nitro-nitrosoguanidine 

were slightly stronger when exposure took place during cell 

proliferation . The genetic activity of aminofluorene was expressed 

only in growing cells . Sterigmatocystin induced comparable qenotoxic 

responses under both treatment conditions . The obtained data 

support the necessity of considering both physiological conditions 

for the assessment of substance induced genetic alterations in 

yeasts. _ 

ADAPTIVE RESPONSE OF HUMAN BLOOD LYMPROCYTES TO LOW CONCENTRATIONS OF BLEOMYCIN . 

VIJAYALAXMI . & W . BURKART . 

Radiation Biology Unit, Paul Scherrer Institute, CH-5232 Villigen PSI, Switzerland . 

609 

Human peripheral blood lymphocytes cultured in the presence of low (adaptive) 

concentrations of bleomycin (BLM), 0 .01 to 0 .1 vg/ml, for 48 hours and then treated 

with a high (challenge) concentration of the same agent (1 .5 Ug/ml), became significantly 

less sensitive to the induction of chromosomel damage than those which did not 

receive the pre-treatment with BLM . They responded with lower frequencies of chromatid 

and ioschromatid breaks . Lymphocytes pre-treated with low concentrations of BLM also 

showed significant reduction in chromatid and chromosome breaks when challenged with 

1 .5 Gy X-rays, as compared with those which were not adapted to BLM . These results lend 

support to the operation of an inducible "adaptive repair" in human blood lymphocytes 

which offers resistance and cross-resistance to the induction of chromosomal damage by 

the same or similar DNA damaging agents . The adaptive repair process was found to be 

negated when 3-aminobenzamide, an inhibitor of poly (ADP) polymerase, was added to the 

cultures immediately after the challenge treatment with BLM or X-rays . The magnitude 

of negation in the adaptive response was greater in the case of lymphocytes challenged 

with X-rays as compared with those challenged with BLM . 

610 

ISOLATION AND STRUCTURE ELUCIDATION OF fAUTAGENS FROfA ROASTED SEEDS OF 

MORINGA OLEIFERA 

Vi esePfor, I . ., C .Y .L . Sylienco, F . Dayrit, P . Finch ; Institute of 

Chem .,Univ . of the Phil . ; Dept . of Chem, Ateneo U . and Dept . of Chem ., 

R .H .B .N .C . (Univ . of London), U .K . 

Several mutaqens have been isolated from the roasted seeds of 

tAorince oleifera, commonly known as drumstick or horseradish tree . 

The extracts of the roasted seeds were purified by solvent partition 

end liquid chromatopraphy . The structures of the mutepens were 13 

elucidated by spectral methods, includinp FT-IR, EI-IAS, 1H-NAiR, C- 

NmR, and 2D-COSY . The Micronucleus Test, an in vivo method, using 

albino mice as test system, was used for monitoring the mutapenicity 

of the various fractions end for studying the structure-activity 

correlations . The structure of an isolated now mutagen has been 

elucidated ea 4(oC,-L-rhamnosyloxy)phenylacetonitrile . A number of 

biosyntheticelly end chemically related compounds were also isolated . 

MUTAGENIC ACTIVITY OF CHILLT-PASTES AND TSEIR INGREDIENTS . 

U .Vinitketkumnuen, M .Suttajit, and T .Matsushim, Dept .of Biochemistry, Fac .of 

Medicine, Chiang Mai University, Chiang Mai 50002, Thailand . and Dept .of Molecular 

Oncology, Inst .of Medical Science, University of Tokyo, Tokyo 108, Japan . 

In Thailand, different kinds of chilli-paste are commonly used as an appetizing 

seasoning for cooking . lie have surveyed the iutagenic activity in some kinds of 

chilli-paste and their ingredients such as dry-chilli, shallot, garlic, coriander 

seeds, caraway seeds, lemon grass, leach lime and greater galangal by Salmonella 

mutation assay . It was found that some but not all preparations of commercial 

chilli-paste and several ingredients vere mutagenic in the Salmonella typhimuriua 

strain TA98 and TA100 with and without metabolic activation . The mutagenicity was 

not due to food preservative (sodium benzeate) but to some other ingredients used 

in chilli-paste preparation . Shallot, exhibited unequivocal mutagenic activity to 

both tester strains with and without 89 mix, greater galangal was weakly mutagenic 

toward TA100 with 39 miz . Chilli, itself and capsaicin, the pungent principle 

611

- 612 

of chilli, were not mutagenic in Salmonella typhimurium TA98 and TA100 with and 

without S9 mix . The mutagenic component(s) in shallot and greater galangal will be 

further isolated and chaxa~cterized . 

This study was supported by the China Medical Board Grant, Fac .of Medicine, 

Chiang Mai University . 

nYVCSncw77omTOwARDS THE Srwra)Aantzw11MorZNB ausaYAtlAxs,TOTTti4i wmr uu+PIDt@rraaC6stnAws 

W . VoDcner• . E .W . M3ller• and H .G . Miltenburger, Institute for Zoology, Technical University, 6100 Darmstadt, F .R.G . 

•Preunt address: Cytotest Cell Research (CCR) GmbH, 6101 RoBdorf, F .R.G. 

The mammalian spot test (MST), developed by Russell and Major (Generfea I2161-175, 1957), is a procedure for the 

detection of chemically induced mutations (especially gene mutations) and/or reeombinational processes (Fahrf$ 

Funkt .Brol.Med5:287-297, 1985) in somatic cells of the mouse. The method uses an exposure of embryonic eella ja utero 

via treatment of pregnant females with mutagens . This can lead to an alteration of wild type aUe4 at different coat colour 

genes in pigment precursor cells of the beterozygous Fl-embryos . Under the precondition of several subsequent 

divisions of the genetically altered precursor cell the recessive phenotype can be expressed, constituting a spot of 

changed colour on a black (nonagouti) background of the F~-~mouse fur 

The aim of our investigations was to compare the suitability of different . crosaes . We expected that the use of a cross 

DBA x NMRI, could minimize the efforts to reach satisfactory Fl-numbers. A possible disadvantage resulting from a 

smaller set of genetic markers as compared to the uosses T x liT and T x C57BI could not be excluded. We performed 

experiments to determine the spontaneous spot rates and to quantify potential sensttiviry differences between the crosses 

T x C57BU61 and DBA x NMRI using the well known point mutagen ethylnitrosourea (ENU) . Neither the spontaneous 

nor the ENU-induced spot frequencies did sub.taatially differ between the ctosses . However, a much lower number of 

pregnant females was required to reach the recommended sample dzea per experimental group of 200 - 300 Fl-anintala 

(Braun, Russel4 Schdteich ; Mutation Research D7:155 161, 1982) in the cross DBA x NMRI . In additional experiments we 

exposed embryos carrying only one or two heterozygous markers in various combinations to an ENU-treatment . It was 

proven by these tests that the value of detectable genetically relevant spots depended on the marker used . The sum of 

spot rates at the loci brown, dilute and albino equalled the value from a comparable treatment of the hybrids of the cross 

DBA x NMRI in which these markers are involved and was in the same range as known from the cross T x C57B1 . 

In conclusion the cross DBA x NMR] can be mommended jor the routine and standardtzed pedormance ojthe MST. 

613 

COMPARISON OF THE PLANT CELL ACTIVATION OF TWU PROMUTAGENS USINC3 ENZYME 

INHIBITORS . E .D. Wagner, M .M. Verdier and MJ . Plewa . Institute for Environmental Studies, University 

of Illinois, Urbana, IL 61801 USA . 

We are studying the mechanisms involved in the plant activation of 2-aminofluorene (2AF) and mphenylenediamine 

(m-PDA). Tbbacco cells (TX1) were the activating system and reversion in SaGnonelta 

ryphimurium (TA98) was the genetic endpoint . Specific inhibitors were introduced into the coincubation 

mixture of TX1 cells at mid-log phase, TA98 cells and a constant amount of 50 µM 2AF or 500 pM m- 

PDA. The endpoints of mutation induction, plant cell viability, and bacteria viability were assayed . 

Diethyldithiocarbamate, a metal chelator, inhibited the activation of both 2AF and m-PDA with no 

concomitant decrease in cell viability . Likewise acetaminophen, a peroxddase inhibitor, also reduced the 

activation of both 2AF and m-PDA . Metyrapone, an inhibitor of cytochrome P-450, had no effect on either 

the activation of 2AF or m-PDA, but was toxic . (+)-Catechin enhanced the plant activation of 2AF at low 

concentrations and inhibited activation at higher concentrations . (+)-Catechin reduced the activation of m- 

PDA Low concentrations of 7,8-bcnzoflavone (a cytochrome P-448 inhibitor) reduced the number of plantactivated 

2AF-induced TA98 revertants . 7,8-benzoflavone had no effect on m-PDA activation . Methimazole, 

a high-affinity flavin-containing monooxygenase substrate did not influence 2AF activation but did inhibit 

the activation of m-PDA . We conclude that cytochrome P-450 is not involved in 2AF or m-PDA activation. 

A peroxidase pathway plays a role in the activation of both promutagens. In addition, a cytochrome R448type 

N-hydroxylase is implicated in 2AF activation, whereas a flavin-containing monooxygenase pathway is 

important in m-PDA activation. Research funded in part by USAF grant tM88-AF-P-0511 . 

614 

IDENTIFICATION OF FOOD MUTAGENS . K. Wakabayashi, National Cancer Center Research 

Institute, Tokyo (Japan) 

Identification of mutagens in food is the first step in determining their 

contribution to human cancer development . By using mutagenicity in Salmonella ss a 

monitoring system, we have found and identified many mutagens and nitrosatable 

mutagen precursors in foods . 

Various kinds of heterocyclic aminea were identified as mutagens in cooked meat 

and fish, and nine of the compounds were proven to be carcinogenic in rodents . IQ 

was shown to be carcinogenic in monkeys . Carcinogenic Trp-P-I and Trp-P-2 were found 

to be very potent inhibitors of monoamine oxidase . Some heterocyclic amines were 

detected in human brain, blood and urine . Thus, heterocyclic'amines may be involved 



Notes 

r

212 1989 EMS Abstract s 

Notes -fn the devel.ofle>srtf6t only of human cancer but also of other disorders, including 

neural diseaser 

Phenol and initle derivatives were identified as nitrosatable mutaRen precursors 

in soybean fer16et4rat7e'n products and vegetables . Broiled meat and fish also contained 

nitrosatable precursors . Pher.olic derivatives reacted with nitrite to produce 

mutagenic diazo compounds . 3-Diazotyramine formed from tyramine and nitrite was 

carcinogenic to rate . In the case of indoles . N-1 and/or C-3 nitrosated compounds 

were formed . 1-Nitrosoindole-3-acetonitrile formed DNA adducts in both the forestomach 

and glandular stomach of rats . A carcinogenicity teat of this ritrosoindole is 

ongoing . 


61 5 

DIFFERENCE BETWEEN INTRAPERITONEAL AND ORAL GAVAGE APPLICATION IN THE MICRONUCLEUS TEST 

The Collaborative Study Group for the Micronucleus Test, CSGMT/JEMS-MMS, Organizers : A . 

Wakata(Yamanouchi Pharm . Co., Ltd . . Tokyo) N . Hayashi(Natl . Inst . Hygienic Sci . .Tokyo), 

S. Sutou(Itoham Foods Inc ., Tokyo), H. Shimada(Daiichi Seiyaku Co ., Ltd., Tokyo) . S.Sato 

(Japan Tobacco Inc ., Kanagaea), and Y .F. Sasaki(lnst . of Env . Toxicol . . Tokyo) 

As a collaborative study of CSGMT/JEMS MMS by 34 participating organizations, the ip 

and po administration routes were compared using 2 mouse strains . MS/Ae and CD-1 . and 17 

chemicals with various modes of action (Ara-C . 6-MP, B[ajP . DMBA . 2AAF . Phenacetin . CYP. 

EMS . ENU . MMS . MMC, COL . VINC, KBrO3, KrCrO„ Benzene and PCZ). On the basis of the 

findings of an acute toxicity and a pilot experiment for dose and sampling time, a full 

scale experiments were performed on each chemical . Almost all chemicals shored a 

positive response in ■icronucleus induction by both routes of administration in both 

mouse strains . Contradictory outcomes were obtained between the ip and po routes on 

potassium chromate in both strains (ip :positive, po :negative) . In the CD-1 ■ice, benzene 

remarkably induced ■icronuclei when administered po, but gave only a marginal response 

when administered ip . Generally the chemicals induced ■icronuclei at lower dose levels 

(mg/kg) by the ip route . This tendency, hotever, was decreased or even reversed when 

dose was expressed as percentage of the LD$ . . Although the ip route, an artificial 

exposure route, is useful to detect the inducibility of ■icronuclei of test chemical per 

se at a small dose, the po route seems sensitive and valuable enough to evaluate the 

test chemicals . When the dose levels of chemicals are adjusted on the basis of the 

LD, ., both routes are acceptable as routes of administration in the ■icronucleus test . 

CHARACTLRIZATION OF METABOLITES OF 

2-AMINO-3,8 DIMETHYL-IMIDAZO(4,S-f)QUINOXALINE . 

HlYsa Wallin, lera A. Holme, Oeorg Becher and Jan Alexander. 

Department of Toxiwlogy, National Institute of Public Health, 

N-0462 Oslo Norway. 

2-Amino-3,8-dimethylimidaxo(4,5-J)quinoxaline was metabolized by Isolated liver 

celle from PCB treated rata to at least 10 metabolites . The five major metabolites 

which were also major metabolites excreted from the rat were etructuraily determined . 

Three of the metabolites contained sulfate, on evidence of the Incorporation of 

radioactive sulphur and the requirement of tultotraasferase for their fotmation . 

Chemical synthesis, NMR, UV and mast tpeciroseopy revealed that the major 

metabolite was 4 or S-MeIQx-sulfate (formed from 4 or S-hydroxy-MeIQx) . The 

other two were MeIQz=2-sulfamate and 8-hydroxymethyl-MdQx-S-sulfate. Two of 

the metabolitea displayed the protons from a glucuronie acid group . The were 

ideatified 4 or S-O-glucoaiduriayl-MeIQx (formed from 4 or S-hydroxy-MeIQx) and 

N-gWcosidurlnyl-MeIQx. We also obtained evidence for the formation of a glutathione 

conjugate . 

ELIfA FOR FOOD MUTAGEN S 

Hlkaa Willis, Karis:,6rEpltierj, 014 Jerges Roulaad, Otorj Becher and Jaa Akxaader . 

Depfrtmest of Toxisology, Natioaal Ieetitnte of Public Health , 

N-0462 Oab Norway . 

To aeseu the risk !oVamiaqimldazo =,azaareaee (AIA) to human popelatiow it Is aeatuery to 

know more about the kveL of t>,eee compounds Is common foods . We'beHeve that immssoawys 

♦dll provide both the ipeoificity and the sensitivity seeded for this task and may be rnn Is 

larae seriet with relative little work effort . 

Our object was to develop its t?LISA for several different AIA. We therefors prepated astigeas 

61 6 

61 7

whicb carry the eommoa regioMe kaowa carcinogenic AIA :s. Tbus, trana-2•amiso-S(•2•urboxy)etheayl-1•metbyl•beaz(midazole 

was synthesized aad coupled to ovalbumia aad boviae albumia . 

Rabbits wert (tqmuaized with tbe bovine albumla•baptea eoajugate, aad aa eompetitivt enayme 

immaaoasuay was developed os the reaction of the antibodies witb the ovalbumia-baptem In 

microtiter ptat6f . 

Fifty percent ishibidoa was demoostrated at 10 pmo11Q, 50 pmol MeIQ, S00 pmo17,8-diMelQx, 

and 2500 pmol Me1Qx, 4,8-diMeIQz aad PhIP . No iahlbitioa was seea for 1 umol ereatiae ot 

ereatiaise . 

618 OBSERVATION ON RENAL HYPERPLASIA CAUSED BY UNILATERAL NEPHRF.CIC)MY 

IN MICE AND ITS SIGNIFICANCE IN SEARCH FOR ANIMAL TESTS FOR 

CHEMICAL CARCINOGENS 

Da Wang 

(Shanghai Institute of Biochemistry, Academia Sinica) 

A series of histological and anatomical changes of different stages was compared 

after unilateral nephrectomy in mice between the remained kidney and the 

sham-operated kidney . Results of the experiment revealed that after unilateral 

nephrectomy in ICR mice a series of changes occurred in the remained kidney : 

there were increase in weight, increase in thicknesses of renal cortex and 

medulla, increases in size of the renal corpuscle, increases in number of cell 

in the glomerulus, hyperplasia of epithelial cells in the renal tubule, dilatation 

of renal blood vessels and stromal hyperplasia . Administration of chemical 

carcinogens to the hypertrophic and hyperplastic kidney may aggravate 

cell hyperplasia and degeneration in the remained kidney, and promote the 

development of tumors . The shortening of induction period of tumors after unilateral 

nephrectomy might help to supply a better model for chronic induction 

of animal experiment in vivo . 

619 • 

MUTAGENIC AND ANTIMUTAGENIC EFFECTS OF 9-AMINOACRIDINE IN THE YEAST SACCNAROYNYCSS 

CsRevISZns . Q . Wang, U .G .G . Hennig, and R .C . von Borstel, Department of Genetics, 

University of Alberta, Edmonton, Alberta (Canada) T6GJE9 

Certain chemicals exhibit the property of reducing the spontaneous mutation rates 

in the yeast SaooharoVoee oerev{eiae . In systems for forward mutations (canavanine 

sensitivity to resistance) and reversions (histidine auxotrophy to prototrophy), 

9-aminoacridine was reported to be antimutagenic during mitosis (Magni et at . 1964, 

!autat . Ree . 1 : 227-230 ; Pugllsi 1967, Mutat . Rea . 4 : 289-294) . Also, there appears to 

be a base sequence specificity involved in the binding of 9-aminoacridine to ONA 

(Young et aZ . 1980, Prod . NatZ . Acad . Soi . (USA) 77 : 6453-6457) . The effects of antimutagenic 

agents can be quite different on various loci . We have found that 9-aminoacridine 

has antimutagenic effects on the hom3-10 frameshift allele but not on the 

hisl-7 and arg4-17 base substitution alleles in strain XV185-14C of yeast . Mutation 

rate measurements were made in growinaq cells of yeast with the 1000-compartment 

fluctuation test (von Borstel 1978, in Methods in Cell Biology", Vol . 20, Academic 

Press, pp . 1-24) . For the hom3-10 allele, 9-aminoacridine, at concentrations of 10-20 

ug/ml, increased the mutation rate 4-fold (mutagenic) and at 50-100 ug/ml it decreased 

the mutation rate 2-fold (antimutagenic) . The concentration range of 50-100 ug/ml 

causes a delay in cell generation time (from 1 .8 hr to 2 .7 hr), which is a decrease in 

the rate of cell division . These results implicate an interaction between DNA repair 

and the delay in the cell cycle in the mechanisr of antimutagenesis when chemicals are 

involved . (This research was supported by a grant from the Natural Sciences and 

Engineering Research Council of Canada) . 

620 

EFFECTS OF CHEMICAL 1eDTAGENESIS ON TREATINO ZYOOTES OF PIANTS . Wang Qinnan . Institute of 

Genetics, Academia Sinioca, Beijing . 

According to the theory of mosaicism, some investigators have considered that treatment 

of zygotes may afford an opportunity to increase the mutation rate . Therefore we have 

designed experiments to study the effect of treating zygotes with a chemical autagen . 

We used wheat zygotes and dry seeds and the mutagen EMS . In addition, the wheat zygotes 

were given bleomycin (BLM) post-treatment so as to enhance the induction of mutations . 

The results obtained indicated that treatment of zygotes with EMS at any stage is 

superior in terms of creating mutations as compared to the control . The treatment of 

zygotes inducas much higher mutation ratios in the eS3 generation as compared with treating 

dry seeds, and brings about a broader mutational spectrum . In accordance with the effects 



Notes 

t71 F9a 

m F, 

00 

I: 

1'


---~- .~-- _- 

Notes- 

of' mutation in ~e [f, the different mutation induction methods used in the experiments 

can be arranged in the following sequence : treating zygotes at late stage > treating 

zygotes at early a~a~treating dry seeds . The results indicated also that the zygotes 

receiving the BL4 post-treatment seem to be equipped with an imperfect DNA-repair system 

of enzymes because their mutation ratio is not obviously higher than that of the variant 

zygotes not receiving Bt2t post-treatment . 


- 621-- 

A cTUDY OF SCE IivDUC'PION BY TRIPTi•,'RYGIUM HYPOGLAUCUM(LEVM . ) HUTCH IN 

HUMAN PERIPHERAL BLOOD LYMPHOCYTES 

Wang Xu, Zhou Rumin and Ceo Neng . Department of Biology, Yunnan 

Normal University, Kunming, Yunnan, Peo le's Republic of China . 

Tripterygium hypoglaucum (Level .) Hutch (pTHH) is a fChinese tranditional 

medicine which has been used for the treatment of various 

diseases such as systemic lupus erythematosus, rheumatoid arthritis etc . 

The medicine showed antifertility effects both in rat and human during 

the medicine taken period . No other cytotoxic effects were reported . 

Our work aimed at the cytogenetic research of THH . The presented 

work emploged the sister chromatid exchanges (SCEs) to assess its 

genotoxic effects in human peripheral blood lymphocytes . 

Blood for all the experiments were obtained from both healthy donors 

and patients cured with TH .9 for over 90 days and treated by THH with 

the doses from 200 to 0 .63mg/ml in vitro . The blood from every donor 

were cultured in two ways of presencing 69 mix or not . 

The results indicated THH didn't induce SCE siRgnificantly in human 

lymphocytes from healthy donors and patients cured with THH before 

sampling . S9 had no effects on SCE induction by THH in all the cases . 

However, THH showed a strong cell cycle delay effects when higher 

treating doses from 200 to 10mg/ml were used in vitro . 

Our results suggest that THH could be a human anti-baby medicine . 

Further research about its other cytogenetic and cytotoxic effects are 

being done in our lab . 

622 

STUDIES ON THE NUTAGENTC . CARCINOGFNIC AND TI]tATOGDU C EFFSCTS OF l,2-UD101 AND ETEPA 

Wang Zhi-qlao, Li Xiu-yin', tlen Si-zhes, Liao Nia'-yaaq 

The studies of mutayeaic effects of 1,E-UD101 and setepa were investi'ated by Anes test,the bone 

narrow micronucleus test of sice,saalysis of bone marrow chranosome aberration of rats,analysls of 

chromosome aberration and SCE test In bn .aa peripheral blood lymphocytes, analysis of chromosone 

aberration and SCE of sice spersatoyenous cetls, the do .inant lethal test of sice . The results 

showed 1,=-UDIDf was re0ative and metepa was positive In all test systens . Nice I drinkiny water 

freely for one year which contained 1,2-I1DIIX at the coaceatratlon of 30-300my/1 highly Induced 

beaangiaatous lesions of liver and adeaotous changes of Lung . The lumoriyenicity of inetepa did 

not be induced at the concentration of 1-10n9i1 during experinrat of one year .The teratoyenic 

test of rats showed setepa maialy Induced limb ∎alfornation whea It was i .p . ad.laistered at 

the dosage of 30sy/kg.Tle malformation frequency was 9f% .aad 1 .=-UDlDI had so teratoyenic effects . 

The further study with rabbits showed metepa had so terato'enic effects . The results of the 

study Indicated that 1,2-UDIOi ∎ibd belong to epioenetic carcinogen and netepa might belong 

to carcinogen with mutaoenesis . 

623 

INDUCTION OF THIOGUANINE RESISTANT MOUSE LYMPHOCYTE VARIANTS BY SUB- 

CHRONIC LoW DOSE INHALATION EXPOSURE TO BENZENE . J . B. Ward Jr ., M . M . 

Ammenheuser, D . L . Morris, V . M . S . Ramanujam, and M . S . Legator Dept . 

of Preventive Medicine and Community Health University of Texas Medical 

Branch, Galveston, TX 77550, USA . 

Spleen lymphocytes of CD-1 male and female mice exposed to benzene 

vapor were evaluated for the frequency of thioguanine resistant variants 

(Vf) . The nice were exposed, by inhalation, 22 hours per day for six 

weeks to benzene concentrations of zero (air only), 40, 100, and 1,000 

parts per billion (ppb) or were housed in standard cages (cage 

controls) . An autoradiographic assay for Vf was used with cryopreserved 

cells from three animals of each sex at each exposure . The mean Vts'were 

zero, 7 .04 ; 40 ppb, 26 .25 ; 100 ppb, 52 .10 ; 1,000 ppb, 20 .39 and cage 

controls 13 .25 . Vts in females were consistently higher than in males in 

benzene exposed animals but not in controls . Confidence intervals (95%) 

50869 3728

i 

on means at 0, 40, and 100 ppb did not overlap . Because the increased 

Vfs could have been due to increased in vivo cell cycling, four 

conditions were evaf'aated in which the frequency of cycling cells was 

determined without In vitro mitogenic stimulation . Benzene exposure did 

not stimulate cell cycling . Because the autoradiographic assay does not 

permit the recovery of variant cells for confirmation of mutant genotype 

these results must be interpreted cautiously . However, they do suggest 

that in vivo benzene exposure at low levels may be mutagenic to motLse 

spleen lymphocytes . Supported by the Texas Air Control Board .(•vf x 10 ) 

624 

0-VANILLIN ENHANCES MUTAGENESIS INDUCED BY MNNG IN ESCHERICHIA COLI . 

K . Watanabe, T . Ohta, T . Kato, M . Watanabe and Y . Shirasu, Institute of Environmental 

Toxicology, Suzuki-cho 2-772, Kodaira, Tokyo 187 (Japan) 

2-hydroxy-3-methoxybenzaldehyde (o-vanillin), the antimutagenic effect of which has 

been reported on mutagenesis induced by 4-nitroquinoline 1-oxide (4NQO) in Escherichia 

coli WP2s, enhanced N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutagenesis in 

the same strain . In order to investigate the mechanism of the enhancing effect of ovanillin 

against MNNG-induced mutagenesis in E . coli, we have carried out experiments . 

Among 7 derivatives of o-vanillin, 2-hydroxy-3-ethoxybenzaldehyde, o-hydroxybenzaldehyde 

and m-methoxybenialdehyde showed an enhancing effect on MNNG-induced 

mutagenesis in E . c-o -li WP2s . A remarkable enhancement of mutagenesis provoked by Nmethyl-N-nitrosourea, 

a methylating agent, was also observed on semi-enriched minimal 

agar plates containing o-vanillin in E . coli WP2s . On the contrary, o-vanillin 

suppressed greatly furylfuramide- and 4NQ0-induced mutagenesis and showed a slight 

suppressing effect against mutagenesis induced by methylmethanesulfonate, N-ethyl-N'nitro-N-nitrosoguanidine 

and N-ethyl-N-nitrosourea . In an investigation employing the 

various repair-deficient mutants of E . coli B/r, clear enhancement effects by ovanillin 

were observed in wild-type atrain E . coli B/r WP2 and its 4 mutants, WP2s 

uvrA, ZA60 recA, ZA12 umuC and ZA160 polA, whereas a weak enhancement was observed in 

E. coli ZA180 ada-5, ZA113 alkA and ZA130 alkB, all of which cannot induce the 

aZcapEive response o alkyla~fon damage . TFiese results may suggest that o-vanillin 

inhibits the inducible adaptive response. • 

625 

NEW MODIFIED STRAINS OF SALMONELLA TYPHIMURIUM TA98, TA100, VERY SENSITIVE TO 

NITROARENES AND AROMATIC AMINES BY CLONING OF ACETYLTRANSFERASE GENE . 

M . Watanabe, M . Ishidate, Jr ., and T . Nohmi, National Institute of Hygienic 

Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158 (Japan) 

Acetyl-CoA :N-hydroxyarylamine 0-acetyltransferase ia known ae an enzyme 

involved in the intracellular metabolic activation of mutagenic nitroarenes and 

aromatic amines . The acetyltranaferase gene of S . t yphimurium TA1538 was cloned 

into pBR322 (Biochem . Biophys . Res . Commun . 147, 974-979 (1987)), and the 

plasmid harboring the gene (pYG219) was introduced into TA98 and TA100 . The 

resulting strains, YG1024 (- TA98(pYG219)) and YG1029 (= TA100(pYG219)), had a 

higher isoniazid- and 2-aminofluorene-N-acetyltraneferaee activity 100 timee 

more than the original strain, TA1538(pBR322) . They showed an extremely high 

mutagenic response to 2-nitrofluorene, 1,8-dinitropyrene, Glu-P-1(+S9) and 2aminoanthracene(+S9) 

. The YG1024 was more sensitive to these chemicals than 

TA1538/1,8-DNP(pYG121 or 122) strains which we previously established, since the 

YG1024 has pKM101 and more acetyltransferase activity . These results indicate 

that the new strains are useful for the detection of mutagenic nitroarenes and 

aromatic amines . 

This work was supported by a Grant-in-Aid from Japan Health Sciences Foundation . 

626 

NITROARENE SENSITIVE SALMONELLA TYPHIMURIUM STRAINS YG1021 AND YG1026 WITH A 

HIGH NITROREDUCTASE ACTIVITY, DERIVED FROM TA98 AND TA100, RESPECTIVELY . 

M . Watanabe, M . Ishidate, Jr ., and T . Nohmi, National Institute of Hygienic 

Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158 (Japan) 

"Classical nitroreductaae" is known as an enzyme involved in the 

intracellular metabolic activation of mutagenic nitroarenes . The nitro :eductase 

gene of Salmonella t himurium TA1538 was cloned into pBR322 (Biochem . Biophys . 

Res . Commun . 147, 974-979 1987)), and the plasmid harboring the gene (pYG216) 

was introduced into TA98 and TA100 . The resulting strains, YG1021 (- 

TA98(pYG216)) and YG1026 (- TA100(pYG216)), had about 50 times higher 

nitrofurazone-reductase activity than TA1538 containing pBR322, and were 


• 


Notes


Notes 


. . ~ 

-- 

_ . .- 

Aztiemely aensi~iVe ~the mutagenio action of 2-nltrofluorene, 1-nitropyrene 

and 2-nitronaphthalene . The YG1021 was more sensitive to these chemicals than 

TA1538NR(pYG111) gtrjAli~w_hich we previously established, since the YG1021 has 

pKM101 . Theae reanlts indicate that the new etrains permit the efficient 

detection of mutagenic nitroarenea in the environment . 

This work was supported by a Grant-in-Aid from Japan Health Sciences Foundation . 

- 627 

A SURVEY OF pCTIVITY PROFILES OF A(~TIMUTAGENS/ANTICARCINOGENS .' Waters, M .1, Brady, 

A ., Stack, F .2, and Brockman, H .3 ; U .S . Environ . Prot . Agency, Res . Tri . Park, NC ; 

2Environ . Hlth . Res . & Testing, RTP, NC ; 3111 . State Univ ., Normal, IL (USA) . 

Experimental evidence suggests that chemical mutagenesis and•carcinogenesis are 

related phenomena such that identification of causative agents and protection from 

exposure might prevent certain human cancers and related diseases . Agents that 

inhibit the processes of mutagenesis and carcinogenesis, particularly naturally 

occurring agents, might be expected to exert a primary protective effect . This paper 

will consider the use of short-term bioassays to identify antimutagenic and 

anticarcinogenic substances and to classify them [cf . Mutation Res . 168 (1986) 47-65] 

according to the locus of their rotective influence, i .e ., intracellular or 

extracellular, and putative mechanismrs) of action . In the extracellular environment, 

inhibition of formation or uptake of mutagens and inactivation of promutagenic or 

mutagenic species are examples of antimutagenic mechanisms . Intracellularly, 

antimutagenic substances have been described as "scavengers" of radicals, "blocking 

agents" (involving at least 3 different mechanisms), and "suppressing agents" . 

Additional intracellular mechanisms include alterations in DNA repair processes and/or 

modification of the genotoxic response to the mutagen/carcinogen . The presentation 

format will be based on the genetic activity profile (GAP) methodology developed by 

the authors . The GAPs of known mutagens/carcinogens will be presented together wi ht 

newly designed profiles for antimutageni/anticarcinogens according to the 

classification scheme outlined above . This is an abstract of a proposed 

presentation and does not necessarily reflect EPA policy . 

628 

MOLECULAR CHARACTERIZATION OF ERCC2- A HUMAN NUCLEOTIDE EXCISION REPAIR GENE WITH 

HOMOLOGY TO THE YEAST RAD3 REPAIR GENE . C.A . Weber, E .P . Salazar, and LH. Thompson, Biomedical 

Sciences Division, Lawrence Livermore National Laborabry, Livermore, CA (USA) 

The UV-sensltNe Chinese hamster ovary (CHO) cell line mutant UV5 has a defect In the Incision step 

of nucleotide excision repair. This same process is defeotive in cells from patients with the cancer-prone 

genetic disorder xeroderma pigmentosum (XP) . Genomlc clones of the human ERCC2 (Exdslon gepair 

~Qross .QomplemenUng) gene that fully complement the UV5 repair defect in a stable manner have been 

isoiated and characterized (Weber et al ., 1988 . Mol. Cell. Biol . 8 :1137-1146) . The genomic clones 

were used to Isolate nine ERCC2 clones from the pcD2 human cDNA expression library (obtained from H . 

Okayama) . One of the cDNA clones, pER2-14 (inserl size . 2.6 kb), was found to confer transient UVresistance 

of an intermediate level to UV5 cells . The nucleolide sequence of pER2•14 and genomic 5'and 

3'-flanking regions was determined . Consensus regulatory signals were Identified for a GC box, CAAT 

box, TATA promoter, and polyadenylation sNe . Translation of the open reading frame and comparison 

with sequenced yeast nucleotide excision repair genes revealed a striking homology between ERCC2 and 

RAD3 (-52% Identical ; 760 aa vs . 778 aa) . This comparison, along with examination of the nucleotide 

sequence for splice junctions, also revealed that pER2-14 contains part of Intron 1 and produces a 

protein that is 24 amino acids short at the amino terminus (738 aa) . This presumably accounts for the 

transient nature of the UV-resistance conferred to UV5 alis by pER2-14 . The RAD3 protein has both a 

repair and an essential function and has both a single-stranded DNA-dependent ATPase activity and an 

ATP-dependent helicase activity . Construction of a cDNA clone with genomic 5'•flankinp sequences that 

will produce a full-length protein Is underway . This clone will be used to test for correction of the 

repair defect in the CHO UV5 mutant and In the XP complsmentatkan groups. Work performed under the 

auspices of the U .S . Department of Energy by LLNL under contract NW-7405-ENG-48 . 

MECHANISMS AND PREVENTION OF FORMATION, AND METABOLISM OF FOOD 

MUTAGENS/CARCINOGEN 2-AMINO-3-METHYLIMIDAZO[4,5-]QUINOLINE (IQ) . 

J .H . Weisburger, R .C . Jones, H .J . Luke, T . Vavrek, American Health 

Foundation, Valhalla, NY 10595 USA . 

We have postulated that IQ and related imidasoasaarenes, formed 

during frying or broiling of meat and fish, may be the genotoxic 

carcinogens associated with important types of cancer in humans, like 

in the breast, colon or pancreas (Prev . Med . 16 :586,1987) . Their 

formation appears to involve Maillard reactions, with creatinine as 

critical reactant . In laboratory models involving high temperature 

50869 3730 

629


reflux of glycine, glucose and creatinine, or in realistic frying of Notes 

meat, competition with creatinine by L-tryptophan, L-proline, or both 

has lowered the formation of IQs considerably . This approach provides 

a feasible, practical means of inhibiting the production of IQs . The 

metabolism of IQ in rats yields evidence of N-acetylation, N-demethylation, 

hydroxylation on carbon 5 (excreted as O-glucuronide and 0sulfate) 

and intestinal flora-mediated formation of 7-OH-IQ . Xanthineoxidase 

reduction of 2-NO2-IQ yielded intermediates reacting with alfthymus 

DNA, and giving 5 products, visualized upon hydrolysis by 3~Ppostlabeling 

. Microsomal metabolism of IQ yielded the same 5 products, 

CAd242i? anathOeAr~21~11fs~rNCampuntg`(supported in part of USPHS grants 

630 

DICHLOROMETHANE-INDUCED CYTOGENETIC DAMAGE IN HICE . B . Westbrook-Collinsl, J .W . 

Allenl, A .D . Kligermanl, J .A . Campbell2, G .L . Erexson2, F . Kari3, and E . 2eiger3 . 

1U .S . Environmental Protection Agency, RTP, NC 27711 USA ; 2Environmental Health 

Research and Testing, Inc ., RTP, NC 27709 USA ; 3National Institute of Environmental 

Health Sciences, RTP, NC 27709 USA . 

Dichloromethane (DCM) or methylene chloride is a widely used industrial solvent 

which causes lung and liver tumors in inhalation-exposed B6C3F1 mice (NTP, 1986) . In 

the present studies, chromosome damage was studied in female 86C3F1 mice exposed to 

DCN by subcutaneous or inhalation treatments . After a single subcutaneous injection 

of either 2,500 or 5,000 mg/kg DCM, no increases in frequencies of sister chromatid 

exchanges (SCEs) or chromosome aberrations (CAS) in bone marrow cells were observed . 

Inhalation exposure to DCM for 10 days at doses of 4,000 or 8,000 ppm resulted in 

significant increases in frequencies of SCEs in lung cells and peripheral blood 

lymphocytes, CAs in lung and bone marrow cells, and micronuclei (MN) in peripheral 

blood erythrocytes . The highest levels of increased damage ware observed for lung 

cell CAs and blood erythrocyte MN (approximately two times control frequencies) and 

bone marrow CAs (approximately four times control frequency) after 10 days . 

Following a 3-month inhalation exposure to 2,000 ppm DCM, mice showed small but 

significant increases in lung cell SCEs and peripheral blood erythrocyte MNr . It was 

concluded that DCM is clastogenic following in vivo inhalation exposure . This 

finding suggests that genotoxicity may play a role in the carcinogenic properties of 

DCM in B6C3F1 female mice . (This is an abstract of a proposed presentation and does 

not necessarily reflect U .S . EPA policy.) A 

631 

CHARACTERIZATION OF THE IN VITRO UNSCHEDULED DNA SYNTHESIS ASSAY USIYG PRIMARY LUNG 

CELLS OF THE RAT . W-2 . Whong, J .D . Stewart, and T . OnB, Division of Respiratory 

Disease Studies, National Institute for Occupational Safety and Health, Morgantown, 

WV (USA) 

Since genotoxic agents can be present in the environment as gases, vapors, and 

airborne particles, inhalation becomes an important exposure route with the lung as a 

major target for such genotoxicants . Therefore, primary lung cells, which retain 

most of their original metabolic ability, may provide a useful cell system for evaluating 

the pulmonary genotoxicity of chemicals with different genetic endpoints . 

Previously, we had established the micronucleus and sister ehromitid exchange assays 

in rat primary lung-cell cultures . In the present study, effort was made to incorporate 

the in vitro unscheduled DNA synthesis (UDS) assay in the same cell culture 

system . The autoradiograph method employed for detecting UDS was characterized, and 

the assay was tested with a direct and an indirect-acting genotoxicants in both 

alveolar macrophages and primary lung cells from rats . Data of a tisas oourse study 

revealed that the optimal radioactive labeling was achieved after a 16-b incubation 

with 3H-thymidine . Hydroxyurea at the concentration of 20 mlK effectively inhibited 

the regular DNA synthesis . With this protocol, a dose-dependent UDS was induced by 

Y-methyl-N'-nitroso-N-guanidine and 2-aminoanthracene in both rat alveolar sucrophages 

and primary lung cells . These results indicate that primary rat lung cells in 

culture possess DNA repair ability and that the UDS assay may be useful for assessing 

the genotoxic effect of chemicals in this cell system . 

632 

:.h-LCCUS LItdI:E.') 'lI^7';'-M?C_-S IPT IN')UC_"OTT OF SCE 'nY °-1tT?C ;a ;w`MTE IN 

f•:CUS : aaE 3^.^ .TE:. IN "IN VIVO" AS CC".?:'AYED TC "IN VI^_RO" T--S'S . S .M, 

WLelgosz Pnd A .L.P?vlak, Inatitute of Hrnman GPnetics, ôlirh Ac^de^!y 

of Sci,~nces, u_.7_9trzeszynskn 32, 60-479 ?ozn .!)A (Polend) 

The e°fect of alleles of Ah lecur on the induction of SC- - as •tudied 

in C57316 and in DBA2 mice +.•r.eeted t .a cs ' . :ith ben~o(^!pyr^nc (EY, 

1C0 m,- -,Pr 1cg, p .o . ) . ='or mea-r.êm^nt^ of ch-n!;eo in fre-uency of SCE 


218 1989 EMS Abstracts . - -- 

: 

Notes ~ t . :o pro':oco?.s '-re uêd : 1 . for "in v!vo" n-,n-uremcnt In hone m~arrorr 

ec' .1c 50 .mg V)iAte of rromo9.coxyuri3inc (BrdU) •.•rre implant^d 2Q4 h beio--'c 

'"±'~-ir~ o`"^n3'~^1^ ; 2 . `.or "in vitro" ~+e?-ttrement ^plecn 1ymFhocvtee 

t•rere cu].tvre :? ~~Str °r' (6/ .tz ~er ml) . The induction of SCy in 

~-tre^.ted ^.^7LJ.C ^nd ^7A^ mice "in vivo" :,^s 1 .21 ^nd ti .07-fold, roo- 

~°etively . "In vitro" th^ re~ictive v^?t .ies •-erc 1,^9 and 2 .73 . 

The ob~-erved ?nter^trpin differences In in3action of SC2, were much 

Qre^.t^r 7n hone n .r=ov~ "in v3vo" ^ .- compared to ^rleen 1•m?hoc?rte3 


ttin vi t'"o" "2re A+rr„r^•tCe T~a•r re .%tlt frOm ~^.~ f^Ct fi .^t At+_ring 'tin 

vi tro" teqt cel?.r P_o not e :~o'rcd tc =1, a- c•ell ?^ from hicher fre- 

ûencieo of SC2 !n ce].13 culturQd " .'.n v!tro" . "_'!•ir• mey Indicate th2t 

z.so in c;m V+.e 'in vivo" tent3 !b :° aasaâment of 7henotype rt Ah locus 

(e . - . antip••r

635 = 

a. 


Notes 

EVALUATION OF FURAN-INDUCED GENOTOXICITY AND CELL PROLIFERATION IN RAT AND HOUSE HEP- 

ATOCYTES . D .H . Wilson, and,. .B .E . Butterworth, CIIT, Research Triangle Park, NC (USA) . 

Initial observations from bioassays by the National Toxicology Program indicate 

that furan produces hepatocellular carcinomas in male F344 rats and in male and female 

B6C3F1 mice . Although furan is negative in the Ames test, the profile of autations 

produced in oncogenes from furan-induced tumors are different from those of spontaneous 

tumors (Reynolds et al, Science 237, 1309 (1987)) . To investigate the possible 

mechanisms by which furan causes tumors, genotoxicity, as indicated by DNA repair, and 

chemically-induced cell proliferation were examined . Prijary hepatocyte cultures 

from male F344 rats were incubated with faran and 10 LCi/ml H-thymidine . DNA repair 

as unscheduled DNA synthesis (UDS) was quantitated autoradiographically as not grains 

per nucleus (NG) . Concentrations from 1 YH to 10 aH furan yielded no UDS in vitro . To 

examine UDS in vivo, furan was administered by gavage to male rats 

(5, 30 and 100 mg/kg) and to male B6C3F1 mice (10 . 50, 100 and 200 ag/kg) twelve hours 

prior to isolation of hepatocytes . No increase in NG was observed in hepatocytea from 

furan-treated animals compared to r3ontrols . Cell proliferation in vivo was evaluated 

by autoradiographic analysis of H-thymidine incorporation in cultures of primary 

hepatocytes isolated 36 hours after a single gavage administration of furan (30 mg/kg) 

to male rats . Hepatocytes from vehicle treated rats had a labeling index (LI) of 

0 .2+0 .1X while that of furan-treated rats was 17+5% . Taken together, available dats 

suggest that the distinct profile of oncogenes from furan-induced tumors may have 

resulted from enhanced susceptibility of those genes to mutations related to forced 

cell proliferation rather than from direct mutagenesis by furan . Puran-induced cell 

proliferation may also have been a driving force in tumor promotion and progression . 

636 

THE CYTOGENETIC EFFECI'S OF INTRODUCING RESTRICTION ENZYMES INTO CHO CELLS 

USING ELECTROPORATION. RA Winegar H.W. Chung, J .W. Phillips and W.F. Morgan, Laboratory of 

Radiobiology and Environmental Health, University of California, San Francisco, CA (USA) 

Studies using restriction enzymes to examine the mechanisms of cytogenetic damage have been hampered 

by the inefficiency of available permeabilization techniques . We have used cell electroporation with great 

success to permeabilize Chinese hamster ovary (CHO) cells for the introduction of restriction enzyptes . Cells 

electroporated in the presence of restriction enzymes show an extremely high frequency (>90%) of aberrant 

njetaphases as well as a dramatic decrease in cell survival. Electroporation itself caused no increase in 

aberrant chromosomes and had only a slight effect on cell survival . The isoschaomer pair Msp I and Hpa II 

was used to determine whether restriction enzymes act with the same sprcificity within a cell as in vitro . Both 

enzymes recognize the DNA sequence CCGG, but whereas Hpa II will not cleave this sequence if the internal 

cytosine is methylated, Msp I will cleave regardless of the methylation status of the internal cytosine . As 

expected, since this sequence is heavily methylated in mammalian cells, Msp I was greatly more effective than 

Hpa II at inducing chromosome aberrations . Although restriction enzymes are very effective at inducing 

chromosome aberrations, experiments using a large number of different enzymes and several different harvest 

times indicated that restriction enzymes do not induce sister chromatid exchanges . This is consistent with 

previous reports that agents that produce double-strand breaks do not induce sister chromatid exchanges . 

Work supported by the Office of Health and Environmental Research of the U .S. Dept of Energy under 

contract DE-AC03-76•SF01012 . 

637 

ISOLATION OF cONAs ASSOCIATED WITH TUMOR SUPPRESSOR GENE FUNCTION IN SYRIAN HAMSTER 

EMBRYO CELLS . R .W . Wiseman, J .C . Montgomery, J . Hosoi, E .W . Hou, J .E . Bisi, and 

J .C . Barrett, Laboratory of Molecular Carcinogenesis, National Institute of Environmental 

Health Sciences, Research Triangle Park, NC 27709 . 

Loss of a tumor suppressor gene function appears to be a critical step in Syrian 

hamster embryo (SHE) cell neoplastic transformation in vitro . In order to understand 

the function of these genes, we have isolated preneoplastic clonal variants 

from 2 imnortal SHE cell lines that have either retained (supB+) or lost (supB-) the 

ability to suppress the tumorigenicity of the BP6T tumor line in cell hybrids . cONA 

probes prepared from polyA+ RNA of supB+ and supB- cells have been used to screen a 

supB+ Lambda Zap cDNA-library for clones that are preferentially expressed in supB+ 

cells . cONAs for at least 4 independent genes have been isolated, and DNA sequence 

analyses revealed that 2 of these encode proal(II) and al(IX) collagens, which are 

normally expressed in chondrocytes . Another cDNA was unrelated to any known gene 

while the fourth cDNA was homologous to the mouse H19 gene, which is developmentally 

regulated in liver and muscle cells . In complementary studies for the identification 

of human tumor suppressor genes . Syrian hamster tumor cells have been utilized as 


220 1989 EMS Abstracts, . ~ -- 

. : "' 


. . . , .. . j. _ - .,r~ ~ 

NOtES gene transfer recipients for a normal human fibroblast cDNA library prepared in the 

pc02 expression y~ect~ Using a low serum/BUDR/nUV light selection, several Independent 

cDNA-induce$BM revertants have been isolated from cultures transfected 

with the pcD2 cDNA library that were greater than 1000-fold suppressed for anchorage 

independence relative to the parental BHKA tumor cells . Progress in characterization 

of these cONAs associated with tumor suppressor gene function will be presented . 

- 638 

SEIECPION OF VF111CL.E MD THE AOfRE OF M14MSTRATICt!1 IN T1B; MD(JSE BfltE MARRQW 

MICRONUCLEUS TEST . J .P . Wojciechowski+, D .K . Gulatil, and M D . Shelb jy 

lEnvironmental Health Research and Testing, Inc ., Lexington, KY ~itre National 

Institute of Environnental Health Sciences, Research Triangle Park, NC . 

Two critical factors that influenoe the swrimum exposure of the target tissue 

with the test material in the bone marrow micranucleus test are the route of 

administration and the vehicle . The purpose of this study was to determine the 

extent to which vehicle and route of administration affect level of effect . 

Dimethylbenza(a)anthraoene (DNIDR), N,N'-methylenebisacrylamide (MR), 

Nynethylolacrylamide (NOA), and 4-aminobiphenyl (ABP) were tested using a variety of 

vehicles and two different routes of ad+ninistration . DlIBA suspended in corn oil and 

administered via lP at 10 ug/kg caused a 2 .9 told increase in the incidence of 

micronucleated polychronatic erythrocytes (Md-FCE) over the vehicle control . D)a3A 

dissolved in Dr9O (lOmq/kg) via lF resulted in an 11 .3 fold increase in t4}-FCE . l81R 

in DMSO via lF had no effect on Mi-FM (p>0 .1) at up to SOsiq/kg . MBA dissolved in 

PBS (50mg/kg) caused a 16 fold increase in FQ}-PCE's . 143ii dissolved/suspended in 

PBS, corn oil, or D6s90 and administered via lP at 112 .5 sq/kg gave a negative 

response for M-FCE. MON in PBS via gavage caused a weak but significant increase 

(p

transformation . The BALB/c 3T3 clone A31-1-13 cells were exposed to coal dust 

extract for 72 hrs . At th:--end of treatment, the coal dust extract was removed, and 

cells were cultivated for 4 weeks with twice weekly medium change . Type III foci 

were scored after fixation and staining . Results showed that coal dust extracts, 

both nitrosated and nonnitrosated, induced eell transformation in a dose response 

manner . However, the transformation frequency was much higher with the nitrosated 

than the nonnitrosated coal dust extracts . All transformed eell lines derived from 

coal dust extract-induced foci showed characteristics of malignant transformation . 

The transforming activity of coal dust extract decreased when chlorophyllin was 

incorporated into the treatment solution .--These results seem to support the coal 

mine dust hypothesis for the causation of gastric cancer in coal miners and indicate 

that chlorophyllin possesses an antitransformation activity . 

641 

RE6P:ARCiI ON MUTAGENICITY OF POLLUT'r.D WATER TREAT6IENT WITH FIOLOGICAL OXIDATION 

:+u Guopine, Hong Huacheng, Lu Ying, and Yue Shenling, ShanPhai Municipal Waterworks 

Co . Shanfhei, China 

In this paper, the efficiency of different water purification processes in 

:emovinF organic muteger.s from raw water was introduced . We use initial "0"-- 

ozonation ; "T"-- trcditional rrocess ( comFiro+ion of coa!-ul .;tion,sedimentetion 

c : .d filtration ) ; "C1"--chlorinQtion ; "F"-- bio_*iltratlon and "C"--bioactive 

cerbon filtration . Thus, the 4 test systems (• combined processes) are 07C1, 

fi9`Cl, FTCC1, and BTOCC1 . 

Reeults showed that OT and FT processes were not al~le to chanre the mute'enic 

nctivities of w~ter . One phenomenon a•e Pre interested in ie that the nurber 

_' tê revertents of the e:nters treated by OT and FT processes is approxime.•el;• 

identical . However, after chlorination, the numFer of OTC1 effluent raised so 

rapiely that it beceme more than thet of rew w .

222 1989 EMS Abstracts «_- ~,,,E:- - - 

Notes gation of the best pez'imental conditions of mouse testes 

chro :aosotnal assajr . .~..~the first part of experiment according 

to an orthogonal design,three factors including dosa„e 

of colchicine,effective time of colchicine .nd time of 

sucrifice of mouse(8time point~ during 24 hoars) are stuuied 

at c: nixin- level of L 32 (8x4 ) design . t)e criteria 

adooted is the number of metaphase I emer . ;,ed under every 

1J'!,) fields of hi„h power version . In the sen - .' r :rt :~.' . 

experiment,Tl,I mice were treated with :Zitomycit . J and then 

sacrificed separately after 1,3,j,9,9 t.nd 11 days,the aberration 

frequency of 100 metaphase I was an,3ljsed of each 

,nouse . The result demu .sir :.tnd that no stu;istical si ;;nificant 

effect upon the frequoncy o° ^let-tphase i were found 

b,j tlte three factors mentioned sbove and -ire-itive result 

could be abt-,ined it 5-1} d--i,is n-fter treltmer.t,but the hi ;-hest 

r-~te o f chroc,osor :r_il .berr .ition is found at tce 1 1 th dkj . 


THE RESEARCH OF LETHAL Nl!TATION OF 

SODII'N FLUORIDE PSI\G SEX-LIM1'KF .D RECESSIVF LETHAL (S1 .RL) 

B .Q .Xieo, N .C,2hu and Q .X .2hang 

Guuugzhou Health and Anti-epidemic Station,Guengzhou,GuangDong,China 

F :uurirle is an e>sential element for human beings,overta{ .ing and undertaking 

wilt rarnse some tadr effect .Nany countries have increased fluoride content in 

drinking water . The purposr of this research is to determine if fluoride can 

imrse leUral mutntion under certain dosage .lhe test use two strains of Drosophiln 

melauognster, Oregon K and Ba>r .The e .

~ 

test,in vivo gnotQxicity test, we found th t GMA i eley tt ~{' the 

sper. bner itY lreq p¢Y t e Qer . 1~ f∎i ~lasal~ P~3?2 

~andoa~y ~~~ed ~ithiM an~ transere~ to np1~for ~~n~ orsat on . 

e re at we trans qrsat on e f1c'e cy ease0 rev a s 

~ resp nse rel t ~sh~D, nt~~o~ T A .p ic[ ~ stent 

sTel racy li e nsqiti e)c~and Ac~~T~r~v th /n er~ta~iS~ty F)ave n 

rhich terd through an~jt otrect g bas~ of ~aaase a~e~&s o luandsinCuce~ieet of terharor-ID4rlonel ~eD~ayr utesen 

~ 647 

aN ToXICITY AND t41fAMMITY OF CFDtCftII[M RICH BR6TaER's YEAST 

Li Xili, Bai Cheng Jiang and Shen Juen, Div'n of Toxicology, Tianjin Medical College 

Schwarz (1957) has postulated that trivalent chromium is an active eonstituent of 

glueose tolerance factor (GTF) . Mertz (1969) denoted that animals with a deficiency 

of trivalent chromium could result in damage of glucose tolerance or develop 

diabetes, hyperlipemia and arteriosclerosis . The supplementation of chromium is 

able to invert or prevent the pherxmena above mentioned . It is reported by China 

Air Force Hospital that a supplesre .nt of 100mg/day of trivalent chraniunrrich yeast 

to patients of diabetes and hyperlipemia induced an evident inprovecrent of glucose 

tolerance and significant decrease of plasma lipids after several months . The 

purpose of the present paper is to study the probably toxicity and nutagenicity of 

of chrenium rich brewer's yeast . The results show that the W50 of chromium yeast 

in rats and mice are all above 21 .5 g/kg, categorized as non-toxic and obtained 

negative responses to micronucleus test, muse-sperm tmrptalogy test, Ames test 

(TA98, 100, 97, 102), Reo-Assay, inductest and chromotest of SOS systan . We consider 

it to be acceptable to use brewer's yeast as carrier of trivalent chromium 

to be the source of chromium supplesnentation for certain populations deficient in 

chromiun . 

648 , 

TRANSPLACENTAL GENOTOXICITY OF TRIETHYLENEMELAMINE, BENZENE AND VINBLASTINE IN HICE . 

S .C . Xing, X . Shi, Z-L . Wu, J-K . Chen, Z,Ona and W-Z . Whong, Division of Respiratory 

Disease Studies, National Institute for Occupational Safety and Health, Morgantown, 

WV (USA) A 

Transplacental cytogenetic effects of triethylenemelamine (TEM), benzene and 

vinblastine on maternal mice and their fetuses hsve been investigated in our 

laboratory . CD1 mice of 12-14 days gestation were exposed to TEM, bensene and 

vinblastine twice by intraperitoneal injection at a 24-h interval and sacrificed 40 

hours after the first injection . Maternal bone marrow and fetal livers (2 to 4) from 

each pregnant mouse were obtained for the micronucleus and the SCE analyses . 

Significant dose-response increases in both micronucleus and SCE following the 

treatment of TEM were found in maternal bone marrow and fetal liver cells . Benzene 

at the highest dose (1 .5 ml/kg) also caused a significant increase in micronuclei and 

SCEs in both maternal bone marrow and fetal liver cells . The data showed that the 

embryonic genotoxic effect of TEM was much higher than that of benzene in both 

genetic endpoints and that the frequency of micronucleus induced by benzene was 

higher in fetal liver than in maternal bone marrow cells . Vinbiastine, a spindle 

poisons agent, induced micronucleus formations but not SCEs . Micronucleus induction 

by vinblastine was 7 folds greater in maternal bone marrow than in fetal liver 

cells . All three chemicals showed cytotoxicity in maternal bone marrow cells, but 

not in fetal liver cells except TEM, which showed a weak toxicity in fetal liver 

cells in the micronucleus assay . These results indicate that TEM, bensene, and 

vinblastine are transplacental genotoxicants in mice . 

649 

DELETION SCREENING AT THE CH ~cSE HAMS E hprt LOCUS USING THE POL - 

MERASE C~AIN REACTION2 7~ . Xu~I , Y . Yu~~~, A .W . Hsiel, C .T . Caskey , B . 

Rossiter , R .A . Gibbs I . Department of Preventive Medicine and 

CommuTity Health, The University of Texas Medical Branch, Galveston2 TX 

77550 Institute of Molecular Genetics, Baylor College of Medicine , 

Laboratories for Genetic Services, Inc .,3 Houston, TX 77030 . 

We have developed a rapid screening method using the polymerase chain 

reaction (PCR) for detecting deletions at the hypoxanthine-guanine 

phosphoribosyltransferase (hprt) locus in Chinese hamster cells, CHO-K1- 

BH4 and V79 . DNA was extracted from the HPRT-deficient mutants and two 

primer sets were used to amplify 274-bp and 334-bp fragments containing 

the exon 3 and exon 9 coding sequence, respectively . The PCR product was 

directly analyzed by electrophoresis on agarose gels stained with 

ethidium bromide . Using this assay, we analyzed 39 independently derived 

HPRT mutants . Four out of ten spontaneous mutants showed deletions at 



Notes

224 1989 EMS Abstracts - f 


r - _ - . - 

F - 

Notes exon 9 on1y . UYtraolet-light-induced mutants produced predominantly 

wild-type amplific ion patterns (10/14), and X-ray induced mostly 

deletion patterns .15) in either exon 9 or exon 3 . These observations 

are consistent with previous data from Southern analyses . Deletions 

occur most frequently at the 3'end of the gene, suggesting the possible 

existence of hot spot(s) for deletions in this region . Supported in part 

by Laboratories for Genetic Services (LGS) and an EPA Distinguished 

visiting Scientists Award to AWH . 

COMPARATIVE STUDIES ON SEVERAL INDICES OF NUCLEAR DAMAGE IN HUMAN 

PERIPHERAL LYMPHOCYTES . K .X . Xue, S . Wang, P . Zhuo, G .J .Ma, Cancer Institute 

of Jiangsu Province, Nanjing ( China ) 

In order to use approperiately NAT and explore possibility of use of the test in 

human cells, authers used Y-rays as mutagenic and carcinogenic irradiated whole blood 

in vitro, prepared directly smears of isolated lymphocytes, studied comparatively 

dose-response relationship of 4 types of nuclear damage . main results of the study 

are given as follows : 

1 . In doses from 0 to 3 Grays MNF ( freqency of micronucleus ), INF ( freqency of 

Irregular nucleus), KNF (frequency of karyorrhetic nucleus), anC PNF ( frequency of 

pyknotic nucleus) increase along with increase of irradiated doses . At 5 grays INF 

still increases, but MNF,KNF and PNF decrease . 

2 . In doses from 0 to 3 grays regression equation of 4 indices of nuclear damage 

are as follows ( D means dose,r means correlative coefficient) : 

MNF : Y-0 .3433+0 .1052D,r-0 .9128,p 0 .05 ; INF : Y-7 .2178+2 .1817D,r-0 .8846,p 0 .05 ; 

KNF : Y-0 .6462+0 .2014D,r-0 .7286,p 0 .05 ; PNF : Y=0 .2774+0 .0728C,r-0 .5484,p 0 .05 . 

Finally according to synthetical analysis of correlative coefficient, intercept, 

regression coefficient and feasibility of indices of nuclear damage,we could 

consider it suitable that nuclear anomalies in human peripheral lymphocytes include 

I' .'ir,INF and KNF . 

650 

651 

SISTER CHROMATID EXCHANGES INDUCED IN PERIPHERAL LYMPHOCYTES OF F10RXERS EXPOSED TO LOW 

CONCENTRATIONS OF STYRENE . J .W . Yager, S .M . Rappaport, and W .M . Paradisin, University 

of California, Berkeley, CA (USA) 

Following inhalation, styrene is absorbed and metabolized to styrene-7,8-oxide (SO) 

which is mutagenic in in vitro test systems ; SO also induces sister chromatid exchanges 

(SCEs) and chromosomal aberrations in human lymphocytes in vitro . However, 

previous occupational studies have not shown increases in lymphocyte SCES of workers 

exposed to styrene at 8-hour time weighted average (TWA) concentrations below about 

40-50 ppm . In this study, relevant exposure data and biological measures were obtained 

longitudinally during one year at a boat manufacturing facility that utilizes styrene 

as a solvent in preparation of reinforced plastics materials . 8KC Model 530 solid 

sorbent diffusion badges were used to measure full-shift breathing zone styrene concentrations 

up to 7 times over the year on the same 46 individual workers . Styrene in 

exhaled air was also determined up to 3 times per day . Blood was obtained for SCE 

analyses ; 80 cells were scored per sample . Health and occupational histories were obtained 

. Mean TWA exposure was 64 .2 m/N3 t 71 .5 (or 15 .1 ppm * 16 .8) styrene in air . 

Overall mean SCE was 6 .41 t 0 .96 per cell (rangee 4 .73 - 9 .47) . Smokers comprised 

54% of the population and smoked an average of 9 .1 cigarettes per day . Statistical 

analysis utilizing a bivariate linear regression model indicates that both smoking and 

exposure to styrene contribute significantly to an increase in SCEs (P < 0 .000011 A~ 

0 .62) . Most strikingly, it can also be shown that these two factors contribute to the 

increase in SCEs relatively free of the influence of one another . Results indicate 

that SCEs may be induced at rather lower styrene exposure concentrations than have 

previously been reported . 

652 

THE ROLE OF GAP JUNCTIONAL INTERCELLULAR COMMUNICATION DURING MULTISTAGE CARCINOGENESIS 

Hiroshi Yamasaki, International Agency for Research on Cancer, 150 cours Albert Thomas, 

69372 Lyon cedex 08, France . 

An important role of gap junctional intercellular communication is to maintain 

homeostasis of cells within a given tissue . Since carcinogenesis involves the breakdown 

of homeostasis in the tissue, it is conceivable that altered gap junctional 

intercellular communication is involved in the multistage carcinogenesia process . A 

possible role of blocked intercellular communication during tumor promotion is strongly 

supported by the fact that various tumor promoting agents inhibit gap junctional communication 

in cultured cells . We have recently extended this finding to in-vivo ; the 

administration of liver-specific tumor promoter phenobarbital reduced theeveT of gap 

50869 3738

junction protein mRNA in liver, but not in the kidney or in the stomach . Gap junctional 

co®unication also plays W important role in the behavior of cancer calls . These 

cells can attain inhibition of intercellular communication with surrounding normal 

cells by one of two different vays : 1) Decrease of intrinsic gap junctional intercellular 

communication among themselves ; 2) Maintainanca of their intrinsic intercellular 

communication capacity, but with selective inhibition of communication with 

surrounding normal cells . Taken together, available evidence suggests that altered 

gap junctional intercellular communication plays an important role in the process of 

carcinogenesis and the maintainance of the phenotype of tumor calls . Supported in 

part by NCI Grant No . ROL CA40534. ' -- 

653 

COMPARING THE FREQUENCY AND SPECTRA OF MUTATIONS INDUCED WHEN DNA CONTAINING 

COVALENTLY-BOUND RESIDUES OF POLYCYCLIC AROMATIC CARCINOGENS REPLICATES IN HUMAN 

CELLS . J .-L . Yang, M .C .-M . Mah, V .M . Maher, and J .J . McCormick, Carcinogenesis 

Laboratory, Michigan State University, ast Lansing, MI (USA) 

To gain insight into the mechanisms by which carcinogens induce mutations in 

human cells, we are using a shuttle vector, pZ189, carrying a bacterial suppressor 

tRNA (juRF) as the target for mutations Induced when the plasmid replicates in 

human cells . We have treated the plasmid with 7,8-diol-9,10-epoxide of 

benzo[a]pyrene (BPDE), 1-nitrosopyrene (1-NOP), and N-acetoxy-2-acetylamnofluorene 

(N-AcO-AAF) and determined the frequency and spectra of mutations induced . BPDE 

binds principally to the N2 position of guanine, 1-NOP binds to CS guanine, and 

N-AcO-AAF forms deacetylated AF adducts at C8 guanine . To obtain AF adducts In 

vitro, we used the trifluoro derivative . Each agent caused a linear increase in 

the frequency of supF mutants as a functi Qn of DNA adducts formed, reachi qg 

frequencies as high as 20 x 10'4 to 40 x 10-4, above a background of 1 .4 x 10-4 . 

When compared on the basis of adducts formed, BPDE was 4 times more mutagenic than 

the other 2 carcinogens . This difference may reflect intrinsic differences in 

the nature of the adducts and/or their location in the gene, but may also reflect 

differences in rate of removal of particular adducts . The majority of mutants 

from untreated plasmids involved large deletions or insertions . Almost all of 

those from treated plasmids involved base substitutions, mainly ` G-C T•A 

transversions, but each agent produced its own spectrum of mutations . The hot 

spots for mutations could not be explained merely by hot spots for adduct formation . 

(Supported by NIH Grant CA21253 and by Contract #87-2 Arom the HEI .) 

654 

MUTAGENESIS BY DNA CROSSLINK PRODUCED AT MULTI-CLONING SITE OF pUC19 

Fumio Yatagai, Sumiyu Hachiya, Kiyomi Nakajima (The Inst . Phys . Chem . 

Res . Saitama351-01 JAPAN) and Barry W . Glickman (York University . 

Toronto M3J 1p3, CANADA) 

To elucidate mutation induced by site-specific DNA damage, we selected 

muticloning-site of plasmid as the target . This site seems relatively 

insensitive for selecting base substitution events, but it is very 

useful for the detection of frameshift, deletion, and rearrangement 

events . Following the treatment of pUC19 with UVA (365nm, 

1 .5mw/cm2)plus 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT, 50 

ug/ml), the 51 bp of EcoRI-HindlII fragment containing the crosslink 

was recovered from a 20% denaturating polyacrylamide gel . This fragment 

was ligated into the large EcoRk-HindlII fragment (2635bp) and 

used to transform E,_c411 JM105 . Amp transformants were rtecovered 

using pre-UV (254nm, 5J/m ) irradiated E~ DII11 host . TrAnsformation 

efficiency was about 13% . Approximately 5 .6% of the Amp transformants 

recovered following UVA plus HMT carried i ;gZ mutations . Dideoxy 

sequencing analysis of these mutations revealed three classes : 

1)minus T frameshift at the EcoRl site, 2) deletion of 302 bp (315- 

616), and 3) rearrangements at position 186 (involving intact 51bptarget 

sequence) . These results indicate the involvement of crosslinks 

in the induction of these SOS-dependent mutations . 

655 

SiUffiES ON THE GENOTOXICI'IY OF DLSIr'FFCI'ANTS WfA-I SOS CMOMOTFS'f 

Yin Muquan, Qxn Yao[u, WangAng 

Departma:t o[ Toxxx:Iogy, Second IvFititary Medical Ucriwrsty, Shanghai, China 

The 906 O:ramotest is a sapd, teqiricing os+ly a singlo stnsin and a'mp1e oolotanctric enzyme as~ay, 

and it doea nottoqwrc survival of tho testa stn~ thus it ooud be wed to d'etact the patooddty af 

di9nfoctanl In the peraent peptr we sttxjiod the grnoto)ddty of 14 disinfoctants utdud~ng 

fm :mldchyd4 glutaraidehYde, ethylaie oxide, sodium diclilaoisocyanurate, peraoetic add, hydropen 

prioxid5 etltianoi, isoprophl aloohol, carbobc add lysd brarno 8asttainum, wdi= 

diddon :socyu :urate mucUmq fungieide and Giangtistt using the SOS dromotest . RasuNs Bnta sttdy 



Notes 

n

226 1989 EMS Abstracts . r -- 


Notes ~' ~~~ts trataa in uris wcpavnent hare no lrigher galactas~e acbviry . 

Among thrac 14 oa1y ethy~e oXdde and C3iangMu gaw posihee nsults We ddmod th 

tcst campo~md to bo ; wkn tk fl-galactaddase activiry was ineeavod 2-toid over ahe beckgro~md 

lerd Ths • rado of ethylrne obdo was 2.39 at ooooernradon 800 ppm in PQ35 witlr 

out metabolic anivation mix4ae (S9 ma) . Asd Cuaaglisu was 2 83 at oon=tration 19 ;q/ml in 

PQ37 witYwut S9 mhc and 2 43 at mime oonornuation in PQ3S with S9 mix cf the nr 

ahs fmm SOS ctuernotat and Ames tat mealod a strrnhg aoncadanoe• S~~vai advan• 

tapes of SOS Chromoteat was discuseod 

dIii; ASS 3S : :T :T 0" 37 :0 .'^ :;TC": I^.As4 :'lIT'T. C-RO:`.'1S(1a,.^ 

. . 3 1, 177 :r:~l' 113 

}~„ - :iu-12.n, ,ao rin, V;, S•'Ao-lin, '.'!is,, -''t,e 

-ie$e^rQl, I,' .~i 1 1ate, "Ile ;'inina 1'v Oo C},oc' 

1 

._ ^.C' . . , . 

Ii? 0?.°êr i:o 4et v - .I 2. n^l4 T94e3R^,en' om 4tn n' n" y p .Pfef`•I S 7f 

~ ;oto ctc c'. :-c^ 1- -)., -e?°r, ce71 e, r me-od for r, r• n nn^-.p nc+ PT 9ri^Cp- 

ìo, of 7o+e^ i.^ .,ouê . . . '!eqcr-ihe•' '•i^ ciôcqAt,r.P tG 'fec'ive 

ò n ^,s,re a. '-i~'• m•ccesa ra+-e of ~ooA ^1i.ren of c rn~o~o~e, 

306 net-'-a.îc e,!- ^niteb'e forr o'-roTn-oê proprrr'i•on tn 774 . 

one-cetl z7r^;ote^ ar-re o`)tînei . ^.hro-noso•e3 of 754 cPt_1-s in 3nA 

e^Tr ca'1 be •,nnt•r?c4e c' .rn ;oô~P î ;des -,•er-e al-ô * . ~e i.n'o ^nrt ^--ba-+A 

for ^n^1 .^, ; .s . 'ex c-•-°o .,oso e C'r,,lt?,-en.' fCr r,^'•tnq in "e 

9 :•C(: a? •r . .-e'1•vle•le-1)tc1 ,4-•, .i',,tt,2nlot 

J'. ;'S3iA-p i,i-Ct .Y,7„~~_o . • .n, .,_ranuinone A .rP.^.•'.U .f.f ;, C^~ .'ti10n 

l. e-r^.7.v.p ' . :el A+t' t'v'ic net'.•o'i , 

n-.-e1i,i•.•^r,r t',e -e .IôO i .s recor 

;~end:ri o. .e of evn1'ie ., :nz mr' :z--en 'o •cerw cells of ma-melian 

. 

656 

THE EXTREMELY POTENT CLASTOGENS, U-73,975 AND U-71,184 IN IN VIVO INDUCTION OF 

CHROMOSOMAL ABERRATIONS IN BONE MARROW CELLS OF CD-1 MICE . R . Yu, C . Aaron, 

S . Wiser and N. Wicnienski . The Upjohn Company, Kalamazoo, Michigan, USA . 

U-73,975 is a member of a novel anticancer drug family under development by The 

Upjohn Company . These materials are structurally related to CC-1065 and have been 

found to be excellent bacterial and mammalian mutagens and exhibit highly selective 

binding to AT regions of DNA (Envir . Mol . Mutag . 11, su pl• 11 . 2, 1988) . U-73,975 and U- 

71,184 were administered in a single I .P . dose at the folPowing doses : U-73,975, 1, S and 

10 ug/kg : U-71,184, 3, 6 and 10 ug/kg . Three animals per sex per group were treated with 

U-73,975 and 5 males per group were treated with U-'1,184 . 24 hours after exposure, the 

percent of cells with chromosomal aberrations were as follows : 

U-73 975 Male F ~inaie U-71 1 4 Male 

u-g`~ ~:6-t 3.5 _5.3 t 3.1 • 3 ylky T3Tt 2 .3*** 

5ug/kg 16.0t4.0*** 31 .3± 6.4*** 6ug/kg 12 .41 4 .6*** 

10 ug/kg 34.7t5.0*** 45.3t12.9••* 9ug/ky 19 .2t10 .3**• 

The percentages compare with untreated control frequency of about 1 X and a mitomycin 

C(MMC) induced frequency of 60-70% at 3 .5 mg/kg . Thus, these compounds are several 

hundred times more potent than MMC in this model . There were decreases in 

chromosomal aberrations observed with Increasing time of exposure . In summary, the 

compounds are potent chromosome breaking agents in rodent bone marrow cells, a 

property shared by many anticancer agents . This study is part of a broader cytogenetic 

evaluation of CC-1065 analogs (* P

prevented by ADPRT inhibitors,3-aminobenzamide or nicotinamide,which were shown to 

exert no influence on,the NAD lowering due to NAD biosynthesis blockade . (4) In pnaphthoflavon,a 

c tocFrome P450 isozyme inducer,induced or unindnced human amnion 

FL cells,AFB1,B(a~P,pyrene,2-AAF,9,10-dimethylanthracene and sthylcarbamate could 

induce the decrease of cellular NAD mediated by ADPRT aotivation,4-4.LF,anthracene, 

isopropyl N-(3-chlorophenyl)-carbamate,(i-propiolactone,Y-butyrolactone,cyclophosphamide 

and safrol couldnot . This preliminary validation results indicate that this 

new assay is specific for detecting DNA damage with a sensitivity and specificity 

at nearly the same grade as well va idated UDS assay with rat liver S9 system for 

metabolic activation of p,romutagens~p_rocarcinogens . 

659 

INITIATION OF CHEMICAL CARCINOGENESIS A ND HYPOMETHYLATION OF CELLULAR DNA REVISITEDe 

Y .N .Yu,Z .Z .Shi,and X .R .Chen,Zhejiang Medical University,Hangzhou(China) 

•The Project supported by National Natural Science Foundation of China 

The extent of enzymatic methylation of newly synthesized DNA labelled with 3H-TdR 

prior to harvesting in human amnion FL cell line after carcino en (two nongenotoxic, 

5-azacytidine and L-ethionine and two genotoxic, MNNG and AFB1~ exposure wes analyer 

ed by the digestibility with restriction endonuclease HpaII . The weight average 

lengths (Lw) of HpaII digest of the DNA isolated from the -azacytidin . (2 x 10-6M 

1-day exposure, DNA isolated at the 5th day after exposure) and L-ethionine (2 x 

10-3M 9-day exposure, DNA isolated at the time of the end of exposure) treated cells 

were significantly lower than those of the controle (Lws8 .0 • 0 .1 kb va 10 .9 i 1 .Okb 

p< 0 .01 and 9 .8 . 0 .3 kb vs 10 .6 + 0 .3 kb p< 0 .05 respectively) . While those from 

the cells exposed to MNNG (5 x 10-6M 1-day exposure, DNA isolated at the 5th day after 

treatment and AFB1 (2 .5 x 10-5M 1-day exposure, DNA isolated at the 6th day after 

treatment) were not different from those of the controls (Lwt 11 . a 0 .7 kb vs 

10 .6 . 0 .8 kb and 10 .6 + 0 .5 kb vs 10 .4 + 0 .7 kb p> 0 .05 respectively~ . Our results 

suggest thpt the stable and heritable alteration of inethylatlon patterns in cellular 

DNA may be a specific mechanism of carcinogenic initiation by some, but not all 

kinds of carcinogens, and the hypomethylation of DNA frequently seen in tumor tissues 

may be formed during the process of initiation as well as promotion and/ or progression 

phase of carcinogenesis. • 

660 " 

BACILLUS SUBTILIS RED-ASSAY AND MUTAGENICITY DETECTION OF METAL COMPOUNDS 

Wang Yuzhi and Tang Tianbao ; Shenyang Res . Institute of Industrial 

Hygiene and Occupational Disease, Shenyang, P .R . CHIA 

The bacterial systems have become efficient and sensitive methods 

for screening mutagenicity and carcinogenicity of chemical compounds . 

in this study, B . subtilis Red-assay was selected to test metal compounds 

with Hiran's spore method . The spores of Rec+ and Rec- strains 

of B . subtilis were prepared in agar and a paper disk, impregnated 

with the chemical solution placed on the surface . After 20 hours incubation 

at 37 C, diameters of inhibition zones are measured with each 

strain . A difference of -3mm is a positive effect . In order to 

compare their sensitivity, the spore and vegetative cell rec-essays 

were performed with chmicals and metals with or without S9 metabolic 

activation . The results show that use of spores in place of vegetative 

cells increased the detective sensitivity 7- to 10-fold . In 24 hours 

incubation at 37°C, BeSOq and V205are negative, but in incubation at 

4°C for 24 hours and at 37°C for another 20 hours, positive results 

are obtained . The B . subtilis spore rec-assay is one of the systems 

which is least expensive, rapid and non-laborious . 

661 

OBSERVATION OF THE EYES IN 53 WORKERS OCCUPATIONALLY EXPOSED TO METHANOL 

SuJuan-Zang . 

Research Institution of Labour Health Sciences in LiaoNing (China) 

The methanol is a solvent of chemical industry . Acute poisoning of 

methsnol may injure the nervous systems and the eyes . Harm of the eyes 

is retrobulbar optic neuritis, optic neuritis and intraocular neuritis . 

cases of acute poisoning of methanol have been reported, however, obser_ 

vation of the eyes in workers occupationally exposed to merhanol of lower 

concentration and long term contact are rather rare . In this paper 

contrast of'the eyes check in 53 workers occupationally exported to me - 

thanol and 49 workers not exposed to it were reported .The lowest concentration 

of methanol in air of the manufacture workshops of Dawa Chemistry 

Factory in Liaoning China is 1 .2rng/m' and the tallest concentration 



Notes

228 1989 EMS Abstracts K~J 

Notes is 165 .5/m: Chockjft-ophthalmology include history,. functional examination 

of the eyes and physical examination of the eyes . The check results 

of these two groups are similar(P :Q,05) . Because every worker will 

change this work frequently, the contacting time, to methanol is short 

for every one, the eyes of workers occupationally exposed to methanol 

do not are harmed . 


STRATEGIES FOR THE IDENTIFICATION OF RODENT CARCINOGENS BY IN VITRO 

SHORT-TERM TESTS . Errol Zeiger, Cellular and Genetic Toxicology Branch, 

National Institute of Envlronmental Health Sciences, Research Triangle 

Park, NC, 27709 (USA) 

The effectiveness of four short-term tests for genetic toxicity 

(induction of mutations in Salmonella (SAL) and mouse lymphoma L5178Y 

(MLA) cells, and induction of sister chromattd exchanges (SCE) and 

chromosome aberrations (ABS) In Chinese hamster ovary cells) for 

predicting rodent carcinogenicity was examined . A total of 114 chemicals, 

including 73 previously evaluated, that had been tested for carcinogenicity 

by the National Toxicology Program were evaluated . The results with the 

114 chemicals confirm and extend the results previously reported with 73 

chemicals (Tennant et al ., Science 236 :933-941,1987) . Salmonella had the 

lowest sensitivity ( .48) and the highest speclficity ( .91), whereas mouse 

lymphoma had the highest sensitivity ( .70) and lowest speclfictty ( .40) . The 

concordances of the tests was .66, .61, .59, and .58, for SAL, ABS, SCE, and 

MLA, respectively . Salmonella had the highest positive predictivity ( .89) . 

None of the other tests complemented Salmonella, and no combination of the 

four tests was more effective than Salmonella, alone, for predicting 

carcinogenicity . 

I9HIBITION Od Tr:6TICUlrin DNA SYNTNiSIS BY CNchIOAL MUThGc:NS 

8ui-Juan Zhang, Ghi-iien We1 and Yu-Zhen Zhu 

Shanghal Institute of Yharmaceutical Industry 

1320 Beijing Xi 8oad, Shanghai, China 

Friedman and staub reported the measurement of the inhibition of testicular DNA 

662 

663 

synthesie by chemical carcinogens and mutagens in sale mice and proposed this method 

as a simple and effective in vivo mammalian screening test . The testicular tissue of 

the mice is capable of utilizing the salvage pathway efficiently when synthesis of 

thymidine is inhibited . It is possible to distinguish between DNA-dasaging agent and 

non-DNA-damaging agent . We now report on the results obtained with eight chemicals . 

Thio-Tepa, Cyclophosphamide and Adriamycin were DNA damaging agents . Fluorouracil and 

Methotrexate were non-DNA-damaaging agents . Fluapyrimidone, Aurapromide and Nonylphe- 

noxypolyethoxyethanol showed no evidence of genotoxicity . 

664 

THE EFFECT OF 5-AZhCYTIDIHE ON REVERSION 0F ISOLATED HPHT MUTANTS IN V79 CHINESE 

HAMSTER CELLS . L-H . Zhang and D . Jenssen, University of Stockholm, Sweden . The 

purpose of the present study was to investigate rare types of spontaneously 

occurring mutational events . Reverse mutation analysis using medium Containing 

L-asaserine (HhsT) of 60 independently isolated HPET- mutants from four groups, 

11 spontaneous mutants, 11 EMS-induced mutants and, 38 1010-induced mutants with or 

without hydro :yurea pretreatment, showed that over 70% of mutant clones of each 

group could be reverted either spontaneously or induced by treatments with Ei1U, 

ICS191 or S-asacytidine (SAC), indicated to be caused by point mutations . Two of 

the revertible mutant clones of spontaneous origin wer* found to be resistant to 

HAT but not HA&T medium . The interestinq result of this study was that these two 

eTGrHATr mutants were the only mutants isolated which could be affected by SAC 

with a significant increase in the reversion frequency . Chromosome aberration 

analysis did not indicate any enhancement in aberration frequency in the 

X-chromosome by 5AC treatment . Studies on the mutagenicity at 00k-locus indicated 

that the SAC- and ENU-induced mutation frequencies in these 2 mutants were 

50869 3742

comparable with the effedEa in the parent wild type cell line . Their cellular 

incorporation of H3-hypoxanthine was enhanced in the presence of aminopterin, 

but decreased with L-asaserine indicating that they were PRPP mutants . According 

to these results, we suggest that reversion of these 2 6TGrAATr mutants may 

occur by a gene amplification mechanism and that this process may be facilitated 

by 5AC treatment . 

665 

MUTAGENICITY AND CARCINOGENICITY OF NITROSATED FISH SAUCE 

R .F .Zhang, D .J .Deng, Y .Chen, H .Y .Wu, S .Jin, S .X .Zhu and Y .P .Liu 

Beijing Institute for Cancer Research, Beijing, China 

Fish sauce, a traditional daily use seasoning, was collected from 

Changle county in Fujian province, where the mortality of gastric cancer 

is the highest in China . To evaluate the risk of taking fish sauce for 

the high incidence of this disease in that area, mutagenicity and carcinogenicity 

of the fish sauce were stidied . By the Ames test(S . typhimurium 

TA100) and in vitro SCE and micronucleus tests in V79 cell 11ne, 

direct mutagenicity(-S9) appeared in the ethyl acetate extract after 

nitrosation with NaN02 . A dose response relationship was obtained . The 

highest response was observed in the extracts of acidic nitrosation(pH 

2 .0) and the samples from the villa e with the highest mortality of 

gastric cancer . After 0,1m1 extract~corresponding to 1 .Oml of nitrosated 

fish sauce) was given to each of the studied new born Wistar rats by 

gava e for 3 consecutive days, marked dysplasia was observed at the 4th 

week~in 5/5 rats) and adenocarcinoma developed(in 1/5 rat) at the 16th 

week in the glandular stomach . It indicated that some nitrosable precursors 

of direct-acting mutagenic/carcinogenic N-nitroso compounds must be 

contained in the fish sauce and it may play important role, after nitrosation, 

in the causes of gastric cancer and its precancerous lesions of 

stomach in Changle county residents . Detection of these compounds is now 

undertaking. f 

666 " 

A METHOD TO GET THE BACTERIOPHACE SPOT CLEAR IN INDIRECT SOS TEST 

Zhao Zezhen, Wel Lirhent Haans Mlntlt Hebet Cancer Instltutes P .R .China 

It has been provod that, among oe many methods te check the mutagens 

In environment, Indirect SOS Test, I,e, bacterlephage Inductlon methed, 

Is a quick, sensitive and reliable ene . The methed is te mix CY6027s a 

lysesenic bacterlat with GY4016s a Indlcatin{ bacteria which Is sensitive 

to the bacterlophage released by Gy60279 In a sub-solid culture 

medium according te a certain preportlon . While meeting mutatens, the 

lysogenic bacteria wlll release bacterlephase dissolution indicating 

bacteria, then bacterlophate spat will appear on the culture medlum 

which Is surrounded by muddy bacterlafur, However, the centrast between 

bacterlophage spot and bacterlafur Is net cloar enough te observe and, 

especlally, to take photo or make lantern slide . In light ef the prlnciple 

that, in the course of grewthr colour bacillus Gy4016 and Gy6027 

can break down glucose and produce acid which can make the Indlcater 

change colour . We make an improvement on the original methed, that los 

add 2m1 of 2% disinfected (low pound) glucose selutlen and iml of 0,6% 

disinfected bromecresel purple solution te 60m1 of ineiting sub-salid 

culture medlumq then put the two test bacteria liquid to the mixture 

selutlon, which will make the culture medium chan{e to blue coleur . 

Other test steps are simillar te the original, Comparing the new method 

with the orisinalt the new test results show to Increase the contrast 

between bacterlephage spot and bacterlafur . And though It, we have succeeded 

in taking phste and making lantern slide, 

667 

OBSERVwTION OF THE EYES ON FIFTY WORKERS OCCUPATIONALLY 

EXPOSBD TO DIMETHYL SULFATE 

rien~iang-Zhao .Electricity centre Hospital in NE China .Shenyang(China) 

ime.thyl sulfate is an important raw material of chemical industry . 

Its decomposing products methanol and sulphuris acid may impair cornea, 

conjunctiva etc . Cases of acute poisoning of dimethyl sulfate have 

been reported,however,observation of the eyes in workers occupationally 

exposed to dimethyl sulfate of lower concentration for a long time 



Notes 

229 

" : . 

I 

t

230 1989 EMS Abstracts . . .- -- 

Notes 


.._~ ,.mercontacting 

is rather rare . In this paper,the involving worker include 

35 males and 15-41 ips,and the control group includs 40 workers no 

exposed to it .Coricentration of dimethyl sulfate in air of the manufacture 

workshop of Petroleum Chemistry Factory in LiaoNing in Ghina is 

2 .89-o .07mg/um .Check of ophthalmology include history,functional examination 

and physical examination of the eyes .In history,many involving 

workers have vision blurred,eyepains,lacrimation,photesthesia etc .The 

most obvious sign is conjunctival congestion and it resed with contacting 

time .By statistics,vision blurred and conjunctival congestion of 

the workers occupationally exposed to . .dimethyl sulfate are higher than 

other workerslPc,0,05 and P

nutber of surviving fetuses decreased significantly with increases in irradiation 

dose, while resorption rsbes increased . Vfien miee were irradiated for 5 consecutive 

days during the preiu+plantation period, the number of surviving fetuses was less than 

in controls but litter weights increased faster within one month of birth, and mice 

fran treated groups remained larger than those from control during 1h years observation 

. The carcinogenic effect of irradiation to embryos showed an incidence of 

leukemia 4 .9 times higher in treated versus control animals . Studies of irradiation 

induced malformations and carosnogegenesis were also conducted by observing inmediate 

and late effects of single or fractionated low dose irradiations . The nssnber of 

living fetuses was significaritly reduoed'with increasing radiation dose . The frequency 

of rnalfornmations in external appearance increased with radiation doses . The imnediate 

ef :ects in the single irradiation group were different fran those in the fractionated 

irradiation groups . In general, all effects fraa single irrad3ati .ans were more significant 

than those in the fractionated irradiation group, except for the frequency 

of cranioschisis . Litters of mice in the fractionated irradiation group were observed 

for late effects for 2 years . Litter weights were lower than in controls and the incidenee 

of leukania was 2 .5 times higher in the treated group . Late effects were also 

studied after 50R of irradiation given at 13-17 days gestation . Litter weights were 

lpwer . ThP incidence of tumr was 8 times higher . 

671 

L .-T . ., ..F n, T', ;="'T~?TY nF L' CTDI!(- : 

Lhû '-hu-,`Zhan~ J+a, -5{a Shu-Zheng, Lfa~Cu+-e,?nd L+an X+n,-3uan ; 

health and ;nt+-ez+damjc Center -f Henan =r-v{nce, Zhan3zh-u, P .RC . 

Jan-derni ]uc+dun, kjn•.i -f tra~+t+-nat Ch+ne :e med+c+nes, had been 

used as a t-n+c f-r th-usands -r" ye,rs . T- evaluate tha Wh-les-mene~!s -f 

3d•±


. . . :-- . 

NOtE'S immunosuppresive agents . Two to three drops of blood were taken from the ear lobes of 

examined persons . TM&,blood was then incubated with 1 .0-2 .0 ml Tris-ammonium chloride 

buffer solution 'to'renxTlyze red blood cells . Monolayer cell smears were made with 

cytocentrifuge and Feulgen stain was applied to detect DNA content . The morphological 

observation and micronuclei counting were done with light microscope . We found that 

the frequencies of micronuclei of lymphocytes in healthy persons were 0 .82 per 

thousand cells but in patients with esophageal carcinoma were 2 .88 per thousand . The 

difference of frequencies of micronuclei of lymphocytes between healthy persons and 

patients with esophageal carcinoma was statastically significant (PA0 .001) . The 

method we used to detect micronuclei of lymphocytes was fast and reliable and easily 

accepted by patients examined . 


674 

4IT' STU7Y i :+CCU' ; L :^_ IO° OF 2.L' :1 .. . . : IN Gr'.Rh: CSLLS ON INDUCTIi . :i 

OF ~.lDICG-_? ;~TC7.L^.OLCGICAL 3F :^r:CT . 

Zhu Bhoup n~, heov Siyinq, -ad lP-n :^- Liuyi, Suzhou Nedical College, Suzhou(China) 

T~j purpose of the present study is to ascertain r,omp,?~rative retontion of 147Pn 

o„- Cs in qerm cells on induction of chromnsome aberraM ns of sparmatogonium and 

eb :iormall+.i .e in sperm . Results it .dicated that after iv Pm throu,h 50 d observstion 

. The rEtr.nt~oPC~~a~s in tastis were well describad by an exponential expression 

13~(t)=0 .1872xe- , where the retention ^=1C5 ( : . 'lhlle th3 .fat~~ntion data of 

Cs in t•?stiF w•er^ described by e-n expreswlofi : =tlt).O,OC15za , where the 

retention T, was on}X75 .2 d, 3xperimsnts indicated that the retention value aQJ4thc 

abs.irption ose of Pn i?A°a is ree sirnificantly high in comparism with C s . 

Internal conta.nins!ion of ?~ or Ce can be induced chromosome aberrations in 

germ cells and abnormalltiT17in spaj~b Atnona the type of chromosome aberrations of 

apermato-onium induc,~d bv _m or Ce includin- â~, chromatid breakage, chromosome 

brea ::age, and tranalocation, as woll as poly :'-oid spermatogonium . },oreover, 

the chromosoma aberrft3on rctt*s and pol,T,:loid cells were elevated when the periods 

of conta^:ination of Pm :rora prolon,? !d . ,1t th : semt_ t#" ehromoso:ae fragment and 

If~nalocations of primary spermatocyte also induced by Pm . By compaTign with 

:m, ho-ver, the induction of cytogenetic effects on germ cells by a wke 

quite low. 

675 

INDUC'TION OF MUTATION IN F-'aihcrichin cofi BY DIMETHYISULFATE IS INFLUENCED BY SOS : 

OBSERVA'11ONS OF THE MUTATIONAL SPF.C IFICI'IY OF DMS AND E7HYL ETHANESULFONATE 

IN WILD-TYPE AND UmuC STRAINS . Maria Zieknaka, Janet Smylie, Jiso Iian Li and Barry W . Olickman . 

Dept . of Biology. York llniversity, Toronto Canada M3J 1P3 . 

In this study we have undertaken to examine how mutagenesis resulting from EMS and DMS is 

influenced by the error-prone misrepair pathway of E. rofi . We Investigated the mutational specificity of 

these alkylating agents by DNA sequencing and oligonucleotide probing methodologies In the first 180 

base pairs of the Inrl gene of F. rnfi in wild-type and UmuC strains . Consistent with the established 

alkylating ability of these agents, the O :C _> A :T transition dominated the resulting spedra . EMS 

elicited an identical mutagenic response in the two strairo ; after treatment with 3mM KMS there was a 

15-fold increase in mutation frequency at 57% survivaL Mutational specificity of EMS in the l1muC 

background was identical to that found in the wild-type strain ; O:C - > A :T transitions accounted for 

over 96% of the mutational events in each spectrum and their distribution was identical . DMS 

mutagenicity decreased in the UmuC background from a 128-fold increase in mutation frequency at 14% 

survival (wild-typel to a 26-fokl increase at 6O/o sunival. The influence of UmuC function was also 

apparent at the DNA sequence level . In the wild-type background t3 :C - > A:T transitions accounted for 

74% of the mutations, which were alan characterised by a significant contribution of additional 

mutatiomd events including Ai r=- (hC' trnnsitkms, all classes of transverskms and framoshifu . In 

eontrast, after DMS treatntent in a UmuC strain 82% of all mutational events were (t :C .• > A:T 

transitions with a different site specificity from that recovered in the wild-type background. The other 

claases of events found consisted of A :T => T :A and C; :C - > T:A tramversions, frameshUts and a 

duplication. We conclude that mutagenesis by EMS Is independent of the UmuC function in contrast to 

mutagenesis by 1)MS which shrn.s a marked Umu inhtence . 

676 

DETERMINATION OF SPONTANEOUS AND ENU-INDUCED MUTANT FREQUENCIES IN 

CYNOMOLOGOUS MONKEYS . D .M .Zimmer, C .S.Aaron, R .J .Albertini, and J .P.O'NeiII. The 

Upjohn Co. and University of Vermont, USA . 

We have undertaken a study of mutagenesis In primates (cynomologous monkeys) to 

determine whether such an assay offers better means of risk assessment than currently 

used in vitro and rodent tests. Using methods similar to those described by Albertini 

(Mutat . Res. 150 (1985), 411-422J, mutation at the HPRT locus was monitored by 

determining the ability of PHA stimulated peripheral T- lymphocytes to form clones In the 

50869 3746


presence of 6-TG . Cells were grown in round bottom 96 well microtiter plates and scored Notes 

visually (microscopicall*l- .for evidence of cell growth . Spontaneous mutant frequencies 

(MF) for a colony of 9 animals (using duplicate samples taken aprox . 2 weeks apart) gave a 

mean of 4 .4 t 5.3/106 cells . The appearance of mutants in peripheral blood after an IF dose 

of 100 mg/kg ENU was followed in a single animal by sampling weekly for 9 weeks . MFs 

for weeks 2-9 were (respectively) 15 .3, 5 .0,16 .5, 9 .9, 20 .6,12 .9,19 .6, and 26.9/106 ;day 0 MF 

was 7 .1/106 cells . In general, both MF and cloning efficiency for given animals were 

reproducible from sample to sam le . TG resistant clones from all experiments are 

currently undergoing molecular analysis to determine the nature of the mutations giving 

rise to TG resistant clones in-cynomoiogoas monkeys . 

677 

AN INSIGHT INTO THE MECHANISMS DETERMINING THE INDUCTION OF GENETIC EFFECTS BY NTA IN 

THE MOUSE AND DROSOPHILA SOMATIC AND GERM CELLS . 

M . Zordanl, A . Russo', R . Costal, C . Beltra .el, F . Pacchierotti=, M . Ostil and A .G . 

Levis' . 1 Dept . of Biology, University of Padova (ITALY) . 2 Lab . Toxicology, ENEA, 

CRE Casaccia ROME (ITALY) . 

Recently we reported the ability of nitrilotriacetic acid (NTA) to induce 

aneuploidy in the germ cells of both Drosophila and the souse . These results prompted 

further research in order to evaluate the response to treatment with NTA of somatic 

cells in the same organisms . The experimental systems adopted consisted in : a) 

chromosomal counting in souse bone sarrow cells after i .p treatment with 138 or 275 

eg/Kg NTA ; b) evaluation of single and twin spots in the somatic recor,bint .tion and 

mutation test (SMART) in Drosophila selanosaster employing the &w_b e.nd (j=3 wing 

∎arkers, after feeding different doses of NTA (Sx10-3, 2 .5x10-2 , Sx10-2 K) for 42 

hrs . to 2nd stage larvae . In the latter case effectively absorbed doses of NTA were 

also evaluated by a .ethod employing 3H-leucine additioned to the treatr.ent sedts . 

The results indicate that NTA does not induce aneuploidy or sister chromatid 

exchanges in souse somatic cells . Positive results were obtained ir .utead :n the SMART 

test (statistically significant dose-dependent increase in the frepuen .:y ui small 

single p_wZi spots) . Extension of the wing spot analysis to include phenotypically AU 

individuals (inversion heterozygotes) revealed that small single spots are virtually 

absent in these flies following treatment with NTA, suggestiug that in the normal 

"w , flrs trans heterozygotes tbese spots originate mainly (90% or more) from 

recosbinational events . 

SUPPORTED BY C .N .R . p .f . "ONCOLOGIA" . 

A 

678 Due to late receipt, abstracts 678-697 are presented out of alphabetical order . 

CURRENT STATUS OF THE GENE-TOX PROGRAM, Angela Auletta, Office of 

Toxic Substance, U .S . EPA, Washington, D .C . 20460 . 

The EPA's Gene-Tox Program is a multi-phased effort to review and 

evaluate published literature in genetic toxicology . Phase I was a 

review by expert work groups of literature published from 1969-1980 

for 23 days . Each group published an evaluation of chemicals tested, 

correlation of results with carcinogenicity, recommended protocols and 

techniques for data analysis, interpretation and presentation . In 

1980-81, a computerized data base of over 2,600 chemicals was 

established ; this data base was analyzed by individuals concerned with 

chemical classification, carcinogenicity, and heritable mutation . At 

present, work groups are evaluating literature published since 1980 

for selected systems . As the update proceeds, the existing data base 

is reanalyzed . Analysis of the data base is affected by several 

factors . The preponderance of results are either positive or 

inconclusive . The high percentage of inconclusive results largely 

reflects the quality of the available literature . Most agents have 

been tested in only one system making cross system comparisons 

difficult . Correlations with carcinogenicity are hampered by the 

nature of the chemicals evaluated and the limited number of validly 

tested noncarcinogens in the data base . The data base is publicly 

available through the NLM TOXNET system . Public availability should 

increase its utility, expand the analysis and affect the manner and 

speed with which it is updated . 

~ 

679 

OD ~ 

DIET AS A SOURCE OF MUTAGENESIS t0 

P .S . Chauhan, Molecular Biology i Agriculture Division, Bhabha Atomic Research Centre, 

Trombay, Bombay-400 085 (INDIA) w 

Diet is a complex mixture of a large number of chemicals of diverse nature and ~ 

continues to remain a major source of human ex posure to exogenous chemicals . In ad- v 

dition to life support constituents, human diet contains a large number of chemicals 


Y


- . . . .r j- . .F0= a}r'- 


Notes which have been i'Nntified as toxicants and those whose biological effects are not 

yet known . While, literAfare is replete with reports showing a number of dietary components 

to be mutag'enit;^-=tirrent information on inhibitors of mutagenesis has been 

recently reviewed (DsFlora, ed ., Mutation Res . 202, No .2, 1988) . However, majority 

of the investigations on antimutagenesis vis-a-vis mutagenesis have emerged from bioassays 

using prokaryotic systems . While, this information provides a basis for further research 

and these assays are suitable for mechanistic studies, the results cannot be directly 

extrapolated to human system . In our laboratory, inhibitory effects of dietary factors 

including vitamins and trace elements, on chemical and radiation mutagenesis have been 

investigated in somatic and germinal cells of mice, These whole animal studies, though . 

support some of the observations emanated from prokaryotes, divergences are apparent 

between the two systems . It is pointed out that the ability of a putative inhibitor 

to survive the physiology of the mammalian gut, its absorption and availability in 

biophase would greatly contribute to its effectiveness in vivo . It is also evident that 

any programme on dietary prevention of genotoxicity wou~t-embark upon, removal of 

mutagens from the diet and incorporation of inhibitors of mutagenesis . Such a dietary 

reciepe, if ever formulated will have the advantage of allowing intervention at the 

community and public health level . 

ENVIRONMENTAL MUTAGENESIS IN DEVELOPING COUNTRIES 

C . cortinas de Nava, instituto de Investigaciones siomidicas, U .N .A .M . 

Ap . Postal 70228, Mexieo 20 D .B ., 04510 Mexico . 

The term developing countries encompasses a wide variety of situations 

: geographical, economical, politioal, soeial, eultural, ete . . A 

common denominator of those countries is the need to optimise efforts 

to stop environmental deterioration and diminish the impact of chemical 

pollution on human health and the eoosystems . This mplies the nesd of 

programs for integral evaluation of environmental pioblems, a~iequate - 

technologies for their control, a rapid multiplication ef 1ooa1 experts 

in the field, integration of multidisciplinary research groups and the 

community participation in activities of environmental protection and 

prevention of health risks . Genetic Toxicology in developing countries 

can only contribute sustantielly to these goals if it is incorporated 

to the global actions intended to set priorities,to characterize and - 

manage the environmental risks . Collaboration of scientists from develoM+d 

countries 1a needed to speed theet, aot•lons, 'in partieul+ir, thnna 

concerning training of personnel, development of technol0q ies and information 

exchange . In addition, the establishment of collaborative - 

research projects to evaluate and control environmental problems of - 

regional interest will make the efforts of scientists from devaloping 

countries, working on the field, more relevant . 

680 

681 

Transgenic mice as a model system for studying gene mutations in vivo . 

Jan A . Gossen, W .F .J . de Leeuw, C .H .T . Tan and Jan Vijg, TNO Institute 

for Experimental Gerontology, P .O . Box 5815, 2280 HV Rijswijk, The 

Netherlands . 

In order to study gene mutations in different organs and tissues of an 

experimental aniT .al, we constructed transgenic nice harbouring 

bacteriophage 1a^:bda shuttle vectors integrated in the qeno^ :e in a headto-tail 

arrangement . As a*.arazt fcr rnutagenesis, the selectable 

bac*.erial LacZ aene was cloned in the vector . The integrated vectors were 

rescued fror total genonic DN1, with high efficiency by in vitro packaging 

and propagation of the phages in an E .cc C Lac2- strain . This systex 

allowed the detection of :-.utation frequencies down to about 5 .10-6, the 

background frequency in different organs . Treatment of adult fenale 

transgenic :rice with fi- ..thyl-N-nitrosourea (ENU) resulted in a dosedependent 

increase of the mutation frequency in the vectors isolated fro^t 

brain D!tA, up to 10-1 at 250 :^g E1N per kg bodyweight . At this dose, in 

liver and bone -arrow Dtd:A of the sa :ê :r:ice, mutation freyu_ncies were 2 .9 

}: 10-' ar.d 8 .5 . . 10-°, respectively . Restriction-enzyme analysis 

indicated that the mutations observed in the LacZ target gene were point 

mutations (

682 


MODULATORY EFFECTS OF WHEAT SEEDLINC,S HGMOjF39ATE (S14) ON (,ENOTOXICITY 

CF SOME SYSPEMIC PE4TICIDES . I .S . GRGVER and S•S .LacYiar, School of Life 

Sciences .Guru Nangk-Dev University, Amritsar-143005, INDIA 

Modulatory effects of wheat seedlings homogenate (S14) on genotoxicity 

of some systemic pesticides viz . benomyl, Ekatin, . . FernoXone, 

and monocrotophos have been assessed employing histidine ceversibn 

assay in Salrtonella yj~urium (Ames assay) and d;romosoiosl aberrations 

ih root meristems inlium =p-a . All these pesticides induced mitotic 

depression and a broad spectrum of physiological (a-mitosis, stickiness, 

vagrant chromosomes, laggards eta .) and .clastogenic (chromosome breaks, 

ring chromosomes and micronuclei) aberrations . However, the frequency 

of induced physiological aberratior.s remain unaltered by S14 homogenate 

but the percentage of cells with clastogenic aberrations was reduced 

significantly with the supplementation of 514 . None of these pesticides 

enhanced significantly the histidine revertants in TA98> TA1V2 and 

TA1535 of .5 . tvnhimurlum . No appreciable effect was observed with the 

supplementation of rat liver S9 homogenate or wheat seedllno S14 

homogenate . 

683 

GENEl`GX EVALUATION OF METASYSTUX IN CHRGMC-SOMAL ABERRATIONS AND CHLORC :- 

YHYLL DEFICIENT MUTATI(~N ASSAY IN HQRDEUM VULGARE, I .S .GFGVER, Vinlta 

and'H .Grover, School of Life Sciences, Guru Nanak Dev University, 

Amritsai anì3 +S .R.Govt .College for Women, Amritsar . INDIA . 

Metasystox-an organophosphorus pesticide is one of the mo4t wic3ely 

used pesticides whose genotoxic nature is uncertain . The present report 

describes its genotoxic effect employing chr'omosoeal aberrations assay 

in root meristems and pollen mother cells and chlorophyll deficient 

mutation assay in }jordeum yylqAy .'It induced a significarit increase in 

chromosomal aberrations in root meristems . The spectrum of chromosomal 

aberrations included chromosome breaks, c-mitotic effects, leading to 

polyploid cells, chromatin'bridgesn laggards .tri- and tetraPolar cells 

and micioruclei . The effect was found to be "dose-dependent• Po'llen mother 

cells with univalents was the moet common aberration encountered at 

metaphase-I .Rarely a quadrivalent attributable to exchanges was also 

noticed . Unequal distr'ibution, chro.matin bridges and laggards were also 

recd rded . M2 analysis at the seedling stage revealed chlorophyll 

deficient mutants 

. Xa ntha, tigrina, maculata, albovirldis and virido- 

chlorophyll deficient mutantsuwarrantTitsifuc~herrstudies~iccaebatteryaof 

assays 

684 

EVOLUTIONARY SIGNIFICANCE OF MUTATION AND REPAIR . Robert H . Haynes, Department of 

Biology, York University, Toronto, Canada, H3J 1P9 . 

Heredity is a manifestation of the stability of genes, chromosomes and genomee 

from one generation of cells and organisms to the next . Heritable variation is a 

manifestation of various types of change in the genetic material of c .lls . In the 

absence of mechanisms to promote an extremely high level of replicational fidelity, 

the long genetic messages of contemporary organisms could not have evolved . In addition, 

were it not for the existence of processes which effect the repair or bypass of 

damage to DNA which arises from many ever-present natural sources, cellular activity 

would collapse from what might be called 'genetic meltdown' . On the other hand, if 

the various mechanisms which promote genetic stability were capable of functioning 

with perfect accuracy, genetic variation, and hence evolution, also would not occur . 

Many different genetic loci are involved in coding and control of the complex array 

of biochemical processes which maintain genetic stability . This is consistent with 

the notion that if very great fidelity is to be achieved with equipmmnt of poor precision, 

extensive checking procedures must be built into the system . For optimum 

economy, the 'cost' of such procedures should be just sufficient to reduce the errorrate 

to a tolerable level . Thus, the genetic machinery of cells can be viewed as a 

remarkable example of a highly reliable, dynamically stable system built from vulnerable 

and unreliable parts . Recent work on the genetic consequences of nucleotide pool 

imbalance suggests that natural selection has fashioned all major aspects of DNA 

metabolism to minimise mortality and mutability . However, the existence of inducible 

error-prone processing of DNA damage indicates that the maintenance of cellular viability 

takes precedence over genetic fidelity . 


Notes

236 1989 EMS Abstracts . :__ __ 

~ 000. ..~ 

685 


Notes tSMROMiNfAI . MJfXMM1S IN GHINA 

J . L . HSUF}I and C . C . TAN~ 

Institute of Cenetics~ Fl~iverstty, Shanghai 200433, China 

China is a developing country, having a population of irarly 1 .2 billion people. Along with the progress 

of modernization, the caaxry is also facing the problen of enviranmental pollution through the release of 

industrial aastes into the air and keterr.mys . This, together with population pressure and agricultural problems, 

has already received the wide attention of the Chinese public . China is prepared to exercise quality 

control and envirarnental monitoring, and a series of regulations have been established . The Chinese Environmental 

lLtagen Society (CElfi) ws found in 1983, it is essential to the development of environnental nutagenesis 

. In the year of 1983, 1985, 1987 and 1988, -the CE2S hdld his annual meetings in Shenghai, Wuhan,Slanghai " 

and Beijing. Over than 100 independent qualified laboratories for identifying autagens are now operating in 

Chira . They are distributed on more than 20 provinces . lbst of these assays are used to help identify nutagens, 

sore can also identify carcinogens . Of these laboratories, t .u-thrids are using nutagenlc assays and 

the other one-third is doing either carcinogenetic or teratogenetic assays . 'kre in vitro assays use a variety 

of cell types ranging form bacterial to huren, fram somatic cells to genn cells ; other tests can be done 

directly on Drosophila, rodents and plants . Recently, the recc.rbinant DNA tecFnique has been introduted into 

the study of environrental nutagenesis . We have developed a shuttle vector system for studying mutational 

specificity, the shuttle vector contains the EB virus origin and E . colt XyIE gene, then introduced into human 

cultured cells by transfection and allowed to replicated autorously in the cell nucleus . During the replication 

period of the vector, the cells are exposed to a nutagen, an increased in nutation frequency above the 

spontaneous background is readily obtained . Induced mutants can be rapidly isolated and characterized by color 

change . Nowadays, all new medtcines, pesticides, food additives, contraceptions and Chinese medicinal 

herbs are subjected to the kres test, micronucleus test, chronosane aberrations and SCE analysis, U06 and 

SOS et al routine screening procedures . 

686 

"THE POTENTIAL ROLE OF CELL DIFFERENTIATION IN TUMOR PROMOTION," E . Nuberman, 

Biological, Environmental, and Medical Research Division, Argonne National Laboratory, 

Argonne, Illinois 60439 

Chemicals that promote tumor formation, including phorbol 12-myristate 13-acetate 

(PMA), in general do not bind to DNA and are devoid of mutagenic activity ; 

nevertheless, many of these promoters induce differentiation in various cell types 

including the human promyelocytic RL-60 leukemia cells . Thus, tumor promotion may 

involve the expression of growth-facilitating genes, which, as a result of prior 

genetic alterations (during tumor initiation), have been placed under the control of 

genes that regulate normal cell differentiation . Consequently, we are studying the 

ability of PHA and related agents to initiate early biochemical events that cause 

alterations in gene expression leading to modulation of differentiation processes . 

These phenomena are analyzed in NL-60 cells that are either susceptible or resistant to 

the induction of cell differentiation by PMA . Differentiation is characterized by 

changes in functional proteins, enzymatic markers, and reactivity with monoclonal 

antibodies generated against maturation-specific cell-surface and nuclear proteins . 

Our results indicate that a number of early events (2-30 m1n) such as subcellular 

translocation and activation of protein kinase C, phosphorylation of a number of 

proteins including nuclear proteins, and modulation of topoisomarase II activity may be 

early steps in the signal transduction that results in the induction of cell 

differentiation and perhaps tumor promotion by PNA and related agents . Work supported 

by the U .S . Department of Energy under Contract No . W-31-109-ENG-38 . 

687 

DETECTION OF THE MUTAGENS IN URINE OF CIVILIANS EXPOSED TO MUSTARD GAS 

M .A .Jabbar ; F .Pourismnili ; G .H .Riazi and M . Nouri Dalawi 

Biochemistry and Biophysic Institute, Tehran University, Box 6479-11365 

Tehran , IRAN . 

Thousands of miliatry and civilian people in Iran were exposed to sublethal 

doses of mustard gas during the Iraqi-Iran war . Therefore, a rapid 

prediction for the hazardous impact of this agent on man and his environment 

is urgently needed to impliment any possible preventive measure . 

Ames and the fluctuation assays were used to detect the mutagenic metabolites 

of mustard gas in urine samples of 20 non-smoker patients who 

were wounded in Sardasht area( west of Iran )in May, 1986 . Salmonellae 

_t~~himurium TA98, TAIOO, and TA202 were used in the screening proce3ures . 

Tfie resul-ts showed that the fluctuation test was more sensitive than 

Ames test in detection the mutagenic agents of mustard gas metabolites . 

More than 701 of the urine samples were mutagenic for TAI02 strain when 

fluctuation assay was implimented . These results reveal that most of the 

mutagenic metabolites in the urine samples of the wounded patients were 

of the oxidative type . Some patients were given garlic Juce to see its 

antimutagenicity in vivo but there was no significant difference in the 

mutagenic activity of their uring concentrates befor or after treatment . 

In conclu sion, more work is needed to spe ify the mutagenic metabolites 

of mustarsd gas and the actual hazard of wh~ch on man or his offspring . 

50869 3750

688 

CYfOGENETIC EFFECTS OF PHORBOL ESTER TUMOR PROMOTERS : POSSIBLE ROLE IN IIULTISTEP TUKORI- 

GENESIS. V . Kinzel . B. Farber, R .T. Petrusevska . N . E . Fusenle . Institute of Exp. Pathology, 

Institute of Biochemistry', German Cancer Research Center . D-8900 Heldelber¢ . PRG 

Tumoriyenesis in mouse skin can be effected in a number of steps : by initiation with DMBA, by 

conversion with one or two applications of TPA (12-0-tetradecanoylphorbol-13-acetate) and by reprated 

treatment with RPA (12-0-retinoylphorbol-13-acetate) . The conversion step effected by TPA 

(but not by RPA) Is characterized by a half-life of 10-12 weeks (in NMRI aice) : it is Sndependent 

from initiation (Fiirstenberger . G . et al ., 1985, Science, L3-0. 76) . '1'his specific effect of TPA 

may be explained best by the clastogenic activity of TPA shown In mouse keratinocytes In culture 

(Petrusevska, R.T. at al . 198fi, Carcirto¢enesis $, 1207) as well as in ex vivo studies (Fiirstenbereer, 

G . et al ., 1988, Carcino¢eneals . In press). Details of the olastoYenic action of TPA were studied in 

HeLa cells which exhibit a rodiuaimetic cell cycle response to TPA (Klnzel, V . at al ., 1980, Science 

210 . 429) . Only TPA exerted a significant elastogenic activity at non-eytotoxic concentrations (10-8 

to 10-6 M) measured after 24 and 48 hrs . Values obtained with RPA were close to those obtained in 

the presence of solvent (acetone 0.2k). The response to TPA was only to some extent correlated with 

the dose. Chromosome aberrations including gaps and breaks were predominantly of the chromatid type ; 

they are earliest measurable after 6-8 hrs, I .e. as the cells recover from TPA-induced 02-inhibition 

(Kinzel . V . et al., 1988. Cancer Res . 40 . 1759). A 30 ∎in exposure to TPA (10-7 N) is sufficient to 

induce aberrations . The data point to an indirect, possibly receptor-mediated action of TPA . If it 

is supposed that TPA-iuduced chromosome lesions represent a key event required for conversion it 

might be further speculated that the absolute requirement for DNA replication In the conversion step 

(Kinsel, V . et a1 ., 1984 . PNAS $1. 5858) is necessary to "fix" a certain degree of chromosome damaQe 

(supported by the DFG) . 

689 

ASSESSMENT OF EXPOSURE AND POTENTIAL RISK FROM MUTAGENIC AIR POLLUTANTS . Joellen 

Levtas, U .S . Environmental Protection Agency, Research Triangle Park, N .C . 27711 

(U .S .A .) . 

Genetic bioassays have been used to identify airborne mutagens and tlieir sources, 

to measure human exposure and to provide comparative assessment of potential cancer 

risk from air pollutants . Recent air pollution studies have integrated the use of 

genetic bioassays into sampling and analysis strategies to seet these goals . Complex 

mixtures of urban air pollutants from vehicles andaresidential heating sources have 

been studied in field investigations in both Western and Eastern cities in the U .S . 

as part of EPA's Integrated Air Cancer Project . Genetic bioassays were applied to 

source emissions and ambient outdoor and indoor air to characterize the exposure and 

potential risk from the gaseous, semi-volatile and particle-bound organic species . 

Source apportionment of the mutagens in these airsheds has been accomplished through 

receptor-modeling of mutagens using multiple linear regression analysis . 

Mutagenesia, tumorigenesis and DNA adduct dosimetry studies of these air pollution 

mixtures have been used in further developing a comparative potency method for 

assessing cancer risk from complex mixtures . This is an abstract of a proposed 

presentation and does not necessarily reflect EPA policy . 

690 

THE GENETIC TOXICITY OF THE HUMAN CARCINOGENS BENZIDINE AND BENZIDINE- 

BASED DYES : CHROMOSOMAL ANALYSIS IN EXPOSED WORKERS 

E .Mirkova and S .Lalchev,The Medical Academy,Sofia (Bulgaria) 

The activities of the human carcinogens benzidine (BENZ) and BENZ-based 

dye Direct Black 38 in the rodent bone marrow micronucleus assays ( BM 

MNA) were established unequivocally (Ashby and Mirkova,1988 ;Beije 1987) . 

The lack of data on their genetic effects in humans acted as the stimulus 

for the present cytogenetic study .Chromosomal analysis was performed 

in the lymphocytes of 23 BENZ-based dyes manufacturing workers and 30 

matched control .individuals . The period of exposure was 7-31 yr . BENZ 

was detecSed in the workplace air at concentration levels of 0 .42 - 

0 .86 mg/m and in the urine of exposed workers respectively at mean level 

of 1 .78 ± 1 .4 µg/1 . The total airborne particulate levels of BENZ - 

based dyes (mainly Direct Black 38) ranged from 7 .8 to 32 .3 mg/m3 . i 

significant increase in the X of aberrant cells (-gaps) was found in the 

exposed group (7 .9 ± 5 .9) as compared with the control incidence of 

0 .87 ± 0 .26X aberrant cells (-gaps) . Aberrations were mainly chromatid 

breaks . The X frequency of polyploid cells in the exposed grou was 

found to be 1 .1 ± 0 .1 and the control incidence was 0 .17 * 0 .5~ . The 

present data "establish that the rodent BM MNA has predicted correctly 

the mutagenicity to humans of the IARC carcinogens BENZ and BENZ-based 

dyes . 



Notes

238 1989 EMS Abstracts _ e _ -- - - 691 


NO LE S REVERSION OF CANCER PR~ENOTYPES BY ALTERATIONS IN GENOTYPE . Minako NAGAO, National 

Cancer Center Research nstitute, Tokyo (Japan) 

NIH3T3 cells Zianè

694 


Notes 

COMPARISON OF HUMAN AND RODENT RESPONSE TO GENOTOEICANTS 

Schm X hl, D ., Institute of Tozicology and Chemotherapy, German Cancer 

Research Center, Im=-Neuenheimer Feld 280, D-6900 Heidelberg, Federal 

Republic of Germany 

On the basis of results found in genetic toxicology, animal experi- 

ments with rodents and human data, two groups of compounds are discussed, 

namely anticancer drugs and N-nitroso compounds . Regarding anticancer 

drugs, there is a good agreement between resulte obtained in 

vitro in tests on genotoxioity and in carcinogenicity ezperimente in 

rodents as well ae in -human-carcinogenesie . Investigations with Nnitroso 

compounds ehowed that many N-nitroso compounde such as die- 

thylnitrosamine are carcinogenic in many laboratory animals, but the 

organotropism of the carcinogenic activity varies considerably . Epide- 

miological studies on people monitoring concerning the extrapolation 

of results obtained in animal experiments to the human situation have 

to take into consideration that the organotropism of the carcinogenic 

activity is often hard to predict . Possible reasons are epeculatively 

discussed . 

695 

GEi:OTOXIC ASSAY OF TI(0 DIF:TARY FURP2dS BY SOW IN VIVO CYTOGENETIC PARAI+R9TERS 

S . Subramanyam, D . SailaJa andD . Rathnaprabha 

Department of Genetics, Osmania University, 

Hyderabad 500007, India 

Furans found in vegetarian and nonvegetarian foods and used to prepare resins, 

varnisiies, pesticides and germicides are suspected to oause respiratory tract 

infections, liver and lung oezicers . In the absence of proper information, the 

genotozic potentials of two furans, Furfural and 2-methyl furan, were evaluated by 

the standard in vivo cytogre netic protocols by employing somatic and molotio tissues 

and nultiple paemeters on 8 to 10 week old Swiss albino mice as test system . In 

somatic system o1L omosome mutatio : .s were scored from bone marrox oells, 24,48 and 

72 hr . after oral feeding at 1000,2000 and 4000 p}m concentrations at 24 hr . intervals 

for five dqyc . Furitiral induced chromosome matatious only with the highest 

dose after 24 and 48 hr . 2-methyl furan did not do so . Both did not inhibit 

synthesis of snindle proteins . There was no retardatiosi of oell division . In 

neiotic test system, the test was . carried out for 24 hr . and from I to V weeks 

at xeeYly intervals to cover one spermatogenetic cycle . Both compounds did not 

induce Genotozio effects . They did not also cause sperm head abnormalities which 

indicete point mutations induced in sex end autosomal genes governing their mospholof•y 

. This analysis illustrates thF .t cumulative doses of the two flurane do not 

cause any genotoxic hazards to the mammalian teet system . 

696 

GENETIC TOXICOLOGY OF COMPLEX MIXTURES--DVERVIEW AND SUMMARY OF THE WASHINGTON 

SATELLITE MEETING . Waters, M ., U .S . Environ . Prot . Agency, Res . Tri . Park, NC (USA) . 

Complex mixture research in genetic toxicology has advanced rapidly since the first 

EPA-sponsored Symposium on "Short-Term Bioassays in the Analysis of Complex 

Environmental Mixtures" held in 1978 . The purpose of this biennial symposium is to 

present state-of-the-art techniques in bioassay and chemical analyses applied to 

complex mixtures and to foster continued advancement of the field . The Washington 

ICEM Satellite Meeting will begin with a presentation on the latest techniques in 

molecular biology applicable to complex mixture research and then will separately 

address complex mixtures in air and in water, and exposure/effects assessment . The 

session on air focuses on the identification and characterization of component 

chemicals and chemical classes found in combustion sources and in ambient and indoor 

air . The thrust of research in this medium is to separately characterize the 

contributions of various combustion sources as volatile and particulate components as 

well as atmospheric transformation products to the observed genotoxicity of the air 

mixture reaching the human receptor . In the water medium, the main emphasis of 

research is on the characterization of mutagenic compounds formed during disinfection 

of drinking water and on the development of new methods to detect genotoxicants in 

various wastewaters . Exposures/effects assessment research deals with the use of DNA 

and protein adduct dosimetry in conjunction with low dose extrapolation models used to 

estimate cancer risk from environmental genotoxicants . Presentations will consider 

cigarette smoking, occupational and industrial exposures as well as environmental 

exposures . A final presentation will address future avenues of research in the field . 


M 

I 

I 

~

240 1989 EMS Abstracts _ ._ -- - . 


Notes "~CHEMOPREVENTI*fOFTMTOXICITY IN VIVO . Wattenber~, L . W . Dept . of Laboratory 

Medicine and Pathology . Univers~iLy' o-Minnesota, Minneapolis, MN 55455, USA . 

Cnemopreventiqp gi56he effects of genotoxic compounds in vivo can be brought about 

by compounds ("bloc~c'Tng agents") that prevent the genotoxi~c agent from reaching or 

reacting with critical cellular targets or by compounds ("suppressing agents") that 

prevent manifestations of genotoxicity in cells in which the target molecules have 

already been hit . Using carcinogens as an example of one type of genotoxic compound, 

blocking agents can be shown to act via three mechanisms : (1) by inhibiting activation 

reactions of carcinogens requiring metabolic activation in order to form reactive 

electrophiles, (2) by enhancing carcinogen detoxification and (3) by trapping 

reactive carcinogenic species . Examples of all three will be discussed . Particular 

emphasis will be on naturally-occurring organosulfur compounds and aromatic 

isothiocyanates that can both induce increased activity of detoxification systems and 

can inhibit carcinogen activation . Trapping agents to be discussed will include aromatic 

thiols and sodium thiosulfate . Data relating to suppressive effects of cruciferous 

vegetables and citrus fruit oils will be presented . Current evidence indicates 

that there are surprisingly large numbers of pure chemicals and natural products 

that can inhibit genotoxicity in vivo . Supported by Grant SIG 5A from the 

American Cancer Society . 

. 697

a- 

Author Index to Abstracts 

Numbers refer to abstract numbers, not page numbers 

AL-Allak, B .M .A ., 539 

Aardema, M .J ., 1, 350 

Aaron, C ., 657 

Aaron, C .S ., 2, 3, 235, 676 

Aaron, S ., 588 

Abarca M ., 133 

Abbondandolo, A ., 66, 371 483 

Abbott, M .G ., 4 

Abdalla, N .A ., 153 

Abe, T ., 420 

Abu-Shakra, A ., 5 

Adair, G, 87 

Adhikari, N ., 6 

Adhvaryu, S .G ., 7 

Adler, L-D ., 8 

Aeschbacher, H .U ., 9 

Afzal, V ., 639 

Agarwal, K ., 526 

Agostinelli, D .A ., 49 

Agostini, J .M .S ., 83 

Ahnstrom, G ., 332 

Ahuja, Y .R ., 10, 35, 463 

Aibara, K ., 490 

Aidoo, A ., 11 

Akintownwa, D .A .A ., 12 

Akiyama, N ., 13 

Al-Ghaith, L .K ., 154 

AlaLeviE, M ., 174 

Albertini, R .J ., 14, 412, 471, 545, 676 

Albertini, S ., 15, 16 

Alc .Intara-Diaz, D ., 17 

Alexander, J ., 28, 616, 617 

Allen, J .W ., 36, 259, 630 

Allen, K .L ., 123 

Alonso, C ., 494 

Alvi, N .K ., 19 

Ammenheuser, M .M ., 20, 623 

An, Y .m . 562 

Anaya, P., 26 

Anderson, D ., 21 

Andrews, A .F ., 246 

Andrews, P .W ., 22 

Antilla, S ., 228 

Anzick, S .L ., 422 

Aranez, A .T ., 23, 24 

Aravindakshan, M ., 25 

Arce, G .T., 49 

Arena, G ., 483 

Arenaz, P ., 26 


Arimoto, S ., 27 

Armstrong, J .D :, 28, 310, 311 

Arras, C .A ., 534 

Arreola, G .G ., 336 

Arrimanoglou, I .I ., 29 

Arumikkili, N ., 528 

Ashby, J ., 30, 81 

Athwal, R .S ., 223 

Atsumi, G ., 31 

Au, W .W ., 20, 32 

Auletta, A ., 678 

Ault, K .T., 241 

Austin, S .J ., 320 

Autrup, H ., 33 

Awogi, T., 565 

Azian, A ., 63 

Baan, R .A ., 34 

. 

Babu, P.P., 35 

Backer, L .C ., 36, 259 

Bai, C .J ., 643, 647 

~Bailey, G ., 122 

Bakale, G ., 38 

Baker, R .S .U ., 245 

Bakke, J .P. 230 

Bala, S ., 221 

Balakrishnan, S ., 53 

Balcgzar, M ., 574 

Balczon, R .D ., 74 

Baldini, M ., 483 

Bali, D ., 541 

Ball, J .C ., 39 

Ball, S .T., 441 

Balwierz, P.S ., 428 

Banerjee, A ., 526 

Banga, S .S ., 70 

Bansal, M .P., 408 

Bansal, M .R ., 40 

Barbosa, H .S., 475 

Bardwell, L ., 176 

Barnes, W.s ., 112 

Barnett, L .B ., 41 

Barrett, J.C., 637 

Baruthio, FL . 157 

Bassani, B ., 431 

Basu, A .K . 337 

Battista, J .R ., 42 

Battula, N ., 43 

Bautista, A .R .P.L., 493 

241 

Bayley, S .T ., 37 

Bayuley, B .C ., 169 

Becher, G . 18, 616, 617 

Beeman, D .K ., 320 

Beije, B ., 379 

Beland, F .A ., 44 

Beland, F .A ., 581 

Bell, D .A ., 45 

Belli, J .A ., 20 

Beltrame, C ., 677 

Bempong, M .A ., 46 

Benasutti, M ., 337 

Benigni, R ., 47, 106 

Benova, D.K ., 48 

Bentley, K .S . 

Berger, N .A . 98 

Berger, S .J ., 98 

Bergtold, D .S ., 50 

Bernini, L .F ., 51 

Bertrand, F., 128 

Bessho, T., 31, 52 

Bewsey, B ., 419 

Beyers, J ., 290, 439 

Beyersmann, D ., 242 

Bhatia, K, 548 

Bhatt, B ., 53 

Bhatt, J ., 602 

Bhattacharjee, S .B ., 194, 518 

Bhattacharya, N .P., 54 

Bhattacharya, R .K, . 54 

Bhaumik, G ., 194 

Bhilwade, H .N., 56 

Bi, D .Q ., 263 

Bieszczad, M .J ., 556, 557 

Bigbee, W .L ., 271, 319 

Bin, Y ., 233, 460 

Bineva, M ., 48 

Bishop, J .B ., 57 

Bisi, J .E ., 637 

Blackburn, G .R ., 58 

Blaich, G ., 59 

Blake, B .W., 60, 61 

Blakey, D .H ., 62 

Blevins, R .D ., 63 

Bloom, S .E., 343, 375 

Bochkov, N .P., 64 

Bodell, W .J ., 65, 634 

Boehm, R .M ., 161 

Bonatti, S ., 66, 371 

I

242 Author Index to Abstracts 

Boobis, A .R ., 206 

Boothman, D .A ., 67 

Boreiko, C .J ., 352 

Borgstedt, H .H ., 60, 61 

Boroffice ; R .A ., 68 

BOrrsen, A .-L., 69 

Bourre, F ., 498 

Boyd, J .L ., 70 

Boyes, B .G ., 71 

Boyley, J .M ., 62 

Brady, A ., 627 

Brandriff, B .F ., 72 

Branstetter, D ., 2 

Brefia-Valle, M ., 17 

Brendler, S .Y ., 449, 450 

Bridges, B ., 111 

Bridges, B .A ., 73 

._Brillinger, R .L ., 410 

Brinkley, B .R ., 74 

Brinson, E .C ., 75 

Brockman, H .E ., 456, 627 

BrOgger, A ., 69 

Brognoli, I ., 83 

Broit, M ., 582 

Bronzetti, G ., 76, 182 

Brookman, K .W ., 579 

Brooks, A .L ., 77, 476 

Brunborg, G ., 78 

Brunny, J ., 79 

Brusick, D .J ., 80, 81 

Bruzzone, E ., 483 

Bryant, M .F ., 82 

Bueno, A .M .S ., 83 

Bulich, A .A ., 84 

Burger, G .T., 144, 322 

Burgess, W .M ., 366 

Burkart, W., 609 

Burkhart-Schultz, K ., 75, 85 

Burlingame, S .F., 65 

Burlinson, B ., 86, 192 

Burrell, M ., 286 

Busch, D . B ., 87 

Busk, L ., 88, 607 

Butler, M .A ., 89 

Butterworth, B .E ., 49, 635 

Cai, G ., 327 

Cai, Y ., 90 

Cain, K .T., 516 

Campbell, J .A ., 259, 630 

Campfield, A ., 583 

Cantelli Forti, G ., 76 

Cao, J ., 91 

Cao, L .F ., 646 

Cao, N ., 621 

Cao, S .-y ., 92 

Caprathe, B.W ., 307 

Carere, A ., 115 


Cariello,_ N', ~=" - - 

Carr, J ., 1~38 

Carr, R., 446 ~ 

Carrano, A.V ., 72 

Carty, M ., 138 

Carvajal de Gil L .A ., 218 

Casciano, D .A ., 93, 388, 406 

Caskey, C .T., 484, 649 

Caspary, W.J ., 123 

Castagnero, M ., 126 

Caterson, C ., 530, 531 

Cattley, R .C ., 451 

Cebula, T .A ., 94 

Cebulska-Wasilewska, A . 95, 96 

Cederberg, H ., 607 

Cercignanni, G ., 66 

Cerqueira, E .M .M ., 475 

Chakraborty, P.K ., 199 

Chandorkar, M ., 209 

Chandrika, K .V., 467 

Chang, C .C ., 277 

Chang, F ., 459, 672 

Chang, S .C ., 117 

Chang, X .-p ., 168 

Chang, Y .-y ., 168 

Chang, Z .-y ., 92 

Channarayappa, J .N ., 97 

Chattetjee, S ., 98 

Chaubey, R .C ., 56 

Chaudhry, M .A ., 99 

Chauhan, P .S ., 56, 679 

Chen, A .T .L ., 472 

Chen, D .s, 132 

Chen, H .-H ., 147 

Chen, J .-K ., 640, 648 

Chen, K .-Z ., 100 

Chen, R ., 101 

Chen, S ., 262 

Chen, S .M ., 263 

Chen, T .D ., 347 

Chen, X .R ., 658, 659 

Chen, Y ., 102, 655, 665 

Chen, Z., 273 

Cheng, M .F., 98 

Cherney, B ., 548 

Chionglo, D ., 568 

Chiu, S .M, 103 

Choi, I .S ., 104 

Chouroulinkov, I ., 425 

Chowdhury, J .B ., 183 

Chu, J .-X ., 168 

Chung, H .W ., 383, 636 

Ciaravino, V ., 575 

Cifone, M .A ., 105, 173 

Citro, G ., 106 

Clapp, D .W ., 148 

Claxton, L .D ., 107 

Cliet, I ., 108 

Clive, D ., 109, 201, 303 

Clonfero, E ., 110 

Cobb, R .R ., 430 

Cochrane, J ., 470, 471 

Coelho, M .C .L .S ., 494 

Cohen, M .D., 549 

Cohen, M .M ., 508, 509 

Coimbriio, C .A ., 494 

Cole, J ., 111 

Coles, B ., 112 

Colyer, S .P., 275, 333 

Combes, R .D ., 521, 522 

Conley, E.C., 114 

Conner, M .K ., 377 

Conti, G ., 115 

Cooper, A .J ., 176 

Cordier, A ., 108 

Corey, L .A ., 369 

Corrie, M ., 379 

Corsale, G ., 162, 434 

Cortinas De Nava, C ., 680 

Costa, R ., 677 

Crebelli, R ., 106, 115 

Crespi, C .L ., 116 

Cross, F .T., 276 

Cui, Y .Q ., 117 

Cunningham, M .L ., 118 

Curren, R .D., 173 

Custer, L ., 105 

Czeizel, A., 119 

de Leeuw, W .F .J ., 681 

de la Rosa, M .E ., 574 

de S . Cblus, I .M ., 113 

de Serres, F.J ., 81, 127, 430 

D'Souza, S .J ., 405 

Dai, Y .F ., 658 

Dalawi, M .N ., 687 

Dallaire, L ., 545 

Damodaran, T.V., 120 

Daniel, F .B ., 367 

Daniels, C .B ., 121 

DaniBre, M .C ., 157 

Danzl, T ., 186 

Das, T., 526 

Dashwood, R ., 122 

Daston, D .S ., 123 

Dave, B .J ., 7 

Davies, D .S ., 206 

Davison, A .J ., 485 

Day, J ., 165 

Daya-Grosjean, L ., 124 

Dayrit, F., 610 

De Carli, L ., 594 

De Ferrari, M ., 66 

De Flora, S ., 125 

De Marini, D.M ., 45, 259 

De MEo, M .P., 126

De Sario, A ., 594 ~ 

De Wals, P ., 128 

De, M., 526 r 

DeAngelo, A .B ., 367 

Dearfield, K .L ., 130, 131 

Deitch, R .A ., 58 

Del Carratore, R ., 76, 182 

Delclos, K .B ., 93 

Della Croce, C ., 182 - 

Demkowicz-Dobrzafiski, K .K ., 248 

Demple, B ., 132 

Deng, D .J ., 665 

Denham, J ., 167 

Descailleauz, J ., 135 

Dewi, D ., 437 

Dhillon, H .S ., 134 

Dhir, H ., 135 

Diggle, 136 

DineshKumar, B ., 10 

Dipple, A ., 137 

Dixon, K ., 138 

Dobbs, R .A . 140 

Dock, L ., 248 

Doehmer, J ., 139 

Doerger, J .U ., 140 

Doerr, C .L ., 130 

Doess, C .L ., 141 

Dolara, P., 142, 143 

Dolk, H ., 128 

Domon, O .E ., 388 

Donahoe, R . M ., 520 

Dong, Z . W . , 117 

Donnelly, C .E ., 42 

Donovan, C ., 148 

Doolittle, D .H ., 144 

Doolittle, D .J ., 322 

Dou, G ., 562 

Douglas, G .R ., 62 

Dragan, Y ., 445 

Drake, J .W ., 145 

Dresp, J .H ., 146 

Driscoll, S ., 283 

Drougard, C ., 124 

Du, Y .-X ., 147 

Dubins, J .S ., 430 

Duker, M .J ., 184 

Dumenco, L .L ., 148 

Dum6nil, G ., 126 

Dunbar, V .G ., 520 

Dunphy, E ., 149 

Dybing, E ., 78 • 

Eastmond, D.A ., 150, 546, 590 

Ebata, J ., 151 

Ehling, U .H . 152 

Eiche, A ., 458 

El-Seehi, M ., 247 

EI-Tarras, A ., 153 


El-Zawahri, M .M ., 154 

El-Zyat, H ., 247 

ElKoweidy, A.H ., 389 

Elespuru, R .E ., 155, 156 

Elias, Z ., 157 

Elmore, E ., 158 

Endo, 0 ., 285, 355, 356 

Ennever, F .K ., 159 

Enslein, K ., 60, 61, 204 

Epe, B ., 160 

Erexson, G .L., 82, 161, 294, 630 

Esaki, S ., 288 

Esancy, J ., 107 

Eskelinen, A ., 344 

Esposito, A ., 162, 434 

Essigmann J .M ., 337 

Etoh, H., 436 

Fahrig, R ., 163 

Falek, A ., 520 

Falta, M .T., 545 

Fan, Q ., 102 

Fan, X ., 264 

Fang, F .D ., 646 

Fang, L., 168 

Fang, M ., 658 

F3irrber, B ., 688 

Favor, J ., 164 

Fedorwicz, G ., 165 

Felton, J .~ 600 

Felton, J .S ., 166, 366, 592 

Fenech, M ., 167 

Feng, P.-C ., 168 

Fengming, G ., 185 

Ferguson, L .R., 169 

Ferguson, R .J ., 246 

Ficsor, G ., 170 

Fielder, 136 

Finch, P., 610 

Fiskesjo, G ., 171 

Fleck, E .W ., 276 

Foiles, P.G ., 19 

Fong, A ., 122 

Fornace, A .J ., Jr, 172 

Forster, R ., 512 

Fort, F.L ., 173 

Fournier, E ., 108 

Fox, M ., 99, 484 

Francis, W., 167 

FranekiE, J ., 174 

Freeman, H ., 107 

Frei, H ., 175 

French, J .E ., 57 

Friedberg, E .C ., 176 

Friedman, L.R ., 103 

Froes, N .C ., 177 

Frohberg, H ., 301 

Frome, E .L ., 275, 333 

. 

Author Index to Abstracts 243 

Fronza, G ., 371, 483 

Frosina, G ., 483 

Fu, J ., 102 

Fu, S ., 562, 673 

Fujika, Y ., 252 

Fukui, S ., 252 

Fullerton, N .F ., 44 

Furihata, C., 178 

Furukawa, A ., 179 

Furukawa, H ., 180 

Fusenig, N .E ., 688 

Gad, S .C ., 428 

Gaidzinsk, K ., 83 

Gairola, C .G ., 578 

Galas, D .J ., 181 

Galati, R ., 106 

Galli, A ., 182 

Gandhi, G ., 183 

Ganguly, T., 184 

Gao, H .L., 646 

Gao, M ., 656 

Gao, N ., 1I 

Gao, Q ., 646 

Garcia, M .V., 177 

Garin, K .E ., 230 

Garriott, M ., 79, 290, 439 

Garry, V., 186 

Gasper, K .P. 276 

Gatehouse, D .G ., 86, 187, 192, 291, 593 

Geard, C .R ., 189, 309 

Geddes, A .D ., 190 

Geethanjali, D ., 404 

Gelboin, H .V ., 343 

Gemmell, M .A ., 197 

Generoso, W .M ., 191, 516 

Gentile, G .J ., 165, 492 

Gentile, J .M ., 165, 301, 492 

Gentile, S .L ., 483 

George, E ., 192 

Gerson, S .L ., 148 

Getman, S .M ., 424 

Ghalib, M .A ., 193 

Ghosh, A ., 135 

Ghosh, A .K ., 135 

Ghosh, B .B ., 571 

Ghosh, R ., 194 

Ghosh, S ., 135 

Gibbs, R .A ., 649 

Gibson, D .P., I 

Gibson, E .S ., 585 

Giesi, R .A ., 276 

Gilbert, A .M ., 482 

Gill, B .S ., 195 

Gille, J .J .,P., 196 

Gilman, J.P.W ., 410 

Gilot-Delhalle, J ., 390 

Ginsberg, L .C ., 170 

e


Giometti, C .S ., 197 

Giordano, G .G ., 162, 434, 553 

Giordano, P.C ., 51 

Giri, A .K ., 198, 199, 542 

Giromini, L ., 182 

Glatt, H .R ., 139 

Glattke, M ., 28, 310, 311 

Glickman, B.W ., 200, 208, 219, 315, 

510, 654, 675 

Glover, P., 201, 237 

Gluecksohn-Waelsch, S ., 197 

Goldstein, B .D ., 428 

Gollapudi, B .B ., 202, 203 

Gombar, V.K ., 60, 61, 204 

Gomes, M ., 205 

Gooderham, N .J ., 206 

Gopalan, H .N .B ., 207 

Gopinath, P.M ., 351, 468 

6ordon, A .J .E ., 208, 315 

Gordon, L .A ., 72 

Gorodetzkaya, N ., 148 

Gossen, J .A ., 681 

Goswami, H .K ., 209 

Goswami, R .P., 209 

Goto, S ., 210, 285 

Goudreau, B ., 335 

Gowri, R ., 528 

Goyle, S ., 211 

Graf, U ., 212 

Grant, W .F ., 213 

Grassi, P ., 142 

Gray, J ., 443 

Green, C .L . 337 

Green, M .R ., 214, 215 

Greenberg, J .T, 132 

Greer, G ., 216 

Griffith, J ., 186 

Grilli, S ., 76 

Grimmer, G ., 450 

Grollman, A .P., 217 

Groot de Restrepo, H ., 218 

Grosovsky, A .J ., 219 

Grover, H ., 683 

Grover, I .S ., 6, 183, 220, 221, 222, 

682, 683, 692 

Grummt, T ., 561 

Grusick, D . J ., 80, 81 

Gryseels, B .J .A.M ., 301 

Grzesiuk, E ., 270 

Gudi, R ., 223 

Guengerich, F .P., 89, 224 

Guevara, M .L ., 133 

Guida, M ., 434 

Guirong, D ., 233 

Gulati, D ., 161, 578 

Gulati, D .K ., 638 

Gupta, R .L ., 225 

Gustaffson, J .A ., 293, 379 


Guttenplan„I .$„r226 

Guzzie; RJ2!r'E 

Hachiya, S .IN6541-5 

Hackman, P., 228 

Hall, B .G . 229 

Halliday, H .A ., 208 

Halperin, E .C ., 294 

Hamamy, H .A., 539 _ 

Hameil'a, M ., 418 

Hamilton, C .M . 230 

Han, J ., 231 

Han, Y ., 452 

Hanawalt, P.C ., 232 

Hanekamp, J ., 577 

Hanmantha, P., 466 

Hanson, R .W ., 148 

Hanxiao, S ., 233 

Hanying, J ., 185 

Haque, J ., 548 

Hara, M ., 234 

Hara, T ., 537 

Harbach, P.R ., 235 

Harbell, J ., 530,531 

Harnois, M .C ., 236, 268 

Harosh, I ., 176 

Harrington-Brock, K ., 130, 237, 381 

Harris, C .H ., 238 

Harris, K ., 298 

Harris, S .B ., 239 

Harrison, D .y ., 369 

Hartman, P .E ., 240, 241 

Hartman, Z ., 241 

Harttig, U .H ., 160 

Hartwig, A ., 242 

Hashimoto, N ., 417 

Hauser, J ., 138 

Hay, D .B ., 557 

Hayashi, M ., 243, 565, 615 

Hayatsu, H ., 27, 31, 52, 244, 269, 407, 

489 

Hayes, A .W ., 144, 322 

Haynes, R .H ., 684 

He, S ., 245 

He, Y ., 90 

Heflich, R .H ., 11, 581 

Heikkila, L ., 228 

Heikkila, P., 418 

Hein, D .W., 246 

Helmi, S ., 247 

Hemminki, K ., 399 

Hemminki, N .F .I ., 504 

Hendricks, J ., 122 

Hennig, E .E ., 248 

Hennig, U .G .G ., 249, 619 

Henry, K .Y ., 324 

Henschler, D ., 289 

Herrera, L .A ., 250 

Hertner, Th ., 457 

Hesso, A ., 418 

Hewer, A ., 504 

Hilliard, C ., 251 

Hinson, W.G ., 388 

Hirayama, T ., 252 

Hisamatsu, Y ., 253 

Hitotsumachi, S ., 536 

Hittelman, W.N ., 254 

Hiyama, K ., 502 

Hofnung, M ., 461, 587 

Holme, J .A ., 18, 78, 616 

Holmes, R .M ., 230 

Hong, H ., 641 

Hong, X .,k, 255 

Hong, Z., 473 

Honorb, G ., 294 

Hook, G .J ., 256, 455 

Horesovsky . G .J ., 257, 455 

Horikawa, K ., 258 

Horiya, N ., 537 

Horsfall, M .J ., 208 

Horvath, J ., 112 

Hosoi, J ., 637 

Hou, E .W ., 637 

Housey, G .M ., 302 

Hovig, E ., 69 

Howard, D .R ., 259 

Howard, P.C ., 260, 361 

Hsiao, W.W .-1 ., 302 

Hsie, A.W ., 649 

Hsu, G .S ., 335 

Hsueh, J .L ., 255, 685 

Hu, Y .-C ., 261 

Huang, J ., 327 

Huang, M ., 666, 668 

Huang, N ., 262 

Huang, S .L ., 367 

Huang, X ., 264 

Huberman, E ., 686 

Hulina, G ., 174 

Hunsicker, P.R ., 488 

Huppi, C ., 548 

Huse, W .D., 300 

Husgafvel-Pursiainen, K ., 228, 330 

Huston, J .L., 161 

Hwang, M ., 600 

Igras, V., 42 

Ikushima, T., 266 

Imanishi, H ., 267 

Inouye, T., 151, 267 

Ipata, P.L ., 66 

Irwin, S .E ., 58 

Ishidate, M ., Jr ., 236, 243, 268, 291, 

355, 550, 624, 625, 626 

Issa, T., 437 

Issac, G .S ., 193, 278

Itoh, S ., 538 - = 

0 

Ivett, 1 .L ., 531 

Iwado, H ., 269 

Iwahara, S ., 490 

Iwasaki, M ., 89, 224 

Iwata, S ., 384 

Jabbar, M .A ., 687 

Jacobs, A ., 190 

Jacobson-Kram, D ., 645 

Jaen, J .C ., 307 

James, S ., 438 

Janion, C ., 270 

Jantunen, K ., 330 

Jarventaus, H ., 418 

Jayaraman, G ., 468 

Jenkinson, G ., 424 

Jensen, R .H ., 271, 319 

Jenssen, D ., 664 

Ji, X .Y ., 117 

Jia, S .-Z ., 671 

Jia, X ., 90 

liang, Z ., 272, 273 

Jin, C ., 274 

Jin, S ., 665 

Joardar, M ., 526 

Joe, C ., 323 

Joenje, H ., 196 

Johnson, E ., 531 

Johnson, M .D ., 302 

Joiner, E .E ., 275, 333 

Jones, I .M ., 75, 85 

Jones, N .J ., 579 

Jones, R .C ., 629 

Jones, R .L ., 534 

Jong, X ., 346 

Jordan, P., 301 

Joshi, V .P., 376 

Jostes, R .F., 276 

1ou, Y .S ., 277 

Juneja, T.R ., 225 

Jung, K .-Y ., 542 

Jurs, P.C ., 391 

Jyothy, A ., 193, 278, 465 

Kadhim, M ., 437 

Kadlubar, F .F., 89, 559 

Kafer, E ., 279 

Kalinowski, D ., 280 

Kalliomaki, P.-L ., 228 

Kamat, J .P., 405 

Kamiya, A ., 281 

Kamiya, S ., 288 

Kanazashi, K ., 490 

Kang, V ., 548 

Kangsadalumpaui, K ., 265 

Kaniguchi, Y ., 372 

Kappas, A ., 282 


Kari, F., 283, 630 

Kashyap, K ., 10, 463 

Kato, T ., 267, 287, 624 

Katoh, M ., 284, 537, 554, 596 

Katz, N ., 301 

Kaur, I .P . 225 

Kaur, R ., 408 

Kaur, S ., 65, 122 

Kauranen, P ., 344 

Kawai, A ., 285 

Kawai, K ., 180, 384 

Kawakishi, S ., 427 

Kawamoto, M ., 234 

Kawatani, N ., 377 

Kelsey, K .T ., 286, 633 

Kemin, B ., 342 

Keohavong, P., 577 

Kerckaert, G .A ., 321 

Kessen, S ., 148 

Khanna, D ., 40 

Kier, L .D ., 411 

Kikugawa, K ., 287 

Kim, J .B ., 104 

Kinae, M ., 288 

Kind, R ., 289 

Kindig, D ., 79 

Kinding, D ., 290 

Kingston, D .G .I ., 598 

. 

Kinzel, V ., 688 

Kirkland, D .J ., 291 

Kirlin, W .10., 246 

Kito, H ., 501 

Klein, C .B ., 292 

Klein, P., 450 

Klein, W.J ., 506 

Kleman, M ., 293 

Klepetka, J .F., 170 

Kligerman, A .D ., 294, 630 

Klopman, G ., 295, 481 

Knasmuller, S ., 296, 359 

Kniewald, J ., 174 

Knize, M .G ., 166, 366, 600 

Knudsen, I ., 297 

Koch, W .H ., 94 

Kochhar, T .S ., 298 

Kodaira, M ., 502 

Kodell, R .L., 388 

Kogiso, S ., 234 

Kohalmi, L ., 28 

Kohalmi, S .E., 299, 310, 311 

Kohler, S.W., 300 

Koop, P.C ., 361 

Kopfler, F .C ., 505 

Koreeda, M ., 542 

Korzeniowski, R ., 96 

Kramers, P.G .N ., 301 

Krauss, R .S ., 302 

Krehl, R ., 201, 303 


Kretz, P.L., 300 

Krishna, G ., 304, 575, 576 

Krishnamoorthy, R ., 405 

Krishnaswamy, K ., 10 

Krishrakumar, A ., 305 

KrOkje, A ., 306 

Kropko, G ., 575 

Kropko, M .L ., 304, 307, 576 

Kryptopoulos, S ., 29 

Kumar, A ., 446 

Kumar, D ., 308 

Kumari, C .K ., 278, 465 

Kumaroo, V ., 2, 3 

Kung, J .S ., 309 

Kunkel, T.A ., 693 

Kunz, B .A ., 28, 299, 310, 311, 374 

Kuo, B ., 443 

Kuo, S, 312 

Kwanyuen, P., 82 

Ladhar, S .S ., 682 

Lafi, A ., 313 

Lafuente, N .M ., 554, 596 

Lagenbach, R ., 318 

Laget, M ., 126 

Lake, R .S ., 314 

Lalchev, S ., 690 

Lambert, I .B ., 315 

Lamela, R ., 633 

Lampelo, S ., 317 

Langenbach, R ., 283 

Langlois, R .G ., 271, 319 

Larimer, F.W ., 280 

Lasher, L ., 508, 509 

Lasko, D .D ., 586 

Lasley, J .A ., 320 

Lasne, C ., 425 

Laufer, C ., 445 

Lavappa, K .S ., 534 

Lave, L .B ., 159 

Le Boeuf, R .A ., 1, 321 

Lechat, M .F ., 128 

Lecona, S .U ., 486 

Lee, C .K ., 144, 322 

Lee, H .G ., 213 

Lee, H .O . 323 

Lee, J .E ., 45 

Lee, J .K ., 534 

Lee, J .S ., 436 

Lee, M .A ., 323 

Lee, P .S ., 324 

Legator, M .S ., 32, 623 

Leonand, B ., 298 

Leopardi, P., 106 

Levin, J .D ., 132 

Levine, A .S ., 138 

Levis, A .G ., 110, 677 

Lewis, P., 166


Lewis, S .E ., 41, 325 

Lewtas, J ., 210, 689 

Li, A .P., 326 

Li, D .S ., 646 

Li, H ., 327 

Li, J .-H ., 328 

Li, J .J ., 675 

Li, X .-y ., 622 

Li, Z ., 90 

Li, Z .-Q ., 669 

Lia, C .-e ., 671 

Lian, X .-g ., 671 

Liang, T ., 90 

Liang, W .-Z ., 147 

Liang, X .-R ., 147 

Liao, M .-y ., 622 

Liber, H ., 470 

Lichtenberger, A ., 498 

Liegibel, U .L ., 506 

Lihua, S ., 342 

Lim, I . K . , 148 

Lim-Sylianco, C .Y . 329 

Lin, F ., 262 

Lin, G ., 346 

Lin, Y ., 668 

Lin, Y .-x ., 92 

Lindahl, T., 586 

Lindeskog, P ., 293 

Linnaimaa, K .I ., 330, 418 

Linscombe, V .A ., 202, 203 

Little, J .B ., 286 

Littlefield, L .G ., 275, 333 

Liu, B ., 102 

Liu, D ., 334 

Liu, P.K ., 335 

Liu, S .B ., 117 

Liu, S .F ., 65 

Liu, Y ., 331 

Liu, Y .P., 665 

Ljungman, M ., 332 

Loarca, F .P., 336 

Lodovici, M ., 142 

Loechler, E .L ., 337 

Lofroth, G ., 338 

Logan, D .M ., 462 

Lohman, P., 81 

London, F ., 390 

Longan, D .M ., 213 

Longid, C .S ., 399, 340, 341 

Longzhan, Y ., 342 

Lon:, N .A ., 343 

Lotjonen, S ., 317, 344 

Loveland, R ., 122 

Lowe, K .W ., 363 

Lu, Y ., 641 

Lucas, J ., 443 

Luks, H .J ., 629 

Lynch, A ., 438 

Lynch, D .W ., 286 


-- 

•-->Ma, G .-Y ., 669 w--` 

Ma, G .J ., 65T 

Ma, T.-H ., 336, 3ft-346, 347, 486 

MacGregor, J .T., 348, 583 

MacPhee, D .G ., 532 

Mackerer, C .R ., 58 

MacInnes, M .A ., 392 

Madden, J .J ., 520 

Maddux, S .C ., 488 

Magnusson, J ., 607 

Mah, M .C .-M ., 653 

Maher, V.M ., 55, 360, 349, 653 

Mahran, H ., 424 

Mailhesi, J .B ., 350 

Maity, S ., 526 

Majeeth, M .A ., 351 

Maki-Paakkanen, J ., 415 

Maness, S .C . 352 

Mara de S . Colus, L, 113 

Marchetti, F ., 431 

Margolin, B .H ., 353 

Marimuthu, K .M ., 120, 351 

Marques, M .M ., 44 

Marr, K ., 548 

Marsman, D .S ., 451 

Martin, M .V ., 224 

Martin, R .H ., 354 

Mason, J ., 57 

Mass, M .H ., 320 

Mass, M .J ., 320 

Matsshita, H ., 210 

Matsuda, H ., 537 

Matsumoto, K ., 31, 267 

Matsuoka, A ., 550 

Matsushima, T., 81, 178, 611 

Matsushita, H ., 253, 285, 355, 356 

Matsushita, H ., Jr ., 355, 356 

Matter, B ., 81 

Matthews, E .J ., 105, 239, 357, 358 

Mattila, S ., 228 

Mattson, K ., 330 

Matula, T.I ., 71 

Mayne, L ., 395 

Mayo, J ., 2, 588 

Mazurek, J ., 2, 3 

McCalla, D.R ., 315, 585 

McCartney, M .A ., 359 

McClintock, M .L ., 203 

McCormick, J .J ., 55, 203, 349, 360, 653 

McCoy, E .C ., 260, 359 

McCoy, G .D ., 361 

McCreary, R .D . 38 

McCune, S .L ., 74 

McDaniels, A .E ., 362 

McDowell, M ., 634 

McFee, A .F ., 4, 477 

McGarrity, L .J ., 388 

McGee, A .F ., 363 

McGregor, D ., 364 

McKelvey, V .J ., 365 

McKenna, P.G ., 365 

McKenzie, W .H ., 591 

McKinley, T .W ., Jr ., 516 

McLaren, K .F., 556, 557 

McLean, J .R ., 62 

McMahon, T .F ., 118 

McManus, M .E ., 366 

McManus, T .P., 170 

McMillin, D ., 121 

McSparrin, L ., 112 , 

Means, J .C ., 121 

Mecca, D .J ., 557 

Meenakshi, N .D ., 528 

Meier, J .R ., 140, 367, 368, 505 

Meiyue, R ., 473 

Melancon, S .B ., 545 

Melchior, W .B ., Jr ., 44 

Melcion, C ., 108 

Meli, C ., 512 

Melluso, G ., 434 

Mendelsohn, M ., 81 

Mendrala, A .L ., 203 

Meng, J .F ., 263 

Merl, T ., 369 

Messerly, E .A ., 199, 542 

Metzler, M ., 59, 160, 507 

Meuth, M ., 370 

Midtvedt, T., 379 

Miele, M ., 371 

Mikalsen, A ., 18 

Mikamo, M ., 372 

Miles, C ., 370 

Miller, G ., 166 

Miller, H ., 343 

Miltenburger, H .G ., 612 

Miranda, A ., 124 

Mirkova, E ., 690 

Mirsalis, J .C ., 3, 230, 373 

Mis, J .R .A ., 310, 374 

Misra, R .R ., 375 

Mitsuoka, T., 427, 567 

Mittelstaedt, R .A ., 581 

Miyamoto, J ., 234 

Miyazawa, T., 180 

Mochizuki, M ., 27, 355, 356 

Mody, R ., 376 

Modzelewski, R .A ., 377 

Mohn, G .R ., 387 

Mohran, Z ., 548 

Mohrenweiser, H.W., 378 

Mbller, L ., 379 

Mollis, S ., 511 

Monach, P ., 132 

Moneti, G ., 143 

Montero, R ., 250, 380 

Montgomery, J .C ., 637 

Moody, W., 298 

Moore, D ., 81

Moore, M .M ., 130, 141,-381 

Moraga, A .A ., 212 

Morales-Ramirez, P., 382r 

Moreira, J ., 83 

Moreno, F ., 380 

Morgan, T .L ., 276 

Morgan, W .F ., 383, 636 

Mori, H., 384 _ 

Morichetti, E ., 76 

Morita, T ., 385 

Morley, A .A ., 167, 386 

Morris, D .L ., 387, 623 

Morris, S .M ., 388 

Mostafa, M .H ., 247, 389 

Mott, K . E . , 301 

Mottus, K .M ., 280 

Moustacchi, E ., 435 

Moutschen, J ., 390 

Moutschen-Dahmen, M ., 390 

Moyer, S .R ., 391 

Mudgett, J .S ., 392 

Mukherjee, A ., 393, 394 

Mullenders, L.H .F ., 395, 604 

Mullenders, P.H .M ., 599 

Miiller, B ., 114 

Miiller, D ., 457 

Muller, E.W., 612 

Mulvihill, J .J ., 301, 396 

Munzy, D.M ., 484 

Murata, M ., 210 

Murli, H., 397 

Murota, T., 537 

Murray, B .P., 206 

Murray, S ., 206 

Murray, T.H ., 398 

Murthy, K ., 

Mustonen, R ., 399 

Nagabhushan, M ., 400, 401, 402 

Nagao, M ., 691 

Nagarajan, B ., 403 

Nagase, H ., 501 

Naito, M ., 269 

Nakajima, K ., 654 

Nakamuro, K ., 489 

Namiki, M ., 427 

Naram, R ., 404 

Narurkar, L.M ., 405 

Narurkar, M .V ., 405 

Natarajan, A .T., 51, 395 

Nauman, C .H ., 22, 583 

Neff, R .E ., 406 

Negishi, K ., 31, 52 

Negishi, T ., 407 

Nehru, B ., 408 

Nelson, R ., 186 

Nesnow, S ., 81, 409 

Nestmann, E .R ., 410, 411 

Nianjun, H ., 263 


Nicklas, 1 .A., 471, 545 

Nillson-Tillgren, T ., 474 

Nishioka, H ., 417 

Nitta, H ., 83 

Nivard, M .J .M, 413, 540 

Nohmi, T., 625, 626 

Nonaka, M ., 414 

Norppa, H ., 243, 415 

Notani, N .K ., 376, 416 

Nunoshiba, T ., 417 

Nylund, L ., 418 

O'Neill, J .P., 412, 471, 545, 676 

Oberly, T ., 419 

Oenick, N .L ., 103 

Oesch, F ., 139 

Ogier, H ., 545 

Ogolla, F ., 246 

Oh, K . C . , 347 

Ohe, T ., 420 

Ohshima, T ., 536 

Ohta, T ., 42, 267, 624 

Ohuchida, A ., 179, 565 

Ohwovoriole, A .E ., 421 

Okeke, G .C .E ., 421 

Okinaka, R .T ., 422 

Okmura, K ., 385 

Oleson, F .B ., 423, 424 

Oliveira, M .D.M ., 493 

Oliveri, G .,,,639 

Olivero, 0 ., 426 

Oliviera, M .D .M ., 475 

Omenn, G.S ., 159 

Ong, T., 97, 535, 631, 640, 648 

Orellana, M .M ., 554 

Orfila, L ., 425 

Osawa, T., 427 

Ose, Y ., 501 

Oshiro, Y ., 227, 428 

Osorio, S ., 198 

Osowole, O.A ., 429 

Osti, M ., 677 

Ostrosky-Wegman, P., 250, 380 

Otsuka, H ., 258 

Ottaggio, L ., 371 

Overton, L .K ., 127, 430 

Overvik, E., 293 

Ozasa, S ., 252 

Pacchierotti, F ., 431, 677 

Padmasani, V ., 497 

Pagano, D .A ., 433, 558 

Pagano, G ., 162, 434, 553 

Palin-Edlund, K ., 330 

Palit, S ., 135 

Panneerselvam, N ., 528 

Paolini, M ., 76 

Papadopulo, D ., 435 

Papapetropoulos, A.,479 

. 


Paradisin, W .M ., 651 

Pardee, A .B ., 67 

Pardo, K . C . , 123 

Paredes, M, 133 

Park, E.-H ., 436 

Park, S .D ., 104 

Park, S . S . , 343 

Parker, C ., 283 

Parker, L., 381 

Parry, E .M ., 437, 438 

Parry, J .M., 313, 437, 438 

Parton, J ., 290, 439 

Pastnik, A ., 413, 540 

Patterson, J ., 440 

Pawlak, A .L ., 632 

Pelaez, V ., 26 

Pellom, A ., 583 

Peng, G .-Y ., 100, 101 

Penman, B .W ., 116 

Perez, A ., 336 

Perry, B .A ., 378 

Peters, J ., 441 

Petrusevska, V., 688 

Petry, T., 2 

Phear, G ., 370 

Phillips, B .J ., 21 

Phillips, D.H ., 442, 504 

Phillips, J .W ., 383, 636 

Picardi, R ., 440 

Ping, L ., 261 

Pinkel, D ., 150, 443 

Piper, C .E ., 428 

Piperakis, S .M ., 444 

Plewa, M .J ., 445, 280, 446, 447, 547, 

613 

Ploem, J .S ., 51 

Pohl, H ., 448 

Poirier, M ., 44, 426 

Poirot, 0 ., 157 

Polverini, P ., 401 

Pongracz, K ., 65 

Pool, B .L ., 449, 450, 506 

Poorman-Allen, P., 237 

Popoff, S .C ., 132 

Popp, J . A, 451 

Popp, R .A ., 41 

Porfirio, B ., 435 

Porter, R ., 437 

Pourismaili, F, 687 

Povrik, L .F., 452, 560 

Prakhya, B ., 453 

Prasad, N .V., 10, 463 

Prasad, V.S ., 35 

Preston, R .H ., 216 

Preston, R .J ., 256, 257, 454, 455 

Provost, G .S ., 300 

Pupatwibul, K ., 456 

Puri, E .C ., 457 

Putman, K .L ., 504


Putnam, D ., 530 

Pyron, M ., 445 

Qian, B .-L ., 458 

Qian, L, 90 

Qingfan, Z ., 233, 459, 460 

Qingxia, Z ., 459 

Qiu, XE, 255 

Quattrochi, L .C ., 366 

Quillardet, P., 461, 587 

Rafter, J ., 379 

Raimondi, E ., 594 

Raj, A .S ., 462 

Rajah, T.T., 463 

Rajendran, M ., 528 

Ramanujam, V .M .S ., 623 

Ramel, C ., 464 

Ramesh, A ., 497 

Randerath, K ., 49, 504 

Randhawa, S .K ., 692 

Rani, A .S ., 278, 465 

Rao, K .P., 467, 555 

Rao, K .R ., 35 

Rao, K .V .S ., 468 

Raposa, T., 469 

Rappaport, S .M ., 65, 651 

Rathnaprabha, D ., 695 

Raychoudhury, A ., 526 

Raymer, G .D ., 516 

Recio, L ., 412, 470, 471, 545 

Reddy, O .S ., 193, 465 

Reddy, P.P., 278, 404 

Reidy, J .A ., 448, 472 

Renli, W ., 572 

Resnick, M .A ., 474 

Rexroat, M ., 419 

Reyes, A .L ., 362 

Riazi, G .H ., 687 

Ribiero, L .R ., 475, 493 

Rice, J ., 195 

Richard, S ., 492 

Richardson, C ., 291 

Richardson, K., 419 

Richmond, F., 111 

Richter, S . P ., 432 

Ringhand, H .P ., 505 

Rithidech, K ., 476 

Rizvi, T., 445 

Roberts, D .W ., 559 

Roberts, J .D ., 693 

Robertson, M .L ., 546 

Robertson, S .D ., 477 

Rocco, M ., 66 

Rodriguez, L .A . 133 

Rodriguez-Reyes, R ., 382 

Rodriguez-Arnaiz, R ., 478 


Rogers, C .Q.,4t _ 

RohrbacheK E~428 

Roilides, E ., 131. 

Rojanapo, W .`26S 

Rokosh, D .A ., 492 

Romagna, F., 479, 564 

Ronen, A ., 582 

Rosellini, D ., 76 

Rosenkranz, H .S ., 159, 295, 359,f180,- 

481 

Rosin, M .P., 482, 485 

Ross, L .S ., 219 

Rossi, 0 ., 483 

Rossiter, 484, 649 

Rossland, O .J ., 617 

Rossman, T .G ., 292, 328 

Rotenberg, S .A . 302 

Rotmensch, J ., 519 

Roupova, I ., 48 

Rousseau, E .J ., 485 

Rowland, I .R ., 521, 522 

Roy, A .K ., 526 

Roza, L ., 34 

Rudd, C .J . 123, 324 

Rudo, K ., 283 

Ruiz, E .F ., 486 

Russell, L .B ., 487 

Russell, W .L ., 488 

Russo, A ., 677 

Sabharwal, P ., 578 

Sadler, B.M ., 127 

Sailaja, D ., 695 

Sakaguchi, K ., 551 

Sakamoto, H ., 489 

Sakamoto, K . 490 

Sakamoto-Hojo, E .T ., 113, 491 

Sakata, Y ., 420 

Salamone, M .F., 213 

Salazar, E .P., 579, 628 

Salmeen, I ., 39 

Salvadori, D .M .F ., 475, 493 

Salvadori, M ., 143 

Samtella, R .M ., 205 

Sancar, A ., 515 

Sanchez, P.S ., 494 

Sandhu, D . 541 

Sandhu, S .S ., 195, 223 

Sandoval, M ., 380 

Sankaranarayanan, K ., 495 

Santella, R.M ., 496, 559 

Santhiya, S .T., 497 

Santos, S .J ., 570 

Sarasin, A ., 124, 498 

Sareen, P.K ., 183 

Sargent, G ., 370 

Sasaki, M .S ., 499, 500 

Sasaki, Y .F ., 267, 615 

Satish, S ., 139 

Sato, M .I .Z ., 494 

Sato, S . 566, 615 

Sato, T., 180, 501 

Satoh, C ., 502 

Savage, J .R .K ., 503 

Savela, K ., 504 

Sawada, M ., 550 

Sayato, Y ., 489 

Scariolo, S ., 594 

Schenck, K .M ., 367, 505 

Schiffmann, D ., 160, 507 

Schlehofer, J .R ., 450 

Schlepegrell, RL, 242 

SchmBl, D ., 694 

Schmezer, P., 449, 450, 506 

Schneider, O . , 157 

Schneiter, S ., 511 

Schnitzler, R ., 507 

Schoeny, R ., 440 

Schol, H .M ., 406 

Schorschinsky, N .S ., 320 

Schreibner, M .G ., 84 

Schreuder-Rotteveel, A .H.M ., 51 

Schut, H .A .J ., 43 

Schwartz, J .L ., 149, 519 

Schwartz, S ., 508, 509 

Schy, W .E., 510 

Scott, B .S ., 77 

Sedwick, W .D ., 511, 602 

Seeberg, A ., 512 

Seed, J .L ., 301 

Sega, G .A ., 513 

Sehgal, S .S ., 514 

Selby, C .P., 515 

Selby, P.B ., 516, 517 

Sen, M .P., 71 

Sen, S ., 525 

Sengupta, L .K ., 209 

Sengupta, S ., 518 

Sera, N ., 258 

Seth, R .K ., 513 

Shaddock, J .G ., 93 

Shadley, J .D ., 519 

Shafer, D .A ., 520 

Shah, A .B ., 521, 522 

Shahin, M .M ., 523 

Shandalis, A ., 219 

Shane, B .S ., 524 

Shankel, D.M ., 312 

Shao, H ., 656 

Sharaf, A .N ., 153 

Sharma, A ., 135, 393, 525, 526, 571 

Sharma, C .B .S .R ., 527, 528, 529 

Sharma, R .K ., 530, 531 

Shaw, T., 532

Shelby, M .D .,57, 161,251, 533, 550,638 

Shen, J ., 647 

Sheu, C .W., 534 

Shi, X ., 535, 648 

Shi, Z.Z ., 659 

Shibuya, T ., 536, 537 

Shimada, H ., 27, 538, 565, 615 

Shimada, T ., 224 

Shiotani, T., 407 

Shirasu, Y ., 267, 624 

Short, J .M ., 300 

Shreiner, C .A ., 58 

Shubber, E.K ., 539 

Shuli, F ., 233, 460 

Shyr, L .-S ., 77 

Siciliano, M .J ., 579 

Siede, W ., 176 

Sierra, L.M ., 540 

Silva, A .R ., 475, 493 

Silvestro, C ., 483 

Simic, M .G ., 50 

Simons, J .W .I .M ., 599 

Simpson, D ., 412, 470, 471, 545 

Simpson, S ., 548 

Singh, H ., 541 

Singh, J .R ., 134, 541 

Singh, V .P., 541 

Sinha, A .K ., 202, 203 

Sinsheimer, J .E ., 198, 199, 542 

Sivaramakrishnan, V .M . 305 

Skelly, M .F ., 367 

Skipper, P .L ., 543 

Skopek, T.R ., 412, 470, 471, 544, 545 

Smillie, R .H ., 532 

Smith, A .L ., 235 

Smith, B .A ., 44, 581 

Smith, M .T ., 546 

Smith, S .J ., 472 

Smith, S .R ., 547 

Smith-Oliver, T ., 49 

Smollinger, J .K ., 591 

Smulson, M .E ., 548 

Smylie, J ., 675 

Snow, E .T., 549 

Snyderwine, E .G ., 43, 366 

Sobrero, A ., 483 

SOderlund, E .J ., 78 

Soelter, S .G ., 428 

Sofumin, T., 550 

Sofuni, T., 236, 243, 268, 565 

Solberg, K .E ., 617 

Solt, D ., 401 

Song, J .-M ., 176 

Sonop, A ., 551 

SOrensen, B .S ., 69 

Sorg, R ., 3 

Sorge, J ., 300 


Soriano, J .D ., 340 

Sorsa, M ., 418, 552 

Soskoline, C .L ., 553 

Soto, J .P., 554 

Souza, S .C ., 570 

Spierto, F .W ., 472 

Sridevi, K ., 467, 555 

Stack, F ., 627 

Stahlberg, M ., 607 

Stankowski, L ., 3 

Stankowski, L.F ., Jr ., 556, 557 

Stark, A .A ., 558 

Stefandis, M ., 559 

Steighner, R .J ., 560 

Steinmetz, K .L ., 3, 230 

Stelma, G .N ., 140 

Stensman, C ., 338 

Stewart, J .D, 631 

Stoltz, S .L ., 37 

Stowesan, G .S ., 230 

Strniste, G .F ., 392, 422 

Strobel, K ., 561 

Strout, C .L., 75, 85 

Stupar, L .L ., 156 

Suarez, H .G ., 124 

Subramanyam, S ., 695 

Sugie, S ., 384 

Sugita, K., 253 

Suhonen, S ., 418 

Sujatha, M ., 465 

Sun, H ., 5b2 

Sun, W ., 346 

Suryakumari, T ., 563, 595 

Suter, W., 564 

Sutou, S ., 566, 615 

Suttajit, M ., 611 

Suzuki, K., 427, 567 

Swartout, J ., 440 

Swierenga, S .H .H ., 410 

Sylianco, C .Y .L., 610 

Szegedi, M ., 569 

Takagi, Y . 210 

Takahashi, C .S ., 113, 491, 570, 606 

Takahashi, N ., 502 

Takahashi, S ., 287 

Takatori, K ., 490 

Takayama, S ., 538 

Takeda, K ., 385 

Talitha, T.R ., 10 

Talukder, G ., 135, 525, 526 

Tammilehto, K ., 330 

Tan, C .C ., 685 

Tan, C .H .T ., 681 

Tan, Y ., 572 

Tanaka, T ., 384 

Tang, W .D ., 589 


Tannenbaum, S .R ., 543 

Tano, S ., 572, 573 

Tateno, H ., 372 

Tates, A ., 51 

Tavera, L ., 574 

Taylor, S ., 446 

Temenak, J .J ., 347 

Terzetti, F., 157 

Theiss, J .C ., 157, 304, 307, 575, 576 

Thilagar, A ., 2, 3, 530 

Thilly, W .G ., 577 

Thomas, D .C ., 693 

Thomas, M .A ., 578 

Thomassen, D.G ., 476 

Thompson, L .H., 87, 149, 579, 628 

Thorgeirsson, S .S ., 43, 580 

Thorton-Manning, J .R ., 581 

Tiah, M ., 582 

Tianbao, T ., 660 

Tice, R .R ., 22, 251, 583 

Tiveron, C ., 431 

ToftgArd, R ., 379 

Tokiwa, H ., 258 

Tollaksen, S .L ., 197 

Tomer, K ., 283 

Tometsko, A .M ., 584 

Tomkins, D .J ., 585 

Tomkinson, A.E ., 586 

Tompa, A ., 449 

Toohill, M ., 519 

Tbrnquist, S ., 379 

Toscano, W ., 633 

Touati, E .,461, 587 

Townsend, L .B ., 198 

Tozzi, M .G ., 66 

Trask, B ., 443 

Traul, K .A., 530, 531 

Travis, C .C ., 432 

Tregerman, L ., 588 

Trevizo, S ., 26 

Trinidad, A ., 246 

Tritscher, A .M ., 59 

Trivedi, A .H ., 7 

Trosko, J .E ., 277 

Troungos, C ., 29 

Tsuda, S ., 420 

Tsujimura, T ., 55 

Tu, Z .H ., 589 

Tucker, J .D ., 590 

Tukey, R .H ., 366 

Tulis, D .A ., 591 

Tuman, W .G ., 556, 557 

Tung, K .-K ., 84 

Turner, D .R ., 386 

Turtletaub, K .W ., 592 

Tutikawa, K ., 536 

Tweats, D .J ., 86, 593 

e


Ueamworapong, C ., 265 

Ukawa, S ., 27 

Uwaifo, A .O ., 429 

van Berkel, C .G.M ., 196 

van Hoffen, A ., 395, 604 

van Loon, A .A .W.M ., 34 

van Rooijen, J ., 599 

van Zeeland, A .A ., 395, 599, 604 

van de Klundert, F .A . J . M ., 196 

van der Schans, G .P ., 34 

von Borstel, R .C ., 619 

Vagnarelli, P., 594 

Vaidyanath, K ., 563, 595 

Vainio, H ., 228 

Valdivia, R .P.A ., 284, 554, 596 

Valencia, D ., 380 

Valent, G .U ., 494 

-Vallarino-Kelly, T., 382 

Valtierra, E.R ., 486 

Van Benthem, J ., 597 

Van Schaik, M ., 212 

Van Tassell, R .L ., 598 

Van der Wulp . C .J .M ., 34 

Vanderlan, M ., 600 

Varkonyi, J ., 469 

Vartiainen, T ., 344 



Vavrek, T., 629 

Veigl, M .L ., 511, 602 

Veleminsky, J ., 603 

Vellosi, R., 76 

Venema, J ., 395, 599, 604 

Venier, P., 110 

Venitt, S ., 605 

Verdier, M .M ., 547, 613 

Verdina, A ., 106 

Veronese, M .E ., 366 

Viaggi, S ., 66, 371 

Victorin, K ., 607 

Vierling, Th ., 608 

Vijayalaxmi, N .F.I ., 608 

Vijg, J ., 681 

Villasenor, I .M ., 610 

Vincenti-Dias, V .E .P., 606 

Vinitketkumnuen, U ., 611 

Vo-Dirh, T ., 205 

Vogel, E.W ., 413, 540 

Vblkner, W ., 612 

Von Borstel, R .C ., 249 

Vrieling, H ., 69, 599 

Vuglenov, A ., 48 

Wagner, E .D ., 547, 613 

Wagner, T., 148 

Wairimu, A .N ., 207 

Wakabayashi, K ., 614 


Wakata, A., W6, 565, 615. 

Walbousn ;~Cs&!f84 

Walker, It, 32 

Walker, G:C,*L-- 

Walker, R .P., 93 

Wallin, H ., 616, 617 

Walsh, D .B ., 107 

Wang, B .-S ., 669 

Wang, D ., 618 

Wang, H .-z., _ 

Wang, J ., 655 

Wang, J .X ., 670 

Wang, L, 273 

Wang, L ., 674 

Wang, L .-h ., 92 

Wang, M .Y ., 589 

Wang, Q ., 619, 620 

Wang, S ., 264 

Wang, S ., 650 

Wang, X ., 327 

Wang, X ., 621 

Wang, X .L ., 255 

Wang, Y ., 117 

Wang, Y ., 55 

Wang, Y ., 562 

Wang, Z .-q ., 622 

Wantanabe, K ., 624 

Wantanabe, M ., 355 

Wantanabe, T ., 252 

Ward, J .B ., Jr ., 20, 623 

Warman, B ., 148 

Warr, T ., 438 

Wasserman, S .S ., 508, 509 

Watanabe, M ., 267, 624, 625, 626 

Waters, M ., 81, 627, 696 

Watkins, B .E., 592, 600 

Wattenberg, L .W ., 697 

Waymack, P.P., Jr., 472 

Weber, C .A ., 579, 628 

Wei, L., 666, 668 

Wei, Z.-W., 663 

Weichoelbaum, R .R ., 149 

Weincke, J .K ., 286, 634 

Weinstein, I .B ., 302 

Weisburger, J .H ., 629 

Wen, S .-z ., 622 

Wenqing, L ., 473 

West, S .C ., 114 

Westbrook-Collins, B ., 630 

Whitlock, J ., 519 

Whong, W .-Z ., 631, 640, 648 

Whorton, E., 186 

Wicnienski, N ., 657 

Wielgosz, S .M ., 632 

Wiencke, J ., 633 

Wierzba, K ., 179 

Wild, D ., 289 

Wilkins, T.D ., 598 

Wilkinson, D ., 62 

William§, G .M ., 19 

Williams, J .R ., 645 

Williams, L ., 583 

Willis, A .E., 586 

Wilmer, J.W .G.M ., 597 

Wilson, D .M ., 635 

Winegar, R .A ., 383, 636 

Winkfield, L ., 26 

Winston, G .W ., 524 

Wise, D ., 198 

Wise, L .D ., 307 

Wiseman, R.W ., 637 

Wiser, S .K ., 235, 657 

Witt, K .L ., 57 

Witz, G ., 428 

Wojciechowski, J .P., 578, 638 

Wold, S .A ., 307 

Wolff, S ., 639 

Working, P.K ., 49 

Wu, G ., 641 

Wu, H .Y ., 665 

Wu, Z .-L ., 640, 648 

Wu, Z .L ., 147 

Wiirgler, F .E ., 175, 642 

Wymer, L .J ., 362 

Xi Li, L ., 643 

Xia, W ., 346 

Xiao, B .Q ., 644 

Xiao, S ., 645 

Xie, D .Y ., 646 

Xie, W ., 90 

Xili, L ., 647 

Xing, S .G, 648 

Xu, E ., 643 

Xu, J ., 346 

Xu, L .S ., 549 

Xu, Z ., 640 

Xue, K .X ., 650 

Xue, L .Y ., 103 

Yager, J .W ., 546, 651 

Yagova, A ., 48 

Yamada, F ., 234 

Yamasaki, H ., 652 

Yamashita, M ., 288 

Yang, B ., 562, 673 

Yang, H .-L. 349 

Yang, H .F ., 646 

Yang, J .-L ., 653 

Yang, W.-l ., 92 

Yannan, Y ., 185 

Yaozhong, W ., 473 

Yatagai, F ., 654 

Yerokun, T., 246 

Yi, A .-K ., 436 

Yin, M ., 655

Yin, S .-1 ., 656 

Yin, X ., 334 

Yongjun, W ., 233 

Yoshimi, N ., 384 

Yoshitake, A ., 234 

Young, R .R ., 531 

Yu, R ., 657 

Yu, Y ., 649 

Yu, Y .-q ., 656 

Yu, Y .N ., 658, 659 

Yuanl, Z .P ., 350 

Yue, S ., 641 

Yuhui, A ., 233 

Yun, J ., 148 

Yuzhi, W ., 660 

Zang, S ., 661 

Zdzienicka, M .Z ., 599 


~ 

Zeiger, E ., 57, 433, 558, 630, 662 

Zeller, U ., 506 

Zempel, J .A ., 203 

Zernik, M ., 138 

Zhang, H .-J ., 663 

Zhang, J ., 671 

Zhang, L .H ., 664 

Zhang, Q .X ., 644 

__Zhang, R .F ., 665 

Zhang, W ., 646 

Zhang, X .-I ., 656 

Zhang, Z ., 572 

Zhao, Q ., 672 

Zhao, W ., 667 

Zhao, Z ., 666, 668 

Zheng, Lin, 342 

Zheng, S ., 674 

Zhong, B .-Z ., 669 


Zhou, J.W ., 670 

Zhou, R ., 621 

Zhou, X ., 347 

Zhou, Z ., 671 

Zhu, M .C., 644 

Zhu, Q ., 672, 673 

Zhu, S .X ., 562, 665, 674 

Zhu, Y .-Z ., 663 

Zhuo, Jian, B ., 91 

Zhuo, P ., 650 

Zielenska, M ., 675 

Zijno, A ., 106 

Zimmer, D .M ., 676 

Zimmering, S ., 478, 574 

Zinkowski, R .P., 74 

Zito, R ., 106 

Zordan, M ., 110, 677

Name 

EMS- 

ENVIRONMENTAL MUTAGEN SOCIETY 

MEUBWSHIP APPLICATION 

Last First Middle Initial Highest degree 

Address 

City State Zip or Postal Code Country 

Affiliation, if above is home address Telephone 

Membership Types : 

Individual : $50 (U .S .A., Canada, Mexico) Patron : $1000 

Individual : $68.00 (Foreign) Sustaining : $500 

Couple : $65 (U .S .A., Canada, Mexico) ; $81 .50 (Foreign) 

Student : $7 .50, $42 .50 with journal (U .S .A., Canada, Mexico) ; $60 .50 with 

journal (Foreign) 

Student sponsor's signature . (Student status must be documented each year .) 

Each type of membership except the $7 .50 student membership includes a single 

subscription to the Society's journal, ENVIRONMENTAL AND MOLECULAR MUTA- 

GENESIS . Foreign subscriptions include partial payment of mailing costs . 

Please make check payable in U .S. funds to the ENVIRONMENTAL MUTAGEN 

SOCIETY . 

Mail to : 

ENVIRONMENTAL MUTAGEN SOCIETY 

P.O. BOX 7925 

BERKELEY, CA 94707-0925 


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are for the current calendar year, provided applications are received by July 1 of that 

year, unless otherwise specified .



INSTRUCTIC/aS TO AUTHORS 

ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 

Publishes original researG'F papers on mutation and mutation-related 

topics; in addition, there are review articles on topics of 

brood interest pertaining to mutagenesis and discussions of issues 

of social concern. 

Among the topics covered ore : mechanisms of mutogenesis, environmental 

modification of DNA, repair of DNA damage, genetic 

and cytogenetic methods forstudying genetic damage, mutogenicity 

screening, methods for estimating mutogenic hazards to humans, 

and epidemiological studies relating to environmental mutagens 

. 

The study of environmental mutagens is a multidisciplinary activity . 

The journal is intended for investigators in such fields as toxicology, 

genetics, microbiology, biochemistry, radiation biology, and 

basic cancer research, as well as other areas of biology, chemistry, 

and medicine that deal with mutagenesis . 

Manuscripts and all editorial correspondence should be sent by 

first-class mail, or air, to the editor : Dr . R .J . Albertini, Vermont Regional 

Cancer Center, 1 South Prospect Street, Burlington, VT 

05401, USA. The manuscript must be typed, double-spaced, on 

one side of white bond paper, approximately 81fi" by 11", with 

generous margins . Begin each section on a separate page . Number 

all pages in sequence beginning with the title page . Submit the 

original and three copies of all elements arranged in the following 

order: 

TITLE PAGE: This should contain an informative title, the names 

and affiliotions of all authors, the name and address for reprint 

requests, and a short running title . 

ABSTRACT : This should be a factual condensation of the entire 

work and include statements of the problem, method of study, results, 

and conclusion . The abstract may not exceed 250 words . 

KEY WORDS: Supply a list of three to six key words (not in the 

title) that will adequately index the subject matter of the article . 

TEXT: Text should follow the format : Introduction, Material!ànd 

Methods, Results, Discussion, Acknowledgments, and References . 

Authors whose "first" language is not English should arrange for 

their manuscript to be written in idiomatic English prior to submission 

. Authors may use either English or American Style ; for the 

former, consult the Oxford Shorter Dictionary; for the latter, consult 

Merriam-Webster's . All unusual abbreviations must be defined 

either at first mention or in a footnote. All measurements must be 

in metric units . All possible areas of misinterpretation such as differentiation 

between letters and numbers (letter I and number 1, 

letter a and number 0) should be marked on the manuscript by 

means of marginal notations . 

MATERIALS AND METHODS: This section of the paper must be 

sufficiently detailed to permit other scientists to evaluate the work 

critically and to repeat the experiments . The source of all chemicals, 

all unusual supplies, the organisms used, and any unusual 

apparatus should be given . Strain designations and relevant information 

on genotypes should be clearly specified . Any deviation 

from published methods should be given. If reference is made to 

o manuscript in press, a thesis, or a publication of limited distribution, 

three copies of the materials and methods from that manuscript 

or publication should be included for use by the reviewers . 

Authors should provide details of plant growth conditions or animal 

husbandry, such as food, bedding, light cycles, et cetero . Details 

of culture conditions and media should be provided for in vitro 

work. If in doubt about whether or not to include any technical 

detail, include it . 

REFERENCES: In the text, cite references by the name and date 

system. When there are more than two authors, use the first name 

and et ol (with period) . In the final list they should be arranged 

alphabetically, and chronologically for more than one reference 

with the same authorship . Use a letter suffix if more than one author 

reference is for the same year . Begin each reference with the 

names of all authors . For references to journals, give titles of articles 

in full, abbreviate journal names according to Index Medicus, 

and provide inclusive pagination . For references to books, include 

all authors' names, year of publication, chapter title (if any), editor 


(if any), book title, city of publication, and publisher's name . In the 

following examples, note the punctuation . Do not use all capitals . 

Do not underline. ' 

Journal Articles : 

Hirao Y, Yanagimachi R (1978) : Effects of various enzymes on 

the ability of hamster egg plasma membranes to fuse with 

spermatozoo . Gamete Res 1 :3-12 . 

Books : 

Austin CR (1968) : "Ultrostructure of Fertilization ." New York : 

Holt, Rinehart, and Winston, ch 2, pp 4-56 . 

Chapters in Books : 

Roth TF, Cutting JA, Atlas SB (1976) : Protein transport : A selective 

membrane mechanism . In Nicolson GL, Raftery MA, 

Rodbell M, Fox CF (eds) : "Cell Surface Receptors ." New 

York: Alan R. Liss, pp 487-508 . 

Contributors are encouraged to use the services of the Environmental 

Mutagen Information Center (EMIC), Oak Ridge National 

Laboratory, PO Box Y, Oak Ridge, TN 37830 . 

TABLES : Each table must have a self-explanatory title, be numbered 

in order of appearance with Roman numerals, and be 

keyed into the text . 

LEGENDS : A descriptive legend must accompany each illustration 

so that it can be understood apart from the text and must define 

all abbreviations used therein . 

ILLUSTRATIONS: Illustrations should be numbered in one consecutive 

series using Arabic numerals and be keyed into the text. 

Name of author, figure number, and an arrow indicating orientation 

should be typed on a gummed label and affixed to the back 

of each illustration. Line drawings must be of high quality ; typewritten 

pr hand lettering is unacceptable. Either the original drawing 

or good quality photographic prints are acceptable . Photographs 

should be high contrast, glossy prints . If possible, use black 

and white; a charge will be made for color reproduction . Photographs 

may be mounted or unmounted ; if mounted they may abut . 

Magnifications may be indicated by a micron bar or in the legend . 

Photographs which are to be reproduced without further reduction 

must be so marked and may not exceed 6 13/16" x 8 7/8" in order 

to fit the journal format. In general practice it is best to restrict 

the length or depth of submitted photographs to 8 1/8" to allow 

for legend placement. Photographs intended for single column 

placement should not exceed 3 5/16" in width . Photomicrographs 

will not be reduced unless they exceed the size of the page . Details 

that are of particular importance should be indicated on 

overlay sheets . 

BRIEF COMMUNICATIONS: Short, original scientific notes may 

be submitted to ENVIRONMENTAL AND MOLECULAR MUTA- 

GENESIS. These should be approximately two printed pages 

(1,000 words) and do not require abstracts . Stylistic requirements 

such as the use of abbreviations, preparation of illustrations, form 

for references, title page, etc, are the same as above . 

LETTERS TO THE EDITOR: Letters should be double-spaced and 

will be subject to review for pertinence and content . 

ALL MANUSCRIPTS submitted to ENVIRONMENTAL AND MO- 

LECULAR MUTAGENESIS must be submitted solely to this journal, 

may not have been published in any part or form, except as an 

abstract for a meeting, in another publication of any type, professional 

or lay. Upon acceptance of a manuscript for publication, the 

author will be requested to sign an agreement transferring copyright 

to the publisher, Alan R . Liss, Inc., 41 East 11th Street, New 

York, NY 10003, USA, who reserves copyright . No published material 

may be reproduced or published elsewhere without the written 

permission of the publisher and the author . The journal will be 

not responsible for the loss of manuscripts at any time. All statements 

in, or omissions from, published manuscripts are the responsibility 

of the authors who will assist the editors by reviewing 

proofs before publication . Reprint order forms will be sent with the 

proofs . A page charge of :15 per printed page will be requested 

from the authors or their institutions for publication in the journal . 

Acceptance and publication, however, are not contingent on the 

ability to pay page charges. 

It

Environmentai and 

Molecular Mutagenesis 


Volume 14, Supplement 15 1989 

Abstracts of the Fifth International Conference on Environmental Mutagens . . . . . . . . . . . . . . . . 3 

Author Index to Volume 14, Supplement 15 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241 


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