[CANCER RESEARCH 39, 131-138, January 1979]0008-5472/79/0039@0O00$02.0O

Oncogenesis, Mutagenesis, DNA Damage, and Cytotoxicity in CulturedMammalian Cells Treated with Alkylating Agents1

A. R. Peterson,2 Hazel Peterson, and Charles Heideiberger

University of Southern California Comprehensive Cancer Center, Cancer Research Laboratory, Los Angeles, California 90033

esis. One is to trace step by step the process of oncogenictransformation in cells assembled from components, DNA,chromatin, nuclei, and cytoplasm, that have been treatedwith carcinogens under carefully controlled conditions invitro ; the other approach is to establish uniform associations between the process of oncogenic transformation andother processes that are better understood at the molecularlevel. We have chosen the latter approach for the studieson the effects of monofunctional methylating and ethylatingagents reported in this series of papers.

A prerequisite for our approach is that the processes thatwe study should have end points that can be measuredaccurately and interpreted with a minimum of ambiguity.For the process of mutation, colonies of AZG3-resistantcells, and for the process of alkali-labile DNA damage,changes in the molecular weight of DNA on alkaline sucrosegradients have been shown to fulfill most of the criteria foraccuracy (3, 8, 19, 22, 24, 32, 33).4 Some difficulties remainin the interpretation of the data of mutation and DNAdamage, but experiments are designed to resolve thosedifficulties in the course of this work.

In the present study, we report associations betweenmutations to AZG resistance, cytotoxicity, and alkali-labileDNAdamage. Mutation to AZG resistance,which could notbe detected in the tetnaploid C3H/10T1/2 cells, was measured in diploid Chinese hamster V79 cells to be relatedthrough associations with DNA damage and cytotoxicity tothe effects of the alkylating agents in the transformablecells. Preliminary accounts of this work have been published (21).

MATERIALSAND METHODS

Chemicals, Radiochemicals,and Cell Culture Media.MNNGand EMS were obtained from Sigma Chemical Co.,St. Louis, Mo. ; ENNG and MMS were obtained from AldrichChemical Co. , Inc. , Milwaukee, Wis. The purity of thesechemicals was 98 to 99.5% as determined by UV andinfrared spectroscopy and by gas-liquid chromatography.Radiochem icals, [2-14C]thymidine (specific activity, 60 mCi/mmoi) and [6-3H]thymidine (specific activity, 5 Ci/mmol),were supplied by Amersham/Seanle Corp. , ArlingtonHeights, ill. Media and sera for cell culture were suppliedby Grand Island Biological Co., Grand Island, N. V. PlasticPetni dishes for many experiments were obtained from

3 The abbreviations used are: AZG, 8-azaguanine; MNNG, N-methyl-N'-

nitro-N-nitrosoguanidine; EMS, ethyl methanesulfonate; ENNG, N-ethyl-N'-nitro-N-nitrosoguanidine; MMS, methyl methanesulfonate; LD, dose lethalto percentage of animals indicated by subscript number; ANNG, N-alkyl-N'nitro-N-nitrosoguanidine.

4 A. A. Peterson and M. Mulkins, Alkali-labile Phosphate Bonds in the

DNA of Mammalian Cells and Bacteriophages, manuscript in preparation.

ABSTRACT

Mutation to 8-azaguanine resistance in Chinese hamstercells and cytotoxicity and production and repair of alkalilabile lesions in the DNA of Chinese hamster V79 andtransformable mouse embryo C3H/10T1/2 fibrobiasts weremeasured after the cells had been treated for 2 hr with themonofunctional alkylating agents, N-methyl-, and N-ethylN'-nitno-N-nitrosoguanidine and methyl and ethyl methanesulfonates. in both cell lines, methylating agents were morecytotoxic than were equimolar concentrations of ethylatingagents, and N-methyl- and N-ethyl-N'-nitno-N-nitrosoguanidine were more cytotoxic than were methyl methanesulfonate, and ethyl methanesuifonate. At equitoxic concentrations, N-methyl-N'-nitno-N-nitnosoguanidine was 14 timesas mutagenic as was methyl methansulfonate and produced400 times as many alkali-labile lesions; while N-ethyl-N'-nitro-N-nitrosoguanidine and ethyl methanesuifonate produced similar numbers of alkali-labile lesions and wereequally mutagenic. A plot of alkali-labile lesions versusmutations was a straight line, but the frequency of alkalilabile lesions was 7 orders of magnitude greater than wasthe frequency of mutations; the rate of repair of the alkalilabile lesions varied inversely as the first power of thenumber of lesions. These findings suggest that mutagenicand alkali-labile lesions are associatedbut not identical andthat neither lesion is associated with cytotoxicity. Theresults are discussed in the light of chemical theories ofalkylation and mutagenesis and are compared with previousstudies that showed that oncogenic and alkali-labile lesionsare not uniformly associated.

INTRODUCTION

Cells from C3H/10T1/2clone 8 of mouse embryo fibroblasts have been transformed with a variety of chemical,radiant, and viral carcinogens to produce foci of cells thatyield malignant fibrosarcomas in syngeneic, irradiated mice(10, 11). Moreover, the promoting effects of phorbol estershave been clearly demonstrated in these cells (16). Therefore, in this cell line under conditions in which no detectable spontaneous transformation occurs the molecularevents associated with oncogenesis can be examined inhomogenous, cloned populations of cells in circumstancesthat can be controlled more effectively than is possible inexperiments with whole animals (10, 11).

We envisage 2 kinds of approaches to obtain from C3H/10T1/2 cells information on the molecular basis of oncogen

1 Supported in part by Grant CA-21036 from the National Cancer Institute,

NIH, and by Grant BC-2H from the American Cancer Society.2 To whom requests for reprints should be addressed.

Received July 17, 1978; accepted October 10, 1978.

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T —Total no of AZG1 colonies from treated cultures— Total no. of dishes after final replating

C —Total AZGrcolonies from acetonetreatedcultures— Total dishes of replated, acetone-treated cultures

Falcon Plastics, Oxnand, Calif. , until 1977 and were supplied by Conning Glass Works, Corning, N. V. , thereafter.Density gradient grade sucrose was obtained fromSchwarz/Mann, Onangeburg,N. Y.

Culture and Treatment of C3H/10T1/2and V79 Cells.C3H/10T1/2cells were grown in Eagle's basal mediumsupplemented with 10% fetal calf serum (26, 27), andChinese hamster V79 cells were grown in Dulbecco's medium supplemented with 10% dialyzed fetal calf serum asdescribed previously (3, 24, 25). Exponentially growingcultures were treated with fresh acetone solutions of alkylating agents for 2 hr at 37°in complete medium. Theconcentration of acetone in the medium was not toxic anddid not slow the growth of the cells. After treatment, thecells were washed with complete medium (5 ml) and coyered with appropriate volumes of fresh medium for measunements of mutagenesis, cytotoxicity, and DNA repair,which required posttreatment incubation of the cells.

CytotoxicityAssays. Cytotoxicitywas assayedas descnibedpreviously (3, 23, 25) by measuring the fraction ofcells that survived treatment with alkylating agents to produce colonies of 50 onmore cells within 7 to 10 days aftertreatment. For controls and treatments with low doses ofalkylating agents (

MMS200 400 600 80020

DNA Damage, Mutation, and Cytotoxicity in Cultured Cells

CONCENTRATIONOF ALKYLATINGAGENT(aiM)

0>>

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Chart 2. The effects of expression time on the frequency of mutation toAZG resistance in @P9cells occurring spontaneously (@) or after a 2-hrtreatment with 600 @a.iMMS (0); 1.7 p@ (•),3.4 @iM(A) and 6.8 @.tM(•)MNNG.

MNNG10 15

60

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LD1@,were constructed with at least 5 data points, morebeing used to delineate shoulder regions, which, particularly in the case of EMS, were subject to considerablevariability. In some cases, survival curves were extendedbeyond LD,,@so as to measure D0, the dose required toreduce the surviving fraction by 1/e in the exponentialportion of the survival curve (7).

Mutation frequencies after MNNG and MMS treatmentwere measured after various expression times. All the datapoints that did not differ significantly from the maximummutation frequency were pooled to produce a mean maximum mutation frequency. Since we (24) and others (9, 18,32) have repeatedly demonstrated that plots of F againstdose of alkylating agent are straight lines under the conditions of these experiments, 3 well-separated data pointswere generally considered sufficient to delineate the doseresponse for mutation. Computer-assisted linear regressionanalysis was used to fit straight lines to the exponentialregion of survival curves and to the plots of F and alkalilabile lesions against dose. S.E. of the slopes and interceptswere calculated by equations given in Ref. 5.

RESULTS

Toxicity of Alkylating Agents In C3H/10T1/2 and V79 Cells

The D0 dose levels of MMS, EMS, and ENNG for V79 cellswere lower than for C3H/10T1/2 cells, but the shoulders ofthe survival curves of the V79 cells suggest that these cellsincurred about twice as much sublethal damage as did theC3H/10T1/2 cells (Chart 1). However, MNNG was not moretoxic to the V79 cells than to the C3H/10T1/2cells andproduced survival curves without shoulders in both celllines (Chart 1), suggesting that the cytotoxicity of MNNG isaffected by factors related to the ploidy and species oforigin of the cells and also to the mechanism of delivery ofthe MNNGmethyl group to targets within the cells.

On the other hand, the ANNG were 1 to 3 orders ofmagnitude more toxic than were equimolar concentrationsof the alkyl methanesuifonates in both lines of cells (Chart1). Furthermore, the ethylating agents were consistentlyless toxic than were the methylating agents.

Mutationin V79cells

Effectsof ExpressionTime. The expressiontime is theinterval between the removal from the cultures of themutagen-containing solution and the addition of selectivemedium containing AZG. During this time, the cultureswere maintained and neplated in fresh medium to allowthem to undergo several doublings, the number of whichwere determined from growth curves. In Chart 2 the expression time is written as the number of doublings.

The spontaneous mutation frequency was unaffected byexpression time, but the mutation frequency induced bytreatment of the cells with MNNG and MMS increased withexpression time until 4 population doublings had occurred,after which further variations in mutation frequency werenot significantly different (p < 0.05; Chart 2). This mimimumrequirement of 4 population doublings for maximumexpression of induced mutants was the same for all concentrations of MNNG, MMS (Chart 2), ENNG, and EMS (unpub

0.10 ‘ 0.100.075@ \@ 0.075

0.050@ I 1.@Chart I . Dose-response curves of the cytotoxicity of alkylating agents.

Colony formation from single cells plated immediately after a 2-hr treatmentwith alkylating agent was determined with Chinese hamster V79 cells (0)and with C3H/1OTV2mouse embryo fibroblasts (•).

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A. R. Peterson et a!.

lished observations). Therefore , the mutation frequencywas determined either by taking the mean of all measurements made after 4 doublings or by measuring the mutationfrequency after a single expression time of 6 to 9 doublings.These 2 methods of determination were compared andfound to yield virtually identical results.

Dose-Response Curves. Chart 3 shows the dose-response curves for mutagenesis in V79 cells treated withmonofunctional alkylating agents. The induced mutationfrequency increased as a linear function of dose withintercepts —0,the ANNG was —102-foldmore mutagenicthan were equimolan concentrations of the correspondingaikyl methanesulfonates and the methylating agents weremore mutagenic than were the ethylating agents. Thesefindings show that the mutagenicities of the alkylatingagents used in this study are influenced both by the structunes of the leaving groups and of the aikyl groups.

20

16

12

8

4

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Alkali-labile Lesions in the DNA of V79 and C3HI1OT'/2Cells

Sedimentation of DNA from C3H/10T1/2 and V79 cellsgave broad symmetrical profiles containing 80% of theradioactivity applied to the gradient (Chart 4).The peaks ofthese profiles were at 1695 and 1545, respectively, fromwhich M,, values of 7.12 ±0.06 x 10@daltons (18 expeniments with C3H/10T1/2 cells) and 4.62 ±0.41 x 10@daitons(8 experiments with V79 cells) were calculated. After treatment of the cells with MNNG (6.8 @tM)the DNA sedimentedmore slowly (—485,0.17 ±0.04 x 10@daltons, 7 expeniments, C3H/10T1/2 cells; —465,0.14 ±0.01 x 10@daltons,3 experiments, V79 cells), showing that single-strand breakage had occurred. The frequencies of alkali-labile lesionscalculated from these examples are 5.74 ±1.35 lesions/i 08daltons of C3H/1OT'/2 cell DNA and 6.93 ±0.62 Iesions/i08daltons of V79 cell DNA. Profiles representative of thesesamples are given in Chart 4.

In both cell lines, alkali-labile lesions increased as a linearfunction of dose of MNNG, EMS, and ENNG, but in V79

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FRACTION (TOP)Chart 4. Alkaline sucrose gradient sedimentation profiles of DNA from

C3H/1OTV2cells (A), V79 cells (•),and from V79 cells treated with 6.8 pMMNNGfor 2 hr, incubatedfor 4 hr, and centrifugedat 13,000rpm for 7 hr(0). A profile of DNA from C3H/1OT'/2 cells treated with 6.8 pM MNNG for 2hr, incubated for 4 hr, and centrifuged at 15,000 rpm for 6 hr is also shown

cells the frequency of alkali-labile lesions produced by MMSwas so low that a significant correlation coefficient couldnot be obtained for a straight line fit of the data (Chart 5). Inall cases the intercepts were —0.Chart 5 also shows thatthe ANNG produced —10k-foldmore lesions than did equimolar concentrations of the corresponding alkyl methanesulfonates and that the methylating agents produced about10 times as many lesions as did the ethylating agents inC3H/10T1/2 cells. However, in V79 cells equimolan concentrations of EMS and MMS produced similar numbers ofalkali-labile lesions.

Comparisons of Charts 3 and 5 show that the effectiveness of the agents in producing mutations parallels theireffectiveness in producing alkali-labile lesions.

DNA Damageand MutationProducedby EqultoxicDosesof AlkylatingAgents

Table 1 shows that at equitoxic concentrations MNNGproduced 6 times as many alkali-labile lesions as did MMSin C3H/iOT'/2 cells and 400 times as many alkali-labilelesions and 14 times as many mutations as did MMS in V79cells. By contrast, ENNG produced only 1.6 times as manyalkali-labile lesions as did EMS in C3H/10T1/2 cells andnumbers of alkali-labile lesions and mutations in V79 cellsthat were not significantly different from those produced byEMS. These results show that mutagenesis and cytotoxicity

200 4 8 12 16ENNG

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Chart 3. Dose-response curves for mutations to AZG resistance producedin V79 cells by MNNG R•),correlation coefficient r = 0.983]; MMS [(0), r =0.882];ENNG[(•),r = 0.9931;and EMS[(0), r = 0.860].

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Table1Mutationsand alkali-labile lesions produced by equitoxic concentrationsofmonofunctional

alkylatingagentsSlopesof dose-response curves of cytotoxicity (lID0) and alkali-labile lesionsproducedin

mouse C3H/10T1/2 and Chinese hamster V79 cells and of mutations to AZGresistanceproducedin the V79cells by methylating (MNNG,MMS)and ethylating (ENNGandEMS)agents.

S.E.'s are calculatedfrom the data points by equations given in Ref.5.10-@x alkali labile 10@xAZGrD0

lesions x cell x mutationsxCellsAgents (p.@i) D01 ce111xD0'V79MNNG 7.02 ± 0.63 4.74 ±0.74 79.2 ±10V79MMS 164.8 ±24 0.011 ±0.04 5.70±1.09V79ENNG 8.13± 1.17 0.714±0.159 19.80±3.3V79EMS 5037 ± 7.00 1.00 ±0.170 17.50 ±3.9C3H/1OT'/2MNNG 4.62 ± 0.75 4.79 ±0.24C3H/10T1/2MMS 271 ±19.0 0.77 ±0.089C3H/10T1/2ENNG 17.8 ± 3.53 2.21 ±0.16C3H/10T1/2EMS 7746 ±19 1 .35 ±0.12

DNA Damage, Mutation, and Cytotoxicity in Cultured Cells

produced by these alkylating agents in V79 cells are notuniformly associated.

A plot of alkali-labile lesions versus mutation in V79 cellsgave a straight line with intercept @0(Chart 6), suggestingan association between mutagenesis and alkali-labile DNAdamage. However, Table 1 and Chart 6 show that alkalilabile lesions occur —i0@times more frequently than domutations, suggesting that the 2 events are not uniformlyassociated, which is supported by further measurements ofthe kinetics of repair of alkali-labile lesions.

3C,)z0@ 2-“C0

@ 1@ 2 ---@-.@-;-o

0 200 400 600 8001000EMS

CONCENTRATION OF ALKYLATING AGENT (,UM)Chart 5. Dose-response curves for alkali-labile lesions produced in V79

cells (0) and in C3H/1OT'/a cells (•)by treatment with alkylating agents for2 hr followed by a 4-hr incubationin fresh medium.Correlationcoefficient,r = 0.95exceptfor r = 0.02for MMSin V79cells.

Chart 7 shows that the molecular weight of DNA fromC3H/10T1/2 cells treated with MNNG, MMS, and EMS increased linearly with time as the cells were incubated infresh medium at 37°,showing that the alkali-labile lesionswere repaired during the incubation. Repair of the alkalilabile lesions produced by ENNG could not be detected inthese experiments. The slopes of the straight lines in Chart7 were plotted against the frequency of alkali-labile lesionsmeasured 4 hr after treatment, and it was found that therates of repair (slopes) varied as the reciprocal of thenumber of alkali-labile lesions (Chart 8). Moreover, ethylation lesions were repaired more slowly than were methylation lesions in the C3H/iOT1/2 cells, and repair of the lesionsproduced by MMS and MNNG in the V79 cells was slowerthan in the C3H/10T1/2 cells (Chart 7). Therefore, for thealkali-labile lesions to be uniformly associated with mutation, the mutation frequency should vary as the first powerof the frequency of alkali-labile lesions, and the ethylating

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A. A. Peterson et a!.

- MNNG

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0 20 40 0 20 40

TIME (HR) AFTER TREATMENTChart7.Theeffectsof posttreatmentincubationin mediumat37°onthe

molecular weight of DNA from C3H/10T/@ cells treated for 2 hr with 0.17 pMMNNG, 45.4 pM MMS, 6.21 pM ENNG, and 161 pM EMS (•);0.34 pM MNNG,90.8 pM MMS, 12.4 pM ENNG, and 322 @iEMS (0); 0.68 @a.iMNNG, 136pM MMS, 24.8 pM ENNG, and 644 pM EMS (A); 1 .36 pM MNNG, and 908 pMMMS (Lx);or 2.72 pM MNNG (s); and 6.80@ MNNG (0).

agents should be more mutagenic than the methylatingagents. These conditions are not met (Chart 6). Therefore,our interpretation of Table 1 and Chart 6 is that the alkalilabile lesions are not mutagenic, but they represent the setof O-alkyIation lesions which includes those that are mutagenic. Furthermore, Table 1 shows that alkylation lesionsthat lead to alkali-labile DNA damage and mutations to AZGresistance are not associated with cytotoxicity.

DISCUSSION

We have measured colony formation as a criterion forcytotoxicity, colony formation in medium containing AZG(40 @.tg/mI)for mutation, and the molecular weight of DNAsedimented through alkaline sucrose gradients for alkalilabile lesions in DNA. Before attempting to interpret thesedose-response data, it was necessary to ensure that theabove criteria do accurately represent cellular lesions thatlead to cytotoxicity, mutagenesis, and alkali-labile DNAdamage.

Experiments with the effects of ionizing radiations onmammalian cells (7) have established that our methods formeasuring cytotoxicity do represent lesions that preventsingle cells from undergoing a sufficient number (-‘-6)ofdivisions to form macroscopic colonies of viable cells.

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0.03

0.02

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Chart 8. The relationship between the rate of repair (@Mn = slopes ofcurvesshownin Chart7)andfrequencyat 4 hr aftertreatmentof alkali-labilelesions produced in C3H/1OT/2 cells by a 2-hr treatment with MNNG (•),MMS (0), ENNG (A), and EMS (Es).Resultsobtained from V79 cells treatedwith MNNG (@)and MMS (0) are also shown.

Those experiments have also been extended to encompassthe effects of alkylating agents on cultured mammaliancells (3, 9, 18, 23, 28, 32). As for the AZG-resistant phenotype, which is our end point for mutagenesis, we showedconcurrently with other laboratories that reproduciblequantitation of mutation at the hypoxanthine:guanine phosphonibosyltransferase locus requires stringent selectionconditions that can be satisfied by using 40 @.tgof AZG perml in medium free of hypoxanthine and by replating mutagen-treated cells at low density prior to selection (8, 18, 19,24, 25, 32). We have now found that expression timeslonger than we had previously used are necessary foroptimal measurement of the mutation frequency, in conformity with other reports (8, 18, 19, 32). The developmentand characterization of our alkaline-sucrose sedimentationprocedure is described in considerable detail4 and providescomplete corroboration for our interpretation that the sedimentation measurements used in the present study represent lesions produced by the alkylating agents in singleDNA strands. That the lesions become single-strand breakswhen exposed to alkali has been deduced from our owncalculations (22) and from other experiments on mammahan cells (33) and bacteriophages (30).

The wide ranges (@-103-fold)in the D0 concentrations ofthe 4 aikylating agents shows that the concentration requined to penetrate to critical targets for cytotoxicity variesconsiderably with the structure of the agents. Therefore, itis to be expected that the concentration required to penetratetotargetsforotherbiologicaleffectswillalsovaryandthat comparisons of mutation and DNA damage at equimoIanconcentrations will reflect differences in the accessibilities of the targets to the agents, while comparisons atequitoxic doses will reflect differences in the mechanisms

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DNA Damage, Mutation, and Cytotoxicity in Cultured Cells

of alkylation. These considerations led us to choose cytotoxicity as a base line for normalizing the results of mutationand DNA damage, but our decision was also guided by thestudies of Roberts et a!. (28), who showed in Chinesehamstercellsthatcytotoxicitywas relatedto the overallextent of alkylation of the DNA by 3 different methylatingagents. Furthermore, by normalizing for cytotoxicity weconsider only lesions that occur in cells that survive treatment with the alkylating agents.

The dose-response curves for mutation are straight lines.This is consistent with the theory (12) that relates themutation frequency to dose according to the formula:

N,,,/N. = Z(G*/G)

where N,,, is the number of mutants, N,, is the number ofsurvivors, Z is the number of alkylations in the genome, G*istargetsize,G isgenomesize,and

z = kD

O-alkyiation, some of which should be mutagenic. Howeven, oncogenic transformation of C3H/10T1/2cells by thesealkylating agents occurs at too low a frequency (i0@survivors) to be accurately measured under the conditionscustomarily used for such experiments (Ref. 26; unpublished observations) but occurs at a frequency >i0@ survivors in synchronized cells (2) that contain the same numbens of alkali-labile lesions as do asynchronous cells (22,23). Therefore, even in cells containing mutagenic lesions(O-alkylation in DNA) produced by potent carcinogens(MNNG and ENNG), expression of the transformed phenotype can be diverted on suppressed.

F We add our findings to the growing weight of evidence( ) from studies on mutagenesis (1, 24) and carcinogenesis (4,

15, 17, 23) thatthe metabolic processes associated with theexpression of genetic damage are at least as important asthe nucleic acid chemistry of the damage itself in detenmining the biological effects of carcinogens and mutagens.

ACKNOWLEDGMENTS

Part of this work was performed at the McArdle Laboratories for CancerResearch, University of Wisconsin, Madison, Wis. 53706. We thank S.Altenbach and D. A. Groom for skilled technical assistance and Dr. H.Campbell and A. Manaka for some of the computer programs.

REFERENCES

1. Auerbach, C. Mutation Research, Problems Results and Perspectives,pp. 291-292. London: Chapman and Hall, 1976.

2. Bertram, J. S., and Heidelberger, C. Cell Cycle Dependency of Oncogenic Transformation Induced by N-methyl-N'-nitro-N'-nitrosoguanidlnein Culture. Cancer Res. 34: 524-537, 1974.

3. Bhuyan, B. K., Peterson, A. A., and Heidelberger, C. Cytotoxicity,Mutations and DNA Damage Produced in Chinese Hamster Cells Treatedwith Streptozoticin , Its Analogs, and N-methyl-N'-nitro-N-nitrosoguanidine. Chem-Biol. Interactions, 13: 173—179,1976.

4. Buecheler, J., and Kleihues, P. Excision of 0-Methylguanine from DNAof Various Mouse Tissues following a Single Injection of N-Methyl-N-nitrosourea. Chem.-BioI. Interactions, 16: 325-333, 1977.

5. Draper, N., and Smith, H. Applied Regression Analysis, pp. 18-21 . NewYork: John Wiley & Sons, 1966.

6. Ehmann, U. K., and Lett, J. T. Review and Evaluation of MolecularWeight Calculations from the Sedimentation Profiles of Irradiated DNA.Radiation Res., 54: 152-162, 1973.

7. Elkind, M. M., and Whitmore, G. F. The Radiobiology of the CulturedMammalian Cell, pp. 7-111 . New York: Gordon and Breach, 1967.

8. Fox, M., Factors Affecting the Quantitation of Dose Response Curves forMutation Induction in W9 Chinese Hamster Cells after Exposure toChemical and Physical Mutagens. Mutation Res., 29: 449-466, 1975.

9. Fox, M., and McMillan, S. Relationship between Caffeine Sensitive andResistant DNA Repair, Cell Lethality and Mutagenesis in MammalianCells after X-Rays and Alkylating Agents. Studia Biophys., 61: 71-79,1977.

10. Heidelberger, C. Chemical Oncogenesis in Culture. Advan. Cancer Res.,18: 217-366, 1973.

11. Heidelberger, C. Chemical Carcinogenesis. Ann. Rev. Biochem., 44: 79-121, 1975.

12. Lawley, P. D. Some Chemical Aspects of Dose-Response Relationshipsin AlkylationMutagenesis.MutationRes.,23: 283-295,1974.

13. Lawley, P. D., and Thatcher, C. J. Methylation of Deoxyribonucleic Acidin Mammalian Cells by N-Methyl-N'-nitro-N-nitrosoguanidine. Biochem.J., 116: 693—707,1970.

14. Lehmann, A. A., and Ormerod, M. G. The Replication of DNA in MurineLymphoma Cells (15178). Biochim. Biophys. Acta, 204: 128-143, 1970.

15. Margison, G. P., Margison, J. M., and Montesanto, A. Accumulation of0-methyl Guanine in Non-Target-Tissue Deoxyribonucleic Acid duringchronic Administration of Dimethylnitrosamine. Biochem J., 165: 463—468, 1977.

16. Mondal, S., Brankow, D. W., and Heidelberger, C. Two-Stage ChemicalOncogenesis in Cultures of C3H/1OT'/2 Cells. Cancer Res., 36: 2254-2260, 1976.

17. Nicole, J. W., Swami, P. F., and Pegg, A. E. The Accumulation of 0'-Methylguanine in the Liver and Kidney DNA of Rats Treated withDimethylnitrosamine for a Short or a Long Period. Chem.-BioI. Interac

(G)

where D = dose and k is a constant. The relationshipbetween D and Z has been established empirically in severallaboratories (13, 28, 33).

Linear dose-response curves for mutation have also beenobtained by others (9, 19, 32), but the view that the measurement of AZG resistance in Chinese hamster cells accurately reflects the mechanism of action of the mutagen (19)is not supported by experiments that showed no differenceinthe mutationfrequenciesproducedbyequitoxiclevelsofMNNG and MMS in V79 cells (29). On the other hand, theconditions used in those early experiments were very different from those that are now considered necessary formeasuring mutation to AZG resistance.

The dose-response curves for alkali-labile lesions arelinear, showing that alkylation lesions in DNA increaselinearly with dose of alkylating agent as demonstrated inprevious studies (13, 28, 33). As to the nature of the lesions,calculations from earlier experiments where the DNA wassedimenting anomalously had suggested that the lesionswere depuninations (22). However, Walker and Ewart (33)and now we have shown that MMS, which would produceapproximately the same number of depuninations as wouldMNNG at equitoxic doses (13, 28), produces fewer alkalilabile lesions than does MNNG. Walker and Ewart (33)provide chromatographic evidence that their alkali-labilelesions primarily result from phosphotniesters, and thisagrees with the results of experiments with bacteriophages(30) and conforms with theory (12, 20) regarding the mechanisms of aikylation. Therefore, we now consider that mostof the alkali-labile lesions that we measure result fromalkylations that give phosphotniesters.

According to chemical theories of the mechanism ofalkylation (12, 20, 31), an agent that aikylates DNA phosphate oxygens would also be expected to alkylate otheroxygens in DNA. Alkylation of 06 of guanine would beexpected to result in base substitution mutations (12, 31).Therefore, mutagenic and alkali-labile lesions should beassociated but not identical, and in dissociating the doseresponses for repair of alkali-labile lesions (Chart 8) andmutagenesis (Chart 6), this is what we have found. The datathat we present here suggest that C3H/10T1/2 cells treatedwith MNNG, ENNG, and EMS contain in their DNA extensive

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A. R. Peterson et a!.

tions,16:301-308,1977.18. O'Neill, J. P., Couch, D. B., Machanoff, R., San Sebastian, J. R., Brimer,

P. A., and Hsie, A. W. A Quantitative Assay of Mutation Induction inChinese Hamster Ovary Cells (CHO/HGPRT System): Utilization with aVariety of Mutagenic Agents. Mutation Res., 45: 91-101 , 1977.

19. O'Neill, J. P., and Hsie, A. W. Chemical Mutagenesis of Mammalian Cellscan be Quantified. Nature, 269: 815—817,1977.

20. Osterman-Golkar, S., Ehrenberg, L., and Wachtmeister, C. A. ReactionKinetics and Biological Action in Barley of Mono-Functional Methanesulfonic Esters. Radiation Botany, 10: 303-327, 1970.

21. Peterson, A. R. A Stratagem for Experiments on Oncogenesis in vitro.In: U.Saffioti andH.Autrup(eds.),In VitroCarcinogenesis,Guideto theLiterature, Recent Advances, and Laboratory Procedures, National Cancer Institute Carcinogenesis Technical Report Series No. 44, pp. 205-211, Washington, D. C.: U. S. Dept. of Health, Education and Welfare,1978.

22. Peterson,A. R., Bertram,J. S., and Heidelberger,C. DNADamageandIts Repair in Transformable Mouse Fibroblasts Treated with N-methylN'-nitro-N-nitrosoguanidine. Cancer Res., 34: 1592-1599, 1974.

23. Peterson, A. R., Bertram, J. S., and Heidelberger, C. Cell Cycle Dependency of DNA Damage and Repair in Transformable Mouse FibroblastsTreated with N-Methyl-N'-nitro-N-nitrosoguanidine. Cancer Res., 34:1600-1607, 1974.

24. Peterson, A. R., Krahn, D. F., Peterson, H., Heidelberger, C., Bhuyan, B.K., and Li, L. H., The Influenceof SerumComponentson the Growthand Mutation of Chinese Hamster Cells in Medium Containing 8-Azaguanine. Mutation Res., 36: 345-356, 1976.

25. Peterson, A. R., Peterson, H., and Heidelberger, C. The Influence ofSerumComponentson the Growth and Mutationof ChineseHamster

Cells in Medium Containing Aminopterin. Mutation Res., 24: 25—33,1974.

26. Reznikoff, C. A., Bertram, J. S., Brankow, D. W., and Heidelberger, C.Quantitative and Qualitative Studies of Chemical Transformation ofCloned C3H Mouse Embryo Cells Sensitive to Post Confluence Inhibitionof Cell Division. Cancer Res., 33: 3239-3249, 1973.

27. Reznikoff, C. A., Brankow, D. W., and Heidelberger, C. Establishmentand Characterization of a Cloned Line of C3H Mouse Embryo CellsSensitive to Post Confluence Inhibition of Division. Cancer Res., 33:3231-3238, 1973.

28. Roberts, J. J., Pascoe, J. M., Plant, J. E., Sturrock, J. E., and Crathom,A. R. Quantitative Aspects of the Repair of Alkylated DNA in CulturedMammalian Cells. I. The Effect on HeLa and Chinese Hamster CellSurvival of Alkylation of Cellular Macromolecules. Chem.-BioI. Interactions, 3: 29—47,1971.

29. Roberts, J. J., Sturrock, J. E., and Ward, K. N. DNA Repair andAlkylation-Induced Toxic, Mutagenic and Cytological Effects in Mammalian cells. In: P. 0. P. T'so and J. A. DiPaolo (eds.), ChemicalCarcinogenesis, Part A. pp. 401-423. New York: Dekker, 1974.

30. Shooter, K. V. The Kinetics of the Alkaline Hydrolysis of Phosphotriestersin DNA.Chem.-BioI.Interactions,13:151—163,1976.

31. Singer, B. All Oxygens in Nucleic Acids React with Carcinogenic Ethylating Agents. Nature, 264: 333-339, 1976.

32. Van zeeland, A. A., and Simons, J. W. I. M. Linear Dose-ResponseRelationships after Prolonged Expression Times in V-79 Chinese Hamster Cells. Mutation Res., 35: 129—138,1976.

33. Walker, I. G., and Ewart, D. F., The Nature of Single-Stranded Breaks inDNA following Treatment of L-CelIs with Methylating Agents. MutationRes.,19:331-341,1973.

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1979;39:131-138. Cancer Res A. R. Peterson, Hazel Peterson and Charles Heidelberger Cultured Mammalian Cells Treated with Alkylating AgentsOncogenesis, Mutagenesis, DNA Damage, and Cytotoxicity in

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