Comparison of 6-Thioguanine-Resistant Mutation and Sister Chromatid Exchanges in Chinese Hamster V79 Cells with Forty Chemical and Physical Agents

[CANCER RESEARCH 44, 3270-3279, August 1984]

Comparison of 6-Thioguanine-resistant Mutation and Sister Chromatid Exchanges in Chinese Hamster V79 Cells with Forty Chemical and Physical Agents

Yoshisuke Nishi,1 Makiko M. Hasegawa, Masako Taketomi, Yoshihiko Ohkawa, and Naomichi Inui

Sectionof Cell Biology and Cytogenetics,Biological Research Center, TheJapan Tobacco and Salt Public Corporation,Hatano,Kanagawa257, Japan

ABSTRACT and reliable indicator of genetic damage of the sort leading to mutation and cancer (1, 22, 33, 45). Exposure of cells to muta- The induction of sister chromatid exchanges (SCE) and mu gens and/or carcinogens can result in SCE, chromosome aber tation at the hypoxanthine-guanine phosphoribosyl transferase rations, mutations, transformation, and cell death. These cellular locus and toxicities of 40 different chemical and physical agents responses can be examined comparatively and more directly by were examined on Chinese hamster V79 cells. These agents use of suitable cell lines. included mono-, di-, tri-, and polyfunctional alkylating agents, With such cell lines, several studies have demonstrated good intercalators, 7-rays, and UV light irradiation. Mutation was mea correlations of SCE with mutations (6, 7, 39), transformation sured as resistance to 6-thioguanine and toxicity as loss of cell- (35), and cell death (29, 47). However, these and other results plating efficiency. SCE were examined 29 hr after treatment. (9, 19, 49) have also indicated that the quantitative relations With the agents examined, a highly positive correlation (r = varied with different types of agents. Available data are insuffi 0.89) exsisted between SCE-inducing and mutagenic potencies, cient to draw any general conclusions regarding the relation of when expressed as increase in the number per a unit dose over SCE to other biological end points. More studies are required to the control values. But the great difference of the ratios of confirm the validity of the SCE test as a reliable indicator of mutagenic potencies versus SCE-inducing potencies among mutagenesis and/or carcinogenesis of mammalian cells. It is also agents was observed, the maximal difference in the ratios being essential to elucidate to what extent SCE formation correlates about 200-fold. with other markers. Such studies should be helpful in under The agents that showed the higher values of the ratio (agents standing the mechanism of SCE formation which still remains producing more mutations than SCE) were bleomycin, cobalt-60 unknown. -y-rays, all ethylating agents (A/-ethyl-/v-nitrosourea, /V-ethyl-AT- V79 cells have properties that are useful in detecting muta nitro-W-nitrosoguanidine, ethyl methanesulfonate, and diethyl- genesis as well as SCE (2, 22); the cells grow rapidly with a sulfate), A/-propyl-A/-nitrosourea, A/-butyl-A/-nitrosourea, isopro- short lag, doubling in 12 to 16 hr, and they have a high cloning pyl methanesulfonate, intercalating acridine compounds (2- efficiency (75 to 85%) and a stable karyotype with a modal methoxy-6-chloro-9-[3-(ethyl-2-chloroethyl)aminopropylamino]- chromosome number of 21 with a narrow range of variation (46). acridine-2HCI and 2-methoxy-6-chloro-9-[3-(chloroethyl)-ami- In addition, these cells respond well to a wider spectrum of nopropylamino]acridine 2HCI) and UV light at 254 nm. The agents mutagens, as judged using the recessive marker for hypoxan that showed the lower values (agents producing more SCE than thine-guanine phosphoribosyl transferase locus, and codominant mutations) were platinum compounds (c/s-diamminedichloro- marker for the Na+-K+-activated ATPase locus (2), and also as platinum and frans-diamminedichloroplatinum), epoxides (epi- judged by assay of SCE (22). chlorohydrin, styrÃ¨ne oxide, and diepoxybutane) and aziri- On the basis of these considerations, we concomitantly ex dines (mitomycin C, decarbamoyl mitomycin C, tris(1-aziridi- amined the induction of SCE and mutation to 6-thioguanine nyljphosphine sulfide, triethylenemelamine, and carboquone). resistance in Chinese hamster V79 cells by a number of chemical The agents that showed the intermediate values included all and physical agents that interact in different ways with DMA, and methylating agents (A/-methyl-A/-nitrosourea, W-methyl-A/'-nitro- compared the results to see the relation between the 2 markers. W-nitrosoguanidine, methyl methanesulfonate, and dimethyl sul fate), A/-(2-hydroxyethyl)ethyleneimine, /3-propiolactone, treat MATERIALS AND METHODS ment of 8-methoxypsoralen plus near-UV light irradiation at 352 nm, 4-nitroquinoline-1-oxide, quinacrine mustard, sodium sÃ³r Cell Line and Culture Conditions. The Chinese hamster V79 cells used in this assay have an average cloning efficiency of more than 85% bate, cigarette tar, and diesel tar. and a doubling time of 14 to 15 hr in Eagle's minimum essential medium For most agents that induced SCE, the toxicity dependency (Nissui Seiyaku Co., Tokyo, Japan), plus 10% fetal bovine serum (Re- of induced SCE was rather biphasic; increase in SCE was steep hatuin F. S., Lots V55403 and V54902; Reheis Chemical Co., Phoenix, at low to moderate toxicity and less at moderate to high toxicity. AZ) and kanamycin (60 pg/m\; Meiji Seika Co., Tokyo, Japan), and At equitoxic doses, the agents showed great difference in induc neomycin (100 /ig/ml; Grand Island Biological Co., Grand Island, NY). tion of SCE. These cells have a stable average modal chromosome number of 21, being karyologically different from normal Chinese hamster cells. All INTRODUCTION Analysis of SCE2 formation has been proposed as a sensitive N-nitrosourea: NM, nitrogen mustard; HY-EI, N-(2-hydroxyethyl)ethyleneimine; MMC, mitomycin C; BLE, bleomycin; ICR 170, 2-methoxy-6-chtoro-9-[3-(ethyl-2- ReceivedFebruary 14,1984; accepted May 3,1984. chloroethyl)arninopropylamino]acridine-2HCI;ICR 191, 2-methoxy-6-chloro-9-[3- 1To whom requests for reprints should be addressed. (chtoroethyl)aminopropylamino]acridine.2HCI; DMP, 1,4-dinitrc-2-methylpyrrole; 2The abbreviations used are: SCE, sister chromatid exchange; QM, quinacrine DDP, diamminedichloroplatinum;PUVA, 8-methoxypsoralen plus near-UV light ir mustard; 4NQO, 4-ni1roquinoline-1-oxide;DMS, dimethyl sulfate; MNU, N-methyl- radiation at 352 nm; DCMMC, decarbamoyl mitomycin C.

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Table 1 sulfonate, NM, and 8-methoxypsoralen from Sigma Chemical Co. (St. Chemical and physical agents tested in SCE and mutation assays Louis, MO); HY-EI and 9-aminoacridine hydrochloride from Eastman Kodak Co. (Rochester, NY); diepoxybutane and /3-propiolactone from N-Nitroso compounds NitrosoamkJes Fulka (Buchs, Switzerland); tris(1-aziridinyl)phosphine sulfide from Sum MNU itomo Chemical Industries (Osaka, Japan); MMC from Kyowa Hakko W-Ethyl-N-nitrosourea Kogyo Co. (Tokyo, Japan); proflavine hemisulfate from Tokyo Kasei N-Propyi-N-nitrosourea N-Butyl-N-nitrosourea Kogyo Co. (Tokyo, Japan); BLE from Nihon Kayaku Co. (Tokyo, Japan); Nitrosoamidines and hydrogen peroxide from Mitsubishi Gas Chemical Co. (Tokyo, Ja N-MethyWV'-nitro-N-nitrosoguanidine N-Ethyl-W' -nitro-N-nitrosoguanidine pan). The other chemicals were kindly provided from sources as follows: 2-methoxy-6-chloro-9-[3-(chloroethyl)aminopropylamino]acridine â€¢2HCI, Alkane sulfonates 2-methoxy-6-chloro-9-[3-(ethyl-2-chloroethyl)aminopropylamino]acri- Methyl methanesulfonate dine-2HCI, and DMP from Dr. T. Kada (National Institute of Genetics, Mi- Ethyl methanesulfonate shima, Shizuoka, Japan); A/-propyl-A/-nitrosourea, isopropyl methanesul Isopropyl methanesulfonate fonate, and triethylenemelamine from Dr. T. Shibuya and Dr. N. Tanaka Alkyl sulfates (Hatano Research Institute, Food and Drug Safety Center, Hatano, QMS Diethyl sulfate Kanagawa, Japan); diesel tar from Dr. K. Kawai (National Institute of Industrial Health, Kawasaki, Kanagawa, Japan); carboquone from Heterocyclic nitrogen compounds Sankyo Co. (Tokyo, Japan); DCMMC from Kyowa Hakko Kogyo Co. Aziridines HY-EI (Tokyo, Japan); and c/s-DDP and irans-DDP from Nihon Kayaku Co. Tris(1-aziridinyl)phosphine sulfide (Tokyo, Japan). Cigarette tar was prepared following the procedure of Triethytenemelamine Mizusaki ef al. (27). Carboquone Assay Protocols. For testing SCE, cells in the substationary phase, MMC DCMMC which had been thawed 1 to 2 days before from a stock culture kept at Acridines -70Â°, were dissociated with 0.05% trypsin (1:250; Difco Laboratories, 9-Aminoacridine hydrochkxide Detroit, Ml) and 0.02% EDTA and transferred to 75-sq cm plastic flasks Proflavine hemisulfate (Miles Laboratories, Napervilte, IL) at 5 x 10s cells/flask. After 24 hr, they ICR 191 ICR 170 were treated with chemical agents for 3 hr. As physical treatments, cells QM were irradiated in flasks or 6-cm plastic dishes (Nunc, Roskilde, Denmark) Others with either "Co 7-rays ("Co teletherapy unit, Theratron 780) or UV at 4NQO EthkJium bromide 254 nm (germicidal lamp; Matsushita Denko Co., Kadoma, Osaka, Ja DMP pan), or for PUVA treatment, they were irradiated with near-UV (352 nm, black light; Matsushita Denko Co.) in the presence of 8-methoxypsoralen. Mustards Chemical agents were either dissolved in distilled water, special-grade (QM) dimethyl sulfoxide (Merck, Darmstadt, Federal Republic of Germany), or NM ethanol (Kanto Chemical Co., Tokyo), and were added to cultures. After Epoxides treatment, the cells were washed with Hanks' balanced salt solution and Epichlorohydrin cultured further for 27 hr in normal medium containing 5-bromodeoxy- StyrÃ¨ne oxide Diepoxybutane uridine (2 fig/ml; Sigma). Metaphases were then arrested by replacing the medium by medium containing Colcemid (0.05 /jg/ml; Grand Island Lactone Biological Co.) but without 5-bromodeoxyuridine and incubating the cells /3-Proptolactone for an additional 2 hr. Chromosome specimens were prepared by the usual air-drying method after hypotonie treatment with 0.075 M potas Metallic compounds CÃ•S-DDP sium chloride for 20 min and fixation with Carnoy's fixative. Before frans-DDP fixation, the cells were kept in the dark or under a safe light. Chromo somes were then differentially stained by the usual FPG (Fluorescence Physical or photodynamically activated agents UV light irradiation at 254 nm plus Giemsa) technique (34) with some modifications. For each dose of "Co -Y-rays agents, SCE were analyzed on 50 well-spread metaphases; the chro PUVA mosome numbers ranged from 19 to 23, but the average chromosome number per cell was 21. SCE were expressed as average numbers per Miscellaneous compounds BLE cell, and control values were subtracted to obtain induced SCE frequen Sodium sorbate cies. Diesel tar For testing mutations and cellular toxicity, cells were plated into flasks Cigarette tar and treated in the same way as for testing SCE. Immediately after Hydrogen peroxide washing with Hanks' balanced salt solution, some cells were plated in dishes at 200 cells/6-cm dish to determine the toxicity of the agents experiments were done in a humidified incubator under 5% CO2 in air at (initial survival). The remaining cells were all replated in fresh flasks at 37Â°. about 0.5 to 1 x 10" cells per 75-sq cm flask, to allow the full expression Agents Tested in the Assay. The chemical and physical agents time. Subculture was usually done twice or 3 times during the expression tested in this experiment are listed in Table 1. The chemicals were time at a split ratio of 1:2 to 1:8, depending on the recovery. The medium purchased from chemical sources as follows: QM, 4NQO, epichlorohydrin was changed every 2 days until the cells reached the stationary phase. and styrÃ¨ne oxide from Wako Pure Chemical Industries (Tokyo, Japan); Usually on Day 6, which is considered to be an adequate expression N-methyl-W'-nitro-N-nitrosoguanidine, N-ethyl-W'-nitro-N-nitrosoquani- time under many conditions (2), some of the treated cells were plated at dine, DMS, and ethidium bromide from Aldrich Chemical Co. (Milwaukee, 200 cells/6-cm dish for survival adjustment (replated survival). The Wl); MNU, N-ethyl-A/-nitrosoruea and N-butyl-W-nitrosoruea from Nakarai remaining cells were plated at 1 x 10s cells/6-cm dish in medium Chemicals (Kyoto, Japan); methyl methanesulfonates, diethylsulfate, and containing 10 /JM 6-thioguanine. On Days 13 to 15, colonies were fixed, sodium sorbate from Kanto Chemical Co. (Tokyo, Japan); ethyl methane stained, and counted. Mutation frequencies were expressed as the

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Chart 1. Dose-response curves for the induction of SCE (O) and 6-thioguanme-resistant mutations (â€¢)andrelative cell survivors P) in V79 Chinese hamster cells of simple aikyiatmg agents. Mote that the abscissa is a log scale and the ordinales are the induced numbers. For abbreviations, see footnotes to Table 2. numbers of mutants per 10" viable cells, and control values were sub were very strong inducers of mutations, but less effective in tracted to obtain induced mutation frequencies. inducing SCE (Chart 2). Two other acridines, proflavine hemisulfate and 9-aminoacridine hydrochloride, were much weaker than those of intercalating acridine compounds, giving similar plots RESULTS between them (Chart 2). QM was the only exception among the Dose-Response Induction of SCE and Mutation by Each acridines; it shows similarity in structure to 2-methoxy-6-chlo- Agent. The dose-response curves of SCE and mutation by the ro-9 - [3 - (ethyl - 2 - chloroethyl)aminopropylamino]acridine â€¢2HCI 40 agents are shown in Charts 1 to 8. Simple alkylating agents and2-methoxy-6-chloro-9-[3-(chloroethyl)aminopropylamino]ac- (nitrosoamides, nitrosoamidines, alkane sulfonates, and alkyl ridine-2HCI, but it induced high incidence of SCE and mutation sulfates) were potent inducers of both SCE and mutation (Chart (Chart 6), which will be shown again later. Another type of 1). Among them, all methylating agents (MNU, A/-methyl-A/ '-nitro- intercalator, ethidium bromide, scarcely induced SCE or mutation /v-nitrosoguanidine. methyl methanesulfonate, and DMS) were (Chart 2). more potent in inducing SCE than their respective ethylating Two classes of agents with specific molecular moieties in their agents, such as A/-ethyl-A/-nitrosourea, A/-ethyl-/v"-nitro-A/-nitro- structures, I.e., aziridines and epoxides, induced SCE at high soguanidme. ethyl methanesulfonate and diethylsulfate, while incidence but mutations at low incidence (Charts 3 and 4). These these ethylating counterparts produced more mutants than the chemicals alkylate DNA bases by a reaction shown in Chart 9. methylating agents. The slopes of the plots of mutation versus Among the aziridines tested, carboquone, tris-(1 -aziridi- SCE for nitrosoamides increased in the order methyl-, propyl-, nyl)phosphine sulfide, and triethylenemelamine, which are anti- butyl-, and ethyl-, and for alkane sulfonates, they increased in neoplastic agents with 2, 3, and 3 aziridinyl residues in their the order methyl-, isopropyl-, and ethyl-. chemical structures, respectively, induced high incidences of The intercalating acridines, 2-methoxy-6-chloro-9-[3-(chloro- SCE but low incidences of mutations (Chart 3), although HY-EI, ethyl)aminopropylamino]acridine-2HCI and 2-methoxy-6-chlo- which has only one aziridinyl residue, induced relatively high ro - 9 - [3 - (ethyl - 2 - chloroethyl)aminopropylamino]acridine â€¢2HCI, incidences of both (Chart 3). Of the epoxides tested, diepoxy-

3272 CANCER RESEARCH VOL. 44

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Chart 3. Dose-response curves for the induction of SCE (O) and 6-thioguanine-resistant mutations (â€¢)andrelative cell survivors (D) in V79 Chinese hamster cells of aziridines. Note that the abscissa is a log scale and the ordinales are the induced numbers. For abbreviations, see footnotes to Table 2. butane having2 epoxideswas a potent inducerof SCE, and alkylatingagent,is structurallyrelatedto MMCbut differsfrom epichlorohydrinandstyrÃ¨neoxidehavingoneepoxidewereless thelatterinitsinteractionswithDNAbases;havingnocarbamoyl effectivein inductionof SCE,whileall3 compoundswereweak moietyat theC-10position,it doesnotcross-linkwithDNA(32). inducersofmutation(Chart4).Theslopesoftheplotsof mutation However,the plots of mutationsversus SCE for MMC and versusSCEfor these aziridines(excludingHY-EI)or epoxides DCMMCwereverysimilar.Boththe platinumisomersalsogave wereverysimilar,/3-propiolactone,whichhasa heterocyclicring similarplotsof mutationversusSCE. with an oxygen atom in its structure,inducedmore mutations Thedose-responsesof2 mustards,QMand NM,werediffer thanepoxides,but its activityin inductionof SCEwas lessthan ent (Chart6). As shown in the previoussection,QM induced thatof diepoxybutaneandsimilarto theseof epichlorohydrinand high incidencesof both mutationsand SCE, whereasNM, a styrÃ¨neoxide(Chart4). moietyof QM, inducedSCEin highincidence,butmutationsin Agents capableof producingDNA-DNAand/or DNA-protein verylow incidence.Themetabolicfateof the mustardsis similar cross-linksaswellas monoadducts,suchas MMCandplatinum to that of aziridinesasshownin Chart9. compounds(c/s-DDPand frans-DDP),were the most potent IrradiationUVwith irradiationat254 nm inducedmutationsin inducersof SCE,but all were weak mutagens(Chart5). PUVA high incidenceand SCE in moderateincidence(Chart 7). By treatment was less effective in inductionof SCE and more analogywith cellularresponses(44),the effect of 4NQOwas effectivein inductionof mutation.DCMMC,a monofunctional alsoexamined.Resultsshowedthat at dose rangesexamined

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4NQO has a similar effect to UV irradiation at 254 nm (Chart 7). DMP, a product of sorbic acid and nitrite (21), was reported to A major product of UV irradiation of DNA is pyrimidine-dimers be mutagenic in bacteria (17) but not to show genotoxicity to (37), while the products of 4NQO are monoadducts (43). mammalian cells. In this work, the effect of sodium sorbate, The effect of '"Co 7-rats and BLE, which are known to cause DMP, and cigarette tar and diesel tar were all very weak in double-strand DNA breaks (41, 50), in inducing SCE and muta induction of SCE and mutation (DMP was negative in mutagen- tion were weak; in particular, BLE induced an increase of SCE icity to V79 cells), and plots of mutation versus SCE were within of only about 0.5/cell (Chart 7). The dose-response characteris the range of those for other compounds (Chart 8). tics for SCE and mutation showed that both agents tended to Quantitative Comparison of SCE and Mutation. To quantify induce mutation much more than SCE. Hydrogen peroxide was the relation of SCE and mutation, we compared the effects of also tested, because it causes only single-strand DNA breaks the agents as ratios of the mutagenic potencies to the SCE- (3). It was found to induce no mutants but a very few SCE (Chart inducing potencies. Mutagenic and SCE-inducing potencies were 7). expressed as increase in the number of mutants and SCE over Among the miscellaneous agents tested, sodium sorbate was the control values per Â¿Â¿gperml, respectively, whose values reported by us to induce both mutations and SCE (15), and were obtained from all the determinations for each treatment

3274 CANCER RESEARCH VOL. 44

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1984 American Association for Cancer Research. Comparison of 6-Thioguanine-resistantMutation and SCE shown in Charts 1 to 8 and expressed as averages (Table 2). pounds, c/s-DDP and frans-DDP, all epoxides, all aziridines ex A highly positive correlation (r = 0.89) exists between SCE- cept for HY-EI, including MMC and DCMMC, and NM gave inducing and mutagenic potencies, when these values or the values of less than one. Agents giving values of between 1 and reciprocals of them are plotted on a logarithmic scale (Chart 10). 10 included all methylating agents, HY-EI, 0-propiolactone, However, as Table 2 shows, the ratios of the mutagenic poten PUVA,4NQO, and QM. cies to the SCE-inducing potencies varied very widely. That is, agents more prone to induce SCE than mutation gave Of the agents that positively induced both SCE and mutation, values of about 10 or more, those inducing SCE much more c/s-DDP had the lowest value of 0.24 and ""Co 7-rays the highest readily than mutation gave a value of less than one, and those of 39, other agents having intermediate values, so the maximum with intermediate effects gave values of 1 to 10. difference between values was approximately 200-fold. Table 2 Relations of SCE Induction and Toxic Effects. There is a also shows that all ethylating agents, A/-propyl-A/-nitrosourea,N- wide variety in the relation between induction of SCE and per butyl-A/-nitrosourea,ICR 170, "Co 7-rays, and BLE gave values centage of survival (Charts 1-8). In many cases, these relations of more than 10, and ICR 191, UV irradiation at 254 nm, and followed biphasic regression lines (data not shown). This means isopropyl methanesulfonate of nearly 10. The platinum com- that the increase in SCE was not largely due or dependent on decrease in cell survival. Increase in SCE tended to be observed --. 100 at almost nontoxic or subtoxic doses and decreased with in crease in toxicity at moderate to high doses. When we compared 5 io the induced SCE at equitoxic dose levels, the results again show that, among the agents that positively induced SCE, differences \ \ in increase of SCE were great (data not shown). ^ DISCUSSION 120 â€¢QM NM The aim of this study was to reach a goal on the quantitative 3000 -S -O value of SCE as an index of mutagenesis in mammaliancells. -5 30 One of important findings in the results was that there was a u highly positive correlation between induction of SCE and muta tion at hypoxanthine-guanine phosphoribosyi transferase locus y with a wide variety of chemical and physical agents (Chart 10). v w When the dose-response characteristics are shown by plots of u 1000 SCE versus mutations on a linear scale, they were generally I 20 U linear or almost linear at low to moderate doses over 50% o* 1 survival (data not shown). These findings are consistent with the Z 0 results of Carrano ef al. (6, 7) on SCE and mutation to 8- i

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EPOXIDES widely among agents under conditions when the lesions induced by agents that lead to SCE were comparable. This suggests that the mutation does not share common lesions with SCE. x This possibility can be better explained by considering differ RrCH-CH-R2 ences in the activities of the agents in inducing SCE and mutation OH in relation to specific modifications or lesions that they produce in DNA. We found that simple alkylating agents are potent inducers of both SCE and mutation. Our results that methylation induce more SCE but less mutations than ethylation are consistent with AZIRIDINES the fact that O6-alkylation of guanine in DNA is closely correlated with induction of mutations in mammalian cells (31), while DNA damage other than 06-alkylation is involved in SCE formation HX R-NH-CHj-CHjX (10,20), if we consider the general fact regarding base alkylation that the preferential reactivity to oxygens of purines is in the order ethyl-, methyl-, while the overall reactivity is in the order methyl-, ethyl- (38). Our results on MNU and DMS are consistent with those of NITROGEN MUSTARDS others (10, 40) that MNU and DMS induced similar numbers of SCE, but that MNU produced 20 times more O6-methylguanine than DMS (10) and was much more mutagenic than DMS, producing a significantly higher O6-guanyl/A/7-guanyl alkylation + Cl ratio than the latter (40). These findings again suggest that alkylation other than at the O6 position of guanine may be the DNA-base modification leading to SCE. More recently, it was HX CHjCHjX + Cl" . reported that no single DNA alkylation products strongly corre lated with SCE induction by ethyl methanesulfonate and A/-ethyl- W-nitrosourea (18) or methyl methanesulfate and MNU (28). HX Â» XCHjCHfN-CHjCHjXR There is evidence, however, that O6-alkylation may be respon sible for SCE (42, 52). Studies on xeroderma pigmentosum Chart 9. Chemical reactions of epoxides, azindines. and nitrogen mustards with XP12RO cells indicated that N7-alkylation is not the lesion that nucleophiles HX, nudeophile. leads to SCE (53). Moreover, the fact that O6-alkylguanine is mutation increase proportionally with increase in the strength of removed much more slowly than A/7-alkylguanine in these cells a given agent, rather than that common lesions are responsible but not in normal cells (14) may be linked to SCE formation (52). for both SCE and mutation. This speculation is supported by a report that the Mer strain of We found that the ratios of mutations to SCE for 40 agents cells isolated from human tumors or SV40-transfomned cells, the tested varied very widely with a 200-fold difference between the phenotype of which is sensitive to methylating agents with smallest and largest values (Table 2). This difference in values respect to cell survival and SCE, has a defect in removing O6- means that the number of lesions leading to mutation varied methylguanine from alkylated DNA (12).

3276 CANCER RESEARCH VOL. 44

Table 2 Summary of 6-thioguanine-resistant mutation and SCE in Chinese hamster V79 cells on various classes of chemical and physical agents

potency*1.80.150.0370.025490121.60.0560.0734.50.151.51515072006203200.653.912Mutagenicpotency6112.80.400.3216002304.40.830.667.82.03.14.3"7835002801700.98e17100Ratio"6.11911133.3202.8159.01.7132.10.290.530.490.450.521.54.39.6Classpotency3292001200.0130.42(200)7100.420.55552.31901.35.8s0.62"0.05390.0048'0.0110.0190.0310.66Mutagenicpotency"1200360540<0.0018'<0.13'(360)2000.230.22244.5450.7249"24Â«0.1190.240.0740.0580.0780.057'Ratioc411.84.4<0.14<0.32(1.8)0.280.530.400.432.00.240.568.5392.1527.13.02.6<0.087 chemicalN-NitrosoClass and chemicalICRand compoundsNitrosoamidesMNUENU"PNUBNUNitrosoamidmesMNNGENNGAlkane 170QMOthers4NQOEtBrDMPMustards(QM)NMEpoxidesECHSOXDEBLactone0-PROMetallic

sulfonatesMMSEMSIPMSAlkyl

sulfatesDMSDESHeterocyclic

compoundsc/s-DDPfrans-DDPPhysical nitrogen com poundsAziridinesHY-EIThiotepaTEMCARMMCDCMMCAcridines9AAPROICR orphotodynamicallyactivated agentsuve""Co 7-raysPUVAMiscellaneous

compoundsBLESOSD-TarC-TarH202SCE-inducing

191SCE-inducing " Values expressed as increase in the number of SCE per cell over the control values per ng per ml. These were determined by the multiple determinants shown in Charts 1-8 and expressed as the average number. '' Values expressed as increase in the number of 6-thioguanine-resistant mutants per 10' viable cells over the control values per ^g per ml- These were determined by the multiple determinants shown in Charts 1-8 and expressed as the average number. c Expressed as the values of mutagenic potency to those of SCE-inducing potency. "END, W-ethyl-N-nitrosourea; PNU, N-propyl-N-nitrosourea; BNU, N-butyl-N-nitrosourea; MNNG, N-methyl-W'-nitro-W-nitrosoguanidine; ENNG, N-ethyl-N'-nitro-N- nitrosoguanidine; MMS, methyl methanesulfonate; EMS, ethyl methanesulfonate; IPMS, isopropyl methanesulfonate; DES, diethyl sulfate; thiotepa tris(1-aziridinyl)phosphine sulfide; TEM, triethylenemelamine; CAR, carboquone; 9AA, 9-aminoacridine hydrochloride; PRO, proflavine hemisulfate; EtBr, ethidium bromide; ECH, epichtorohydrin; SOX, styrÃ¨ne oxide; DEB, diepoxybutane; 0-PRO, 0-propiolactone; UVC, UV-light irradiation at 254 nm; SOS, sodium sorbate; D-Tar, diesel lar; C-Tar, cigarette tar; H.,0.., hydrogen peroxide. 8 A dose-response was obtained, but it was not mutagenic according to the criteria of Bradley et al. (2). ' No mutants over the spontaneous numbers were obtained. For these agents, increase in the number of mutants is shown as the maximally presumed number calculated from treated concentrations and the background values. 9 Values expressed per J per sq m. h Values expressed per Gy. ' Increase in SCE over the control number was not significant by the ( test.

It Is puzzling why epoxides, aziridines, and mustards are date for SCE is cross-linking induced by MMC, c/s-DDP, trans- potent Â¡nducersof SCE but weak Â¡nducersof mutation (Charts DDP, and PUVA. Our results on these cross-linking agents (Chart 3, 4, and 6), since these agents are known to alkylate DNA 5), however, support the idea proposed previously that DNA- bases (4, 5, 23, 24, 30, 36, 51). No systematic study of the DNA or DNA-protein cross-links are not the main course of SCE reaction products of these agents with nucleic centers in DNA (8, 54). DCMMC, which cannot induce cross-linking (32), was as has been carried out. But it seems likely that, depending on effective as MMC in inducing SCE and mutations (Chart 5), nucleophilicity, substrate constants and steric factors, the same suggesting that monoadducts rather than cross-links are sites centers in DNA will be alkylated as with simple alkylating agents, for lesions leading to SCE and mutation. Likewise, frans-DDP, following the reaction shown in Chart 9. Several studies have which can form several times as many DNA-protein cross-links shown that, as with simple alkylating agents, the A/7-position of as c/s-DDP but comparable amounts of DNA-DNA cross-links to guanine is the most reactive center in nucleic acids (4,5, 23,24, c/s-DDP (3), was similar to c/s-DDP in inducing many SCE but 30,36,51 ). A possible explanation for the difference in inductions few mutations (Chart 5). This indicates that, as for mutation, of SCE and mutation is that these agents may form alkylated DNA-protein cross-links are not necessary for SCE formation. In bases whose lesions lead to SCE more readily than to mutagenic a more recent study on the reactivity of MMC with DNA, only 3 events, because some steric factors resulting from the modifi monoadducts at the A/2-and Oppositions of guanine and the W6- cation may affect the mutation frequency. This idea is based on position of adenine were obtained under conditions that reflect the fact that specific base modifications leading to mutation, the physiological state more closely (16), although this finding such as O6-alkylguanine, can cause mispairing or miscoding (13), was not compatible with a previous observation that the ratio of but that this will not occur if the alkyl residues reacting with mono- to bifunctional alkylations of MMC was approximately bases are too large or not suitable to be recognized as sites for 10:1 (26, 48). Our results together with other findings (8) and mispairing or miscoding. The mechanism leading to SCE may this biochemical data all suggest that monoadducts, rather than not be affected by such steric factors. DNA-DNA cross-links, may be chiefly associated with the induc Another lesion that has been considered as a possible candi tion of both SCE and mutation by MMC. And this may also be

AUGUST 1984 3277

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1984 American Association for Cancer Research. Y.Nishietal. io3io2_fio'i.rÂ«111Â«io11Wi'io'3.X*10â€”rsurvival indicated a linear correlation at low to high toxic doses of some simple alkylating agents (29,47). However, we observed a linear relation only within narrow ranges of those alkylating â€¢â€¢"r =0-89 agents, and this relation was not general for all agents. In fact, 'S05Â«EtBrâ€¢ D-Torâ€¢8NU our results demonstrated that SCE do not necessarily share a T_EMS . r Â»PNUâ€” common element of toxic damage as a whole. ENU â€¢â€¢DESECH

Â»DMP_ 9AA Â» ACKNOWLEDGMENTS ,^5Â»H2029 â€¢ SOX We thank Dr. T. Kada (National Institute of Genetics. Mishima, Shizuoka. Japan), -proÂ«â€¢Â«O .DMSE.NNÃÃa Dr. N. Tanaka and Dr. T. Shibuya (Hatano Research Institute, Food and Drug Safety Center, Hatano, Kanagawa, Japan), and Dr. K. Kawai (National Institute of .rh!o_IEPAâ€¢.91 Industrial Health, Kawasaki, Kanagawa, Japan) for providing chemical samples. We thank Ninon Kayaku Co. (Tokyo, Japan), Sankyo Co. (Tokyo, Japan), and Kyowa Â»DEIMNNG^J^'c"1"â€¢â€¢NMâ€¢CARâ€”1ICK ITO Hakko Kogyo Co. (Tokyo, Japan) for gifts of antineoplastic drugs We also thank Professor T. Mori and Dr. C. Murayama (Department of Radiation Oncology, School of Medicine, Tokai University, Isobara. Kanagawa, Japan) for kindly allowing the use of a "Co teletherapy unit.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1984 American Association for Cancer Research. Comparison of 6-Thioguanine-resistant Mutation and Sister Chromatid Exchanges in Chinese Hamster V79 Cells with Forty Chemical and Physical Agents

Yoshisuke Nishi, Makiko M. Hasegawa, Masako Taketomi, et al.

Cancer Res 1984;44:3270-3279.

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