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

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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, styrène 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 sór 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. 3270 CANCER RESEARCH VOL 44 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1984 American Association for Cancer Research. Comparison of 6-Thioguanine-resistant Mutation and SCE 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 •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 -70°, 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
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