Relationship between ease of reduction and mutagenicity of dinitroarenes in Salmonella typhimurium strains TA98 and TA98NR

245 Osaka Prefecture, Mozu-umemachi, Sakai, Osaka 591 (Japan)

Mutagenicity of newly synthesized 6-azabenzo[ a ] pyrene

Four major trihalomethanes as inducers sister-chromatid exchanges in vitro

Mutagenicity and carcinogenicity of aza-polycyclic aromatic hydrocarbons are a matter of concern in these years. As aza-analogs of benzo[a]pyrene (azaBaP), 4-, 7-, 8-, 10- and 12-azaBaPs have been synthesized and a few of their mutagenic properties have been reported. No compounds are known which have a nitrogen atom in the 1, 3 or 6 position of BaP. Since these positions are involved in the metabolism of BaP, 1-, 3- and 6-azaBaPs should be of particular interest. Recently we synthesized 6-azaBaP and its N-oxide using perinaphthenone and 1-iodo-2-nitrobenzene as starting materials (Fukuhara et al., Tetrahedron Lett., 31 (1990) 3743). Now we report their mutagenic property. The mutagenic activities of 6-azaBaP and its N-oxide were tested on Salmonella typhimurium strains TA98 and TA100 with or without $9 mix. 6-AzaBaP, like BaP, showed strong mutagenicity in both strains in the presence of $9 mix, and the activity in TA100 was higher than in TA98. The mutagenicity of 6-azaBaP-N-oxide was higher than that of 6-azaBaP. Even in the absence of $9 mix, 6-azaBaP-N-oxide showed weak mutagenicity in TA98. Our results show that 6-azaBaP is a potent mutagen activated by metabolic oxidation and the activity is further increased by N-oxidation. The metabolic study of 6-azaBaP is now in progress.

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The concentration of trihalomethanes (THMs) in drinking water has become higher as the amount of chlorine infused into the water as a disinfectant increased, because of the advance of water pollution. The THMs are suspected carcinogens-mutagens. We have previously investigated the effects of THMs on rat bone marrow cells in vivo with the chromosome aberration test and obtained positive results. An in vitro sisterchromatid exchange (SCE) assay using rat erythroblastic leukemia ceils, D25_la , given by the Department of Pathology, Kobe University School of Medicine, was conducted with 4 major THMs (chloroform, CHC13; bromodichloromethane, CHC12Br; chlorodibromomethane, CHC1Br2; bromoform, CHBr3). Cells were added to 5 ml of a-MEM (Gibco) containing 15% fetal calf serum and 2 / z l / m l BrdU (Sigma), and each chemical to be tested was added to the cultures to give the final concentrations of 10-5-10 -3 M. The cultures were incubated at 37 °C for 26 h in complete darkness. Colcemid was added at a concentration of 3 /zl/ml for the final 2 h of culture. CHBr 3, CHCI2Br, CHC1Br 2 but not CHC13 showed positive effects and dose responses at these doses. The THMs differed greatly in their ability to induce SCEs in vitro with CHBr 3 being most active and CHC13 least active, whereas CHC13 showed a positive result similar to CHBr 3 in ability to induce chromosome aberrations in vivo in rat bone marrow cells. The above results suggest that CHC13 needs microsomal activation in inducing SCEs. L-Ascorbic acid showed positive suppression of the ability of CHBr 3 to induce SCEs.

9 Fukuhara, K., N. Miyata and S. Kamiya, Division of Organic Chemistry, National Institute of Hygienic Sciences, Kamiyoga, Setagaya, Tokyo 158 (Japan)

10 Fukuhara, K, 1, N. Miyata 1, M. Takei 1, M. Matui 1, K. Matui 1, T. Nohmi 1, T. Sofuni 1, K. Ohmori 2 and S. Kamiya 1, i National Institute of Hygienic Sciences, Tokyo 158 and 2 Kanagawa Prefectural Public Health Laboratory, Yokohama 241 (Japan) Relationship between ease of reduction and mutagenicity of dinitroarenes in Salmonella typhimurium strains TA98 and TA98NR

1,6- and 3,6-dinitrobenzo[a]pyrenes are potent mutagens in Salmonella typhimurium strains TA98 and TA98NR (Fukuhara et al., Chem. Pharm.

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Bull., 38 (1990) 3158). 1- and 3-nitro-6-cyanobenzo[a]pyrenes also showed strong mutagenicity in both strains (Fukuhara et al., Presented at the 18th Annual Meeting of the Environmental Mutagen Society of Japan, 11 November 1989, Tokyo). As they do not require a metabolic activation system ($9 mix) and their mutagenicity in TA98 is not reduced even in TA98NR, they are classified as direct-acting mutagens which are reduced by non-classical nitroreductase like 1,6and 1,8-dinitropyrenes. Reduction of the nitro substituent to nitroso function is known as one of the key metabolic steps for mutagenesis. To clarify the reduction properties related to the mutagenicity of dinitroarenes in TA98NR, the ionization potentials (IPs) for the 2-electron reduced state of 1-nitropyrenes with a substituent (H, CN or NO 2) at the 3, 6 or 8 position and 1- (or 3-) nitrobenzo[a]pyrenes with a substituent (H, CI, CN or NO 2) at the 6 position were calculated by the MNDO (modified neglect of diatomic overlap) method packed in MOPAC program version 5.0 using standard parameters. Linear relationships between the second reduction potentials and the IPs were established. The mutagenicity in TA98NR of a series of 1-nitrated pyrenes and 1(or 3-) nitrated benzo[a]pyrenes correlated well with the calculated ionization potentials for the 2-electron reduced state. These results indicate that the ease of reduction of nitroarene anion radical to nitrosoarene should predict the potent mutagenicity of these environmental mutagens in TA98NR. 11 Furihata, C. L, M. Yamashita 2, N. Kinae 2 and T. Matsushima ~, ~ Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108 and 2 Laboratory of Food Hygiene, School of Food and Nutritional Sciences, University of Shizuoka, 395 Yada, Shizuoka 422 (Japan)

Possible tumor-initiating and -promoting activities of 3-chloro-4-(dichloromethyl)-5-hydroxy2(5H)-furanone (MX) in rat glandular stomach MX is a strong direct-acting mutagen on Salmonella typhimurium TA100 and is present in

chlorinated tap water in big cities throughout the world. MX was administered orally to 7-week-old male Fischer F344 rats, and its possible tumorinitiating activity in the pyloric mucosa was examined by examination of DNA single-strand scissions by the alkaline elution method. The possible tumor-promoting activity of MX was examined with replicative DNA synthesis and ornithine decarboxylase as markers. MX at doses of 20-50 mg/kg body weight induced DNA singlestrand scissions dose-dependently (p < 0.05) in the pyloric mucosa of the stomach 2 h after its administration. MX at doses of 10-60 mg/kg body weight induced an up to 21-fold increase in replicative DNA synthesis (p < 0.01) 16 h after its administration. MX at doses of 30-60 mg/kg body weight induced an up to 100-fold increase in ornithine decarboxylase activity with a maximum 16 h after its administration. These results suggest that MX has tumor-initiating and -promoting activities in the glandular stomach of rats.

12 Furukawa, H., and K. Kawai, Laboratory of Biological Science, Faculty of Pharmaceutical Sciences, Meijo University, Yagoto-urayama 15, Tempaku-cho, Tempaku-ku, Nagoya 468 (Japan)

Mutagenic activities of nitropyrenes with the Drosophila wing spot assay 1,6- and 1,8-dinitropyrene (DNP) are potent mutagens in Ames test. But 150 /zg of 1,6-DNP was required to induce lung cancers (Maeda et al., J. Natl. Cancer Inst., 76 (1986) 696). The Drosophila wing spot assay, which is a somatic mutation and recombination test system, is able to detect several genetic damages, and effectively detects mutagenic activities of various genotoxic carcinogens. Hence the mutagenic activity of nitropyrenes was tested with the Drosophila wing spot test. Larvae for the wing spot test were obtained from crosses between mwh jL,;spap°l (virgin female) and flr3/TM3, Ser (male). 72 + 6-h-old larvae were transferred to 5.2 g medium (Carolina Biological Supply Company, U.S.A.) containing 3 mg of nitropyrene (1-NP, 1,6-DNP or 1,8-DNP). Larvae were given