Environmental mutagen society of Japan

Mutation Research, 216 (1989) 353-385 Elsevier

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MUTENV 08734

Environmental Mutagen Society of Japan Selected abstracts of the 17th Annual Meeting of the Environmental Mutagen Society of Japan 4 - 5 November 1988, Tokyo (Japan) (Received18 April 1989) (Accepted28 April 1989)

Keywords: Japanese Environmental Mutagen Society;Abstracts; Annual Meeting 1988 5 Fujiki, H., M. Suganuma, H. Suguri, S. Contents Yoshizawa, K. Takagi, N. Uda, T. Sassa, K. Yamada 2, T. Yasumoto 3 and T. Sugimura 1, 1 Abu-Zeid, M., Y. Yamazoe and R. Kato, DeCancer Prevention Division, National Cancer partment of Pharmacology, School of MediCenter Research Institute, 1National Cancer cine, Keio University, Tokyo 160 (Japan), Center, Tsukiji 5-1-1, Chuo-ku, Tokyo 104, Contribution of externally added hepatic and 2Department of Chemistry, Faculty of Science, bacterial cytosols in the Ames mutagenesis test Nagoya University, Nagoya 464, and 3Faculty 2 Arimoto, S., H. Shimada, A. Yoshida 1, S. of Agriculture, Tohoku University, Sendal 980, Ukawa 1, M. Mochizuki i and H. Hayatsu, (Japan), Tumor promotion with diarrhetic Faculty of Pharmaceutical Sciences, Okayama shellfish toxins University, Tsushima, Okayama 700, and 6 Fujimoto, Y., P.J. Wirth, K. Dempo 1, M. 1Kyoritsu College of Pharmacy, Shibakoen 1Mori 1 M. Nagao and T. Sugimura, Carcino5-30, Minato-ku, Tokyo 105 (Japan), Chargenesis Division, National Cancer Center Reacterization of a direct-acting mutagen formed search Institute, Tsukiji, Chuo-ku, Tokyo 104, from N-nitrosopiperidine and phosphate with and tSapporo Medical College, Sapporo, HokUVA irradiation kaido 060 (Japan), Protein analysis of heredi3 Ebata, J., and T. Inoue, Faculty of Science of tary hepatitis in rats by 2D-PAGE electroLiving, Osaka City University, Osaka 558 phoresis (Japan), Antimutagenic activity of vegetables 7 Furihata, C., and T. Matsushima, Department harvested in different growing conditions of Molecular Oncology, Institute of Medical 4 Fujie, K., and T. Aoki a, Laboratory of EnScience, University of Tokyo, Shirokanedai, vironmental Science, Department of Natural Minato-ku, Tokyo 108 (Japan), Possible antiScience, Osaka Women's University, Daisentumor promoter in the glandular stomach cho, Sakai, Osaka 590 (Japan) and 1Labora8 Hachiya, N., T.-H. Quan and Y. Takizawa, tory of Environmental Chemistry, College of Department of Public Health, Akita UniverEngineering, University of Osaka Prefecture, sity School of Medicine, Hondo 1-chome, Akita Mozuumemachi, Sakai, Osaka 591 (Japan), 010 (Japan), Mutagen adsorption effects of a Acute cytogenetic effects of alternative disindietary fiber: difference between in vitro and fectants on rat bone marrow cells in vivo in vivo situations 9 Hara, T., K. Ishihara, N. Horiya and T. Shibuya, Laboratory of Genetic Toxicology, Correspondence: Dr. Hikoya Hayatsu, Faculty of Hatano Research Institute, Food and Drug Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700 (Japan). Safety Center, 729-50chiai, Hatano, Kanaga-

0165-1161/89/$03.50 © 1989 ElsevierSciencePublishers B.V. (BiomedicalDivision)

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wa 257 (Japan), The proportion of recombination spots in the Drosophila wing spot test Hasegawa, J., M. Hosokawa, F. Okada and H. Kobayashi, Laboratory of Pathology, Cancer Institute, Hokkaido University School of Medicine, Sapporo 060 (Japan), Inhibition of mitomycin C-induced sister-chromatid exchanges in mouse bone marrow cells by the immunopotentiators Krestin and Lentinan Collaborative Study Group for the Micronucleus Test (CSGMT/JEMS MMS): Chief Organizer, M. Hayashi, Division of Mutagenesis, National Institute of Hygienic Sciences, Tokyo 158 (Japan), Difference between intraperitoneal and oral gavage application in the micronucleus test Higashimoto, M. 1,2, K. Matano 2 and Y. Ohnishi 1, i School of Medicine, The University of Tokushima, Tokushima 770, and 2 Tokushima Bunri University, Tokushima 770 (Japan), Augmenting effect of a non-mutagenic fraction in soy sauce on mutagenicity of 3-diazotyramine produced in the nitrite-treated sauce Hirayama, T., S. Ogawa, S. Kumo, T. Yamada, T. Watanabe and S. Fukui, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607 (Japan), Comutagenic and desmutagenic effect of spices in the Salmonella typhimurium mutagenicity assay Hirayama, T., T. Watanabe, S. Shimomura, Y. Fujioka, S. Ozasa and S. Fukui, Kyoto Pharmaceutical University, Yamashina, Kyoto 607 (Japan), Relationships between structure of nitrated arenes and their mutagenicity in Salmonella typhimurium; 2- and 2,7-nitrosubstituted fluorene, phenanthrene and pyrene Hisaoka, S., and N. Muraoka, Sanyo Gakuen Women's Junior College, Hirai, Okayama 703 (Japan), Mutagenicity in cooked foods. Effect of seasoning method and cooking oils on the formation of mutagenicity in pan-fried chicken Imaeda, T., K. Takahashi and Y. Kawazoe, Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya 467 (Japan), Inhibitory effect of the cadmium ion on induction of the adaptive response Imanishi, H., Y.F. Sasaki, K. Matsumoto, T.

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Ohta and Y. Shirasu, Institute of Environmental Toxicology, Suzuki-cho 2-772, Kodaira, Tokyo 187 (Japan), Suppression by flavorings of 6-TG-resistant mutations in V79 cells and of recessive spot formations in mice Ishii, Y., Y. Samejima, F. Saji and T. Nomura, Faculty of Medicine, Osaka University, Kitaku, Osaka 530 (Japan), Effect of alcohol sulfate and linear alkylbenzene sulfonate on the development of fertilized eggs of the mouse Iwado, H., M. Naito and H. Hayatsu 1, Okayama Prefectural Research Center of Environmental and Public Health, Uchio, Okayama 701-02, and i Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700 (Japan), Mutagenicity and antimutagenicity in air-borne particulates Kamiya, A., Y. Ose and T. Sato, Department of Public Health, Gifu Pharmaceutical University, 5-6-1, Mitahora-higashi, Gifu City (Japan), Detection of 1,6-dinitropyrene in municipal incinerator emissions Karube, T., Y. Odagiri, K. Takemoto and S. Watanabe a, Department of Public Health, Saitama Medical College, Moro, Irumagun, Saitama 350-04, and 1Epidemiology Division, National Cancer Center Research Institute, 51-1, Tsukiji, Chuo-ku, Tokyo 104 (Japan), Analysis of transplacentally induced sisterchromatid exchanges and micronuclei in mouse fetal liver cells. Effect of maternal exposure to cigarette smoke Kasai, H., Y. Okada, S. Nishimura, M.S. Rao 1 and J.K. Reddy 1, Biology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104 (Japan), and 1 Department of Pathology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611 (U.S.A.), Formation of 8hydroxydeoxyguanosine in liver DNA of rats following long-term exposure to a peroxisome proliferator, ciprofibrate Kato, F., A. Araki, K. Nozaki and T. Matsushima 1, Japan Bioassay Laboratory, Hatano 257, and i Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108 (Japan), Mutagenicity of aldehydes and diketones

355 24 Kinae, N., M. Yamashita, T. Ozawa 1, K. Mataki I and I. Tomita 1, Food and Nutritional Sciences, and 1 Pharmaceutical Sciences, University of Shizuoka, Yada, Shizuoka 422 (Japan), Mutagenicity of the reaction products from D-glucose and bovine serum albumin 25 Kong, Z.-L., K. Shinohara 1, H. Murakami, M. Nonaka and H. Omura, Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, Fukuoka 812, and 1 National Food Research Institute, 2-1-2 Kannondai, Tsukuba, Ibaraki 305 (Japan), Inhibitory effects of melanoidins on the nitroblue tetrazolium (NBT)-reducing ability of murine peritoneal macrophages treated with phorbol myristate acetate 26 Matsui, M., K. Matsui, T. Nohmi, H. Mizusawa and M. Ishidate Jr., Division of Mutagenesis, National Institute of Hygienic Sciences, Setagaya-ku, Tokyo 158 (Japan), Detection of deletion mutations in pSV2-gpt plasmid induced by metabolically activated steviol 27 Matsumoto, K., Y.F. Sasaki, T. Ohta and Y. Shirasu, Institute of Environmental Toxicology, Kodaira, Tokyo 187 (Japan), Suppressing effects of tannic acid on chromosome aberrations induced by mitomycin C in mouse bone marrow cells 28 Matsuoka, A., N. Miyata 1, T. Sofuni and M. Ishidate Jr., Division of Mutagenesis and 1 Division of Synthetic Chemistry, National Institute of Hygienic Sciences, Setagaya-ku, Tokyo 158 (Japan), Clastogenicity of BHA and its metabolites in cultured mammalian cells 29 Miura, K., and K. Morimoto, Department of Hygiene and Preventive Medicine, School of Medicine, Osaka University, Nakanoshima, Kita-ku, Osaka 530 (Japan), Effects of liquid holding on sister-chromatid exchange frequencies in human lymphocytes 30 Nakatsuka, S., S. Arimoto 1 and M. Namba 2, Departments of Urology and 2 Pathology, Kawasaki Medical School, Kurashiki City, Okayama 701-01, and I Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700 (Japan), Tumor-promoting effects of Fe3+-nitrilotriacetic acid (NTA) 31 Nishifuji, K., T. Kinouchi and Y. Ohnishi,

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Department of Bacteriology, School of Medicine, The University of Tokushima, Tokushima 770 (Japan), Metabolism of glutathione conjugates of 1-nitropyrene oxides in the intestinal tract Nozaka, T., F. Watanabe, M. Ishino, I. Morimoto, J. Kunitomo 1, H. Ishii 2 and S. Natori 3, Saitama Institute of Public Health, Urawa, Saitama 338, 1 Faculty of Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya 663, 2 Faculty of Pharmaceutical Sciences, Chiba University, Yayoi, Chiba 260, and 3 Meiji College of Pharmacy, Yato, Tanashi 188 (Japan), Mutagenicity of isoquinoline alkaloids Ochiai, M., N. Nitta, H. Shima, P.J. Wirth, M. Suzuki, S. Nagase 1, T. Sugimura and M. Nagao, Carcinogenesis Division, National Cancer Center Research Institute, Tsukiji, Tokyo 104 and 1 Sasaki Institute, Kandasurugadai, Tokyo 101 (Japan), In vivo mammalian cell mutation assay using albumin + as a marker in analbuminemic rats Ohara, Y., S. Arimoto, T. Hayatsu and H. Hayatsu, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700 (Japan), Classification of 62 compounds according to their ability to adsorb to blue cotton and blue rayon Ohshima, H., M. Friesen, C. Malaveille, I. Brouet, A. Hautefeuille and H. Bartsch, International Agency for Research on Cancer, Lyon (France), Formation of direct-acting genotoxic substances by the reaction of smoked foods with nitrite Ohuchida, A., A. Furukawa and R. Yoshida, Drug Safety Laboratory, Taiho Pharmaceutical Co., Kawauchi, Tokushima 771-01 (Japan), Micronucleus test of polyploidy inducers Ohyama, K., R. Endo and H. Kawahara, Tokyo Metropolitan Research Institute for Environmental Protection, Koto-ku, Tokyo 136 (Japan), Mutagenicity of surface soil Okuda, H., S. Yoshioka and T. Watabe, Tokyo College of Pharmacy, 1432-1 Horinouchi, Hachioji-shi, Tokyo 192-03 (Japan), Sulfotransferase-mediated activation of 9-hydroxymethyl-10-methylanthracene, a major metabolite of the carcinogen 9,10-dimethylanthracene

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39 Sai, K., A. Takagi, T. Umemura, Y. Kurokawa and H. Kasai 1, Division of Toxicology, National Institute of Hygienic Science, Setagayaku, Tokyo 158, and I Biology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104 (Japan), Detection of 8hydroxydeoxyguanosine (8-OH-dG) in rat kidneys treated with renal carcinogens 40 Sakagami, Y., H. Yamazaki, N. Ogasawara, H. Yokoyama, Y. Ose 1 and T. Sato 1, Osaka Prefectural Institute of Public Health, Nakamichi, Higashinari-ku, Osaka 537, and 1Gifu Pharmaceutical University, Mitahora-higashi, Gifu 502 (Japan), Evaluation of genotoxic activities of disinfectants and their metabolites by the u m u test 41 Sasagawa, C., Y. Kodama and T. Matsushima, Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Tokyo 108 (Japan), Formation of mutagenic diazo compounds on treatment of derivatives of tyramine and bamethan with nitrite 42 Sasaki, K., H. Mizusawa 1, M. Ishidate Jr. 1 and N. Tanaka, Department of Cell Biology, Food and Drug Safety Center, Hatano Research Institute, Hatano, Kanagawa 257, and 1 Division of Mutagenesis, National Institute of Hygienic Sciences, Setagaya-ku, Tokyo 158 (Japan), A screening system for tumor promoters using v - H a - r a s - t r a n s f e c t e d BALB 3T3 cells (Bhas 42) 43 Sawada, M., T. Sofuni and M. Ishidate Jr., Division of Mutagenesis, National Institute of Hygienic Sciences, Setagaya-ku, Tokyo 158 (Japan), Induction of chromosomal aberrations in active oxygen-generating systems, VI. Selection and use of menadione-resistant cells in culture 44 Sawada, S. 1.2, C. Furihata 1 and T. Matsushima 1, 1 Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108, and 2 Department of Drug Safety Research, Ei.sai Co., Kawashima-cho, Hashima, Gifu 483 (Japan), In vivo short-term assay of unscheduled DNA synthesis (UDS) in rat liver 45 Sengoku, Y., M. Kamiya, K. Takahashi, K. Kohda and Y. Kawazoe, Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabedori, Mizuho-ku, Nagoya 467 (Japan),

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Effect of substituents on the mutagenicity and metabolism of quinoline Sera, N., K. Morita, K. Horikawa, H. Tokiwa, K. Wakabayashi 1 and Y. Ohnishi 2, Fukuoka Environmental Research Center, Fukuoka 81801, 1 National Cancer Center Research Institute, Tokyo 104, and 2 Department of Bacteriology, School of Medicine, University of Tokushima, Tokushima 770 (Japan), Binding of mutagens to rice fiber Shibahara, T., H. Ryo 1, G. Tsushimoto and T. Nomura 1, Otsuka Pharmaceutical Co. Ltd., Tokushima 771-01, and a Department of Fundamental Radiology, Faculty of Medicine, Osaka University, Osaka 530 (Japan), DNAdamaging potency in vivo of hepatocarcinogenic aflatoxins as measured in the Drosophila DNA repair test Shibuya, T., T. Murota 1, N. Horiya and T. Hara, Hatano Research Institute, Food and Drug Safety Center, Ochiai 729, Hatano, Kanagawa 257, and a Toxicology Laboratory, Mitsubishi Kasei Co., Midori-ku, Kanagawa 227 (Japan), Induction of recessive mutations by N-ethyl-N-nitrosourea in mouse primordial germ cells Shimada, H., T. Sato, C. Hattori, S. Satake and S. Itoh, Research Institute of Daiichi Seiyaku Co., Tokyo 134 (Japan), Induction of micronuclei by benzene and its metabolites Shimizu, H., M. Akiyama, Y. Suzuki and K. Hayashi, Department of Public Health, Jikei University School of Medicine, Minato-ku, Tokyo 105 (Japan), The effects of magnetic field on mutagenic activity Shioya, M., K. Wakabayashi, T. Sugimura and M. Nagao, Carcinogenesis Division, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo 104 (Japan), Formation of 8-hydroxydeoxyguanosine in DNA treated with coffee Sofuni, T., A. Matsuoka, M. Sawada, M. Ishidate Jr. and M.D. Shelby 1, National Institute of Hygienic Sciences, Setagaya-ku, Tokyo 158 (Japan), and 1 National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (U.S.A.), A comparison of chromosome aberration induction in the CHO and CHL cell systems by 25 chemicals Sugimura, K., S. Iwamoto 1, S. Hashimoto and

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T. Ohnishi, Nara Medical University, Kashihara, Nara 634, and 1 Prefectural Institute of Public Health, Ohmori-cho, Nara City, Nara 630 (Japan), UVA radiation and chemicals induced umu gene expression in Escherichia coli Sutou, S., and S. Sato, Central Research Institute, Itoham Foods Inc., 1-6-21 Mita, Meguro, Tokyo 153 (Japan), Maternal inheritance of the ms gene Suzuki, Y., S. Toda 1 and H. Shimizu, Department of Public Health, Jikei University School of Medicine, Minato-ku, Tokyo 105, and 1 Mutagenicity Test Division, Sohgo Biochemical Laboratory Inc., Kawagoe, Saitama 305 (Japan), Effect of prostaglandin E 2 and calcium in the micronucleus test in mice Takagi, A., K. Sai, T. Umemura, Y. Kurokawa and H. Kasai 1, Division of Toxicology, National Institute of Hygienic Sciences, Kamiyoga, Setagaya-ku, Tokyo 158, and i Biology Division, National Cancer Center Research Institute, Chuo-ku, Tsukiji, Tokyo 104 (Japan), Production of 8-hydroxydeoxyguanosine in rat liver DNA by oral administration of peroxisome proliferators Takahashi, S., T. Kato and K. Kikugawa, Tokyo College of Pharmacy, 1432-1 Horinouchi, Hachioji, Tokyo 192-03 (Japan), Formation and content of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) in roasted coffee beans Takayama, K., K. Yamashita, K. Wakabayashi, M. Nagao and T. Sugimura, Carcinogenesis Division, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo 104 (Japan), Formation of PhIP-DNA adduct in F344 rats Takenaka, S., N. Sera, H. Tokiwa, I. Hirohata and T. Hirohata 2, Fukuoka Environmental Research Centre, Dazaifu, Fukuoka 818-01, 1 Kurume Shinai Women's College, Kurume 830, and 2 Department of Public Health, School of Medicine, Kyushu University, Maedashi, Fukuoka 810 (Japan), Identification of mutagens in the pickles produced in Akita and Fukuoka Tamakawa, K., K. Matsumoto, Y. Takahashi, T. Seki, A. Tsunoda and J. Lewtas, Sendai Municipal Institute of Public Health, 2-5-10, Oroshimachi-higashi, Sendai 983 (Japan), Ap-

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plication of the sensitive Ames test (accelerated microsuspension procedure) to assess indoor air pollution. Mutagenicity of dust on filters in air-conditioners Tatsumi, K., S. Kitamura, H. Amano 1, K. Ueda 2, F. Okumura and T. Fujimoto, Institute of Pharmaceutical Science, Hiroshima University School of Medicine, Kasumi, Minami-ku, Hiroshima 734, 1 Wakunaga Pharmaceutical Co., Koda-cho, Takada-gun, Hiroshima 729-64, and 2 Santen Pharmaceutical Co., Shimoshinjyo, Higashiyodogawa-ku, Osaka 533 (Japan), Studies on metabolic activation of carcinogenic arylamines. N-Formylation in animals Tatsumi, K., M. Toyoda, A. Fijimori, A. Tachibana, I. Arita and H. Takebe, Faculty of Medicine, Kyoto University, Sakyo, Kyoto 606 (Japan), Mutation assay at the adenosine phosphoribosyltransferase locus in lymphoblastoid ceils derived from obligate heterozygotes of 2,8-dihydroxyadenine urolithiasis Tsuda, M., T. Kato, Y. Kurashima and T. Sugimura, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104 (Japan), Studies on nitrosation dynamics in the human body and in rats using thioproline, an effective nitrite-trapping agent Ueda, Y., K. Wakabayashi, T. Sugimura and M. Nagao, Carcinogenesis Division, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo 104 (Japan), Trapping of mutagens/carcinogens in cigarette smoke by fibrillated composite fibers Wakabayashi, K., K. Yamashita, Y. Kitagawa, M. Suzuki, M. Nagao and T. Sugimura, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo 104 (Japan), DNA modification by 1-nitrosoindole-3-acetonitrile in the rat stomach Watanabe, K., T. Ohta and Y. Shirasu, Institute of Environmental Toxicology, Suzukicho 2-772, Kodaira, Tokyo 187 (Japan), oVanilin enhances mutagenesis induced by M N N G in Escherichia coli Watanabe, M., T. Nohmi and M. Ishidate Jr., Division of Mutagenesis, National Institute of Hygienic Sciences, Setagaya-ku, Tokyo 158 (Japan), Establishment of new strains of S.

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typhimurium highly sensitive to nitroarenes and aromatic amines: TA98 and TA100 derivatives with high levels of nitroreductase or acetyltransferase activities Watanabe, T., Y. Hanasaki, T. Hirayama and S. Fukui, Kyoto Pharmaceutical University, 5-Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607 (Japan), Mutagenicity of nitro- and amino-substituted phenazines in Salmonella typhimurium Yamakage, K., Y. Iwata, M. Oshimura 1 and N. Tanaka, Department of Cell Biology, Hatano Research Institute, Food and Drug Safety Center, Hatano, Kanagawa and 1 Department of Cytogenetics, Kanagawa Cancer Center, Yokohama, Kanagawa (Japan), Development of an in vitro screening system for agents causing aneuploidy: use of h u m a n / mouse hybrid cells Yamanaka, K., K. Hakomori, A. Someya, A. Hasegawa, R. Sawamura and S. Okada 1, College of Pharmacy, Nihon University, Tokyo, and 1 School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka (Japan), DNA strand breaks in rat lung induced by dimethylarsinic acid Yasunaga, K., N. Asai and K. Yoshikawa, Toxicology Laboratory, Life Science Research Sector, Research Center, Mitsubishi Kasei Corp., 1000, Kamoshida-cho, Midori-ku, Yokohama, Kanagawa 227 (Japan), Relation between uvrB and umuDC gene functions and DNA cross-linking damages induced by mitomycin C in Salmonella typhimurium strain G46/pSK1002

cent studies on the metabolism of arylamines in bacterial cells suggest the possibility that the exogenously added cytosol plays a role not only in N-hydroxylation, but also in the esterification of the N-hydroxy derivatives formed. In the present study, we have examined the contribution of the cytosol in the Ames mutagenesis test of the N-hydroxy derivatives of Glu-P-1, IQ, 2-AF and Trp-P2. The mutagenicity of N-hydroxy Glu-P-1 towards Salmonella typhimurium TA98/1,8-DNP 6 was enhanced 6-fold by the addition of rat hepatic cytosol and 9-fold by the addition of bacterial cytosol, both in the presence of 0.25 mM acetyl CoA (acetyltransferase-mediated activation). Similarly, the mutagenicity of N-hydroxy IQ towards TA98/1,8-DNP 6 was enhanced 9-fold by rat hepatic cytosol and 12-fold by the bacterial cytosols. Lower enhancing effects were observed for the cytosols when the TA98 strain was used for the assay. On the other hand, addition of PAPS (sulfotransferase-mediated activation) showed no significant effect on the original mutagenic activities of the N-hydroxy arylamines studied. Our results show that externally added cytosol has a minor, but significant, role in the mutagenic activation of arylamines, and that the enhancement is probably caused by the esterification of the hydroxylamino groups.

Abu-Zeid, M., Y. Yamazoe and R. Kato, Department of Pharmacology, School of Medicine, Keio University, Tokyo 160 (Japan)

Characterization of a direct-acting mutagen formed from N-nitrosopiperidine and phosphate with UVA irradiation

Contribution of externally added hepatic and bacterial cytosols in the Ames mutagenesis test

Previously, we found that direct-acting mutagens 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

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In the Ames mutagenesis test of arylamines, addition of hepatic cytosol often results in an increase in the number of induced revertants. Re-

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Arimoto, S., H. Shimada, A. Yoshida 1, S. Ukawa a, M. Mochizuki I and H. Hayatsu, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700, and 1 Kyoritsu College of Pharmacy, Shibakoen 1-5-30, Minatoku, Tokyo 105 (Japan)

359 with UVA (313-400 nm, 10 /~W/mm2) for 3 h. Direct-acting mutagenicity of the solution on Salmonella typhimurium TA1535 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 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 tx-phosphonooxy-NPIP in terms of the retention time in HPLC, UV absorption spectrum, sensitivity to Escherichia coli alkaline phosphatase and the mutagenic potency as determined on the basis of A231 units. 3 Ebata, J., and T. Inoue, Faculty of Science of Living, Osaka City University, Osaka 558 (Japan) Antimutagenic activity of vegetables harvested in different growing conditions

By employing the streptomycin-dependent strain SD100 of Salmonella typhimurium (T. Kada et al., Environ. Mutagen., 5 (1983) 9-15) the antimutagenicity of the homogenates of fresh vegetables against AF2 has been examined. The influence of growing conditions on the antimutagenicity of 5 kinds of vegetables cultured in an open field at the Agricultural Experiment Station, Nara Prefecture, was studied. The antimutagenic activities were compared with those of vegetables of comparable maturity and size. The antimutagenicity of spinach, Spinacia oleracea L., increased in the order of harvesting time, summer < autumn < winter. The activity of cabbage, Brassica oleracea L. var. capitata L., was more potent in autumn than in summer, and greater in the outer layer than in the inner layer in both seasons. In the case of egg-plant, Solanum melongena L., the maximum activity was found in September, during the harvest time July to October. The activity of cucumber, Cucumis sativis L., increased in the order of low < medium < high fruit-bearing positions on the stem. After sowing at the end of March, great burdock, Arctium lappa L., was harvested 6 times between June and November

and homogenized. The 99th-day and 172th-day samples showed activities higher than those of the other samples. 4

Fujie, K., and T. Aoki 1, Laboratory of Environmental Science, Department of Natural Science, Osaka Women's University, Daisen-cho, Sakai, Osaka 590 (Japan) and 1 Laboratory of Environmental Chemistry, College of Engineering, University of Osaka Prefecture, Mozuumemachi, Sakai, Osaka 591 (Japan) Acute cytogenetic effects of alternative disinfectants on rat bone marrow cells in vivo

Chlorine (C12) is the most widely used disinfectant of drinking water. However, trihalomethanes (THMs) are formed during the chlorination of water containing precursor compounds, most commonly humic substances. The maximum allowable level for total THMs (TTHM) in the source water in Japan and U.S.A. is 100 ppb. We have studied acute cytogenetic effects of C12 and 2 alternative disinfectants, monochloramine (NH2C1) and chlorine dioxide (C102) , on rat bone marrow cells in vivo, using the chromosome aberration test. Female Long-Evans rats were used, aged 4-5 weeks and weighing 50-100 g. Dose-response relationships were clearly observed for these 3 chemicals. Statistically significant positive results were obtained 6 h after intraperitoneal treatment with 20 mmole NH2C1/kg , 80 mmole C102/kg and 20 mmole C12/kg body weight. The percentage of aberrant metaphase cells induced with 20 mmole NH2C1/kg , 80 mmole C102/kg and 40 mmole C12/kg increased rapidly, reaching the significantly positive level 6 h later and reaching maximum levels after 12 h, 12 h and 6 h, respectively. The percentages decreased to the control level within 24 h. These results indicate that the mutagenic activity of NH2C1 is similar to that of C12 and higher than that of C102. A sample of water from Yodo River was treated with 0.5 mM C12, 0.5 mM NH2C1 and 0.5 mM C102, and the TTHM formed were measured. 45 ppb TTHM were formed with C12, but no TTHM were formed with NH2C1 or C102.

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Fujiki, H., M. Suganuma, H. Suguri, S. Yoshizawa, K. Takagi, N. Uda, T. Sassa, K. Yamada 2, T. Yasumoto 3 and T. Sugimura ~, Cancer Prevention Division, National Cancer Center Research Institute, 1 National Cancer Center, Tsukiji 5-1-1, Chuo-ku, Tokyo 104, 2 Department of Chemistry, Faculty of Science, Nagoya University, Nagoya 464, and 3 Faculty of Agriculture, Tohoku University, Sendai 980 (Japan) Tumor promotion with diarrhetic shellfish toxins Our recent study revealed the presence of various kinds of tumor promoters in marine organisms. Okadaic acid and dinophysistoxin-1 (35-methylokadaic acid), which are products of marine dinoflagellates, are tumor promoters of the okadaic acid class. Their tumor-promoting activity is as strong as those of TPA, teleocidins and aplysiatoxins. The potency of okadaic acid and dinophysistoxin-1 in diarrhetic shellfish poisoning correlates well with their tumor-promoting activity in mouse skin. Acylated derivatives of okadaic acid and pectenotoxins are also associated with diarrhetic shellfish poisoning. Pectenotoxin 2, like okadaic acid and dinophysistoxin-1, induces ornithine decarboxylase (ODC) in mouse skin. Furthermore, okadaic acid is able to induce ODC in rat stomach. The tumor-promoting activity of okadaic acid in rat stomach is now under investigation. The possible distribution of potent tumor promoters through the food chain is discussed.

Fujimoto, Y., P.J. Wirth, K. Dempo ~, M. Mori 1, M. Nagao and T. Sugimura, Carcinogenesis Division, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo 104, and 1 Sapporo Medical College, Sapporo, Hokkaido 060 (Japan) Protein analysis of hereditary hepatitis in rats by 2D-PAGE electrophoresis The LEC rat strain exhibits 100% incidence of hepatitis associated with severe jaundice. Hepatitis

occurs spontaneously in adult rats at 3-4 months of age, after which 50% die from hepatic failure. Of the surviving animals, 100% develop liver cancer. In an attempt to elucidate the pathogenic mechanism(s) responsible for the development of hepatitis, the qualitative and quantitative differences in hepatic protein expression in normal (LEA) and LEC rats were compared using 2-dimensional electrophoresis. Approximately 1000 cytosolic and 1000 particulate polypeptides were detected over the pI range 4.5-7.0 and molecular weight range of 18-120 kDa. Polypeptide patterns from age-matched (1 day, 2, 8, 12, 15, 16, 24 weeks) LEA and LEC livers were very similar, although both qualitative and quantitative differences were noted. Two constitutively expressed cytosolic polypeptides (30 kDa, pI 6.70 and 29.5 kDa, pI 6.72) were not detected in LEC livers at any age. Analysis of the nature of these proteins should provide insight into their involvement in hepatitis development in LEC rats. The LEC rat provides a good animal model for detecting environmental mutagens/carcinogens which may be involved in human hepatocarcinogenesis.

Furihata, C., and T. Matsushima, Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108 (Japan) Possible anti-tumor promoter in the glandular stomach Previously we found that all 6 glandular stomach tumor promoters examined (NaC1, taurocholate, glyoxal, catechol, K2S205 and formaldehyde) and all 5 glandular stomach carcinogens examined (MNNG, ENNG, PNNG, 4NQO and N-nitroso-N-methylurethane) induced about a 10-fold increase in replicative DNA synthesis (RDS) in the proliferative zone of the stomach pyloric mucosa of F344 male rats 16 h after their administration. In the present study, we looked for chemicals that can inhibit the increase in RDS induced by glandular stomach tumor promoters in the pyloric mucosa. Administration of 1 ml of

361 100-400 mM CaC12 to rats 1-2 h before administration of I ml of 3.3 M NaC1 resulted in dose-dependent inhibition of the increase in RDS in the pyloric mucosa 17 h after the NaC1 administration. 400 mM CaCl 2 caused 80-100% inhibition of the increase in RDS by NaC1. However, 13-cis-retinoic acid at doses of 10 /xg-10 mg/kg body weight did not inhibit the NaCl-induced increase in RDS in the pyloric mucosa. These results suggest that CaC12, but not 13-cis-retinoic acid, may have anti-tumor promoter activity in the pyloric mucosa of the rat stomach.

8 Hachiya, N., T.-H. Quan and Y. Takizawa, Department of Public Health, Akita University School of Medicine, Hondo 1-chome, Akita 010 (Japan) Mutagen adsorption effects of a dietary fiber: difference between in vitro and in vivo situations The efficiency of mutagen adsorption to a dietary fiber, refined corn bran (RCB), was determined in vitro and in vivo to examine the possibility that the binding effect participates in the suppression of carcinogenesis by dietary fibers. RCB was tested for adsorption efficiency on 16 chemical mutagens in vitro. It was shown that RCB can bind heterocyclic amines, dinitropyrene and benzo[a]pyrene, dissolved in distilled water, at more than 95% efficiency, but simple alkylating agents, MNNG and ENNG, at less than 20%. Mutagens such as Trp-P-1 and dinitropyrene were successfully eluted with methanol:NHaOH (50 : 1) from RCB. Ionic strength and pH also affected not only the mutagenicity binding, but also its retention on RCB. In experiments in vivo, Trp-P-1, IQ or dinitropyrene was adsorbed to RCB, and these RCB preparations were given orally to rats, together with a fiber-free diet. Feces of these rats were fractionated to separate RCB, the residues and the aqueous parts. Mutagenicity was assayed for extracts of these fractions and urine after removal of mutagenicity inhibitors. The mutagenic activity recovered from the RCB in feces was 0.47% of the ingested activity for Trp-P-1, 0.24%

for IQ and 0.04% for dinitropyrene. We conclude that the mutagen-adsorbing property of RCB is not effective as a mechanism inhibitory to the physiological effects of mutagens in vivo.

Hara, T., K. Ishihara, N. Horiya and T. Shibuya, Laboratory of Genetic Toxicology, Hatano Research Institute, Food and Drug Safety Center, 729-50chiai, Hatano, Kanagawa 257 (Japan) The proportion of recombination spots in the Drosophila wing spot test The Drosophila wing spot test is a sensitive in vivo short-term assay for detecting many kinds of genetic lesion, such as gene mutations and recombinations, in somatic cells. A study was intended to estimate the proportion of the recombination spots, spots caused by recombinations, in total wing spots induced by various mutagens. The mutagens used were 3 alkylating agents, EMS, MMS and ENU, a cross-linking agent, mitomycin C (MMC), a spindle poison, vinblastine (VB), and an inducer of DNA breaks, bleomycin (BM). Females of the mwh strain were mated to males of the fir strain, and the 2nd and 3rd instar F1 larvae were treated with a mutagen. Two types of F1 flies emerged. In the yellow flies, recombinations between 3rd chromosomes are suppressed because of the heterozygosity for complex inversions, TM1. In wild-type flies, recombinations are not suppressed. From comparison of the spot frequency between these 2 types, the proportion of recombination spots was estimated. 55-60% of the wing spots were recombination spots for EMS, MMS, ENU or MMC and 84% for BM, whereas VB induced almost no recombination spots. From these results it was concluded that this test of Drosophila wing spots is useful for detecting mitotic recombinations.

10 Hasegawa, J., M. Hosokawa, F. Okada and H. Kobayashi, Laboratory of Pathology, Cancer In-

362 stitute, Hokkaido University School of Medicine, Sapporo 060 (Japan) Inhibition of mitomycin C-induced sister-chromatid exchanges in mouse bone marrow cells by the immunopotentiators Krestin and Lentinan

We have carried out an investigation to determine whether the sister-chromatid exchanges (SCEs) observed in bone marrow cells in mice treated with mitomycin C (MMC) can be inhibited by the immunopotentiators Krestin and Lentinan. We found that mitomycin C (2 mg/kg, i.v.)-induced SCEs were inhibited in 27% of the mice treated with Krestin (300 mg/kg, i.p.) and in 23% of the mice treated with Lentinan (1 mg/kg, i.p.). The effects of Krestin were dose-dependent, while those of Lentinan were not. Our findings suggest that Krestin and Lentinan are not only useful for cancer treatment as immunopotentiators in combination with anticancer drugs, but may also prevent the chromosomal damage inducible by the anticancer drugs.

11 Collaborative Study Group for the Micronucleus Test ( C S G M T / J E M S MMS): Chief Organizer, M. Hayashi, Division of Mutagenesis, National Institute of Hygienic Sciences, Tokyo 158 (Japan) Difference between intraperitoneal and oral gavage application in the micronucleus test

In the third collaborative study organized by the Collaborative Study Group for the Micronucleus Test, a task group of the Environmental Mutagen Society of Japan, the intraperitoneal (i.p.) and oral (p.o.) administration rbutes were compared using 2 mouse strains, M S / A e and CD-1, and 17 chemicals with various modes of action, (Ara-C, 6-MP, B[a]P, DMBA, 2AAF, phenacetin, CYP, EMS, ENU, MMS, MMC, COL, VINC, KBrO3, K2CrO 4, benzene and PCZ). On the basis of pilot tests designed to determine LDs0, doses and sampling times, full-scale experiments were performed on each chemical using 4 mice per group. Almost all chemicals induced micronuclei

by both routes of administration in both mouse strains. Generally, the chemicals induced micronuclei at lower dose levels (mg/kg) by the i.p. route. This tendency, however, is lessened or even reversed if the dose is expressed as a percentage of the LDs0. When the dose levels of chemicals are moderate, both routes are acceptable in the micronucleus test.

12 Higashimoto, M. 1.2, K. Matano 2 and Y. Ohnishi 1, 1 School of Medicine, The University of Tokushima, Tokushima 770, and 2 Tokushima Bunri University, Tokushima 770 (Japan) Augmenting effect of a non-mutagenic fraction in soy sauce on mutagenicity of 3-diazotyramine produced in the nitrite-treated sauce

When 25 kinds of Japanese soy sauce at a concentration of 5% were incubated with 50 mM sodium nitrite (pH 2) at 37 ° C for 1 h, the reaction mixtures induced 34-834 (average 368 + 228) revertants per /~1 of soy sauce equivalent from Salmonella typhimurium strain TA100 in the absence of $9 mix. The mutagen(s) formed was very unstable under natural daylight or under fluorescent light, but was stable under a yellow lamp or in the dark. In addition to the known precursors, i.e., tyramine and 1-methyl-l,2,3,4-tetrahydro-fl-carboline-3-carboxylic acid, 1-methyl1,2,3,4-tetrahydro-fl-carboline, which led to weak mutagenesis, was found in the soy sauce. However, the sum of the activity of the 3 mutagen precursors after nitrite treatment accounted for only part of the mutagenicity of nitrite-treated soy sauce. In the soy sauce, there was a factor which can enhance 9-fold the mutagenicity of 3-diazotyramine, a product of nitrite-treated tyramine. These 3 precursors and their mutagenicity augmentation accounted for all the mutagenicity of the nitrite-treated sauce. The mutagenicity-augmenting factor in the soy sauce was non-mutagenic before and after nitrite treatment, and was stable to heat and light.

363 13 Hirayama, T., S. Ogawa, S. Kumo, T. Yamada, T. Watanabe and S. Fukui, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607 (Japan) Comutagenic and desmutagenic effect of spices in the Salmonella typhimurium mutagenicity assay The effects of 14 kinds of popular spice on 9 kinds of mutagen were investigated in the Salmonella typhimurium mutagenicity test. Methanol extracts of spices (100 ttg/plate) were applied with or without mutagens to TA98 and TA100 strains with or without $9 mix. The methanol extracts themselves were not mutagenic in these strains. The mutagenicity of AAF was increased 1.6-6.3-fold by 9 kinds of spice. The mutagenicity of 4NQO and MNNG was also increased 1.6-2.4-fold by 6 of the spices and 1.7-2.9-fold by 10 of the spices. The mutagenicity of B[a]P was not affected. The mutagenicity of amino acid pyrolysates (Trp-P-1, Trp-P-2, Glu-P-1, Glu-P-2 and IQ) was apparently depressed: 0.1-0.5-fold by allspice, clove and sage. We found that eugenol, one of the main essential oils contained in these spices, depressed the mutagenicity of the amino acid pyrolysates and AAF, and inhibited the $9 mix-mediated N-hydroxytation of AAF. Thus, the desmutagenic effect of allspice, clove and sage on the amino acid pyrolysates may be due to the presence of eugenol as a component.

14 Hirayama, T., T. Watanabe, S. Shimomura, Y. Fujioka, S. Ozasa and S. Fukui, Kyoto Pharmaceutical University, Yamashina, Kyoto 607 (Japan) Relationships between structure of nitrated arenes and their mutagenicity in Salmonella typhimurium; 2- and 2,7-nitro-substituted fluorene, phenanthrene and pyrene In order to elucidate the mechanisms of mutagenic activation of nitroarenes, we studied the relationships between the mutagenic potency and

chemical structure of 2-nitro- and 2,7-dinitroarenes, including nitrated fluorene (F1), dihydrophenanthrene (DHPh), phenanthrene (Ph), tetrahydropyrene (THPy), dihydropyrene (DHPy) and pyrene (Py) together with 9-NO2-Ph, 1-NO2-Py and 1,3-diNO2-Py. The mutagenicity tests were carried out on Salmonella typhimurium TA98, TA98NR and TA98/1,8-DNP 6 in the absence of $9 mix. The order of mutagenicity of mononitroand dinitro-arenes in TA98 is: 2-NO2-THPy < 2NO2-F1 < 2-NO2-DHPh < 9-NO2-Ph < 2-NO2-Ph < 2-NO2-DHPy < 1-NO2-Py < 2-NO2-Py, and 2,7diNO2-DHPh < 2,7-diNO2-F1 < 2,7-diNO2-ThPy < 2,7-diNO2-Ph < 2,7-diNO2-DHPy < 2,7-diNO 2Py < 1,3-diNO2-Py. 9-NO2-Ph and 1-NO2-Py, which have been detected in environmental samples, are not as potent mutagens as 2-nitrated phenanthrene and pyrene, respectively. 2-NO 2THPy (37.7 rev./nmole) was a weak mutagen, but 2,7-diNO2-THPy (3197 rev./nmole) was as potent a mutagen as 2,7-diNO2-Ph (3934 rev./nmole). Tetrahydropyrene has a twisted form in its structure. 1,3-diNO2-Py (99660 rev./nmole) was more mutagenic than 2,7-diNO2-Py (37960 rev./nmole), and their mutagenicities were correlated with the behavior of the K-band in their UV spectra by the introduction of the nitro group on pyrene.

15 Hisaoka, S., and N. Muraoka, Sanyo Gakuen Women's Junior College, Hirai, Okayama 703 (Japan) Mutagenicity in cooked foods. Effect of seasoning method and cooking oils on the formation of mutagenicity in pan-fried chicken Pan-fried chickens were tested for mutagenicity by the blue-cotton extraction method coupled with the Ames test (TA98, + $9). It was found that the potency of mutagenicity in the pan-fried chicken was dependent on the method of seasoning and also on the kind of oil or fat used in the heating step. Generally high levels of mutagenic activity were obtained with the soy sauce/sake/sugar and ginger/soy sauce/sake/sugar treatments. We also observed that the fiber material of ginger can adsorb the mutagens present in heated sauce. On

364

the other hand, ginger fiber enhanced the soy sauce mutagenicity when it was present during heating of the soy sauce. Pan-fried chicken pretreated with ginger/soy sauce/sake/sugar showed higher mutagenic activity than that pre-treated without ginger. The mutagenic activity of chicken pan-fried in oils and fats was lower than that of chicken cooked without oils and fats. Among the 5 different oils and fats tested, vegetable oil gave the lowest mutagenicity in chicken.

16

Imaeda, T., K. Takahashi and Y. Kawazoe, Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya 467 (Japan) Inhibitory effect of the cadmium ion on induction of the adaptive response

Among the heavy metal ions, Cd 2+ strongly inhibited the adaptive response induced by Nmethyl-N-nitrosourea (MNU) in Eseherichia coli. In the present study, we examined the effect of C d 2+ o n adaptive responses induced by a variety of methylating agents, such as methyl methanesulfonate and methyl iodide, in comparison with its effect on SOS responses induced by MNU and UV irradiation. The plasmids pYM3 (ada'-lacZ') and pMCP1000 (alkA'-lacZ') were used for quantification of the induced adaptive response, and the plasmid pSK1002 (umu'-lacZ') for that of the induced SOS response. C d 2+ inhibited the ada expression induced by any of the methylating agents examined to a similar extent. Cd 2+ also inhibited the MNU-induced alkA expression as well as the ada expression. On the other hand, the UV-induced umu expression was not affected by Cd 2+, while the MNU-induced umu expression was enhanced up to 150% in a CdZ+-dose-dependent manner. The mutagenicity of MNU was potentiated by the presence of Cd 2+ in an E. coli strain. These results suggest that the mechanism for inhibition of the adaptive response in E. coli involves a specific inhibitory interaction of Cd 2+ with O6-methylguanine-DNA methyltransferase.

17 Imanishi H., Y.F. Sasaki, K. Matsumoto, T. Ohta and Y. Shirasu, Institute of Environmental Toxicology, Suzuki-cho 2-772, Kodaira, Tokyo 187 (Japan) Suppression by flavorings of 6-TG-resistant mutations in V79 cells and of recessive spot formations in mice

The in vitro antimutagenic effects of flavorings, the antimutagenic effects of which were previously elucidated in bacterial systems, were investigated by use of cultured Chinese hamster V79 cells. The frequency of 6-TG-resistant mutations induced by UV or X-ray irradiation was decreased by treatment with anisaldehyde, cinnamaldehyde, coumarin, or vanillin during the expression time. These decreases were not due to cytotoxic effects on cellular growth or killing effects on damaged cells. An in vivo antimutagenic effect of vanillin was also investigated in the mouse spot test using male PW and female C57BL/10 mice. Female mice were injected intraperitoneally with ethylnitrosourea (ENU) on the 10th day of pregnancy and received 3 oral administrations of vanillin. The frequency of recessive carrier offspring induced by ENU was decreased by the post-administration of vanillin. These results indicate that the flavorings act as bio-antimutagens in mammalian cells, both in vitro and in vivo.

18

Ishii, Y., Y. Samejima, F. Saji and T. Nomura, Faculty of Medicine, Osaka University, Kita-ku, Osaka 530 (Japan) Effect of alcohol sulfate and linear alkylbenzene sulfonate on the development of fertilized eggs of the mouse

Eggs from B6C3F1 female mice, which were fertilized in vitro with sperm from C3101F~ male mice, were treated with alcohol sulfate (AS) and linear alkylbenzene sulfonate (LAS), major ingredients of cleansers, for 1 h at the pronuclear

365 stage, and then cultivated for 5 days. Eggs treated with AS or LAS at concentrations of < 0.025% developed well up to the blastocyst stage, as did the untreated eggs. At concentrations of AS or LAS higher than 0.03%, no eggs developed beyond the single-cell stage. There appeared to be a threshold concentration of AS or LAS for the development of the mouse egg, between the concentrations of 0.025% and 0.03%. When AS or LAS was added to the culture medium during the cultivation of fertilized eggs for 5 days, there also appeared to be a threshold concentration, between 0.01% and 0.025%. The results support our previous observations that AS and LAS can interrupt pregnancy in mice by killing fertilized eggs.

contrast, (2) gave a linear dose response up to 270 m 3. Although the numbers of revertant colonies found were about 2 times greater with (1) than with (2) in the range lower than 27 m 3, 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. These phenomena were reproducible using other particulate samples. Furthermore, in some preparations of (1), cytotoxic effects were observed, whereas no cytotoxicity was detected in preparations of (2). The antimutagenic factors present in (3) were capable of inhibiting the mutagenicity of benzo[a]pyrene and of 2-nitrofluorene.

19

20 Iwado, H., M. Naito and H. Hayatsu 1, Okayama Prefectural Research Center of Environmental and Public Health, Uchio, Okayama 701-02, and I Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700 (Japan)

Mutagenicity and antimutagenicity in air-borne particulates Air-borne particulates collected by a highvolume 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 dimethyl sulfoxide and the solution was divided into 2 portions. One was subjected as such to the Ames test for 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 in the Ames test. We found that (1) showed mutagenicity towards S. typhimurium TA98 in the presence of $9 mix in a dose-responding linearity up to 27 m 3 air volume equivalent, and that the colony number stayed constant in the dose range of 27-270 m 3. In

Kamiya, A., Y. Ose and T. Sato, Department of Public Health, Gifu Pharmaceutical University, 5-6-1, Mitahora-higashi, Gifu City (Japan)

Detection of 1,6-dinitropyrene in municipal incinerator emissions It has been observed that there are direct-acting mutagens for S. typhimurium TA98 in the emission gas from municipal incinerators, and a mutagenic component has been identified as 1,6-dinitropyrene (1,6-DNP) by capillary gas chromatography with a nitrogen-selective detector. 0.044/~g/1 of 1,6-DNP was detected in extracts of batch-type combustion gases. No 1,6-DNP was detectable in continuous-type combustion gases. The potency of the mutagenic extract can be accounted for by the 1,6-DNP in the extract. The formation of 1,6-DNP and 1-nitropyrene (1-NP) by photochemical reaction of pyrene with NO 2 gas was studied. It was found that 1-NP is readily formed from pyrene and NO2, and that the formation does not require irradiation with light. In contrast, the formation of 1,6-DNP from pyrene and NO 2 does require irradiation with light, as well as high temperature and a catalytic amount of nitric acid.

366 21 Karube, T., Y. Odagiri, K. Takemoto and S. Watanabe ~, Department of Public Health, Saitama Medical College, Moro, Irumagun, Saitama 350-04, and 1 Epidemiology Division, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuoku, Tokyo 104 (Japan) Analyses of transplacentally induced sister-chromatid exchanges and micronuclei in mouse fetal liver cells. Effect of maternal exposure to cigarette smoke

ment of hepatocellular carcinoma. However, unlike the majority of chemical carcinogens, none of these drugs interacts with DNA to form adducts. In this presentation we show a significant increase in the formation of 8-hydroxydeoxyguanosine (8OH-dG) in rat liver DNA following chronic administration of ciprofibrate, a potent carcinogenic peroxisome proliferator, but not after a single large dose. These results clearly demonstrate, for the first time, that persistent proliferation of peroxisomes leads to specific oxidative DNA damage.

23 The effect on the fetus of maternal exposure to cigarette smoke was studied by analyses of sister-chromatid exchange (SCE) and formation of micronuclei in fetal liver cells of ICR/Jcl mice at the 16th day of gestation. Mice were exposed to mainstream and sidestream smoke. Each stream was used for 2 exposure schedules: one short-term and one long-term. The number of SCEs in fetal river cells was significantly increased in all exposed groups. In the mainstream experiments, the long-term exposure group showed a significant increase in the number of SCEs in comparison to that in the short-term exposure group. Exposure to sidestream smoke increased the number of SCEs more strongly than exposure to mainstream smoke. On the other hand, no significant changes were observed in the micronucleus test.

22

Kasai, H., Y. Okada, S. Nishimura, M.S. Rao 1 and J.K. Reddy 1, Biology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104 (Japan) and a Department of Pathology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611 (U.S.A.) Formation of 8-hydroxydeoxyguanosine in liver DNA of rats following long-term exposure to a peroxisome proliferator, ciprofibrate

Long-term administration of peroxisome proliferators to rats and mice results in the develop-

Kato, F., A. Araki, K. Nozaki and T. Matsushima 1, Japan Bioassay Laboratory, Hatano 257, and 1Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108 (Japan) Mutagenicity of aldehydes and diketones

The mutagenicity of 16 structurally related aldehydes and 3 diketones was tested in Salmonella typhimurium TA98, TA100, TA104 and Escherichia coli WP2uorA/pKMI01 by the preincubation assay (37 ° C, 20 min) with or without metaboric activation. The compounds which gave positive results were as follows (chemical n a m e / bacterial strain/$9 mix ( - or + )/specific activity of test compounds (rev.//~g)): formaldehyde/ 98/ - (+)/3.08(1.33), 100/ - (+)/10.0(9.49), 1 0 4 / - (+)/50.1(35.3), W P / - (+)/1.24(3.05); m e t h a c r o l e i n / 9 8 / - / 0 . 0 3 6 8 , 1 0 0 / - (+)/0.390 (0.341), 1 0 4 / + / 1 1 . 1 , W P / + / 0 . 1 0 9 ; pyruvica l d e h y d e / 9 8 / - (+)/1.20(0.568), 1 0 0 / (+)/58.1(10.9), 1 0 4 / - (+)/44.1(19.4), W P / ( + )/35.5(14.3); g l y o x a l / 9 8 / - ( + )/0.218(0.184), 1 0 0 / - (+)/4.29(3.11), 1 0 4 / - ( + )/3.63(4.51), W P / - (+)/1.17(3.77); crotonaldehyde/100/ - (+)/1.84(1.36), 1 0 4 / - (+)/7.01(1.09), W P / - (+)/0.166(0.564); diacetyl/100/ + / 0.204, 1 0 4 / - (+)/0.987(1.79), W P / + / 0 . 3 5 1 ; acetyla c e t o n e / 1 0 4 / - / 2 . 2 5 , W P / + / 7 . 7 3 ; acrolein/ 9 8 / - / 7 9 2 . trans-2-Hexenal was suspected to be positive. The following compounds showed no mutagenicity: acetaldehyde, propionaldehyde, phenylacetaldehyde, cyclohexanecarboxaldehyde,

367 n:capronaldehyde, trans-cinnamaldehyde, a-methylcinnamaldehyde, 2-ethylcrotonaldehyde, furfural and acetonylacetone.

24 Kinae, N., M. Yamashita, T. Ozawa 1, K. Mataki and I. Tomita l, Food and Nutritional Sciences and ~ Pharmaceutical Sciences, University of Shizuoka, Yada, Shizuoka 422 (Japan) Mutagenicity of the reaction products from D-glucose and bovine serum albumin A mixture of D-glucose and bovine serum albumin (10-100:1) was dissolved in phosphatebuffered saline and the solution was kept at 37 °C for 2 months. At appropriate times, aliquots of the reaction mixture were taken and the intensity of the color (A, 420 nm) and the fluorescence (Ex 370 nm, Em 440 nm) were measured. After dialysis of the reaction solution, which had been kept for 2 months, against distilled water, the non-dialyzable fraction was lyophilized and then subjected to the Ames test using S. typhimurium TA100 without metabolic activation. The mutagenic activity of the lyophilisate was weak (450 rev./10 mg), but it increased with an increase in the molar ratio of D-glucose in the original solution. The lyophilisate was hydrolyzed with HC1 and extracted with ethylacetate after alkalization. When the solvent was evaporated, a yellow fluorescent compound was isolated as crystals. The compound induced 281 rev./2 mg, but its structure is unknown.

25 Kong, Z.-L., K. Shinohara 1, H. Murakami, M. Nonaka and H. Omura, Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, Fukuoka 812, and 1 National Food Research Institute, 2-1-2 Kannondai, Tsukuba, Ibaraki 305 (Japan)

Inhibitory effects of melanoidins on the nitroblue tetrazolium (NBT)-reducing ability of murine peritoneal macrophages treated with phorboi myristate acetate Melanoidins, a mixture of polymeric pigments having reductone structures, are produced in the last stage of Maillard reactions, and are abundantly present in foods. These compounds are also considered to be physiologically important. We studied the action of melanoidins on the: NBT-reducing ability of mouse peritoneal macrophages that had been treated with a tumor promoter, phorbol myristate acetate (PMA). The mixtures of sugars (1 M) such as glucose or xylose, and amino acids (1 M) such as monosodium glutamate (MSG) or glycine were heated in the presence of sodium bicarbonate (0.25 M) at 100 ° C. Melanoidins were prepared from the mixtures. Macrophages exuded from the peritoneal cavity of mice were treated with PMA (100 ng/ml) and melanoidins simultaneously at 37 °C for 1 h in a CO 2 incubator, and then the NBT-reducing ability was measured. The number of cells out of 150 having formazan deposits was scored. NBT-reducing ability of macrophages was enhanced by PMA. Melanoidins prepared from the mixture of xylose and MSG or glycine decreased the NBT-reducing ability of macrophages treated with PMA, suggesting that the melanoidins inhibited the activity of PMA. The molecular weight of the effective melanoidins was about 16 000. No significant inhibitory effect on NBT-reducing ability was found in the melanoidins prepared from the mixture of glucose and MSG or glycine.

26 Matsui, M., K. Matsui, T. Nohmi, H. Mizusawa and M. Ishidate Jr., Division of Mutagenesis, National Institute of Hygienic Sciences, Setagaya-ku, Tokyo 158 (Japan) Detection of deletion mutations in pSV2-gpt plasmids induced by metabolically activated steviol Steviol is the aglycone of stevioside, which is a non-caloric sugar substitute commonly used in

368 Japan. We and others have demonstrated that steviol is mutagenic after metabolic activation in the forward-mutation assay using Salmonella typhimurium TM677, whereas it is non-mutagenic in the reverse-mutation assay using S. typhimurium TA100, TA98, TA102 and TA97. There is a possibility, therefore, that the activated steviol selectively induces a deletion in the xgprt gene of TM677. We have explored this possibility using pSV2-gpt plasmids carrying the xgprt gene. The plasmids were treated with steviol in the presence of $9 mix, and the DNA of xgprt mutants was analyzed by polyacrylamide gel electrophoresis following digestion with restriction endonucleases Sau3AI, HhaI and HpaII. The frequency of mutations in this system showed a 5-fold increase in the group treated by steviol with $9 mix as compared to the controls without steviol. 7 xgprt mutants in pSV2-gpt were obtained, and all these mutants showed deletions of various sizes ranging from 20 bp to 2 kb. These results suggest that the metabolically activated steviol induces deletion in chromosomal genes of S. typhimurium.

27 Matsumoto, K., Y.F. Sasaki, T. Ohta and Y. Shirasu, Institute of Environmental Toxicology, Kodaira, Tokyo 187 (Japan) Suppressing effects of tannic acid on chromosome aberrations induced by mitomycin C in mouse bone marrow cells

It has been reported that tannic acid suppresses the induction of mutagenesis in E. coli and causes a reduction in the frequency of chromosome aberrations in cultured Chinese hamster cells. In this study, we examined the anticlastogenic effect of tannic acid by means of in vivo chromosome aberration test. To find out the most effective period for tannic acid treatment, tannic acid (500 mg/kg) was given orally to mice at various intervals before and after mitomycin C (MMC, 2 mg/kg) intraperitoneal injection. When tannic acid was given to mice 6 h prior to the MMC injection, the most marked decrease in the frequency of aberrant cells, from 40 to 22.1%, was

observed. However, the post-treatment with tannic acid or simultaneous administration of tannic acid and MMC caused no effect on the frequencies of aberrant cells. This suppressing effect of tannic acid was dose-dependent up to 500 mg/kg. In a time-course study, tannic acid was given to mice 6 h before the MMC injection and the bone marrow cells were sampled at 6-42 h after the administration of MMC. The fact that induction of aberrant cells by MMC was reduced by the administration of tannic acid at any sampling time indicates that this effect of tannic acid never reflects the delay in the cell cycle of the bone marrow cells due to the toxicity of tannic acid. These results suggest that tannic acid acts as an anticlastogenic factor in vivo.

28

Matsuoka, A., N. Miyata 1, T. Sofuni and M. Ishidate Jr., Division of Mutagenesis and 1 Division of Synthetic Chemistry, National Institute of Hygienic Sciences, Setagaya-ku, Tokyo 158 (Japan) Clastogenicity of BHA and its metabolites in cultured mammalian cells

Chromosome aberration tests were carried out on 3-tert-butyl-4-hydroxyanisole (BHA) and 6 metabolites using a Chinese hamster lung fibroblast cell line, CHL. BHA induced chromosomal aberrations (11% at 0.02 m g / m l ) only in the presence of $9 mix. Of the 6 metabolites, 3-tert-butyl4,5-dihydroxyanisole (BHA-OH; 25% at 0.02 mg/ml), 3-tert-butyl-4,5-quinone anisole (BHA-oQ; 24% at 0.02 m g / m l ) and 3-tert-butylquinone oxide (BQO; 11% at 0.002 m g / m l ) induced chromosomal aberrations only in the absence of $9 mix, but tert-butylhydroquinone (BHQ; 19% at 0.04 m g / m l ) and tert-butylquinone (BQ; 11% at 0.02 m g / m l ) did so only in the presence of $9 mix. 2,2'-Dihydroxy-3,3'-di-tert-butyl-5,5'-dimethoxy diphenyl (diBHA) showed neither cytotoxicity nor clastogenicity even at the highest dose of 0.2 m g / m l (maximum solubility) with and without $9 mix. BHA and its metabolites (except diBHA) were clastogenic to C H L cells, although their cytotoxic effects were found at relatively low

369 dose levels. The present results suggest that the clastogenicity of BHA might be due to BHA-OH, BHA-o-Q a n d / o r BQO.

29 Miura, K., and K. Morimoto, Department of Hygiene and Preventive Medicine, School of Medicine, Osaka University, Nakanoshima, Kita-ku, Osaka 530 (Japan) Effects of liquid holding on sister-chromatid exchange frequencies in human lymphocytes Human peripheral blood lymphocytes were treated with the bifunctional agent mitomycin C (5 >( 10 -7 M) for 3 h at 37°C. After removal of the drug with washing, the cells were stored in a complete culture medium without addition of phytohemagglutinin for up to 72 h at temperatures of 4, 15, 32 and 37°C, and then cultured for 72 h according to the usual microculture method for detecting sister-chromatid exchanges (SCEs). As a control, mitomycin C-treated, but unstored, cells were assayed. The results showed that the storage in culture medium had no significant influence on SCE frequencies, suggesting the apparent absence of repair of SCE-causing DNA damage in mitomycin C-treated cells, at least up to 72 h.

30 Nakatsuka, S., S. Arimoto 1 and M. Namba 2 Departments of Urology and 2 Pathology, Kawasaki Medical School, Kurashiki-City, Okayama 701-01, and 1 Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700 (Japan) Tumor-promoting effects of Fe3+-nitrilotriacetic acid There are reports that Fe3+-nitrilotriacetic acid (Fe3+-NTA) causes renal cell carcinoma in rats and that the carcinogenesis of Fe3+-NTA might be mediated by active oxygen. We examined the biological effects of Fe3+-NTA in more detail.

The treatment of V79 cells with 100 /~g/ml of Fe3+-NTA caused marked cytolysis. Its cytotoxicity was partially inhibited by the presence of superoxide dismutase (SOD) and catalase. No significant induction of chromosomal aberrations or mutations ( 6 T G S ~ 6 T G R) was observed on treatment of V79 cells with Fe3+-NTA. No gene reversions were induced by Fe3+-NTA in 8 strains of Salmonella typhimurium (TA97, TA98, TA102, TA1537, TA100/1,8-DNP6, TA100NR and TA102) With or without H202. The assay of the inhibition of metabolic cooperation demonstrated that Fe3+-NTA inhibited the decrease of the cloning efficiency of 6TG R V79 cells co-cultured with a large number of 6TG s cells. This inhil~ition was dose-dependent and partially diminished by SOD and catalase. The present results suggest that the carcinogenic effects of Fe3+-NTA depend on its promoter action and that the active oxygens generated by Fe3+-NTA participate in the promotion of carcinogenesis.

31 Nishifuji, K., T. Kinouchi and Y. Ohnishi, Department of Bacteriology, School of Medicine, The University of Tokushima, Tokushima 770 (Japan) Metabolism of glutathione conjugates of 1-nitropyrene oxides in the intestinal tract When 1-nitropyrene (1-NP) was administered to rats, oxidatively activated metabolites, 1-NP oxides, were excreted into the bile as glutathione conjugates. Therefore we investigated the metabolism of glutathione conjugates in the intestinal tract. Enzymatically synthesized glutathione conjugates of 1-NP 4,5- or 9,10-oxide (4,5- or 9,10GSH) (29/~M) were incubated anaerobically in 50 mM phosphate buffer (pH 7.4) with cell-free extracts from intestinal contents (I.C.), intestinal bacteria and intestinal mucosa at 37 °C and then analyzed by HPLC. More than 85% of the 4,5-GSH and 9,10-GSH was degraded by I.C. after 24 h incubation. Of the anaerobes tested, Fusobacterium nucleatum metabolized 83.5% of the 4,5GSH and 32% of the 9,10-GSH within 20 h, but major components of the intestinal microflora,

370

such as Bacteroides, Eubacterium, Bifidobacterium and Peptostreptococcus had very weak metabolic activity. On the other hand, among the aerobes, Proteus mirabilis, Klebsiella pneumoniae and Pseudomonas aeruginosa showed strong activity. The main metabolite showed a retention time identical to that of cysteinylglycine conjugates of the 1-NP oxides, which were prepared by treatment with 3,-glutamyltranspeptidase (TGTP). The mucosa of the small intestine also metabolized 18-59% of the glutathione conjugates within 20 h, and 2 metabolites appeared. One was the same as the bacterial metabolite and the other had the same retention time as that of cysteine conjugates of 1-NP oxides, which were made by treatment with both ~,GTP and aminopeptidase M. These results indicate that 1-NP oxide-GSHs are degraded within the intestinal tract by enzymes from the intestinal mucosa and intestinal bacteria.

32 Nozaka, T., F. Watanabe, M. Ishino, I. Morimoto, J. Kunitomo 1, H. Ishii 2 and S. Natori 3, Saitama Institute of Public Health, Urawa, Saitama 338, 1 Faculty of Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya 663, 2 Faculty of Pharmaceutical Sciences, Chiba University, Yayoi, Chiba 260 and s Meiji College of Pharmacy, Yato, Tanashi 188 (Japan) Mutagenicity of isoquinoline alkaloids

43 isoquinoline alkaloids were assayed for mutagenicity by Ames' method with the preincubation technique using Salmonella typhimurium strains TA98 and TA100 with and without the addition of $9. The alkaloids included: (a) morphine type; (b) hasubanan type; (c) tetra(hexa)hydroisoquinoline type; (d) benzylisoquinoline type; (e) bisbenzylisoquinoline type; (f) berberine type; (g) aporphine type; (h) monoterpene isoquinoline type; and (i) benzo[c]phenanthridine type compounds. Three compounds (berberine, chelerythrine chloride, 5,6,10-trimethoxy-7Hdibenzo[de, h]quinolin-7-one) showed weak mutagenicity when tested without $9 on TA98. With $9, papaverine, steporphine, dicentrine, O-methyl-

domesticine, ushinsunine, O-nornuciferine, roemerine, dehydrocrebanine, N-demethyl-Nformyldehydronuciferine, liriodenine, lysicamine, 9-methoxy-l,2-methylenedioxy-7-oxoaporphine, 1,2,9-trimethoxy-7-oxoaporphine, 5,6,9-trimethoxy-7H-dibenzo[ de, h ]-quinolin-7-one and oxychelerythrine were mutagenic for TA100, and 13 of the 43 alkaloids were mutagenic for TA98. In the assay on TA100 with $9 mix, liriodenine was the most potent mutagen among these alkaloids, and on TA98 with $9 mix roemerine was the most potent. It seems to be essential for the potent mutagenicity of aporphine in TA100 with $9 that the 10,11 positions of the aporphine nucleus are not substituted.

33 Ochiai, M., N. Nitta, H. Shima, P.J. Wirth, M. Suzuki, S. Nagase 1, T. Sugimura and M. Nagao, Carcinogenesis Division, National Cancer Center Research Institute, Tsukiji, Tokyo 104 and 1Sasaki Institute, Kandasurugadai, Tokyo 101 (Japan) In vivo mammalian cell mutation assay using albumin + as a marker in analbuminemic rats

Analbuminemic rats (NARs) were administered diets containing 0.06% 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB), 0.05% Glu-P-1 or 0.04% MelQx. After 1-30 weeks, albumin-producing hepatocytes were detected by immunohistochemical analysis. Most of the albumin-positive (alb +) foci were composed of single cells, but some were composed of multiple cells. In male rats, 3'-MeDAB, Glu-P-1 and MelQx induced a similar number of alb ÷ foci, i.e., about 15/103 hepatocytes after 19 weeks, though only 3'MeDAB induced liver cancer. These 3 mutagens, however, showed different activities in the production of alb + clusters, which are composed of more than 20 cells. 3'-MeDAB, Glu-P-1 and MelQx induced 4.3, 1.3 and 0 clusters/10 s hepatocytes, respectively. Glutathione-S-transferase-P (GST-P) foci were induced by these 3 chemicals in the order 3'-MeDAB > GIu-P-1 > MelQx. Female NARs were less sensitive to 3'-MeDAB hepato-

371 carcinogenesis although they developed a similar number of alb ÷ foci to male NAILs. Female NARs, however, developed far fewer alb ÷ clusters than the males. Analysis of features of the alb + foci induced by hepatocarcinogens should give information on the initiating and promoting activities of hepatocarcinogens.

34 Ohara, Y., S. Arimoto, T. Hayatsu and H. Hayatsu, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700 (Japan) Classification of 62 compounds according to their ability to adsorb to blue cotton and blue rayon Blue cotton and blue rayon have been used to adsorb mutagens in the environment. Our earlier studies indicated that this adsorption was selective for polycyclic structures. We have now increased the number of compounds examined for their ability to adsorb to blue cotton and blue rayon. Of the 40 compounds with 3 or more fused rings, 36 showed strong (> 50% under specified conditions), 3 medium (30-50%) and 1 no adsorption. The exceptional, unadsorbed single compound was 1,N6-ethenoadenosine, which is known to have a structural anomaly. Of the 12 2-ring compounds, 3 were medium adsorbers and 7 were poorly adsorbed (< 10%); but 2 compounds, PhlP and quercetin, were strongly adsorbed, probably due to the fact that the 2-ring and 1-ring systems in these compounds are conjugated and as a result the coplanar sizes of these compounds are comparable to a 3-ring system. 7 1-ring compounds and 3 no-ring compounds tested were all medium or poor adsorbers. Among them, fecapentaene-12 was a medium (20%) adsorber. These results have confirmed that blue cotton and blue rayon can adsorb polycyclic compounds in a selective manner.

35 Ohshima, H., M. Friesen, C. Malaveille, I. Brouet, A. Hautefeuille and H. Bartsch, International Agency for Research on Cancer, Lyon (France)

Formation of direct-acting genotoxic substances by the reaction of smoked foods with nitrite Consumption of smoked food products has been epidemiologically associated with an increased risk of stomach cancer. In the present study, the reaction of such smoked foods with nitrite under acidic conditions was shown to produce potent direct-acting genotoxic substances as detected by the SOS Chromotest. Since similar genotoxic activity was observed in nitrosated samples of various wood-smoke condensates, precursors of the genotoxic substances were isolated and identified in commercial hickory smoke condensate. The precursors were extractable with ethyl acetate under neutral conditions, but did not adsorb to blue cotton. Various simple phenolic compounds were identified by GC-MS in HPLC fractions showing genotoxicity after nitrosation. 27 out of 70 different phenols present in wood-smoke condensate and their related compounds exhibited directacting genotoxicity after nitrosation. On the basis of the concentrations of these phenolic compounds and their genotoxicity after nitrosation, phenol, 3-methoxycatechol, vanillin and hydroquinone were found to contribute mainly to the genotoxicity of nitrosated wood-smoke condensate. The TLC and GC-MS analyses of azocoupling products formed with N-ethyl-naphthylamine revealed that nitrosation of smoke condensates generates various diazonium compounds, including diazoquinones, which largely accounts for the genotoxicity of nitrosated smoked foods.

36 Ohuchida, A., A. Furukawa and R. Yoshida, Drug Safety Laboratory, Taiho Pharmaceutical Co., Kawauchi, Tokushima 771-01 (Japan) Micronucleus test of polyploidy inducers Micronucleus tests of polyploidy inducers were carried out in order to prove a relationship between the frequency of numerical aberrations (polyploidy) in the in vitro chromosomal aberration test and the frequency of micronucleated polychromatic erythrocytes (MNPCEs) compared

372 to polychromatic erythrocytes (PCEs). Five chemicals, i.e., ethyl vanillin, diethylstilbestrol, pnitrotoluene, noscapine, and thiabendazole, were selected as specific in vitro polyploidy inducers (Ishidate, 1983), none of which are thought to be spindle poisons. They were suspended in olive oil, and were injected intraperitoneally into male BDF 1 mice (8 weeks old, 23.3-28.5 g). To comprehend the overall trend of frequency of MNPCEs and to determine the doses and the treatment time in the main test, pilot tests, using 2 animals per group, were carried out at 4-5 different dosages of each chemical. The animals were killed at 24 h, 48 h and 72 h after administration. The frequencies of MNPCEs varied from 0 to 0.4%, depending on the dose and sampling time. The main tests, using 5 animals per group, were carried out with a treatment time of 24 h. The frequencies of MNPCEs in the test chemicals showed no increase at any of the doses tested. The inducers of polyploidy, which are not spindle poisons, did not induce MNPCEs.

37 Ohyama K., R. Endo and H. Kawahara, Tokyo Metropolitan Research Institute for Environmental Protection, Koto-ku, Tokyo 136 (Japan)

Mutagenicity of surface soil There is a possibility that contamination of surface soil indirectly reflects air pollution. Six soil samples (black soil, 2 kinds of manured soils, red ball clay, Kanuma clay and leaf mold), collected from roadside verges, were extracted and the extracts were tested for mutagenicity in the Ames Salmonella assay. In addition, mixtures of 2-nitrofluorene (2-NF) or benzo[a]pyrene (BaP) with the soil extracts were investigated for recovery rate of mutagenicity in order to evaluate the modulating effects of the extracts. The mutagenic activities of the extracts dissolved in dimethyl sulfoxide were tested by the preincubation method, using Salmonella typhimurium TA100, TA98 (with or without $9 mix), TA98NR and T A 9 8 / 1 , 8 D N P 6 (without $9 mix). The mutagenicity of the soil samples was low in TA98 without $9 mix, but high in TA98NR and TA100 without $9

mix. There was a difference in the mutagenicity recovery for 2-NF or BaP among the mixtures. Mutagenic activities of the mixtures of 2-NF with the soil extracts were high in TA98 without $9 mix but low in TA98NR.

38 Okuda, H., S. Yoshioka and T. Watabe, Tokyo College of Pharmacy, 1432-1 Horinouchi, Hachioji-shi, Tokyo 192-03 (Japan)

Sulfotransferase-mediated activation of 9-hydroxymethyl-lO-methylanthracene, a major metabolite of the carcinogen 9,10-dimethylanthracene 9-Hydroxymethyl-10-methylanthracene (9HMA), a major oxidative metabolite of the carcinogen 9,10-dimethylanthracene in rat liver, induced His + reverse mutation in Salmonella typhimurium TA98 to a marked extent in the presence of dialyzed rat liver cytosol ($105) fortified with 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and to a small extent in the presence of the hepatic post-mitochondrial fraction fortified with NADPH. Incubation of 9-HMA with the PAPS-S105 system in 0.1 M phosphate buffer, pH 7.4, gave 9-HMA sulfate and 9-HMA phosphate, which were isolated and identified with synthetic specimens. 9-HMA sulfate, which showed potent intrinsic mutagenicity toward TA98 in the phosphate buffer, decomposed rapidly in the same buffer to the parent alcohol and 9-HMA phosphate in a ratio of 4 : 1 . 9-HMA phosphate appearing in the activation system was assumed to be formed non-enzymatically from 9-HMA sulfate by nucleophilic attack of the phosphate anion on the sulfate ester. The rate of sulfation of 9-HMA was estimated as 90 p m o l e / m g p r o t e i n / m i n from the 9-HMA-dependent formation of 3'-phosphoadenosine 5'-phosphate from PAPS in the activation system. 9-HMA phosphate was also found to be as mutagenic toward TA98 as 9-HMA sulfate. These results indicate that 9-HMA is activated to the intrinsic mutagen 9-HMA sulfate by the PAPS-S105 system, although a proportion of the apparent mutagenicity induced by the metabolite 9-HMA sulfate found in the phosphate buffer is

373 due to its decomposition product, 9-HMA phosphate.

39 Sai, K., A. Takagi, T. Umemura, Y. Kurokawa and H. Kasai 1, Division of Toxicology, National Institute of Hygienic Science, Setagaya-ku, Tokyo 158 and ~ Biology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104 (Japan) Detection of 8-hydroxydeoxyguanosine in rat kidneys treated with renal carcinogens

Recently, it was demonstrated that active oxygen species can produce 8-hydroxydeoxyguanosine (8-OH-dG) on reaction with DNA and its components. The production of 8-OH-dG is considered to be closely related to carcinogenic mechanisms of the oxygen species. Indeed, after oral administration of potassium bromate (KBrO3), which is an oxidizing agent and a renal carcinogen, an increase in 8-OH-dG levels was observed in rat kidneys. Therefore, it seemed interesting to examine whether 8-OH-dG production can be observed in rat kidneys treated with various renal carcinogens. We chose 3 mutagenic compounds (dimethylnitrosamine, ethylhydroxyethylnitrosamine and lead acetate) and 9 non-mutagenic compounds (p-dichlorobenzene (p-DCB), chloroform, bis-(2,3-dibromopropyl)phosphate, tris-(2-chloroethyl)phosphate, dioxane, decalin, ferric nitrilotriacetate (Fe-NTA), trisodium nitrilotriacetate and 2,2,4-trimethylpentane (TMP)). Rats (F344, male, 6 weeks old) were given these carcinogens by a single intragastric administration at a dose of approx, half LDs0 values. Kidneys were extirpated 12, 24 and 48 h after treatment, and the levels of 8-OH-dG in DNA were measured. A tendency for 8-OH-dG levels to increase was observed on administration of p-DCB, Fe-NTA and TMP. Furthermore, a significant increase was evident on intraperitoneal treatment of male Wistar rats with Fe-NTA.

40 Sakagami, Y., H. Yamazaki, N. Ogasawara, H. Yokoyama, Y. Ose 1 and T. Sato 1, Osaka Prefectural Institute of Public Health, Nakamichi, Higashinari-ku, Osaka 537, and 1 Gifu Pharmaceutical University, Mitahora-higashi, Gifu 502 (Japan)

Evaluation of genotoxic activities of disinfectants and their metabolites by the u m u test

The genotoxic potential of 6 disinfectants and their 9 metabolites was investigated by use of the umu test. Glutaraldehyde showed positive genotoxicity, independent of the metabolic activation system, and acrinol was positive only in the presence of $9 mixture. Alkyldiaminoethylglycine hydrochloride, benzalkonium chloride, chlorhexidine digluconate and methylrosaniline chloride were negative in the presence or absence of $9 mixture. In some metabolites of benzalkonium chloride, chlorhexidine digluconate or glutaraldehyde, only pyrogallol showed direct genotoxicity. Aniline, pchloroacetoanilide, p-chloroaniline, p-chlorophenol, decabutyldimethylamine, glutaric acid, phenol and pyrocatechol did not induce u m u gene expression. The relative genotoxic potency was estimated to be glutaraldehyde > acrinol > pyrogallol on the basis of dose-response curves. This evaluation agrees with that previously reported on the basis of the liquid rec assay. The u m u test, in combination with the liquid rec assay a n d / o r the Ames test, is suitable for detecting genotoxicity of these classes of disinfectants, which have killing effects.

41

Sasagawa, C., Y. Kodama and T. Matsushima, Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Tokyo 108 (Japan)

374 Formation of mutagenic diazo compounds on treatment of derivatives of tyramine and bamethan with nitrite

Tyramine and bamethan are reported to be converted to mutagenic diazo compounds by treatment with nitrite in acidic conditions. 11 tyramine analogues and 6 bamethan analogues at a concentration of 50 mM were treated with 500 mM nitrite at pH 3 for 1 h at 37 o C, and then the mutagenicities of the products on Salmonella typhimurium TA98 and TA100 were tested by preincubation assay (30°C, 30 min) with and without $9 mix. The compounds that showed mutagenicity were as follows (chemical name, specific mutagenic activity (revertants per #mole) on TA98 and TA100, without and with $9 mix, respectively): tyramine, 4830, 5820, 9120, 9220; tyrosine, 296, 96, 948, 552; 4-coumaric acid, 2290, 1140, 1730, 672; 4-hydroxyphenyl pyruvate, 2160, 224~ 3370, 496; 4-hydroxyphenethyl alcohol, 1820, 2410, 4540, 5580; 2-hydroxyphenethyl alcohol, 1740, 1580, 6080, 11400; 3-hydroxyphenethyl alcohol, 0, 1050, 0, 0; 4-propylphenol, 318, 368, 0, 0; bamethan, 8860, 10200, 11000, 14500; synephrine, 2270, 3340, 6340, 9150; phenylephrine, 1740, 2350, 3940, 5200. The following compounds did not show mutagenicity: 3-(4-hydroxyphenyl)propionic acid, 3-(4-hydroxyphenyl)lactic acid, dopamine, epinephrine, octopamine and et-(methylaminomethyl)benzyl alcohol. The relation between the chemical structures of these compounds and the formation of mutagenic diazo compounds was considered. An amino group in the side chain of tyramine analogues was necessary as a primary amine, but an amino group in the side chain of bamethan analogues was necessary as a secondary amine, o,p-Dihydroxy compounds did not show any mutagenicity after treatment with nitrite in acidic conditions. 42

Sasaki, K., H. Mizusawa 1, M. Ishidate ! and N. Tanaka, Department of Cell Biology, Food and Drug Safety Center, Hatano Research Institute, Hatano, Kanagawa 257, and ~ Division of Mutagenesis, National Institute of Hygienic Sciences, Setagaya-ku, Tokyo 158 (Japan)

A screening system for tumor promoters using v-Ha-ras-transfected BALB 3T3 cells (Bhas 42)

Several studies suggest that activation of ras genes can function as the initiation step in 2-stage carcinogenesis. We cloned v-Ha-ras-transfected BALB 3T3 cells (Bhas 42) by co-transfection with pSV2-neo genes. Bhas 42 cells showed contact inhibition, and showed a transformation-like morphology on treatment with TPA. These results imply that Bhas 42 could be a model for the initiated cells in 2-stage transformation. We plated 100 cells of Bhas 42 together with 10 4 BALB 3T3 cells in a single dish, and then the cells were treated with promoters. At 6 weeks after inoculation, transformed foci were scored. The system had advantages over standard transformation assays, in: (1) objective criterion of foci; (2) no variation with serum lots; and (3) high reproducibility of transformation frequency. This study shows that the Bhas 42 transformation assay system is useful for screening promoters.

43 Sawada, M., T. Sofuni and M. lshidate Jr., Division of Mutagenesis, National Institute of Hygienic Sciences, Setagaya-ku, Tokyo 158 (Japan) Induction of chromosomal aberrations in active oxygen-generating systems, VI. Selection and use of menadione-resistant cells in culture

Cells with elevated activities of active oxygen scavenging enzymes are very useful for investigating the mechanism of cytotoxicity and mutagenicity of oxygen radicals in cultured mammalian cells. Previously, we selected Chinese hamster cells (CHL) with an elevated catalase activity by repeated treatments with H202 (Mutation Res., 197 (1988) 133). In the present study, we tried to obtain cells with increased activity of superoxide dismutase (SOD) by repeated treatments with menadione. The resistance of CHL cells to menadlone was slowly elevated by the treatment with menadione at stepwise-increasing concentrations. The LCs0 of the resistant cells (MN cells) reached approximately twice that of the parental cells.

375 Both SOD and catalase activities of the MN cells were also 1.5-2 times higher than those of parental cells. With regard to the induction of chromosomal aberrations, MN cells were about twice as resistant to menadione, and about 1.5 times more resistant to H202. However, no differences in the frequencies of aberrations were observed between these cells when they were treated with adriamycin, bleomycin or soft X-ray.

44

Sawada, S. 1,2, C. Furihata 1 and T. Matsushima 1, 1 Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108, and 2 Department of Drug Safety Research, Eisai Co., Kawashima-cho, Hashima, Gifu 483 (Japan) In vivo short-term assay of unscheduled D N A synthesis ( U D S ) in rat liver

An in vivo short-term assay of repair and replication of rat liver DNA was developed by which possible hepatocarcinogens could be identified in a few days. F344 rats were treated orally with carcinogens and their hepatocytes were isolated by an in situ 2-step collagenase perfusion technique and incubated with [3H]dThd with or without hydroxyurea (HU), which inhibits DNA replication. Then the DNA was extracted and the incorporation of [3H]dThd into nuclear DNA was determined by radioactivity measurement. UDS induced by N-nitroso-dimethylamine at doses of 2.5-10 mg/kg body weight and 2AAF at doses of 12.5-50 mg/kg body weight could be detected as up to 5.8- and 6.0-fold increases in DNA synthesis in the presence of HU, 2 h and 4 h, respectively, after their administration. Replicative DNA synthesis (RDS) induced by CC14 at a dose of 200 mg/kg could be detected 48 h after its administration as a 23-fold increase in DNA synthesis in the absence of HU. RDS induced by 2AAF could be detected 16 h after 2AAF administration as a 6.8-fold increase in DNA synthesis in the absence of HU. These results show that by simultaneous measurement of the incorporation of [3H]dThd into nuclear DNA in the presence and absence of

HU in the culture medium, UDS and RDS could be clearly distinguished.

45 Sengoku, Y., M. Kamiya, K. Takahashi, K. Kohda and Y. Kawazoe, Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabedori, Mizuho-ku, Nagoya 467 (Japan) Effect of substituents on the mutagenicity and metabolism of quinoline

3-Fluoro, 2-chloro and 3-chloro derivatives of the potently mutagenic quinoline were deficient in mutagenicity in Salmonella typhimurium TA100, whereas all the other fluoro and chloro derivatives examined, including 4-chloroquinoline, were mutagenic. These results suggest that the ultimate structure of genotoxic quinoline may be the 2,3-dihydro-2,3-epoxide of the 1,4-hydrate form of quinoline. This is supported by the finding that, among methyl-substituted quinolines, the mutagenicity of the 2-methyl isomer was weakest and that of the 4-methyl isomer highest. The present study provides support for the view that a fluorine substitution at the site of the oxidation leading to the genotoxic metabolite may deprive the aromatic hydrocarbons of their genotoxicity without affecting other biological properties of the parent hydrocarbons.

46 Sera, N., K. Morita, K. Horikawa, H. Tokiwa, K. Wakabayashi 1 and Y. Ohnishi 2, Fukuoka Environmental Research Center, Fukuoka 818-01, National Cancer Center Research Institute, Tokyo 104, and 2 Department of Bacteriology, School of Medicine, The University of Tokushima, Tokushima 770 (Japan) Binding of mutagens to rice fiber

The binding of mutagens to rice fiber was investigated. Rice fiber was prepared enzymatically from rice bran. Each chemical (1/~g/ml) was

376

mixed with rice fiber (10 m g / m l ) and after incubation at 37 ° C for 30 min, the unbound chemical in the supernatant was quantitated by highperformance liquid chromatography or gas chromatography. The percentage of chemicals not bound to rice fiber was 7-16% for nitrated aromatic compounds, 10-18% for heterocyclic amines, 10-16% for benzo[a]pyrene, 4-nitroquinoline-N-oxide and flavonols, and 6-10% for polychlorinated biphenyls (PCBs). Binding of these chemicals to rice fiber was complete within 10 rain. 1-Nitropyrene (1-NP) and PCB binding to rice fiber were not influenced by change in pH, the presence of various metal ions or preincubation with bacterial cell cultures. In contrast, the binding of heterocyclic amines to rice fiber was pH-dependent. The binding site of the chemicals to rice fiber is probably associated with lignin. Rice fiber may be effective for accelerated excretion of mutagens and carcinogens from the gastrointestinal tract.

47 Shibahara, T., H. Ryo 1, G. Tsushimoto and T. Nomura 1, Otsuka Pharmaceutical Co. Ltd., Tokushima 771-01 and a Department of Fundamental Radiology, Faculty of Medicine, Osaka University, Osaka 530 (Japan) DNA-damaging potency in vivo of hepatocarcinogenic aflatoxins as measured in the Drosophila DNA repair test Aflatoxin B 1, Bz, G 1 or M 1 was administered orally via a medium to a larval Drosophila stock consisting of recombination-deficient ( R e c - ) double mutant mei-9 a mei-4105 males, and Rec ÷ attached-X females at the third-instar stage. The maximum dose was set at 1 tzg/ml for all test compounds. After the adults emerged, the ratio of males to females was determined at each dose level. The sex ratio in each Ba-, G1- and Ma-treated group showed a drastic, dose-dependent decrease from the control ratio. The dose required to produce a R e c - / R e c + ratio of 0.5 was estimated to be 0.02/~g/ml for G1, 0.03/~g/ml for B 1 and 0.14 /~g/ml for M~. No appreciable reduction in the

ratio occurred with B2 under the conditions used. These data correlate well with the data reported for the potency of hepatocarcinogenicity in rats (Butler et al., 1969; Wogan et al., 1974). Hence, the results of the present study lend further support to the notion that the Drosophila D N A repair test may provide a simple in vivo system for predicting relative carcinogenic potency of structural analogues (Fujikawa, 1988).

48 Shibuya, T., T. Murota a, N. Horiya and T. Hara, Hatano Research Institute, Food and Drug Safety Center, Ochiai 729, Hatano, Kanagawa 257, and 1Toxicology Laboratory, Mitsubishi Kasei Co., Midori-ku, Kanagawa 227 (Japan) Induction of recessive mutations by N-ethyl-Nnitrosourea in mouse primordial germ cells Previously, we found that N-ethyl-N-nitrosourea (ENU) can induce recessive mutations in somatic cells and can kill primordial germ cells in mouse embryos (Shibuya et al., 1982). We have now carried out a specific-locus test on mouse primordial germ cells with ENU. Male and female C 3 H / H e mice were mated, and pregnant females were treated with 30 or 50 m g / k g ENU on day 10.5 of pregnancy. The F 1 male mice obtained were grown and mated with tester female PW mice. The mice of the next generation were checked for coat color and ear shape to evaluate whether or not primordial germ cells had been mutated by ENU. The fertility of male mice was 99% and 38%, at 30 and 50 m g / k g ENU, respectively. So far, no mutants have been obtained from 15190 non-treated control male offspring. Three mutants were obtained in the 30 m g / k g group from 5832 offspring; 2 were recovered from the same male, showing a cluster mutation. Fifteen mutants were gained from 2837 offspring in the 50 m g / k g group, and most of those had originated from cluster mutations. All mutated genes thus obtained are viable in homozygous states. This result is similar to that of previous experiments in which the effect of ENU on stem-cell spermatogonia was examined.

377

49 Shimada, H., T. Sato, C. Hattori, S. Satake and S. Itoh, Research Institute of Daiichi Seiyaku Co., Tokyo 134 (Japan) Induction of micronuclei by benzene and its metabolites The relationships between DNA single-strand breaks (SSBs) and micronucleus formation induced by benzene and its metabolites were investigated. Micronucleated polychromatic erythrocytes in the bone marrow cells of male mice increased markedly 42 h after a single oral administration of benzene. SSBs detected by the alkaline elution assay were high at 36 h after the administration of benzene. In the tests for the benzene metabolites, micronuclei were induced by phenol and hydroquinone but not by catechol, hydroxyhydroquinone, 1,4-benzoquinone, 2,2'-biphenol, 4,4'-biphenol or muconic acid. In the in vitro studies using Chinese hamster cells and mouse bone marrow cells, hydroquinone, 1,4-benzoquinone, 2,2'-biphenol and hydroxyhydroquinone induced SSBs, and hydroquinone, catechol and muconic acid induced metaphase arrest as detected by metaphase/anaphase analysis. Moreover, hydroquinone, catechol, hydroxyhydroquinone and 4,4'-biphenol exhibited clastogenicity in in-vitro cytogenetic studies. These results suggest that the induction of miclonuclei by benzene is an event occurring subsequent to the SSBs, and that several metabolites play important roles in inducing micronuclei and myelotoxicity.

50 Shimizu, H., M. Akiyama, Y. Suzuki and K. Hayashi, Department of Public Health, The Jikei University School of Medicine, Minato-ku, Tokyo 105 (Japan) The effects of magnetic field on mutagenic activity Strong static magnetic fields (MF) are used widely in research and in industrial processes, e.g., nuclear magnetic resonance, magnetic resonance

imaging, linear motor car and aluminium production processes. However, the biological effects of MF on humans are not well understood. The aim of this experiment is to explore the effects of strong MF on mutagenesis, using Salmonella tester strain TA98. The static MF used was the FTNMR GS & FX series of the Japan Electron Optical Laboratory. Plate incorporation assays without metabolic activation were performed for 3 mutagens, with exposure of the bacteria-mutagen mixture to MF for 10 min. No effect was observed when TA98 alone was exposed to MF at 0.15, 0.5, 1, 2, 6.34 and 11.75 T. A suppressive effect of MF on the mutagenic activity of AF2 was observed, and this effect was dependent on the MF dose. On the other hand, the mutagenicity of 5nitroacenaphthene was enhanced by MF, and this enhancement was MF-dose-dependent. No such effects of MF were observed on the mutagenicity of 1-nitropyrene.

51 Shioya, M., K. Wakabayashi, T. Sugimura and M. Nagao, Carcinogenesis Division, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo 104 (Japan) Formation of 8-hydroxydeoxyguanosine in DNA treated with coffee 8-Hydroxydeoxyguanosine (8-OH-dG) is formed in DNA by oxygen radical-forming substances. It is known that coffee shows strong direct-acting mutagenicity in S. typhimurium TA104, a strain that had been developed to detect oxidative mutagens, and contains hydrogen peroxide and polyphenols. We therefore examined 8-OH-dG formation in DNA on treatment with coffee. When the samples of 0.01-0.3 mg instant coffee were incubated with 0.5 mg of calf thymus DNA in 1 ml of 0.1 M Tris-HC1 buffer (pH 7.4) at 37°C for 2 h, 8-OH-dG was formed in a dose-dependent manner, reaching levels of 0.93.2/104 dG. 8-OH-dG was also formed with freshly brewed coffee at similar levels to those obtained with instant coffee. The level of 8-OH-dG in non-treated DNA was 0.5/104 dG. Further-

378

more, the formation of 8-OH-dG was suppressed on the addition of catalase by 65%, and with EDTA by 98%. These results indicate that hydrogen peroxide and metal ions are involved in the formation of 8-OH-dG in DNA by coffee.

52 Sofuni, T., A. Matsuoka, M. Sawada, M. Ishidate Jr. and M.D. Shelby 1, National Institute of Hygienic Sciences, Setagaya-ku, Tokyo 158 (Japan), and 1 National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (U.S.A.) A comparison of chromosome aberration induction in the CHO and CHL cell systems by 25 chemicals The present study was conducted to compare the results of chromosome aberration tests using the C H L and CHO cell systems on 25 test chemicals supplied by NTP, U.S.A. In the CHL system, cells were exposed to the test chemical for 24 h and 48 h without $9 mix, and for 6 h with $9 mix. In the CHO system, cells were exposed for 10.5-20.5 h without $9 mix and for 2 h with $9 mix. In the absence of $9 mix, 8 (32%) of the 25 test chemicals showed different results in the 2 test systems: 6 were positive in CHL only, 2 were positive in CHO only. In the presence of $9 mix, 13 (52%) of the chemicals again showed inconsistent results: 9 were positive in CHL only and 4 positive in CHO only. According to the combined results with and without $9 mix, 11 (44%) showed qualitatively inconsistent results between the 2 test systems. One of the reasons for this inconsistency may be the difference in the experimental protocol used. However, the possibility remains that, even using the same experimental protocol, chromosome aberration induction by some chemicals might be qualitatively a n d / o r quantitatively different in the CHL and CHO cell systems.

53 Sugimura, K., S. Iwamoto 1, S. Hashimoto and T. Ohnishi, Nara Medical University, Kashihara,

Nara 634, and 1 Prefectural Institute of Public Health, Ohmori-cho, Nara City, Nara 630 (Japan) UVA radiation and chemicals induced expression in E s c h e r i c h i a c o l i

umu

gene

In Escheriehia coli, an u m u operon plays an important role in inducible mutations, and the gene expression of the operon is inducible by a number of DNA-damaging agents. In the present experiments, we showed that irradiation with UVA in the presence of acetophenone, which is known to produce D N A damage, induced u m u gene expression in E. coli and higher induction was observed in a p h r strain than in the parental strain ( p h r + ) . These results suggest that pyrimidine dimers induce the gene expression. Irradiation with UVA and acetophenone produced preferentially thymine-thymine (TT) dimers among the pyrimidine dimers in DNA molecules, although TT dimers are unable to induce mutations. Hence, we assume that the induction of umu gene expression does not always bring about inducible mutation. We also presume that the induction of mutations depends on the kind of D N A damage produced even after umu gene products have been fully induced.

54 Sutou, S., and S. Sato, Central Research Institute, Itoham Foods Inc., 1-6-21 Mita, Meguro, Tokyo 153 (Japan) Maternal inheritance of the

ms

gene

M S / A e mice, which are mutagen-sensitive in both the dominant lethal test and the micronucleus test, and CD-1 mice, which are the parental strain of M S / A e , were mated in all 4 possible combinations. The male and female offspring were subjected to the micronucleus test using mitomycin C (MMC), colchicine (Col), and 6-mercaptopurine (6-MP) as inducing agents. Col gave equivocal results. However, MMC and 6-MP clearly showed differential responses: both male and female offspring from CD-1 dams had a lower incidence of micronucleated polychromatic eryth-

379

rocytes (MNPCEs) than offspring from M S / A e dams, regardless of the sire strains. In addition, the body weights of offspring from M S / A e dams were lower than those of offspring from CD-1 dams, regardless of the sires. These results strongly suggest that the m s gene (or genes), which regulates the characteristics of MS/Ae, is maternally inherited.

in the actions of vincristine and 5-fluorouracil. The Ca 2+ concentrations in the serum of the mice used and in the culture media decreased proportionately with the increase in the EGTA dose. These results show that the decrease in MPCE is attributable to the inhibition of erythropoiesis by Ca 2+ deficiency. Our present findings suggest that the acceleration of erythropoiesis can result in an increase in mutagen-inducible MPCE.

55 56

Suzuki, Y., S. Toda ~ and H. Shimizu, Department of Public Health, Jikei University School of Medicine, Minato-ku, Tokyo 105, and 1 Mutagenicity Test Division, Sohgo Biochemical Laboratory Inc., Kawagoe, Saitama 305 (Japan) Effect of prostaglandin E z and calcium in the micronucleus test in mice

The aim of this experiment is to investigate a mechanism of micronucleus formation in relation to erythropoiesis. We have reported that acceleration of erythropoiesis increased the frequency of micronuclei (MPCE) inducible by certain mutagens. It is known that prostaglandin E 2 (PGE2), an arachidonate metabolite, may increase the release of renal erythropoietin in vivo, giving rise to acceleration of erythropoiesis. It is possible that PGE 2 pretreatment might increase the frequency of MPCE inducible by mutagens. PGE 2 itself did not induce any MPCE in BALB/c mice. The highest frequency of MPCE and a dose-response relationship between PGE 2 doses and MPCE frequency were seen 30 h after the injection of mitomycin C (MMC) to mice which had been treated 24 h before with PGE 2. The induction of MPCE by vincristine, 5-fluorouracil, 2-naphthylamine and 1,1-dimethylhydrazine was also increased by pretreatment with PGE 2. We also studied the effect of the Ca2+-chelating agent EGTA on micronucleus formation, since it is known that erythroid colony formation induced by erythropoietin can be inhibited by EGTA. The frequency of MPCE inducible by MMC decreased in proportion to the EGTA dose in the pretreatment. The same effect of EGTA was also observed

Takagi, A., K. Sai, T. Umemura, Y. Kurokawa and H. Kasai ~, Division of Toxicology, National Institute of Hygienic Sciences, Kamiyoga, Setagaya-ku, Tokyo 158, and 1 Biology Division, National Cancer Center Research Institute, Chuoku, Tsukiji, Tokyo 104 (Japan) Production of 8-hydroxydeoxyguanosine in rat liver D N A by oral administration of peroxisome proliferators

A number of peroxisome proliferators have been shown to induce liver tumors in rats and mice, but they have no direct interaction with DNA and have no activity in established in vitro genotoxicity assays. This study was undertaken to investigate the hypothesis that peroxisome proliferation is linked to increased cellular levels of H202 and subsequent DNA damage in vivo. As an index of DNA damage caused by oxygen radicals, levels of 8-hydroxydeoxyguanosine (8OH-dG) were examined in the liver and kidney DNA of male F344 rats (6 weeks old) given single or daily intragastric administration (8 days) of peroxisome proliferators (aluminium clofibrate, simfibrate, di-(2-ethylhexyl)phthalate). In liver DNA, 8-OH-dG levels were significantly increased after treatment with the 3 compounds. In kidney DNA, on the other hand, 8-OH-dG levels were not increased. These results suggest that formation of 8-OH-dG in tissue DNA is closely related to the organ specificity observed in peroxisome proliferator carcinogenesis.

380

57

Formation of PhIP-DNA adduct in F344 rats

Takahashi, S., T. Kato and K. Kikugawa, Tokyo College of Pharmacy, 1432-1 Horinouchi, Hachioji, Tokyo 192-03 (Japan)

2-Amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP) is a mutagen found in fried ground beef. Its level is around 15 n g / g (based on uncooked weight), which is 750 and 15 times higher than the amounts of IQ and MelQx, respectively. We studied the in vivo formation of PhlP-DNA adduct using the 32p-postlabeling method. Male F344 rats were fed PhlP in the basal diet at a concentration of 0.01, 0.02 or 0.05%. Rats were killed after 2 or 4 weeks' administration and major organs (liver, kidney, lung, stomach, colon and pancreas) were removed. PhlP-DNA adducts were detected in all the organs examined. Interestingly, the highest levels of DNA adducts were found in the pancreas, unlike other heterocyclic amines. For example, the adduct level in the pancreas after 4 weeks' administration of 0.05% PhlP was 12.25 per 107 nucleotides, which was 20 times higher than that in the liver. It was also demonstrated that the level of D N A adducts increased with increasing PhlP dose and feeding period in all organs tested.

Formation and content of 2-amino-3,4-dimethylimidazol4,5-f ]quinoline in roasted coffee beans Heterocyclic amine mutagens were extracted from regular coffee beans, which were either hotair-roasted, charcoal-roasted or high-temperatureroasted. The mutagens could be extracted with methyl alcohol/ammonium hydroxide and partitioned into acidic water. The mutagens were extracted into chloroform after the acid solution was made alkaline, and then purified with blue cotton. They gave positive responses to Salmonella typhimurium strains TA98 and TA100 with metabolic activation. The numbers of His + revertant colonies/10 g beans with TA98 strain with $9 mix were 120 for hot-air-roasted, 200-700 for charcoal-roasted, and 2700 for high-temperatureroasted coffee beans. The mutagens were separated into 2 mutagenic fractions, A and B, by HPLC. Fraction A, of high temperature-roasted beans, was purified by successive XAD-2 column chromatography and HPLC, and was suggested to be 2-amino-3,4-dimethylimidazo[4,5-f ]quinoline (MelQ) by comparison with authentic MelQ in co-chromatography, ultraviolet absorption spectrum and activity. The mutagens in fraction B were purified by successive Sephadex LH-20 column chromatography and HPLC into 5 unknown heterocyclic amine-like mutagens. The MelQ content in coffee beans was estimated to be 0.16 ng (hot-air-roasted), 0.32 ng (charcoal-roasted) and 1.5 n g / 1 0 g (high-temperature-roasted coffee beans).

58 Takayama, K., K. Yamashita, K. Wakabayashi, M. Nagao and T. Sugimura, Carcinogenesis Division, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo 104 (Japan)

59 Takenaka, S., N. Sera, H. Tokiwa, I. Hirohata ~ and T. Hirohata 2, Fukuoka Environmental Research Centre, Dazaifu, Fukuoka 818-01, 1 Kurume Shinai Women's College, Kurume 830, and 2 Department of Public Health, School of Medicine, Kyushu University, Maedashi, Fukuoka 810 (Japan) Identification of mutagens in the pickles produced in Akita and Fukuoka A total of 108 samples of pickles, which were produced in districts in Japan with high and low incidence of stomach cancer, were extracted with chloroform methanol. The extracts were bioassayed with Salmonella tester strains. The pickles produced in the high-cancer-incidence district were more mutagenic than those produced in the lowincidence district. The most mutagenic sample among 20 pickle specimens collected in the high-incidence district induced 130 revertants per

381 mg of the crude extract for strain TA98. The mutagenic compounds were purified, and 2 flavonols, quercetin and rhamnetin, were identified as the major mutagens in the pickles by gas chromatography-mass spectrometry. The quantities of the 2 compounds were determined to be 6.60 mg for quercetin and 1.96 mg for rhanmetin per g of crude extract. The mutagenic activities of crude extracts of 12 samples, the pickles produced in the 2 districts named, were closely related to the amounts of quercetin identified. 60 Tamakawa, K., M. Matsumoto, Y. Takahashi, T. Seki, A. Tsunoda and J. Lewtas, Sendai Municipal Institute of Public Health, 2-5-10, Oroshimachihigashi, Sendai 983 (Japan) Application of the sensitive Ames test (accelerated microsuspension procedure) to assess indoor air pollution. Mutagenicity of dust on filters in airconditioners The sensitive Ames test (the modified Kado microsuspension procedure) was applied to assess the mutagenicity of dust collected on filters in air-conditioners. The major modification consists of using 'amino acid-enriched minimal glucoseagar base', developed by S. Arimoto to accelerate the growth of His + revertants and shorten the incubation period (18-24 h). This procedure was approximately 4-18 times more sensitive than the modified Ames test (preincubation method by Yahagi et al.) for detecting mutagens in air particulate extracts. Dust collected from air-conditioners of rooms in which cigarettes had been smoked showed higher mutagenicity than those of rooms without smoke. It was suggested that the mutagenic activity, tar content and B[a]P content were generally related to the frequency of using fire in the rooms. The microsuspension procedure is useful for testing samples of limited mass. 61 Tatsumi, K., S. Kitamura, H. Amano 1, K. Ueda 2, F. Okumura and T. Fujimoto, Institute of Phar-

maceutical Science, Hiroshima University School of Medicine, Kasumi, Minami-ku, Hiroshima 734, 1Wakunaga Pharmaceutical Co., Koda-cho, Takada-gun, Hiroshima 729-64, and 2Santen Pharmaceutical Co., Shimoshinjyo, Higashiyodogawa-ku, Osaka 533 (Japan) Studies on metabolic activation of carcinogenic arylamines. N-Formylation in animals When 4-amimobiphenyl, 2-aminonaphthalene, 2-aminofluorene or 1-aminopyrene was given orally to rabbits, the corresponding N-arylformamide could be isolated from the urine together with the corresponding N-arylacetamide. Metabolic conversion of the arylamines to the N-arylformamides and N-arylacetamides was also observed in guinea pigs and rats. The quantitative experiments showed that only minor amounts of N-arylformamides and N-arylacetamides were excreted in the urine or feces of rats and rabbits given the arylamines. This seemed to be due to almost complete further metabolism of these Nacyl derivatives in vivo. Liver cytosols from several mammalian species exhibited a significant N-formylating activity toward the arylamines in the presence of N-formyl-L-kynurenine and Nacetylating activity in the presence of acetyl-CoA; the formylation is due to formamidase and the acetylation is due to arylamine acetyltransferase.

62 Tatsumi, K., M. Toyoda, A. Fujimori, A. Tachibana, I. Arita and H. Takebe, Faculty of Medicine, Kyoto University, Sakyo, Kyoto 606 (Japan) Mutation assay at the adenosine phosphoribosyltransferase locus in lymphoblastoid cells derived from obligate heterozygotes of 2,8-dihydroxyadenine urolithiasis An assay system has been developed to measure the frequency of mutations at the adenine phosphoribosyltransferase (APRT) locus on the human autosomal chromosome No. 16. An EBV-transformed B lymphoblastoid cell line,

382 WR010/B, was chosen for the assay among cell lines derived from heterozygous carriers of hereditary 2,8-dihydroxyadenine urolithiasis (APRT deficiency). 100 /xM of 2,6-diaminopurine (DAP) was found to be appropriate for selecting out aprt - / - mutants from aprt +/- WR010/B cells. DAP-resistant (DAP R) mutants grew as rapidly as phenotypically wild-type W R 0 1 0 / B cells, and the reconstruction experiments indicated that the mutant fraction is stable for at least 14 days. There was no metabolic cooperation, and the recovery of DAP R mutants was essentially 100%. Exposure to "/-rays of W R 0 1 0 / B resulted in a dose-dependent increase in the DAP R mutant fraction, as measured by the limiting dilution technique using microtiter plates. 2 Gy induced 2.5 X 10 - 4 DAP R frequency with a background frequency of 2.5 × 10 -5, while 4.7 × 10 -5 was induced for the 6-thioguanine-resistant (TG R) mutation with a background of 1 × 10 -6.

63 Tsuda, M., T. Kato, Y. Kurashima and T. Sugimura, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104 (Japan)

Studies on nitrosation dynamics in the human body and in rats using thioproline, an effective nitritetrapping agent N-Nitrosothioproline (NTPRO), one of the major N-nitroso compounds in human urine, is non-mutagenic and probably non-carcinogenic, like N-nitrosoproline. Thioproline (TPRO) is nitrosated about 1000-fold faster than proline in vitro, and N T P R O is excreted in urine without metabolism. We examined the urinary amount of N T P R O by giving nitrate a n d / o r TPRO to a male volunteer. When the subject ingested 340 mg of N a N O 3 or 10 mg of TPRO alone, the amounts of N T P R O in 12 h urine were 8.7 +_ 6.5/~g (mean _+ SD) and 6.5 + 4.5 /~g, respectively. The urinary level of N T R P O increased to 165.1 _+ 137.6 /~g when the subject took both of the precursors, and the level was decreased to 21.5 _+ 12.7/~g by additional ingestion of ascorbic acid (300 mg × 2). Thus, we concluded that T P R O is an effective

nitrite-trapping agent in the human body. When rats (F344, male) were administered 20 m g / k g b.w. of NaNO 3 followed by 20 m g / k g of T P R O by gastric intubation, the urinary N T P R O increased to 2.3 _+ 0.7/~g/24 h from 0.7 _+ 0.0 # g / 2 4 h (TPRO alone), but increased only to 1.8 + 0.5 /~g/24 h when KSCN (15 mg/kg) was co-administered. The suppression of the in vivo formation of N T P R O is probably due to competitive inhibition by the S C N - of nitrate secretion into the oral cavity via the salivary glands, a mechanism which would result in a decrease in nitrite formation in the saliva.

64 Ueda, Y., K. Wakabayashi, T. Sugimura and M. Nagao, Carcinogenesis Division, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo 104 (Japan)

Trapping of mutagens/carcinogens in cigarette smoke by fibrillated composite fibers For the development of new materials for cigarette filter tips, we tested the ability of fibrillated polystyrene-polypropylene fibers to trap mutagens/carcinogens in cigarette smoke. The composite fibers, which were recently developed by Toray Ind. Inc., were inserted between the filter tip and tobacco leaves in commercial cigarettes. When 35 mg (08 mm × 6 mm) of the fibers were inserted into cigarettes, the amounts of cigarette smoke condensates and their mutagenicity to S. typhimurium TA98 with $9 mix were reduced by 30-50%. In concordance with the reduction in the tar content, the amounts of carcinogens, such as tobacco-specific nitrosamines, namely N N N and N N K , N-nitroso-dimethylamine, N-nitrosopyrrolidine and 2-aminoc~-carboline in mainstream smoke were reduced by 50-70%. On the other hand, cellulose acetate fibers, which currently find widespread use in filter tips, removed only 10% of the tar when included at the same level as the fibrillated fibers. Thus, these newly developed fibrillated fibers are highly effective in trapping the tar and m u t a g e n s / carcinogens in cigarette smoke.

383 65

Wakabayashi, K., K. Yamashita, Y. Kitagawa, M. Suzuki, M. Nagao and T. Sugimura, National Cancer Center Research Institute, Tsukiji, Chuoku, Tokyo 104 (Japan) DNA modification by l-nitrosoindole-3-acetonitrile in the rat stomach

Indole-3-acetonitrile is present in various kinds of vegetables. This compound reacts with nitrite under acidic conditions and produces a directacting mutagen, 1-nitrosoindole-3-acetonitrile (NIAN). We studied DNA modification by NIAN in the stomach of male F344 rats using a 32P-postlabeling method. NIAN was given by gavage at 100 mg/kg body weight, and animals were killed after 2 h. Six DNA adducts, including 3 major adduct spots, were observed in the DNA from both the forestomach and glandular stomach. The total adduct levels in the forestomach and glandular stomach were about 1 per 107 nucleotides each (K. Yamashita et al. (1988) Carcinogenesis, 9, 1905-1907). The 3 major adducts were also found to be produced following in vitro reaction of N I A N with calf thymus DNA. We are now investigating the carcinogenicity of N I A N in rats dosed by gavage.

66

Watanabe, K., T. Ohta and Y. Shirasu, Institute of Environmental Toxicology, Suzuki-cho 2-772, Kodaira, Tokyo 187 (Japan) o-Vanillin enhances mutagenesis MNNG in Escherichia coli

induced

by

2 - H y d r o x y - 3 - m e t h o x y b e n z a l d e h y d e (ovanillin), of which the antimutagenic effect on the 4-nitroquinoline 1-oxide (4NQO)-induced mutagenesis in Escherichia coli WP2s has been reported, enhanced the N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutation of the same strain. Among 7 derivatives of o-vanillin, 2-hydroxy-3-ethoxybenzaldehyde, o-hydroxybenz-

aldehyde and m-methoxybenzaldehyde showed an enhancing effect on the MNNG-induced mutagenesis in E. coli WP2s. A marked enhancement of N-methyl-N-nitrosourea mutagenesis by ovanillin was also observed, as assayed on E. coli WP2s. On the other hand, o-vanillin greatly suppressed furylfuramide- and 4NQO-induced mutagenesis and showed a slight suppressing effect against mutagenesis inducible by methyl methanesulfonate, N-ethyl-N'-nitro-N-nitrosoguanidine and N-ethyl-N-nitrosourea. The enhancing activity of o-vanilhn on mutagenesis induced by MNNG or MNU in E. coli WP2s may arise from inhibition of the inducible adaptive response.

67 Watanabe, M., T. Nohmi and M. Ishidate Jr., Division of Mutagenesis, National Institute of Hygienic Sciences, Setagaya-ku, Tokyo 158 (Japan) Establishment of new strains of S. typhimurium highly sensitive to nitroarenes and aromatic amines: TA98 and TA100 derivatives with high levels of nitroreductase or acetyltransferase activities

The nitroreductase (NRase) and acetyltransferase (ATase) genes of S. typhimurium TA1538 are cloned into pBR322 (see Watanabe et al. (1987) Biochem. Biophys. Res. Commun., 147, 974, for details). The cloned genes (pYG216 and pYG219) were introduced into TA98 and TA100. TA98(pYG216), TA98(pYG219), TA100(pYG216) and TA100(pYG219) were called YG1021, YG1024, YG1026 and YG1029, respectively. YG1021 and YG1026, both of which have high levels of NRase, were highly sensitive to 2-nitrofluorene (2-NF), 1-nitropyrene (1-NP) and 2-nitronaphthalene (2NN), while YG1024 and YG1029, both of which have high levels of ATase, were highly sensitive to 2-NF, 1-NP, 1,8-dinitropyrene, 2-NN, Glu-P-1 and 2-aminoanthracene. These strains should be very useful for detecting nitroarenes and aromatic amines that are present in the environment in tiny amounts, and also for estimation of the metabolic pathways of chemical mutagens.

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68 Watanabe, T., Y. Hanasaki, T. Hirayama and S. Fukui, Kyoto Pharmaceutical University, 5Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607 (Japan) Mutagenicity of nitro- and amino-substituted phenazines in Salmonella typhimurium Nitro- and amino-substituted phenazines were synthesized and assayed for their mutagenicity in Salmonella typhimurium strains TA98 and TA98NR. Of 7 nitrophenazines tested, 4 were mutagenic in the absence of a microsomal metabolic activation system ($9 mix) and. were more mutagenie in TA98 than in TA98NR. The order of mutagenicity of nitrophenazines in TA98 is 1,7- < 2- < 2,8- < 2,7-substituted phenazine. Of 7 amino derivatives tested, 4 exhibited mutagenic activity with $9 mix in TA98. 1-Nitro-, 1-amino-, 1,6-dinitro-, 1,9-dinitro-, 1,6-diamino- and 1,9-diaminophenazine were not mutagenic. On the study of the relationship between mutagenic potency and chemical structure of phenazines, the result suggested that structural requirements favoring mutagenic activity were the presence of substituents at the 2 a n d / o r 7 position. Furthermore, 2,7-disubstituted phenazines were extremely mutagenie; 2,7-dinitrophenazine and 2,7-diaminophenazine induced 36450 and 12110 rev./nmole, respectively. In the preliminary study, 2,7-diaminophenazine was identified by gas chromatographymass spectrometry from the reaction mixture of m-phenylenediamine and hydrogen peroxide. 69 Yamakage, K., Y. Iwata, M. Oshimura 1 and N. Tanaka, Department of Cell Biology, Hatano Research Institute, Food and Drug Safety Center, Hatano, Kanagawa, and 1 Department of Cytogenetics, Kanagawa Cancer Center, Yokohama, Kanagawa (Japan) Development of an in vitro screening system for agents causing aneuploidy: use of human/mouse hybrid cells To develop an in vitro screening system for agents causing aneuploidy, 3 human/mouse

monochromosomal hybrid cell lines, 1533, 3552 and 7151, which had been cloned after fusing mouse A9 and X/autosome translocated human cells, were used. Cells were treated with colcemid for 24 h, then chromosome specimens at various recovery times were prepared. The human chromosome present in the mouse cells was readily identified by Q-band or Giemsa 11 staining. Cells containing 0 or 2 human chromosomes were counted as aneuploids. The frequencies were compared with the induction of polyploid cells. The profile of the induction of aneuploids found by treatment with colcemid was highly consistent with that of polyploids. The frequency increased with the increase in colcemid dosage and showed a peak at the doubling time of the cells. These results show that this short-term assay is useful for the screening of aneuploidy-causing agents.

70 Yamanaka, K., K. Hakomori, A. Someya, A. Hasegawa, R. Sawamura and S. Okada 1, College of Pharmacy, Nihon University, Tokyo, and 1 School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka (Japan) DNA strand breaks in rat lung induced by dimethylarsinic acid Epidemiological studies have shown that inorganic arsenics are human lung carcinogens, although their carcinogenicity and genotoxicity have not been fully demonstrated in experimental studies. Inorganic arsenics are known to be metabolized to methanearsinic acid and dimethylarsinic acid (DMAA) in mammals. However, the carcinogenicity or genotoxicity of the methylated arsenics has been little investigated so far. In the present study, we have found that DNA-strand breaks occur specifically in lungs of rats orally administered DMAA. Experiments on the mode of strand breaks, the pulmonary metabolites of DMAA and the effects of SOD and catalase indicated that the ultimate inducers of the breaks were active oxygens, which were produced by the reaction of pulmonary oxygen with dimethylarsine produced metabolically from DMAA. These results suggest

385 that the metabolic methylation of inorganic arsenics may activate their genotoxicity in the lung.

71 Yasunaga, K., N. Asai and K. Yoshikawa, Toxicology Laboratory, Life Science Research Sector, Research Center, Mitsubishi Kasei Corp., 1000, Kamoshida-cho, Midori-ku, Yokohama, Kanagawa 227 (Japan)

Relation between uvrB and umuDC gene functions and DNA cross-linking damages induced by mitomycin C in Salmonella typhimurium strain G46/pSK1002 A DNA cross-linking agent, mitomycin C, has never induced mutations in uvr-deficient strains ( u o r - ) of Escherichia and Salmonella. This agent,

however, has proved to induce u m u D C gene, a gene responsible for SOS repair, in TA1535/ pSK1002. To analyze the relation between uor and umu gene functions on the DNA cross-linking damage by mitomycin C, the induction level of the u m u C - l a c Z fusion gene was examined in uvr + strains, G46/pSK1002 (uorB + rfa+), TA1535/ pSK1002 (uorB + r f a - ) and u o r - strain TA1535/ pSK1002 ( u o r B - r f a - ) . These strains were treated with mitomycin C for 2 h at 37°C at concentrations ranging from 0.005 to 5 /~g/ml, and flgalactosidase activities were measured using onitrophenyl-fl-D-galactoside as substrate. The resuits showed that the induction level of the u m u C - l a c Z gene in the uvr + strains was higher than in the uor- strain. These findings suggest that uorB plays a role in the induction of u m u D C gene caused by the D N A cross-links.