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Induction of mutations by hepatitis B virus X protein in Escherichia coli
JEMS Abstracts/Mutation Research 334 (1995) 385-427
ELF magnetic fields (83-160 mT, 7-50 Hz). After 20-25 h, when the cells reached mitotic phase following two rounds of DNA synthesis, the cells were collected and fixed and slides were prepared. The metaphase slides were examined for cell cycle kinetics and sister-chromatid exchanges (SCE). Exposure to ELF magnetic fields did not alter cell cycle kinetics or SCE frequencies. The effect of long-term exposure was also evaluated with serially cultured cells under ELF magnetic field exposure for 2 weeks; no significant difference in the frequencies of SCE was observed when compared to control groups. 56 Noguchi, T., M. Asakura, T. Sugiyama and T. Matsushima, Japan Bioassay Laboratory, 2445 Hirasawa, Hadanoshi, Kanagawa 257, Japan In vivo micronucleus test in hepatocytes of rats treated with N-nitrosoamines
N-Nitrosoamines are known to produce unstable ultimate mutagens/carcinogens when metabolized in liver. The mouse bone marrow micronucleus test is a widely used in vivo mutagenicity test but is not sensitive to N-nitrosoamines, because these metabolites are too short-lived to reach the target cells. So, we carried out the micronucleus test on rat hepatocytes. Two-thirds of the liver was partially hepatectomized (PH) under ether anesthesia 24 h after the last injections of nitrosoamines and hepatocytes were isolated 4 days after PH by perfusing with collagenase medium. Hepatocytes were fixed with buffered formalin, spread on a glass slide stained with 4',6-diamidino-2-phenylindole (DAPI) and covered with a glass. N-Nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosodi-n-propylamine, and N-nitrosodi-n-butylamine induced micronuclei dose-dependently in hepatocytes but N-nitrosodiphenylamine did not. Similar responses have been obtained from mutagenicity tests of bacteria, chromosomal aberration tests of cultured mammalian cells and carcinogenicity tests. The micronucleus test of PH rat liver is thus a useful
409
method for detecting clastogenicity of chemicals which are metabolically activated in the liver. 57 Nohmi, T. a, M. Katoh b, M. Watanabe a, M. Yamada a, M. Matsui a, N. Ishihara b and T. Sofuni a, a Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158 and b Laboratory of Genetic Toxicology, Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano, Kanagawa 257, Japan Novel phage lambda Cre-lox vectors designed for development of gene mutation assays using transgenic mice
Novel phage lambda vectors, EG3 and EG7, were constructed. The vectors possess linearized plasmid DNAs between the right and left arms of lambda gt6. The linearized plasmid DNA regions include the gpt gene of Escherichia coli (Ecogpt), chloramphenicol resistance gene and origins of plasmid replication. Lambda EG3 and EG7 have different replication origins derived from plasmids pACYC184 and pBR322, respectively. The plasmid DNA regions are located between the two direct repeats of loxP, a recognition sequence of the site-specific recombinase Cre. When a strain of E, coli YG6020 expressing Cre recombinase was infected with lambda EG3 or EG7, the phage DNAs were automatically converted to the plasmids carrying Ecogpt. The recognition sites of HindlII were created at the junction between the plasmid DNA regions and both arms of lambda EG3 and EG7. Lambda EG3 and EG7 could be useful for the development of new gene mutation assays using transgenic mice, which allow the positive selection of mutant colonies, multiple DNA rescue methods and rapid DNA sequencing. 58 Oda, Y. and Y. Minekawa, Laboratory of Virology, Osaka Prefectural Institute of Public Health, 3-69 Nakamichi 1-chome, Higashinari-ku, Osaka 537, Japan
410
JEMS Abstracts/Mutation Research 334 (1995) 385-427
Induction of mutations by hepatitis B virus X protein in Escherichia coli Hepatitis B virus (HBV) causes acute and chronic hepatitis and is associated with the development of hepatocellular carcinoma. In particular, the X protein of HBV has been shown to develop tumors in the liver of transgenic mice and to transactivate transcription of cellular or virus gene promoters. But little is known about whether this protein has mutagenic effects in cell. To examine mutagenesis mediated by plasmid harboring the X gene, plasmid pBR322, carrying an X gene-containing region, was introduced into Escherichia coli ABl157 and KMBL3835, for the detection of base-substitution mutations and frameshift mutations, respectively. The plasmid carrying the X gene showed a mutation frequency 10-fold higher than the background level in the frameshift strain, though significantly lower in the base-substitution strain. In addition, the induced mutation frequency was inhibited by phosphonoacetic acid, an inhibitor of virus replication. These findings indicate that X protein induces frameshift-type mutation in E. coli. 59 Oguri, A. a, K. Wakabayashi a, H. Kushida a, W.R. Kusamran b, T. Watanabe c and T. Sugimura a, a National Cancer Center Research Institute, Tokyo 104, Japan, b National Cancer Institute, Bangkok, Thailand and c Kyoto Pharmaceutical University, Kyoto 607, Japan Mutagenicities of river water in Bangkok, Tokyo and Kyoto River pollution is a serious problem in many countries. From rivers in Bangkok, Tokyo and Kyoto, we collected mutagens having multicyclic planar structures with absorbent blue rayon and compared mutagenicities. Three grams of blue rayon was suspended in river water for 24 h and the adsorbed material was eluted with a methanol-ammonia solution, then tested for mutagenicity in S. typhimurium YG1024. All the samples tested showed mutagenicity with $9 mix but not without $9 mix. The mutagenicities ob-
tained from samples taken from four sections of the Chao Phraya river gave 714-1850 revertants/ 0.1 g blue rayon equivalent extract. Regarding the mutagenicities of samples from the two canals, Pra-pa and Rungsir, which flow into Chao Phraya, two samples from the former were lower than those of Chao Phraya, being 174 and 268 revertants/0.1 g blue rayon, and one sample from the latter higher, being 2426 revertants/0.1 g blue rayon. The mutagenicity of five samples from the Sumida river was 472-804 revertants/0.1 g blue rayon, which is almost the same as that of samples from Chao Phraya. As reported by Hayatsu's group, we could confirm that the mutagenicity at the point under the Toba sewage plant of the Katsura river in Kyoto was extremely high, being 80-1060-fold more potent than those of Chao Phraya and Sumida rivers. Characterization of mutagens in the three rivers and their sources requires further studies. 60 Ohmori, K. a, K. Fukuhara b, N. Iwata c, y. Horiguchi a, T. Endo c and N. Miyata b, a Kanagawa Prefectural Public Health Laboratory, Asahi-ku, Yokohama, Kanagawa 241, b National Institute of Health Sciences, Setagaya-ku, Tokyo 158 and c Tokyo Medical College, Shinjuku-ku, Tokyo 160, Japan Characteristics of nitroreductase activity in S. typhimurium strains TA98, TA98NR and YG1021 Enzymatic nitroreduction is the first step in the metabolic activation of nitrated polycyclic aromatic hydrocarbons (nitroPAHs). We have already reported that 1,3,5-trinitronaphthalene (1,3,5-trinitroNP) is a potent mutagen in the Ames test using S. typhimurium strains TA98, TA98NR (nitroreductase deficient mutant), and YG1021 (nitroreductase overproducing strain). Nitroreductase activities toward 1,3,5-trinitroNP in three strains were examined to characterize nitroreductases related to the metabolic activation of nitroPAHs. Nitroreductase activity toward 1,3,5-trinitroNP in YG1021 was more than 20-fold higher than that in TA98. The 1,3,5-trinitroNP reduction of K m values was almost the same among the
ELF magnetic fields (83-160 mT, 7-50 Hz). After 20-25 h, when the cells reached mitotic phase following two rounds of DNA synthesis, the cells were collected and fixed and slides were prepared. The metaphase slides were examined for cell cycle kinetics and sister-chromatid exchanges (SCE). Exposure to ELF magnetic fields did not alter cell cycle kinetics or SCE frequencies. The effect of long-term exposure was also evaluated with serially cultured cells under ELF magnetic field exposure for 2 weeks; no significant difference in the frequencies of SCE was observed when compared to control groups. 56 Noguchi, T., M. Asakura, T. Sugiyama and T. Matsushima, Japan Bioassay Laboratory, 2445 Hirasawa, Hadanoshi, Kanagawa 257, Japan In vivo micronucleus test in hepatocytes of rats treated with N-nitrosoamines
N-Nitrosoamines are known to produce unstable ultimate mutagens/carcinogens when metabolized in liver. The mouse bone marrow micronucleus test is a widely used in vivo mutagenicity test but is not sensitive to N-nitrosoamines, because these metabolites are too short-lived to reach the target cells. So, we carried out the micronucleus test on rat hepatocytes. Two-thirds of the liver was partially hepatectomized (PH) under ether anesthesia 24 h after the last injections of nitrosoamines and hepatocytes were isolated 4 days after PH by perfusing with collagenase medium. Hepatocytes were fixed with buffered formalin, spread on a glass slide stained with 4',6-diamidino-2-phenylindole (DAPI) and covered with a glass. N-Nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosodi-n-propylamine, and N-nitrosodi-n-butylamine induced micronuclei dose-dependently in hepatocytes but N-nitrosodiphenylamine did not. Similar responses have been obtained from mutagenicity tests of bacteria, chromosomal aberration tests of cultured mammalian cells and carcinogenicity tests. The micronucleus test of PH rat liver is thus a useful
409
method for detecting clastogenicity of chemicals which are metabolically activated in the liver. 57 Nohmi, T. a, M. Katoh b, M. Watanabe a, M. Yamada a, M. Matsui a, N. Ishihara b and T. Sofuni a, a Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158 and b Laboratory of Genetic Toxicology, Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano, Kanagawa 257, Japan Novel phage lambda Cre-lox vectors designed for development of gene mutation assays using transgenic mice
Novel phage lambda vectors, EG3 and EG7, were constructed. The vectors possess linearized plasmid DNAs between the right and left arms of lambda gt6. The linearized plasmid DNA regions include the gpt gene of Escherichia coli (Ecogpt), chloramphenicol resistance gene and origins of plasmid replication. Lambda EG3 and EG7 have different replication origins derived from plasmids pACYC184 and pBR322, respectively. The plasmid DNA regions are located between the two direct repeats of loxP, a recognition sequence of the site-specific recombinase Cre. When a strain of E, coli YG6020 expressing Cre recombinase was infected with lambda EG3 or EG7, the phage DNAs were automatically converted to the plasmids carrying Ecogpt. The recognition sites of HindlII were created at the junction between the plasmid DNA regions and both arms of lambda EG3 and EG7. Lambda EG3 and EG7 could be useful for the development of new gene mutation assays using transgenic mice, which allow the positive selection of mutant colonies, multiple DNA rescue methods and rapid DNA sequencing. 58 Oda, Y. and Y. Minekawa, Laboratory of Virology, Osaka Prefectural Institute of Public Health, 3-69 Nakamichi 1-chome, Higashinari-ku, Osaka 537, Japan
410
JEMS Abstracts/Mutation Research 334 (1995) 385-427
Induction of mutations by hepatitis B virus X protein in Escherichia coli Hepatitis B virus (HBV) causes acute and chronic hepatitis and is associated with the development of hepatocellular carcinoma. In particular, the X protein of HBV has been shown to develop tumors in the liver of transgenic mice and to transactivate transcription of cellular or virus gene promoters. But little is known about whether this protein has mutagenic effects in cell. To examine mutagenesis mediated by plasmid harboring the X gene, plasmid pBR322, carrying an X gene-containing region, was introduced into Escherichia coli ABl157 and KMBL3835, for the detection of base-substitution mutations and frameshift mutations, respectively. The plasmid carrying the X gene showed a mutation frequency 10-fold higher than the background level in the frameshift strain, though significantly lower in the base-substitution strain. In addition, the induced mutation frequency was inhibited by phosphonoacetic acid, an inhibitor of virus replication. These findings indicate that X protein induces frameshift-type mutation in E. coli. 59 Oguri, A. a, K. Wakabayashi a, H. Kushida a, W.R. Kusamran b, T. Watanabe c and T. Sugimura a, a National Cancer Center Research Institute, Tokyo 104, Japan, b National Cancer Institute, Bangkok, Thailand and c Kyoto Pharmaceutical University, Kyoto 607, Japan Mutagenicities of river water in Bangkok, Tokyo and Kyoto River pollution is a serious problem in many countries. From rivers in Bangkok, Tokyo and Kyoto, we collected mutagens having multicyclic planar structures with absorbent blue rayon and compared mutagenicities. Three grams of blue rayon was suspended in river water for 24 h and the adsorbed material was eluted with a methanol-ammonia solution, then tested for mutagenicity in S. typhimurium YG1024. All the samples tested showed mutagenicity with $9 mix but not without $9 mix. The mutagenicities ob-
tained from samples taken from four sections of the Chao Phraya river gave 714-1850 revertants/ 0.1 g blue rayon equivalent extract. Regarding the mutagenicities of samples from the two canals, Pra-pa and Rungsir, which flow into Chao Phraya, two samples from the former were lower than those of Chao Phraya, being 174 and 268 revertants/0.1 g blue rayon, and one sample from the latter higher, being 2426 revertants/0.1 g blue rayon. The mutagenicity of five samples from the Sumida river was 472-804 revertants/0.1 g blue rayon, which is almost the same as that of samples from Chao Phraya. As reported by Hayatsu's group, we could confirm that the mutagenicity at the point under the Toba sewage plant of the Katsura river in Kyoto was extremely high, being 80-1060-fold more potent than those of Chao Phraya and Sumida rivers. Characterization of mutagens in the three rivers and their sources requires further studies. 60 Ohmori, K. a, K. Fukuhara b, N. Iwata c, y. Horiguchi a, T. Endo c and N. Miyata b, a Kanagawa Prefectural Public Health Laboratory, Asahi-ku, Yokohama, Kanagawa 241, b National Institute of Health Sciences, Setagaya-ku, Tokyo 158 and c Tokyo Medical College, Shinjuku-ku, Tokyo 160, Japan Characteristics of nitroreductase activity in S. typhimurium strains TA98, TA98NR and YG1021 Enzymatic nitroreduction is the first step in the metabolic activation of nitrated polycyclic aromatic hydrocarbons (nitroPAHs). We have already reported that 1,3,5-trinitronaphthalene (1,3,5-trinitroNP) is a potent mutagen in the Ames test using S. typhimurium strains TA98, TA98NR (nitroreductase deficient mutant), and YG1021 (nitroreductase overproducing strain). Nitroreductase activities toward 1,3,5-trinitroNP in three strains were examined to characterize nitroreductases related to the metabolic activation of nitroPAHs. Nitroreductase activity toward 1,3,5-trinitroNP in YG1021 was more than 20-fold higher than that in TA98. The 1,3,5-trinitroNP reduction of K m values was almost the same among the