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Analysis of data from in vitro chromosomal aberration tests on 951 chemical substances
378 toxic dose. These results suggest that the clastogenicity inducible by sodium chloride is related to the inhibition of DNA synthesis and subsequent physiological changes of cells.
56 Shinkawa, K., K. Morimoto and A. Koizumi, Department of Public Health, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113 (Japan) Micronuclei in human lymphocytes exposed to mitomycin C and colchicine The frequencies of micronuclei (MN) were examined in human lymphocytes treated with mitomycin C (MMC) or colchicine. MMC is a bifunctional alkylating agent while colchicine is a spindle-fiber poison. These chemicals caused doseand time-dependent increases in MN when the cells were treated for the entire culture time of 72 h. To elucidate the relation between the appearance of MN and culture time, cells were pulse-treated for 1 h with the chemicals at G 0, and subsequently stimulated with phytohemagglutinin. MN frequencies were examined in cultures sampled at 6-h intervals up to 96 h after stimulation. With increasing sampling times, the frequencies of MMC-induced MN increased up to 60 h, and stayed almost constant at subsequent times, whereas those of colchicine-induced MN increased gradually and continuously. Further analyses of the frequency of cells having more than 1 MN in a cell (multi-nucleated cells) revealed that only colchicine treatment led to a dose-related increase in the frequency of multi-nucleated cells. This difference in the induction patterns of MN by MMC and colchicine might be due to the difference in the action mechanisms of these two chemicals.
57 Shudo, K., Y. Hashimoto, E. Kawachi and T. Sekiya 1, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, and I National Cancer Research Institute, Chuo-ku, Tokyo 104 (Japan)
Transforming activity of human c-Ha-ras-I protooncogene chemically modified with Glu-P-I and 4NQO: Direct evidence of cellular transformation by chemically modified DNA 2-Amino-6-methyldipyrido[1,2-a: 3',2'-d ]imidazole (Glu-P-1) and 4-nitroquinoline N-oxide (4NQO) are carcinogens found in Japan. The pathways of chemical modification of DNA with these carcinogens were established; the ultimate form(s) of these carcinogens are the corresponding N-acyloxy derivatives. In vitro modification of plasmids containing the human c-Ha-ras protooncogene with the synthesized ultimate carcinogen, 2-acetoxy-amino-6-methyldipyrido[1,2-a:3', 2'-d]imidazole (N-OAc-Glu-P-1), generated an activity to transform NIH 3T3 cells. Genomic DNAs isolated from the transformants were analyzed by restriction fragment length polymorphism (RFLP) assay using a restriction enzyme Msp I. Of 14 transformants studied, 6 contained a mutation in the region of CCGG sequence in which GG corresponds to the first two nucleotides of the 12th codon. Chemical modification of the plasmids with 4-acetoxyaminoquinoline N-oxide (N-OAc-4AQO) also generated a transforming activity. Since the plasmid is the only cellular macromolecule present in the reactions, the results clearly show that chemical modification of DNA can lead to transformation of mammalian cells.
58 Sofuni, T., M. Hayashi, A. Matsuoka, M. Sawada, M.C. Harnois I and M. Ishidate Jr., Division of Mutagenesis, Biological Safety ReSearch Center, National Institute of Hygienic Sciences, Tokyo 158, and 1 Japan Foundation for Promotion of Cancer Research, Tokyo 104 (Japan) Analysis of data from in vitro chromosomal aberration tests on 951 chemical substances Approximately 250 original reports on the in vitro clastogenicity of chemical substances were selected from those published since 1971 in major scientific journals. Data on 951 substances were entered into the division's computerized data files and analyzed. Of the 951 substances, 780 had been
379 tested only without metabolic activation system; 153 both with and without metabolic activation; and the remaining 18 only with metabolic activation. Results of the tests were that 447 were consistently positive either with or without activation systems; 417 were negative without activation but not tested with activation; 4 were negative with activation but not tested without activation; and 30 were clearly negative with and without activation. The remaining 53 substances gave variable results because of different protocols or different cell types, but all were positive in at least one test. Quantitative comparisons of the minimum effective concentration (mM) were also carried out on substances which were positive in at least one test. The most potent substance had 4.3 × 10 -8 mM as the minimum effective dose, the majority of chemicals were positive at levels between 0.1 and 1.0 raM, and several chemicals were positive at only very high concentrations (100-1000 mM). In addition, the results were compared with those obtained from other biological assays, namely, the Ames test, the in vivo micronucleus test and the rodent carcinogenicity assay.
zinc, hydrazine sulfonate, hydrazine hydrate, 2-hydroxyethylhydrazine, ethylhydrazine oxalate, 2-methyl-4-chlorophenoxyacetic acid hydrazide HC1, N-methyl-N-formylhydrazine, fl-phenylisopropylhydrazine, 1-acetyl-2-phenylhydrazine, benzoylhydrazine, carbamylhydrazine HC1, 1-carbamyl-2-phenylhydrazine, 1,2-diformylhydrazine, formic acid hydrazide, guanylhydrazine HC1, isonicotinic acid hydrazide, succinic acid 2,2-dimethylhydrazide and thiosemicarbazide, were generally negative in this assay. It is known that there is little correlation between the mutagenicity and carcinogenicity of hydrazine derivatives. In the present study too, the correlation between the genotoxicity and carcinogenicity in this class of compounds is not high.
6O The Collaborative Study Group for the Micronucleus Test (The Mammalian Mutagenicity Study Group, The Japanese Environmental Mutagen Society), Chief organizer: S. Sutou, NRI Life Science, 4-7-1 Kajiwara, Kamakura, Kanagawa 247 (Japan)
Strain difference in the micronucleus test 59 Sugie, S. 1, N. Yoshimi 1, H. Mori 1 and H. Shimizu 2, 1 Department of Pathology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu-city 500, and 2 Jikei University School of Medicine, Nishi-Shinbashi, Minato-ku, Tokyo 105 (Japan)
Genotoxicity of hydrazine derivatives in the hepatocyte primary culture/DNA repair test using rat hepatocytes 31 hydrazine derivatives were examined in the hepatocyte primary culture (HPC)/DNA repair test using ACI rats. Chemicals positive in this assay were 1-hydrazinophthalazine HC1, 4-methylphenylhydrazine HC1, p,p'-oxybisbenzenedisulfonylhydrazide, phenylhydrazine HC1, 1,2-dimethylhydrazine 2HC1, methylhydrazirte sulfate and N-acetyl-4-(hydroxymethyl)phenylhydrazine. The other 24 chemicals: 4-allylthiosemicarbazide, butylhydrazine oxalate, 2,4-dinitrophenylhydra-
Our collaborative study group investigated sex difference in the micronucleus test last year (Mutation Res., in press, 1986). This year we studied strain difference in this test. Strains of mice were chosen mainly from a practical viewpoint, i.e., ddY (Shizuoka Agricultural Cooperative Association for Experimental Animals (SLC)), BDF (SLC), and CD-1 (Nippon Charles River Inc.), since these 3 strains are most commonly used by the members of our study group. In addition, ms (mutagen sensitive, derived from CD-1, Hitachi Medical Animals Laboratories) was compared with the other 3 strains. 6 test compounds were used: colchicine, 7,12-dimethylbenz[a]anthracene, ethyl methanesulfonate, N-ethyl-N-nitrosourea, potassium chromate, and 6-mercaptopurine. 24 laboratories took part in this study. One test compound was assigned to each laboratory, which performed the micronucleus test using 3 strains of mice. One dose group consisted of 4 male mice, which were given physiological saline or olive oil (negative
56 Shinkawa, K., K. Morimoto and A. Koizumi, Department of Public Health, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113 (Japan) Micronuclei in human lymphocytes exposed to mitomycin C and colchicine The frequencies of micronuclei (MN) were examined in human lymphocytes treated with mitomycin C (MMC) or colchicine. MMC is a bifunctional alkylating agent while colchicine is a spindle-fiber poison. These chemicals caused doseand time-dependent increases in MN when the cells were treated for the entire culture time of 72 h. To elucidate the relation between the appearance of MN and culture time, cells were pulse-treated for 1 h with the chemicals at G 0, and subsequently stimulated with phytohemagglutinin. MN frequencies were examined in cultures sampled at 6-h intervals up to 96 h after stimulation. With increasing sampling times, the frequencies of MMC-induced MN increased up to 60 h, and stayed almost constant at subsequent times, whereas those of colchicine-induced MN increased gradually and continuously. Further analyses of the frequency of cells having more than 1 MN in a cell (multi-nucleated cells) revealed that only colchicine treatment led to a dose-related increase in the frequency of multi-nucleated cells. This difference in the induction patterns of MN by MMC and colchicine might be due to the difference in the action mechanisms of these two chemicals.
57 Shudo, K., Y. Hashimoto, E. Kawachi and T. Sekiya 1, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, and I National Cancer Research Institute, Chuo-ku, Tokyo 104 (Japan)
Transforming activity of human c-Ha-ras-I protooncogene chemically modified with Glu-P-I and 4NQO: Direct evidence of cellular transformation by chemically modified DNA 2-Amino-6-methyldipyrido[1,2-a: 3',2'-d ]imidazole (Glu-P-1) and 4-nitroquinoline N-oxide (4NQO) are carcinogens found in Japan. The pathways of chemical modification of DNA with these carcinogens were established; the ultimate form(s) of these carcinogens are the corresponding N-acyloxy derivatives. In vitro modification of plasmids containing the human c-Ha-ras protooncogene with the synthesized ultimate carcinogen, 2-acetoxy-amino-6-methyldipyrido[1,2-a:3', 2'-d]imidazole (N-OAc-Glu-P-1), generated an activity to transform NIH 3T3 cells. Genomic DNAs isolated from the transformants were analyzed by restriction fragment length polymorphism (RFLP) assay using a restriction enzyme Msp I. Of 14 transformants studied, 6 contained a mutation in the region of CCGG sequence in which GG corresponds to the first two nucleotides of the 12th codon. Chemical modification of the plasmids with 4-acetoxyaminoquinoline N-oxide (N-OAc-4AQO) also generated a transforming activity. Since the plasmid is the only cellular macromolecule present in the reactions, the results clearly show that chemical modification of DNA can lead to transformation of mammalian cells.
58 Sofuni, T., M. Hayashi, A. Matsuoka, M. Sawada, M.C. Harnois I and M. Ishidate Jr., Division of Mutagenesis, Biological Safety ReSearch Center, National Institute of Hygienic Sciences, Tokyo 158, and 1 Japan Foundation for Promotion of Cancer Research, Tokyo 104 (Japan) Analysis of data from in vitro chromosomal aberration tests on 951 chemical substances Approximately 250 original reports on the in vitro clastogenicity of chemical substances were selected from those published since 1971 in major scientific journals. Data on 951 substances were entered into the division's computerized data files and analyzed. Of the 951 substances, 780 had been
379 tested only without metabolic activation system; 153 both with and without metabolic activation; and the remaining 18 only with metabolic activation. Results of the tests were that 447 were consistently positive either with or without activation systems; 417 were negative without activation but not tested with activation; 4 were negative with activation but not tested without activation; and 30 were clearly negative with and without activation. The remaining 53 substances gave variable results because of different protocols or different cell types, but all were positive in at least one test. Quantitative comparisons of the minimum effective concentration (mM) were also carried out on substances which were positive in at least one test. The most potent substance had 4.3 × 10 -8 mM as the minimum effective dose, the majority of chemicals were positive at levels between 0.1 and 1.0 raM, and several chemicals were positive at only very high concentrations (100-1000 mM). In addition, the results were compared with those obtained from other biological assays, namely, the Ames test, the in vivo micronucleus test and the rodent carcinogenicity assay.
zinc, hydrazine sulfonate, hydrazine hydrate, 2-hydroxyethylhydrazine, ethylhydrazine oxalate, 2-methyl-4-chlorophenoxyacetic acid hydrazide HC1, N-methyl-N-formylhydrazine, fl-phenylisopropylhydrazine, 1-acetyl-2-phenylhydrazine, benzoylhydrazine, carbamylhydrazine HC1, 1-carbamyl-2-phenylhydrazine, 1,2-diformylhydrazine, formic acid hydrazide, guanylhydrazine HC1, isonicotinic acid hydrazide, succinic acid 2,2-dimethylhydrazide and thiosemicarbazide, were generally negative in this assay. It is known that there is little correlation between the mutagenicity and carcinogenicity of hydrazine derivatives. In the present study too, the correlation between the genotoxicity and carcinogenicity in this class of compounds is not high.
6O The Collaborative Study Group for the Micronucleus Test (The Mammalian Mutagenicity Study Group, The Japanese Environmental Mutagen Society), Chief organizer: S. Sutou, NRI Life Science, 4-7-1 Kajiwara, Kamakura, Kanagawa 247 (Japan)
Strain difference in the micronucleus test 59 Sugie, S. 1, N. Yoshimi 1, H. Mori 1 and H. Shimizu 2, 1 Department of Pathology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu-city 500, and 2 Jikei University School of Medicine, Nishi-Shinbashi, Minato-ku, Tokyo 105 (Japan)
Genotoxicity of hydrazine derivatives in the hepatocyte primary culture/DNA repair test using rat hepatocytes 31 hydrazine derivatives were examined in the hepatocyte primary culture (HPC)/DNA repair test using ACI rats. Chemicals positive in this assay were 1-hydrazinophthalazine HC1, 4-methylphenylhydrazine HC1, p,p'-oxybisbenzenedisulfonylhydrazide, phenylhydrazine HC1, 1,2-dimethylhydrazine 2HC1, methylhydrazirte sulfate and N-acetyl-4-(hydroxymethyl)phenylhydrazine. The other 24 chemicals: 4-allylthiosemicarbazide, butylhydrazine oxalate, 2,4-dinitrophenylhydra-
Our collaborative study group investigated sex difference in the micronucleus test last year (Mutation Res., in press, 1986). This year we studied strain difference in this test. Strains of mice were chosen mainly from a practical viewpoint, i.e., ddY (Shizuoka Agricultural Cooperative Association for Experimental Animals (SLC)), BDF (SLC), and CD-1 (Nippon Charles River Inc.), since these 3 strains are most commonly used by the members of our study group. In addition, ms (mutagen sensitive, derived from CD-1, Hitachi Medical Animals Laboratories) was compared with the other 3 strains. 6 test compounds were used: colchicine, 7,12-dimethylbenz[a]anthracene, ethyl methanesulfonate, N-ethyl-N-nitrosourea, potassium chromate, and 6-mercaptopurine. 24 laboratories took part in this study. One test compound was assigned to each laboratory, which performed the micronucleus test using 3 strains of mice. One dose group consisted of 4 male mice, which were given physiological saline or olive oil (negative