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Induction of chromosomal aberrations by the short-term treatment with cadmium chloride in cultured Chinese hamster cells
374 complex which affects the susceptibility to the mutagen-induced micronuclei and chromosome breaks. In the B10.A recombinant strains, B10.A(3R), B10.A(5R) and B10.S(9R) were more sensitive than B10. These sensitive strains similarly have the A strain-derived chromosome segment between S and D regions in the H-2 complex. These results strongly suggested that the susceptibility to the various mutagen-induced micronuclei and chromosome breaks is affected by the gene closely linked with the H-2 complex on chromosome 17. This control gene is probably located between S and D regions in the H-2 complex.
41 Nohmi, T., K. Yoshikawa, M. Ishidate Jr., A. Hiratsuka i and T. Watabe 1, National Institute of Hygienic Sciences, Tokyo, and 1 Tokyo College of Pharmacy, Tokyo (Japan) Mechanism of species difference in bucetin mutagenicity: A key mechanistic role of deacylation by rat and hamster liver microsomes The mutagenicity of bucetin, a component of analgesic drugs, in Salmonella typhimurium TA100 was detected with $9 fractions prepared from hamsters, but not rats. A mechanism of the species difference in bucetin mutagenicity was investigated. By using high-performance liquid chromatography analysis, it was found that bucetin was activated to a direct-acting mutagen, i.e., pnitrosophenetole, through deacylation and N-hydroxylation by hamster liver microsomes. Although there was no significant species difference in N-hydroxylation, the activities of bucetin deacylation were about 140 times higher in hamsters than in rats. From these results, we suggested that the species difference in bucetin mutagenicity is due to the difference in deacylation activities between rat and hamster liver microsomes.
42 Nunoshiba, T., K. Minamisako and H. Nishioka, Biochemistry Laboratory, Doshisha University, Kyoto (Japan)
Effect of human saliva on mutagenicity of environmental carcinogens After finding the inactivating effect of human saliva on mutagenicity of carcinogens, we reported further research on this subject including the individual difference in the capacity, the role of saliva components and the inactivation mechanism. We report this time regarding (1) inactivation for the mutagenicity of amino acid-pyrolysate mutagens, and (2) a simple method for determination of inactivating capacity of individual saliva. Mutagenicity was assayed with the Ames test. Experimental procedures were similar to those described in our paper (Mutation Res., 85 (1981) 323 333). (1) Saliva obtained from a healthy donor inactivated almost 100% of Trp-P-1 mutagenicity and 30-50% of Trp-P-2 and AaC, while no significant effects were seen in Glu-P-1, Glu-P-2 and MeAaC. It seems that a peroxidase in saliva may be involved in the inactivation because this enzyme showed similar specificity in the inactivation of these pyrolysate mutagens. (2) The finding that saliva with the higher inactivating capacity possesses higher peroxidase activity led us to develop a simple method for determining the inactivating capacity of saliva; i.e., the measurement of the peroxidase activity.
43 Ochi, T., M. Mogi, M. Watanabe and M. Ohsawa, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa (Japan) Induction of chromosomal aberrations by the short-term treatment with cadmium chloride in cultured Chinese hamster cells Inducibility of chromosomal aberrations and cytotoxicity in cultured Chinese hamster cells by cadmium chloride was investigated under 3 different conditions: (1) 2-h treatment with CdC12 in MEM medium with 10% fetal bovine serum (MEM + 10% FBS) and continuous treatment for 22 h in MEM + 10% FBS, (2) 2-h treatment with CdC12 in HEPES-buffered Hanks' solution and
375 continuous treatment for 22 h in MEM + 10% FBS, and (3) 24-h treatment with CdC12 in MEM + 10% FBS. Treatments 2 and 3 were 2 and 3 times more cytotoxic, respectively, than treatment 1. Treatment 3 with CdC12 over 5 x 10 -6 was too toxic to the cells to perform chromosomal analysis and moreover only a little increase in the incidence of chromosomal aberrations was observed at 5 x 10 -6 M CdC12. In contrast, a marked and concentration-dependent increase in the incidence of chromosomal aberrations was observed in treatments 1 and 2 with 1 × 10-6-5 x 10 -6 M CdC12. Treatment 2 was approximately 3 times more potent for the induction of chromosomal aberrations than treatment 3. The 2-h treatment with CdC12 to induce chromosomal aberrations was comparable to that of benzo[a]pyrene activated with $9 at equitoxic concentrations. A study on thymidine incorporation into these cells suggests that the restoration of DNA synthesis after cadmium exposure is required for efficiently detecting the metal-induced chromosomal aberrations.
44 Oda, Y., S. Nakamura, I. Oki, K. Sugita ~, T. Kato 1, K. Makino 2, A. Nakata 2 and H. Shinagawa 2, Osaka Prefectural Institute of Public Health, Osaka, 1 Osaka University Medical School, Osaka and 2 Research Institute for Microbial Diseases, Osaka University, Osaka (Japan)
A simple test system (umu test) for the detection of environmental mutagens. II. Improvement of sensitivity by employing Salmonella typhimurium TA1535 as a host strain A rapid, simple and quantitative system ( u m u test) for the detection of environmental mutagens was developed by introducing the plasmid pSK1002 carrying u m u C ' - l a c ' Z fusion gene into S. typhimurium. The urnu test is based on in vitro assay of the fl-galactosidase synthesized from the lacZ gene fused to the umu operon responsible for inducible mutagenesis in E. coli. With this system, a variety of mutagens were examined for their induction of fl-galactosidase. Among those so far tested, base change mutagens
(methyl methanesulfonate, ethyl methanesulfonate, fl-propiolactone); frameshift mutagens (ICR191, 9-aminoacridine, benzo[a]pyrene); oxidative m u t a g e n s (bleomycin, h y d r o g e n peroxide, paraquat); mitomycin-C, 4-nitroquinoline 1-oxide, formaldehyde, a pyrolysate of tryptophan and UV light were positive. In terms of dose response, this test was approximately as sensitive as the Ames test. Polychlorinated biphenyl, DDT, safrole, thioacetamide and actinomycin D were negative. These results show that the umu test is useful as a short-term screening of environmental mutagens.
45 Odagiri, Y., and K. Takemoto, Saitama Medical School, Saitama (Japan)
Application of inhalation technique to the micronucleus assay Administration of test substances by inhalation was applied to the micronucleus assay in order to investigate the mutagenic effect of inhaled substances in vivo. The mist of ethylcarbamate (urethane) generated from an ultrasonic nebulizer was administered to mice by means of inhalation at 4 doses ranging from 67 m g / m 3 x 30 min to x 1 5 0 min. The frequency of micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow increased in a dose-dependent way by the inhalation for 24 h (1.70% of MNPCEs induced at the highest dose). The amount of mist inhaled during the period of 150 min was estimated at ca. 200 mg/kg. These results are compared with those obtained in intraperitoneal injection methods (1.78% of MNPCEs induced at 1000 mg/kg) and are discussed with regard to the high sensitivity and the availability of the micronucleus assay as a rapid in vivo test for inhaled substances.
46 Ohara, A., G. Danno, K. Kanazawa and M. Natake, Department of Agricultural Chemistry, Faculty of Agriculture, Kobe University, Kobe (Japan)
41 Nohmi, T., K. Yoshikawa, M. Ishidate Jr., A. Hiratsuka i and T. Watabe 1, National Institute of Hygienic Sciences, Tokyo, and 1 Tokyo College of Pharmacy, Tokyo (Japan) Mechanism of species difference in bucetin mutagenicity: A key mechanistic role of deacylation by rat and hamster liver microsomes The mutagenicity of bucetin, a component of analgesic drugs, in Salmonella typhimurium TA100 was detected with $9 fractions prepared from hamsters, but not rats. A mechanism of the species difference in bucetin mutagenicity was investigated. By using high-performance liquid chromatography analysis, it was found that bucetin was activated to a direct-acting mutagen, i.e., pnitrosophenetole, through deacylation and N-hydroxylation by hamster liver microsomes. Although there was no significant species difference in N-hydroxylation, the activities of bucetin deacylation were about 140 times higher in hamsters than in rats. From these results, we suggested that the species difference in bucetin mutagenicity is due to the difference in deacylation activities between rat and hamster liver microsomes.
42 Nunoshiba, T., K. Minamisako and H. Nishioka, Biochemistry Laboratory, Doshisha University, Kyoto (Japan)
Effect of human saliva on mutagenicity of environmental carcinogens After finding the inactivating effect of human saliva on mutagenicity of carcinogens, we reported further research on this subject including the individual difference in the capacity, the role of saliva components and the inactivation mechanism. We report this time regarding (1) inactivation for the mutagenicity of amino acid-pyrolysate mutagens, and (2) a simple method for determination of inactivating capacity of individual saliva. Mutagenicity was assayed with the Ames test. Experimental procedures were similar to those described in our paper (Mutation Res., 85 (1981) 323 333). (1) Saliva obtained from a healthy donor inactivated almost 100% of Trp-P-1 mutagenicity and 30-50% of Trp-P-2 and AaC, while no significant effects were seen in Glu-P-1, Glu-P-2 and MeAaC. It seems that a peroxidase in saliva may be involved in the inactivation because this enzyme showed similar specificity in the inactivation of these pyrolysate mutagens. (2) The finding that saliva with the higher inactivating capacity possesses higher peroxidase activity led us to develop a simple method for determining the inactivating capacity of saliva; i.e., the measurement of the peroxidase activity.
43 Ochi, T., M. Mogi, M. Watanabe and M. Ohsawa, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa (Japan) Induction of chromosomal aberrations by the short-term treatment with cadmium chloride in cultured Chinese hamster cells Inducibility of chromosomal aberrations and cytotoxicity in cultured Chinese hamster cells by cadmium chloride was investigated under 3 different conditions: (1) 2-h treatment with CdC12 in MEM medium with 10% fetal bovine serum (MEM + 10% FBS) and continuous treatment for 22 h in MEM + 10% FBS, (2) 2-h treatment with CdC12 in HEPES-buffered Hanks' solution and
375 continuous treatment for 22 h in MEM + 10% FBS, and (3) 24-h treatment with CdC12 in MEM + 10% FBS. Treatments 2 and 3 were 2 and 3 times more cytotoxic, respectively, than treatment 1. Treatment 3 with CdC12 over 5 x 10 -6 was too toxic to the cells to perform chromosomal analysis and moreover only a little increase in the incidence of chromosomal aberrations was observed at 5 x 10 -6 M CdC12. In contrast, a marked and concentration-dependent increase in the incidence of chromosomal aberrations was observed in treatments 1 and 2 with 1 × 10-6-5 x 10 -6 M CdC12. Treatment 2 was approximately 3 times more potent for the induction of chromosomal aberrations than treatment 3. The 2-h treatment with CdC12 to induce chromosomal aberrations was comparable to that of benzo[a]pyrene activated with $9 at equitoxic concentrations. A study on thymidine incorporation into these cells suggests that the restoration of DNA synthesis after cadmium exposure is required for efficiently detecting the metal-induced chromosomal aberrations.
44 Oda, Y., S. Nakamura, I. Oki, K. Sugita ~, T. Kato 1, K. Makino 2, A. Nakata 2 and H. Shinagawa 2, Osaka Prefectural Institute of Public Health, Osaka, 1 Osaka University Medical School, Osaka and 2 Research Institute for Microbial Diseases, Osaka University, Osaka (Japan)
A simple test system (umu test) for the detection of environmental mutagens. II. Improvement of sensitivity by employing Salmonella typhimurium TA1535 as a host strain A rapid, simple and quantitative system ( u m u test) for the detection of environmental mutagens was developed by introducing the plasmid pSK1002 carrying u m u C ' - l a c ' Z fusion gene into S. typhimurium. The urnu test is based on in vitro assay of the fl-galactosidase synthesized from the lacZ gene fused to the umu operon responsible for inducible mutagenesis in E. coli. With this system, a variety of mutagens were examined for their induction of fl-galactosidase. Among those so far tested, base change mutagens
(methyl methanesulfonate, ethyl methanesulfonate, fl-propiolactone); frameshift mutagens (ICR191, 9-aminoacridine, benzo[a]pyrene); oxidative m u t a g e n s (bleomycin, h y d r o g e n peroxide, paraquat); mitomycin-C, 4-nitroquinoline 1-oxide, formaldehyde, a pyrolysate of tryptophan and UV light were positive. In terms of dose response, this test was approximately as sensitive as the Ames test. Polychlorinated biphenyl, DDT, safrole, thioacetamide and actinomycin D were negative. These results show that the umu test is useful as a short-term screening of environmental mutagens.
45 Odagiri, Y., and K. Takemoto, Saitama Medical School, Saitama (Japan)
Application of inhalation technique to the micronucleus assay Administration of test substances by inhalation was applied to the micronucleus assay in order to investigate the mutagenic effect of inhaled substances in vivo. The mist of ethylcarbamate (urethane) generated from an ultrasonic nebulizer was administered to mice by means of inhalation at 4 doses ranging from 67 m g / m 3 x 30 min to x 1 5 0 min. The frequency of micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow increased in a dose-dependent way by the inhalation for 24 h (1.70% of MNPCEs induced at the highest dose). The amount of mist inhaled during the period of 150 min was estimated at ca. 200 mg/kg. These results are compared with those obtained in intraperitoneal injection methods (1.78% of MNPCEs induced at 1000 mg/kg) and are discussed with regard to the high sensitivity and the availability of the micronucleus assay as a rapid in vivo test for inhaled substances.
46 Ohara, A., G. Danno, K. Kanazawa and M. Natake, Department of Agricultural Chemistry, Faculty of Agriculture, Kobe University, Kobe (Japan)