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The effects of cAMP on the micronucleus test in mice
295 84 Suzuki, T., M. Hayashi, M. Honma, B. Myhr a and T. Sofuni, National Institute of Hygienic Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158, Japan and a Hazleton Washington, Inc., Vienna, VA 22182, USA Comparison of clastogenicity and gene mutagenicity of ENU and EMS using transgenic mice
Ethyl nitrosourea (ENU) and ethyl methanesulfonate (EMS), which are both ethylating agents, differ in their chemical reactivity and mutagenic potency. ENU possesses higher reactivity toward the 0 6 position of guanine residues through an SN1 type reaction, which is considered the basis of its stronger carcinogenicity. The correlation between the chemical reactivity of alkylating agents and their clastogenic potency, however, has not been elucidated completely. We have examined the genotoxic specificities of ENU and EMS in vivo using lacZ transgenic mice (Muta'rMMouse). The MutaMice were i.p. treated with ENU (50, 100, 200 mg/kg) and EMS (200, 400 mg/kg). Micronucleus induction in peripheral blood was examined with a small amount of blood collected from the tail after 48 h, and gene mutations in bone marrow and liver were examined after a 7-day expression period. Both ENU and EMS induced micronucleated reticulocytes and gene mutations in bone marrow. Both gene mutagenic and clastogenic potency were higher for ENU than for EMS. MFs in liver were weak for both agents, suggesting tissue specificity. This assay system enabled us to compare clastogenicity with gene mutagenicity for the test compounds and to obtain tissue-specific mutation data. 85 Suzuki, H., K. Ohsawa, C. Watanabe, M. Yamashiro, H. Watanabe, S. Nakane, J.C. Mirsalis a C.M. Hamilton a and K.L. Steinmetz a, Taisho Pharmaceutical Co., Ltd., 1-403 Yoshino-cho, Ohmiya-shi, Saitama 330, Japan and a SRI International, Menlo Park, CA 94025, USA
Mutagenicity of a new chemical with a carbazole structure
We have assayed the mutagenicity of a new psychotropic drug with a carbazole structure using the Ames Salmonella mutagenesis test, chromosome aberrations in Chinese hamster lung (CHL) cells, the mouse peripheral blood and rat bone marrow micronucleus assays, the in vivo-in vitro unscheduled DNA synthesis (UDS) assay in rat hepatocytes, and the in vitro UDS assay in human hepatocytes. Ames and chromosome aberration assays were negative in the absence of metabolic activation ($9), but were positive in the presence of $9. The effect of metabolic activation may be due to sulfate conjugation as is observed for metabolism of 7,12-dimethylbenz[a]anthracene. Both micronucleus tests were negative, but both UDS tests produced very strong positive responses. Hepatic toxicity was also observed in short-term repeat-dose toxicity studies. These results indicate that this chemical is genotoxic in both in vitro and in vivo test systems, and that the liver appears to be a principal target organ for these effects. Furthermore, these resuits demonstrate that evaluation of effects in multiple tissues is important to properly identify genotoxicity of chemicals. 86 Suzuki, Y., H. Shimizu, M. Fukumoto, H. Okonogi, Y. Seki and M. Ishii, Department of Public Health and Environmental Medicine, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-ku, Tokyo 105, Japan The effects of cAMP on the micronucleus test in mice
The aim of this experiment was to determine the mechanism of the micronucleus test in relation to erythropoiesis. We have reported that an acceleration of erythropoiesis increased the frequency of micronuclei induced by mutagens. It is reported that N6-2'-O-dibutyladenosine-3':5'cyclic monophosphate (cAMP) may accelerate the release of renal erythropoietin in vivo, giving rise to an acceleration of erythropoiesis. It may be possible that cAMP pretreatment will increase
296 the frequency of micronucleated polychromatic erythrocytes (MPCE) induced by mutagens. cAMP itself did not induce any MPCE in BALB/c mice. The highest frequency of MPCE and a dose-response relationship between cAMP concentration and MPCE frequency were observed 30 h after an injection of mitomycin C to mice which had been administered cAMP 24 h previously. The induction of MPCE in the bone marrow was also increased with three other compounds, vincristine (spindle poison), 5-fluorouracil (metabolic antagonist) and carboquone (alkylating agent) in mice pretreated with cAMP. Our present findings suggest that the increase of MPCE induced by mutagens is attributable to the acceleration of erythropoiesis. 87 Takasawa, H., Y. Uno, M. Miyagawa, Y. Inoue, T. Murata and K. Yoshikawa, Toxicology Laboratory, Life Science Research Sector, Research Center, Mitsubishi Kasei Co., 1000 Kamoshidacho, Midori-ku, Yokohama 227, Japan Rapid detection of non-mutagenic hepatocarcinogens in an in vivo-in vitro replicative DNA synthesis (RDS) test using mouse livers
We have concentrated attention on whether a mouse hepatocyte replicative DNA synthesis (RDS) test could detect non-mutagenic (the Ames-negative) mouse liver carcinogens. For this purpose male B6C3F1 mice (8 weeks old) were treated with one of 10 non-mutagenic mouse liver carcinogens by single gavage at the maximum tolerated dose (MTD, less than 2000 mg/kg) and ½MTD levels. After various time periods (0, 24, 39 and 48 h), hepatocytes were prepared by the collagenase perfusion method and incubated for 4 h in WE medium containing 3H-thymidine (370 kBq/ml). After autoradiography, the RDS incidences were monitored by counting 3H-thymidine-labeled hepatocytes. The RDS test gave positive results for 7 of the compounds: benzyl acetate, 1,4-dichlorobenzene, hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane, 1,1,2-trichloroethane and trichloroethylene (RDS incidence > 0.4%). Tetrachloroethylene, 2,4,6-trichlorophenol and tris(2-ethylhexyl) phosphate
gave negative responses (RDS incidence < 0.4%). The present results indicate that the mouse hepatocyte RDS test has application as a rapid screening method to detect non-mutagenic mouse liver carcinogens. 88 Tamakawa, K., N. Ioritani a, T. Seki, M. Kuwahara a and A. Tsunoda, Sendai Municipal Institute of Public Health, 2-5-10 Oroshimachihigashi, Wakabayashiku, Sendai 983 and a Tohoku University, School of Medicine, 2-1 Seiryocho, Aobaku, Sendai 983, Japan Effect of shock wave on the Ames test
Extracorporeal shock wave lithotripsy (ESWL) is well recognized to be one of the important clinical treatments for destruction of calculi in kidney and bladder by employing underwater shock wave focusing. It has been reported that human tissue damage occurs near the focal region of shock waves. As for the genotoxic effects of shock waves on the tissues, however, no clear evaluation has been made. In this study, mutagenic and mutagenicity modifying effects of shock waves were examined using the Ames test. Shock waves were generated by specially designed piezoceramics (diameter 300 mm, maximal pressure 100 MPa, size of half maximal pressure 1 x 1 x 19 mm). Each bacterial tester strain (S. typhimurium TA98, TA100, TA97, TA102) and a mixture of tester strain and a mutagen (cis-diaminedichloroplatinum (CDDP)) were shot by shock waves by standing at the focal point of an ESWL machine. No mutagenic response of shock waves was detected between maximal pressures of shock waves of 0 and 100 MPa, regardless of strains, with and without $9 mix. 89 Tamura, H., Y. Yamashita and K. Iwakura, Toxicology Laboratories, Nippon Shinyaku Co., Ltd., Nishiohji-Hachijo, Kyoto 601, Japan The micronucleus test with 5-nitroacenaphthene (5-NA) in peripheral blood reticulocytes
Ethyl nitrosourea (ENU) and ethyl methanesulfonate (EMS), which are both ethylating agents, differ in their chemical reactivity and mutagenic potency. ENU possesses higher reactivity toward the 0 6 position of guanine residues through an SN1 type reaction, which is considered the basis of its stronger carcinogenicity. The correlation between the chemical reactivity of alkylating agents and their clastogenic potency, however, has not been elucidated completely. We have examined the genotoxic specificities of ENU and EMS in vivo using lacZ transgenic mice (Muta'rMMouse). The MutaMice were i.p. treated with ENU (50, 100, 200 mg/kg) and EMS (200, 400 mg/kg). Micronucleus induction in peripheral blood was examined with a small amount of blood collected from the tail after 48 h, and gene mutations in bone marrow and liver were examined after a 7-day expression period. Both ENU and EMS induced micronucleated reticulocytes and gene mutations in bone marrow. Both gene mutagenic and clastogenic potency were higher for ENU than for EMS. MFs in liver were weak for both agents, suggesting tissue specificity. This assay system enabled us to compare clastogenicity with gene mutagenicity for the test compounds and to obtain tissue-specific mutation data. 85 Suzuki, H., K. Ohsawa, C. Watanabe, M. Yamashiro, H. Watanabe, S. Nakane, J.C. Mirsalis a C.M. Hamilton a and K.L. Steinmetz a, Taisho Pharmaceutical Co., Ltd., 1-403 Yoshino-cho, Ohmiya-shi, Saitama 330, Japan and a SRI International, Menlo Park, CA 94025, USA
Mutagenicity of a new chemical with a carbazole structure
We have assayed the mutagenicity of a new psychotropic drug with a carbazole structure using the Ames Salmonella mutagenesis test, chromosome aberrations in Chinese hamster lung (CHL) cells, the mouse peripheral blood and rat bone marrow micronucleus assays, the in vivo-in vitro unscheduled DNA synthesis (UDS) assay in rat hepatocytes, and the in vitro UDS assay in human hepatocytes. Ames and chromosome aberration assays were negative in the absence of metabolic activation ($9), but were positive in the presence of $9. The effect of metabolic activation may be due to sulfate conjugation as is observed for metabolism of 7,12-dimethylbenz[a]anthracene. Both micronucleus tests were negative, but both UDS tests produced very strong positive responses. Hepatic toxicity was also observed in short-term repeat-dose toxicity studies. These results indicate that this chemical is genotoxic in both in vitro and in vivo test systems, and that the liver appears to be a principal target organ for these effects. Furthermore, these resuits demonstrate that evaluation of effects in multiple tissues is important to properly identify genotoxicity of chemicals. 86 Suzuki, Y., H. Shimizu, M. Fukumoto, H. Okonogi, Y. Seki and M. Ishii, Department of Public Health and Environmental Medicine, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-ku, Tokyo 105, Japan The effects of cAMP on the micronucleus test in mice
The aim of this experiment was to determine the mechanism of the micronucleus test in relation to erythropoiesis. We have reported that an acceleration of erythropoiesis increased the frequency of micronuclei induced by mutagens. It is reported that N6-2'-O-dibutyladenosine-3':5'cyclic monophosphate (cAMP) may accelerate the release of renal erythropoietin in vivo, giving rise to an acceleration of erythropoiesis. It may be possible that cAMP pretreatment will increase
296 the frequency of micronucleated polychromatic erythrocytes (MPCE) induced by mutagens. cAMP itself did not induce any MPCE in BALB/c mice. The highest frequency of MPCE and a dose-response relationship between cAMP concentration and MPCE frequency were observed 30 h after an injection of mitomycin C to mice which had been administered cAMP 24 h previously. The induction of MPCE in the bone marrow was also increased with three other compounds, vincristine (spindle poison), 5-fluorouracil (metabolic antagonist) and carboquone (alkylating agent) in mice pretreated with cAMP. Our present findings suggest that the increase of MPCE induced by mutagens is attributable to the acceleration of erythropoiesis. 87 Takasawa, H., Y. Uno, M. Miyagawa, Y. Inoue, T. Murata and K. Yoshikawa, Toxicology Laboratory, Life Science Research Sector, Research Center, Mitsubishi Kasei Co., 1000 Kamoshidacho, Midori-ku, Yokohama 227, Japan Rapid detection of non-mutagenic hepatocarcinogens in an in vivo-in vitro replicative DNA synthesis (RDS) test using mouse livers
We have concentrated attention on whether a mouse hepatocyte replicative DNA synthesis (RDS) test could detect non-mutagenic (the Ames-negative) mouse liver carcinogens. For this purpose male B6C3F1 mice (8 weeks old) were treated with one of 10 non-mutagenic mouse liver carcinogens by single gavage at the maximum tolerated dose (MTD, less than 2000 mg/kg) and ½MTD levels. After various time periods (0, 24, 39 and 48 h), hepatocytes were prepared by the collagenase perfusion method and incubated for 4 h in WE medium containing 3H-thymidine (370 kBq/ml). After autoradiography, the RDS incidences were monitored by counting 3H-thymidine-labeled hepatocytes. The RDS test gave positive results for 7 of the compounds: benzyl acetate, 1,4-dichlorobenzene, hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane, 1,1,2-trichloroethane and trichloroethylene (RDS incidence > 0.4%). Tetrachloroethylene, 2,4,6-trichlorophenol and tris(2-ethylhexyl) phosphate
gave negative responses (RDS incidence < 0.4%). The present results indicate that the mouse hepatocyte RDS test has application as a rapid screening method to detect non-mutagenic mouse liver carcinogens. 88 Tamakawa, K., N. Ioritani a, T. Seki, M. Kuwahara a and A. Tsunoda, Sendai Municipal Institute of Public Health, 2-5-10 Oroshimachihigashi, Wakabayashiku, Sendai 983 and a Tohoku University, School of Medicine, 2-1 Seiryocho, Aobaku, Sendai 983, Japan Effect of shock wave on the Ames test
Extracorporeal shock wave lithotripsy (ESWL) is well recognized to be one of the important clinical treatments for destruction of calculi in kidney and bladder by employing underwater shock wave focusing. It has been reported that human tissue damage occurs near the focal region of shock waves. As for the genotoxic effects of shock waves on the tissues, however, no clear evaluation has been made. In this study, mutagenic and mutagenicity modifying effects of shock waves were examined using the Ames test. Shock waves were generated by specially designed piezoceramics (diameter 300 mm, maximal pressure 100 MPa, size of half maximal pressure 1 x 1 x 19 mm). Each bacterial tester strain (S. typhimurium TA98, TA100, TA97, TA102) and a mixture of tester strain and a mutagen (cis-diaminedichloroplatinum (CDDP)) were shot by shock waves by standing at the focal point of an ESWL machine. No mutagenic response of shock waves was detected between maximal pressures of shock waves of 0 and 100 MPa, regardless of strains, with and without $9 mix. 89 Tamura, H., Y. Yamashita and K. Iwakura, Toxicology Laboratories, Nippon Shinyaku Co., Ltd., Nishiohji-Hachijo, Kyoto 601, Japan The micronucleus test with 5-nitroacenaphthene (5-NA) in peripheral blood reticulocytes