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Micronucleus test in rat hepatocytes in vivo
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22 Hayashi, M., T. Suzuki, T. Sofuni and B. Myhr i, National Institute of Hygienic Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158 (Japan) and ~ Hazleton Washington, Inc., Kensington, MD 20895 (USA) A simultaneous in vivo gene mutation and micronucleus assay on mitomycin C using transgenic mice
The micronucleus (MN) assay with peripheral blood using the acridine orange supravital staining method was combined with an in vivo gene mutation assay using transgentc mice to develop a more global genotoxicity assay system with two major endpoints, namely gene mutation and chromosomal aberrations. Here we evaluated the assay system using mitomycin C (MMC) as a model chemical. Transgenic MutaMouse animals, carrying the bacterial lacZ gene as a target, received i.p. injections of MMC on either a single occasion or once daily for 5 days, with doses of 1.0 and 2.0 mg/kg fl~r each injection. The MN and gene mutation assays were pertbrmed on the same animals. The frequencies of micronucleated reticulocytes increased in a dose-dependent manner and showed comparable sensitivity to other stains of mice. in contrast, induced mutations in the lacZ gene were not detected in either bone marrow or liver DNA after a single treatment. However, after repeated treatments, a 2-3-fold increase in mutant plaques was observed in bone marrow cells compared to control animals. These results revealed that MMC has strong clastogenic activity but is only a weak inducer of gene mutations. These results contrast with those of ethyinitrosourea which induced both MN and gene mutations. These results show that this method can be used to provide a profile of genotoxin characterization of chemicals.
23 Higashiguchi, T., Y. Kondo, S. Nito, Y. Asano and F. Ariyuki, Safety Research L:'.b~r,itory, Tan-
abe Seiyaku Co., Ltd., 16-89 Kashima, 3-chome, Yodogawa-ku, Osaka 532 (Japan) Micronucleus test in rat hepatocytes in vivo
As it is well established that the liver is the most active organ involved in the metabolism of most drugs, we postulated that concentrations of metabolites would be higher in the liver than in other organs. It has been reported that two liver carcinogens, DMN and DEN, which showed no or weak cytogenetic effects in bone marrow, showed a positive effect in the rat liver micronucleus test. In this study, we investigated whether this assay is useful in assessing cytogenicity of other chemicals. Crj : CD(SD) rats aged 7-9 weeks were administered 5 mg/kg i.p. of MMC, 50 mg/kg p.o. of CPA, 100 mg/kg i.p. of DMBA, 2.5 mg/kg i.p. of AFBI and 10 mg/kg p.o. of DMN 24 h after partial hepatectomy. Hepatoc~es (1000/rat) isolated 24-72 h after drug administration were stained with acridine orange for the micronucleus test. All chemicals showed a positive effect, with the number of cells with micronuclei showing a 2-5qbld increase over that of the respective vehicle cuntrol group. These results suggest that this assay is useful in the detection of cytogenetic effects in vivo.
24 Higashikuni, N., T. Nakamura and S. Sutou, Itoham Central Research Institute, I-2 Kubogaoka, Moriya, lbaraki 302-01 (Japan) A protocol of d.ouble sampling of peripheral reticulocytes after double treatment as the primary choice for in vivo mouse micronucleus assay: a proposal based on experiments wit.~ phenacetin (PHEN) and nitrogen mustard (NM)
As part of a collaborative study (JEMS • MMS) to develop a simple and universal protocol for the micronucleus test, PHEN or NM was given to CD-1 and MS/Ae mice; induction of micronuclei in the bone marrow (BM assay) and the peripheral reticulocytes (RET assay) was examined at
22 Hayashi, M., T. Suzuki, T. Sofuni and B. Myhr i, National Institute of Hygienic Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158 (Japan) and ~ Hazleton Washington, Inc., Kensington, MD 20895 (USA) A simultaneous in vivo gene mutation and micronucleus assay on mitomycin C using transgenic mice
The micronucleus (MN) assay with peripheral blood using the acridine orange supravital staining method was combined with an in vivo gene mutation assay using transgentc mice to develop a more global genotoxicity assay system with two major endpoints, namely gene mutation and chromosomal aberrations. Here we evaluated the assay system using mitomycin C (MMC) as a model chemical. Transgenic MutaMouse animals, carrying the bacterial lacZ gene as a target, received i.p. injections of MMC on either a single occasion or once daily for 5 days, with doses of 1.0 and 2.0 mg/kg fl~r each injection. The MN and gene mutation assays were pertbrmed on the same animals. The frequencies of micronucleated reticulocytes increased in a dose-dependent manner and showed comparable sensitivity to other stains of mice. in contrast, induced mutations in the lacZ gene were not detected in either bone marrow or liver DNA after a single treatment. However, after repeated treatments, a 2-3-fold increase in mutant plaques was observed in bone marrow cells compared to control animals. These results revealed that MMC has strong clastogenic activity but is only a weak inducer of gene mutations. These results contrast with those of ethyinitrosourea which induced both MN and gene mutations. These results show that this method can be used to provide a profile of genotoxin characterization of chemicals.
23 Higashiguchi, T., Y. Kondo, S. Nito, Y. Asano and F. Ariyuki, Safety Research L:'.b~r,itory, Tan-
abe Seiyaku Co., Ltd., 16-89 Kashima, 3-chome, Yodogawa-ku, Osaka 532 (Japan) Micronucleus test in rat hepatocytes in vivo
As it is well established that the liver is the most active organ involved in the metabolism of most drugs, we postulated that concentrations of metabolites would be higher in the liver than in other organs. It has been reported that two liver carcinogens, DMN and DEN, which showed no or weak cytogenetic effects in bone marrow, showed a positive effect in the rat liver micronucleus test. In this study, we investigated whether this assay is useful in assessing cytogenicity of other chemicals. Crj : CD(SD) rats aged 7-9 weeks were administered 5 mg/kg i.p. of MMC, 50 mg/kg p.o. of CPA, 100 mg/kg i.p. of DMBA, 2.5 mg/kg i.p. of AFBI and 10 mg/kg p.o. of DMN 24 h after partial hepatectomy. Hepatoc~es (1000/rat) isolated 24-72 h after drug administration were stained with acridine orange for the micronucleus test. All chemicals showed a positive effect, with the number of cells with micronuclei showing a 2-5qbld increase over that of the respective vehicle cuntrol group. These results suggest that this assay is useful in the detection of cytogenetic effects in vivo.
24 Higashikuni, N., T. Nakamura and S. Sutou, Itoham Central Research Institute, I-2 Kubogaoka, Moriya, lbaraki 302-01 (Japan) A protocol of d.ouble sampling of peripheral reticulocytes after double treatment as the primary choice for in vivo mouse micronucleus assay: a proposal based on experiments wit.~ phenacetin (PHEN) and nitrogen mustard (NM)
As part of a collaborative study (JEMS • MMS) to develop a simple and universal protocol for the micronucleus test, PHEN or NM was given to CD-1 and MS/Ae mice; induction of micronuclei in the bone marrow (BM assay) and the peripheral reticulocytes (RET assay) was examined at