Hiroyasu Shimada

Evaluation of the rodent micronucleus assay in the screening of IARC carcinogens (Groups 1, 2A and 2B): The summary report of the 6th collaborative study by CSGMT/JEMS·MMS

To assess the correlation between micronucleus induction and human carcinogenicity, the rodent micronucleus assay was performed on known and potential human carcinogens in the 6th MMS/CSGMT collaborative study. Approximately 100 commercially available chemicals and chemical groups on which there was little or no micronucleus assay data were selected from IARC (International Agency for Research on Cancer) Groups 1 (human carcinogen), 2A (probable human carcinogen) and 2B (possible human carcinogen). As minimum requirements for the collaborative study, 5 male mice were treated by intraperitoneal injection or oral gavage once or twice with each chemical at three dose levels, and bone marrow and/or peripheral blood was analyzed. Five positives and 2 inconclusives out of 13 Group 1 chemicals, 7 positives and 5 inconclusives of 23 Group 2A chemicals, and 26 positives and 6 inconclusives of 67 Group 2B chemicals were found. Such low positive rates were not surprising because of a test chemical selection bias, and we excluded well-known micronucleus inducers. The overall evaluation of the rodent micronucleus assay was based on the present data combined with published data on the IARC carcinogens. After merging, the positive rates for Groups 1, 2A and 2B were 68.6, 54.5 and 45.6%, respectively. Structure-activity relationship analysis suggested that the micronucleus assay is more sensitive to the genetic toxicity of some classes of chemicals. Those to which it is sensitive consist of (1) aziridines and bis(2-chloroethyl) compounds; (2) alkyl sulfonate and sulfates; (3) acyl-type N-nitroso compounds; (4) hydrazines; (5) aminobiphenyl and benzidine derivatives; and (6) azo compounds. Those to which it is less sensitive consist of (1) dialkyl type N-nitroso compounds; (2) silica and metals and their compounds; (3) aromatic amines without other functional groups; (4) halogenated compounds; and (5) steroids and other hormones. After incorporation of structure-activity relationship information, the positive rates of the rodent micronucleus assay became 90.5, 65.2 and 60.0% for IARC Groups 1, 2A and 2B, respectively. Noteworthy was the tendency of the test to be more sensitive to those carcinogens with stronger evidence human carcinogenicity.

Mouse Lymphoma thymidine kinase locus gene mutation assay : International Workshop on Genotoxicity Test Procedures Workgroup Report

The Mouse Lymphoma Assay (MLA) Workgroup addressed and reached consensus on a number of issues. Discussion focused on five areas: (1) acceptable assay versions; (2) cytotoxicity measure; (3) 24‐hr treatment; (4) microwell colony counting and sizing; and (5) data acceptability/statistical analysis. Although the International Conference on Harmonisation (ICH) indicated a preference for the microwell over the soft agar method, all of the workgroup members agreed that both versions of the MLA are equally acceptable. The workgroup agreed that it is desirable for both assay versions to use the same measure of cytotoxicity to define the acceptable and required concentration range. Currently, laboratories using the microwell version use the relative survival (RS) determined by cloning immediately after the treatment. Laboratories using the soft agar method do not obtain an RS but use the relative total growth (RTG), a combination of the relative suspension growth (RSG) during the expression period and the relative cloning efficiency determined at the time of mutant selection. The workgroup agreed to investigate the RSG, the RS, and the RTG and to develop further guidance. In the interim, the workgroup reached consensus that the RTG be used as the standard measure of cytotoxicity. The ICH recommended a 24‐hr treatment in the absence of S9 when negative results are obtained with short (3–4 hr) treatments. The workgroup agreed to retain this requirement but acknowledged that more data are needed prior to making final recommendations concerning the need for and the specific protocol for the 24‐hr treatment. Environ. Mol. Mutagen. 35:185–190, 2000 Published 2000 Wiley‐Liss, Inc.

Effect of aging on spontaneous micronucleus frequencies in peripheral blood of nine mouse strains: the results of the 7th collaborative study organized by CSGMT/JEMS · MMS*

The spontaneous frequencies of micronucleated reticulocytes (MNRETs) were examined monthly over the life spans of animals belonging to nine mouse strains for the 7th collaborative study organized by the CSGMT/JEMS.MMS. Both sexes of the BDF1 strain and females of the A/J strain showed a statistically significant increase in mean spontaneous MNRET frequency in their last month of life, suggesting the possibility of strain-specific, age-dependent chromosomal instability. SAMP6/Tan, an accelerated senescence-prone strain, showed the same tendency, although it was not statistically significant. The other strains studied, ddY, CD-1, B6C3F1, SAMR1, and MS/Ae, did not show significant age-related differences in mean of MNRET frequencies. More extensive statistical analyses are underway, and the outcomes will be reported separately.

In vitro photochemical clastogenicity of quinolone antibacterial agents studied by a chromosomal aberration test with light irradiation.

The photochemical clastogenic potential of 12 quinolone antibacterial agents with or without light irradiation was assessed by an in vitro chromosomal aberration test using cultured CHL cells. Exposure to all test compounds, except for DK-507k, increased the incidence of cells with structural aberrations excluding gap (TA) following light irradiation. Test compounds used in the present study under light irradiation were divided into three groups based on their ED(50) values, doses inducing chromosomal aberrations in 50% of cells. The first group with ED(50) values below 30 microg/ml includes sparfloxacin (SPFX), clinafloxacin (CLFX), gemifloxacin (GMFX), lomefloxacin (LFLX), sitafloxacin (STFX), grepafloxacin (GPFX) and fleroxacin (FLRX); the second group with ED(50) values of 100 microg/ml, enoxacin (ENX) and levofloxacin (LVFX); the third group with little or no potency, moxifloxacin (MFLX), trovafloxacin (TVFX) and DK-507k. The photochemical clastogenicity of these compounds correlates well with their reported in vivo phototoxic potentials. In the chemical structure and clastogenicity relationships, substitution of a methoxy group at the C-8 position in the quinolone nucleus was confirmed to reduce not only photochemical clastogenicity, but also the clastogenic potential of quinolone antibacterial agents.

Ethyl nitrosourea and methyl methanesulfonate mutagenicity in sperm and testicular germ cells of lacZ transgenic mice (Muta Mouse).

The germ cell mutagens ethyl nitrosourea (ENU) and methyl methanesulfonate (MMS), were tested for their genotoxicity in sperm cells and testicular germ cells using lacZ transgenic mice (Muta Mouse). Eight- to 10-week-old Muta mice were treated with ENU (150 mg/kg) or MMS (40 mg/kg) by intraperitoneal injection. Three and 14 days after treatment, testes and sperm were collected for lacZ mutation analysis. Sperm were isolated from the epididymis and vas deferens by washing out the minced tissue. Germ cell DNA was isolated from testicular germ cells and sperm with the help of 2-mercaptoethanol, and the target lacZ gene, which is integrated into a lambda shuttle vector, was recovered by in vitro packaging. The resultant phages were allowed to infect to E. coli C (galE), and the lacZ mutant plaques were dominantly selected on a plate containing phenyl-beta-D-galactoside. Spontaneous mutant frequencies (MF) in vehicle-treated control mice were approximately 1 x 10(-5) and 3 x 10(-5) in testicular germ cells and sperm, respectively, at both sampling times. ENU treatment increased the MF in the testicular germ cells to 5 x 10(-5) on days 3 and 14, but did not affect sperm MF. MMS was not mutagenic in either tissue. The peripheral blood micronucleus assay was performed on the same animals 48 h after treatment, and strong inductions of micronucleated reticulocytes (MNRETs) were observed in both ENU- and MMS-treated mice. These data suggest that agents mutagenic to premeiotic germ cells, e.g., ENU, can be detected by transgenic mutation assay system using germ cells isolated from the testis. On the other hand, those mutagenic to postmeiotic cells, e.g., MMS, are insensitive in the assay system.

Bone marrow and liver mutagenesis in lacZ transgenic mice treated with hexavalent chromium

The mutagenic effects of the hexavalent chromium compound K2CrO4 in lacZ transgenic mice (Muta Mouse) were investigated at two sampling times. K2CrO4 was administered intraperitoneally to five male mice per treatment group at a single dose of 40 mg/kg. The animals were sacrificed on days 1 and 7 after the treatment. Mutant frequencies in the bone marrow and liver were analyzed by the positive selection method using Escherichia coli C (galE-) strain and phenyl beta-D-galactoside. K2CrO4 induced a significant increase in mutant frequency in the bone marrow on day 1, but not on day 7 after the treatment. In the liver, on the other hand, a significant induction in the mutant frequency was seen on day 7, whereas no induction was observed on day 1. The reason for the different responses to the mutagenic activity of K2CrO4 between these organs may be related to their cell turnover rates. The mutations induced by K2CrO4 in the bone marrow may have occurred in more differentiated cells than stem cells, and the rapid proliferative activity may have caused a rapid decrease in mutated cells by day 7. These results suggest that experiments on mutagenesis should be done with more than one sampling point, a short expression time in addition to a longer one, so as to detect mutations induced in organ with high cell proliferation.

Clastogenicity and mutagenicity of hexavalent chromium in lacZ transgenic mice

The clastogenic and mutagenic effects of the hexavalent chromium compound K2CrO4 in lacZ transgenic mice (Muta Mouse) were investigated. Male Muta mice were administered an intraperitoneal dose of 40 mg/kg of K2CrO4 once on each of 2 consecutive days. The K2CrO4 induced a significant increase in the peripheral blood micronucleated reticulocyte count. Also, K2CrO4 induced a statistically significant increase in mutant frequency in the liver but not in the bone marrow on day 7 after the second treatment. The reason for the failure to increase the mutant frequency in the bone marrow may have been the rapid cell turnover rate there. The mutation induced by K2CrO4 in the bone marrow may have occurred in more differentiated cells than stem cells, and the rapid proliferative activity may have caused a rapid decrease in mutated cells by day 7. Further study with a sampling point earlier than day 7 is needed. The results obtained in the present study indicate that K2CrO4 has clastogenic and mutagenic potential in vivo.

Germ cell mutagenesis in lacZ transgenic mice treated with methyl methanesulfonate.

Mutagenesis induced by methyl methanesulfonate (MMS), a germ cell mutagen, in the testis and the sperm isolated from epididymis and vas deferens have been investigated using lacZ transgenic mice (Muta Mouse). Male Muta Mice were injected intraperitoneally with MMS at a dose of 80 mg/kg, a potent dominant lethal dose. Animals were killed on days 3 and 7 (Experiment 1) or days 10 and 14 (Experiment 2) after the treatment. Mutant frequencies (MFs) in the testis, sperm and spleen (Experiment 2 only) were analyzed by the positive selection system using E. coli C (GalE-) strain and phenyl beta-D-galactoside. The spontaneous MFs in the testis and sperm were 2.0-3.1 x 10(-5). No induction of mutation in the testis or sperm of the MMS-treated groups was observed at any sampling point. In the spleen, the spontaneous MF was approximately twice as high as that in the germ cells although the MF at each sampling point was almost the same as the spontaneous MF. MMS is known as a potent clastogen from the results of the dominant lethal assay and the micronucleus assay. The reason for the discrepancy between the results of these assays and the present results may have been insensitivity of the in vitro packaging to large deletion due to the failure to rescue the large deleted gene. It is suggested that the transgenic mouse assay using the in vitro packaging can not replace the dominant lethal assay in the case of MMS.

N-Nitrosodi-n-propylamine induces organ specific mutagenesis with specific expression times in lacZ transgenic mice

The mutagenic and clastogenic effects of N-nitrosodi-n-propylamine (NDPA) in lacZ transgenic mice (MutaMouse) were investigated as a part of the second collaborative study of the transgenic mouse mutation assay by a subgroup of the Mammalian Mutagenesis Study Group, a suborganization of the Environmental Mutagen Society of Japan. Male MutaMouse mice were administered NDPA intraperitoneally at a dose of 250 mg/kg, which is half of the LD(50) of the compound. The clastogenicity of NDPA was examined by the peripheral blood micronucleus test just before and at 24, 48 and 72 h after the treatment. The mutant frequencies in the bone marrow, liver, lung, kidney and urinary bladder were examined by the positive selection method for lacZ kidney. These findings demonstrate that NDPA induces organ-specific mutagenesis with specific expression times, and that the mutagenicity of NDPA in lacZ transgenic mice is consistent with its carcinogenicity.

Effect of aging on spontaneous micronucleus frequencies in peripheral blood of nine mouse strains: the results of the 7th collaborative study organized by CSGMT/JEMS · MMS: Mutation Res. 338 (1995) 51–57☆☆☆

Abstract The spontaneous frequencies of micronucleated reticulocytes (MNRETs) were examined monthly over the life spans of animals beloning to nine mouse strains for the 7th collaborative study organized by the CSGMT/JEMS · MMS. Both sexes of the BDF1 strain and females of the A/J strain showed a statistically significant increase ni mean spontaneous MNRET frequency in their last month of life, suggesting the possibility of strain-specific, age-dependent chromosomal instability. SAMP6/Tan, an accelerated senescence-prone strain, showed the same tendency, although it was not statistically significant. The other strains studied, ddY, CD-1, B6C3F1, SAMR1, and MS/Ae, did not show significant age-related differences in mean of MNRET frequencies. More extensive statistical analyses are underway, and the outcomes will be reported separately.