Mammalian Mutagenicity Study Group Research Articles

Evaluation of a repeated dose liver micronucleus assay in rats treated with two genotoxic hepatocarcinogens, dimethylnitrosamine and 2-acetylaminofluorene: The possibility of integrating micronucleus tests with multiple tissues into a repeated dose general toxicity study

As part of a collaborative study by the Collaborative Study Group for Micronucleus Test (CSGMT) of the Mammalian Mutagenicity Study Group (MMS) in the Japanese Environmental Mutagen Society (JEMS), the present study evaluated the effectiveness of the repeated dose liver micronucleus (RDLMN) assay. Two genotoxic hepatocarcinogens, dimethylnitrosamine (DMN) and 2-acetylaminofluorene (2-AAF), were administered orally to male rats (6 weeks old at the initial dosing) once daily for 14 and 28 days to evaluate the micronucleus (MN) inducibility in the liver. In addition, these chemicals were evaluated for MN inducibility in the bone marrow (BM) and gastrointestinal (GI) tract, i.e. glandular stomach and colon of the same animals used in the RDLMN assay. As a result, both chemicals produced positive results in the liver, although a weak positive response was given by 2-AAF. DMN gave negative results in the tissues other than the liver. 2-AAF produced positive responses in the BM and glandular stomach, and a prominent response was particularly observed in the glandular stomach, which is directly exposed to the test chemicals by gavage. The present results suggest that the RDLMN assay is a useful method for detecting genotoxic hepatocarcinogens, and that it is especially effective for evaluating test chemicals, such as DMN, undetectable by the BM and GI tract MN assay. Moreover, the results in this investigation indicate that the use of multiple tissues in the study integrating the MN tests is more effective than using a single tissue, for detection of the MN induction produced by chemical exposure to rats, and helps to determine the characteristics of the test chemicals.

Assessment of methyl methanesulfonate using the repeated-dose liver micronucleus assay in young adult rats

A repeated-dose liver micronucleus assay using young adult rats was conducted with methyl methanesulfonate (MMS) as a part of a collaborative study supported by the Collaborative Study Group for the Micronucleus Test/the Japanese Environmental Mutagen Society–Mammalian Mutagenicity Study Group. MMS is a classical DNA-reactive carcinogen, but it is not a liver carcinogen.In the first experiment (14-day study), MMS was administered per os to 6-week-old male Crl:CD (SD) rats every day for 14 days at a dose of 12.5, 25, or 50mg/kg/day. In the second experiment (28-day study), 6-week-old male SD rats were treated with MMS at 7.5, 15, or 30mg/kg/day for 28 days, because the highest dose used in the 14-day study (50mg/kg/day) caused mortality. Hepatocyte and bone marrow cell specimens were prepared on the day after the final dose. The frequency of micronucleated hepatocytes (MNHEPs) in the liver and that of micronucleated immature erythrocytes (MNIMEs) in the bone marrow were evaluated. Exposure to 50mg/kg/day MMS for 14 days resulted in an increased frequency of MNHEPs, but MMS had no effect on the frequency of MNHEPs in the rats exposed to the chemical for 28 days at doses up to 30mg/kg/day. MMS induced MNIMEs production at doses of 25 and 50mg/kg/day in the 14-day study and at doses of 15 and 30mg/kg/day in the 28-day study. Overall, the effect of MMS on the frequency of MNHEPs was considered to be equivocal.

Evaluation of the repeated-dose liver micronucleus assay using N-nitrosomorpholine in young adult rats: Report on collaborative study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Japanese Environmental Mutagen Society (JEMS)—Mammalian Mutagenicity Study (MMS) Group

The present study was conducted to evaluate the suitability of a repeated-dose liver micronucleus (LMN) assay in young adult rats as a collaborative study by the Mammalian mutagenicity study (MMS) group. All procedures were performed in accordance with the standard protocols of the MMS Group. Six-week-old male Crl:CD(SD) rats (5 animals/group) received oral doses of the hepatocarcinogen N-nitrosomorpholine (NMOR) at 0 (control), 5, 10, and 30mg/kg/day (10mL/kg) for 14 days. Control animals received vehicle (water). Hepatocytes were collected from the liver 24h after the last dose, and the number of micronucleated hepatocytes (MNHEPs) was determined by microscopy. The number of micronucleated immature erythrocytes (MNIMEs) in the femoral bone marrow was also determined. The liver was examined using histopathologic methods after formalin fixation. The results showed statistically significant and dose-dependent increases in the number of MNHEPs in the liver at doses of 10mg/kg and greater when compared with the vehicle control. However, no significant increase was noted in the number of MNIMEs in the bone marrow at doses of up to 30mg/kg. Histopathology of the liver revealed hypertrophy and single cell necrosis of hepatocytes at doses of 5mg/kg and above. These results showed that the induction of micronuclei by NMOR was detected by the repeated-dose LMN assay, but not by the repeated-dose bone marrow micronucleus assay.

Evaluation of repeated dose micronucleus assays of the liver and gastrointestinal tract using potassium bromate: A report of the collaborative study by CSGMT/JEMS.MMS

The food additive potassium bromate (KBrO3) is known as a renal carcinogen and causes chromosomal aberrations in vitro without metabolic activation and in vivo in hematopoietic and renal cells. As a part of a collaborative study by the Mammalian Mutagenicity Study group, which is a subgroup of the Japanese Environmental Mutagen Society, we administered KBrO3 to rats orally for 4, 14, and 28 days and examined the micronucleated (MNed) cell frequency in the liver, glandular stomach, colon, and bone marrow to confirm whether the genotoxic carcinogen targeting other than liver and gastrointestinal (GI) tract was detected by the repeated dose liver and GI tract micronucleus (MN) assays. In our study, animals treated with KBrO3 showed some signs of toxicity in the kidney and/or stomach. KBrO3 did not increase the frequency of MNed cells in the liver and colon in any of the repeated dose studies. However, KBrO3 increased the frequency of MNed cells in the glandular stomach and bone marrow. Additionally, the MNed cell frequency in the glandular stomach was not significantly affected by the difference in the length of the administration period. These results suggest that performing the MN assay using the glandular stomach, which is the first tissue to contact agents after oral ingestion, is useful for evaluating the genotoxic potential of chemicals and that the glandular stomach MN assay could be integrated into general toxicity studies.

Tissue Sample Preparation for In Vivo Rodent Alkaline Comet Assay

The Japanese Environmental Mutagen Society/the Mammalian Mutagenicity Study group conducted a collaborative study to investigate whether cell nuclei or whole cells might be more suitably used to correctly detect genotoxic chemicals in the in vivo rodent alkaline Comet assay. Four participating laboratories applied four sample processing methods, i.e., three homogenization methods using the usual Potter-type shaft, a customized (loose) Potter-type shaft, or a Downs-loose-type shaft, for preparing cell nuclei, and the mesh membrane method for preparing whole cells, to the male rat liver. Homogenization with the usual Potter-type shaft clearly produced damage of the cell nuclei and DNA, while the other three methods seemed to provide similar conditions of the tissue samples. The proportion of cell nuclei: whole cells was 80-90%: 10-20% in all laboratories when the samples were prepared by homogenization using a Downs-loose-type shaft or by the mesh membrane method. The %DNA in tail were comparable in both samples among the negative control groups (single oral administration with physiological saline) of all laboratories, and showed an equal degree of increase in both samples of the ethyl methanesulfonate groups (single oral administration at 250 mg/kg) in all laboratories. In conclusion, the homogenization method using a loosely customized Potter-type shaft or a Downs-loose-type shaft, and the mesh membrane method would be equally acceptable for the in vivo rodent alkaline Comet assay.

Evaluation of a liver micronucleus assay in young rats (IV): A study using a double-dosing/single-sampling method by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Japanese Environmental Mutagen Society (JEMS)–Mammalian Mutagenicity Study Group (MMS)

A collaborative study was conducted to evaluate whether a liver micronucleus assay using four-week-old male F344 rats can be used to detect genotoxic rat hepatocarcinogens using double-dosing with a single-sampling 4 days after the second dose. The assay methods were thoroughly validated by the seven laboratories involved in the study. Seven chemicals, 2,4-diaminotoluene, diethyl nitrosamine, p-dimethylaminoazobenzene, 1,2-dimethylhydrazine dihydrochloride, 2,4-dinitrotolunene, 2,6-dinitrotoluene and mitomycin C, known to produce positive responses in the single-dosing/triple-sampling method were selected for use in the present study, and each chemical was examined in two laboratories with the exception of 2,4-dinitrotolunene. Although several of the compounds were examined at lower doses for reasons of toxicity than in the single-dosing/triple-sampling method, all chemicals tested in the present study induced micronuclei in liver cells indicating a positive result. These findings suggest that the liver micronucleus assay can be used in young rats to detect genotoxic rat hepatocarcinogens using a double-dosing/single-sampling procedure. Further, the number of animals used in the liver micronucleus assay can be reduced by one-third to a half by using the double-dosing/single-sampling method. This reduction in animal numbers also has significant savings in time and resource for liver perfusion and hepatocyte isolation.

Evaluation of a liver micronucleus assay in young rats (III): A study using nine hepatotoxicants by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Japanese Environmental Mutagen Society (JEMS)–Mammalian Mutagenicity Study Group (MMS)

We have been investigating a liver micronucleus assay to detect genotoxic chemicals using young rats for several years, and had established its advantages with respect to using autonomous proliferation of young rat hepatocytes. Nine chemicals known to induce hepatotoxic effects such as necrosis (2,6-dinitrotolune, bromobenzene, isoniazid, phenacetin, allyl alcohol and thioacetamide), cholestasis (chlorpromazine hydrochloride and α-naphthyl isothiocyanate) and oxidative stress (clofibrate) were selected for this study. A liver micronucleus assay was conducted in 4-week-old male F344 rats using two or three dose levels of test chemicals given orally by gavage to evaluate the compound's ability to induce micronucleated hepatocytes. Several of these test chemicals were additionally examined in a peripheral blood micronucleus assay conducted concurrently and in the same animals. The genotoxic rodent hepatocarcinogen, 2,6-dinitrotoluene showed a positive result in the liver micronucleus assay, but the nongenotoxic hepatocarcinogens, clofibrate and thioacetamide gave negative responses. Bromobenzene, known to produce DNA adducts but is noncarcinogenic in rodent liver, was judged equivocal in this assay. α-Naphthyl isothiocyanate is noncarcinogenic and showed negative response in the liver. The other four chemicals, known to be either noncarcinogenic or carcinogenic in other non-liver target organs, showed negative results in the liver micronucleus assay. Based on the results in the present study and previous report described above, it was concluded that this technique is able to effectively predict genotoxic rodent hepatocarcinogenicity, and does not give false positives due to hepatotoxicity.

Evaluation of liver and peripheral blood micronucleus assays with 9 chemicals using young rats: A study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Japanese Environmental Mutagen Society (JEMS)—Mammalian Mutagenicity Study Group (MMS)

We conducted simultaneous liver and peripheral blood micronucleus assays in young rats with seven rodent hepatocarcinogens—4,4′-methylenedianiline (MDA), quinoline, o-toluidine, 4-chloro-o-phenylenediamine (CPDA), dimethylnitrosamine (DMN), p-dimethylaminoazobenzene (DAB), and di(2-ethylhexyl)phthalate (DEHP)—and two mutagenic chemicals—kojic acid and methylmethanesulfonate (MMS).Quinoline, DMN, and DAB were positive in the liver assay, while o-toluidine, kojic acid, DAB, and MMS were positive in the peripheral blood assay. o-Toluidine, kojic acid, and DAB are reportedly negative in mouse bone marrow micronucleus assays, indicating a species difference.Our results revealed a correlation between micronucleus induction in hepatocytes and hepatocarcinogenicity. This technique can be useful for the detection of micronucleus-inducing chemicals that require metabolic activation, and it enables simultaneous comparison of the micronucleus-inducing potential of chemicals in the liver and peripheral blood in the same individual.

What is required of JEMS?

The constitution of the Japanese Environmental Mutagen Society (JEMS) says, “The society aims to encourage basic research on mutagens in humans, other organisms, and the environment, especially mutagens that affect public health. It also aims to further good communication and the transfer of research techniques in related subjects” (Chapter 4). I believe that JEMS should follow these written objectives of the society precisely and honestly. The main theme of the 31st JEMS annual meting is, “Challenges to Internationalization and Humanization”, and it is the current requirement to the society that member want. On the matter of “Internationalization”, we held the 8th ICEM at Shizuoka last year under the most difficult circumstances and had great success, providing evidence that JEMS is effective internationally. We also played an important role in having our research in this field reflected in regulation considered in international harmonizing activities (i.e., International Workshop on Genotoxicity Testing). On the other hand, to take care global problems, e.g., air and water, international cooperation is important. Our society, however, has made little effort to cooperate with the Asian EM societies with except for a few individuals. JEMS should seek ways to collaborate on problems we share with these countries. We could possibly be a hub for communication between societies that are located in Asian countries. In an effort to do that, we organized the “Asian Environmental Mutagen Societies Forum”, hoping that it would eventually head to an Asian Association of Environmental Mutagen Societies.The topic of “Humanization” is a most important one. The ultimate goal of our field is the maintaining the highest possible quality of life (QOL), and that includes maintains the environment that affects our QOL directly or indirectly in good condition. For this purpose our society should focus more on human risk assessment, including exposure assessments of environmental mutagens, and molecular epidemiology.In addition to the main issues mentioned above, our society should orient our research more to the genome than to the detection of environmental mutagens. Also, I think that the society should contribute to regulatory requirement. This includes organizing validation studies of novel technology and establishing strategies on how to evaluate and interpret test data.From the administrative viewpoint, the organization of JEMS should be more transparent and the responsibilities of the executive and the council should be clarified. Moreover, we need to establish a mechanism for gathering opinions from the membership. On the web, we have started to improve our Home Page and mailing system to permit better communication between the executive board and society members and also among members.Lastly, I would like to mention the society’s important research activities. Our sub-organizations include the Mammalian Mutagenicity Study Group (MMS), the Bacterial Mutagenicity Study Group (BMS), the Nongenotoxic Carcinogen Study Group (NGCS), the Antimutagenesis and Anticarcinogenesis Study Group (AAS), the FISH Study Group, and Mutag 21. Activation of JEMS, we support all thee groups as official activities of JEMS. Finally, I believe, the most important factor for an active society is the individual contribution of each society member.