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The usefulness of the rat repeated-dose liver and gastrointestinal (GI) tract micronucleus (MN) tests to detect site-specific carcinogens has been shown previously using 22 chemicals in a study conducted by the Mammalian Mutagenicity Study group in the Japanese Environmental Mutagen Society. However, in the 6th International Workshop on Genotoxicity Testing, the need for further data to identify the sensitivity and specificity of the GI tract MN test and the specificity of the liver MN test, for the purpose of regulatory use, was mentioned. In the present study, we conducted additional studies to validate the performance of the 28-day repeated-dose GI tract and liver MN tests using genotoxic stomach carcinogens, N-nitroso-N-methylurea (MNU) and N-methyl-N-nitrosourethane (NMUT); genotoxic colon carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine hydrochloride (PhIP), and non-carcinogens, sodium chloride, sucrose, and amaranth. Male Crl:CD (SD) rats were administered with each chemical by oral gavage for 28 days and the micronucleated cell frequencies in the glandular stomach, colon, and liver were monitored. MNU and NMUT showed positive results in the glandular stomach, and PhIP did so in the colon. These carcinogens showed negative results in the liver, which is not a target organ for these chemicals. Negative results were obtained for all three non-carcinogens in the glandular stomach, colon, and liver. Therefore, it was shown that the glandular stomach and colon MN tests with 28-day repeated-dose regimen have a good sensitivity for detecting genotoxic GI tract carcinogens as positive and have a good specificity to determined non-carcinogens as negative. The negative results with these six chemicals in the liver provide additional evidence supporting the good specificity of the 28-day repeated-dose liver MN test., (Copyright © 2019 Elsevier B.V. All rights reserved.)

At the 6th International Workshop on Genotoxicity Testing, the liver micronucleus test (MNT) working group briefly discussed the MNT using tissues other than liver/erythrocytes. Many tissues other than liver/erythrocytes have been studied, primarily for research purposes. They have included the colon and intestinal epithelium, skin, spleen, lung, stomach, bladder, buccal mucosa, vagina, and fetal/neonatal tissues. These tissues were chosen because they were target sites of carcinogens, and/or relevant to a specific route of exposure. Recently, there has been particular focus on the gastrointestinal (GI) tract as it is a contact site associated with high exposure following oral gavage. Furthermore GI tumors are observed with high frequency in human populations. A collaborative study of the rat glandular stomach and colon MNT was conducted in conjunction with a collaborative study of the repeated-dose liver MNT. Based on limited data currently available, the rodent MNT using the glandular stomach and/or colon seems to detect genotoxic carcinogens with GI tract target-organ specificity. The working group concluded that the GI tract MNT would be a promising method to examine clastogenicity or aneugenicity of test chemicals in the stomach and/or colon. Further data will be needed to fully establish the methods, and to identify the sensitivity and specificity of the GI tract MNT., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a direct-acting mutagen that induces tumors in the glandular stomach, but not in the liver or colon, of rats after oral administration. To evaluate the performance of repeated dose liver and gastrointestinal tract micronucleus (MN) assays in young adult rats, MNNG was administered by oral gavage to male CD (SD) rats aged 6 weeks at doses of 0 (vehicle; 2.5% DMSO aqueous solution), 3.125, 6.25, 12.5, and 25mg/kg/day once daily for 14 and 28 days, and the MN frequencies were examined in the hepatocytes, glandular stomach cells, and colonic cells. The MN induction in immature erythrocytes in the bone marrow of these animals was also simultaneously evaluated. The frequencies of micronucleated (MNed) glandular stomach cells were significantly increased in all MNNG treatment groups in a dose-dependent manner in both repeated dose studies. In contrast, the frequencies of MNed hepatocytes and colonic cells were not significantly increased compared to the vehicle control. In the bone marrow, a small but significant increase in the frequency of MNed immature erythrocytes was observed only at the highest dose in the 28-day study. Since a clear positive result in the glandular stomach agrees with the tissue specificity of tumor induction by this chemical, the MN assay with the glandular stomach, which is a direct contact site with high concentrations of test substances administered by oral gavage, may be useful for detecting genotoxic compounds that are short-lived in vivo, such as MNNG., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Our aim was to develop a multi-tissue micronucleus (MN) test method for the simultaneous analysis of rat glandular stomach, colon, and bone marrow. We have evaluated the multi-tissue MN test method with a regimen in which rats were administered chemicals orally once per day for four days and the cells of each tissue were collected 24 h after the final dose. The following compounds were studied: N-nitroso-N-methylurea (MNU), 4-nitroquinoline-1-oxide (4NQO), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N-nitrosourethane (NMUT), 1,2-dimethylhydrazine 2HCl (DMH), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine HCl (PhIP), KBrO(3), amaranth (AM), and quercetin (QN). The gastrointestinal tract carcinogens increased the frequencies of micronucleated (MNed) cells in target tissue in a dose-dependent manner: MNU in gastric- and colonic-cells; 4NQO, MNNG, and NMUT in gastric cells; DMH and PhIP in colonic cells. In immature erythrocytes, MNU, 4NQO, DMH, and PhIP increased the frequency of MNed cells but MNNG and NMUT did not. The food additive KBrO(3), which is known to be a renal carcinogen, increased the frequencies of MNed cells in the glandular stomach and bone marrow. The food additive AM and the plant flavonoid QN, which are non-carcinogenic in most studies, did not cause increased MNed cells in any of the three tissues. Our results indicate that this multi-tissue MN test method is useful for the comprehensive evaluation of the genotoxicity of orally administered compounds., (Copyright © 2013 Elsevier B.V. All rights reserved.)

To assess the genotoxicity of chemicals on the stomach, we developed in vivo assays that can detect micronucleus induction and gene expression changes in epithelial cells of the glandular stomach in mice. Male BALB/c mice were orally given a single dose (100 mg/kg) of N-nitroso-N-methylurea (MNU) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as stomach-targeted carcinogens. The glandular stomach was excised at 4h, 3 and 4 days after administration, and a single cell suspension of epithelial cells was prepared from the everted glandular stomach by EDTA treatment. For determination of micronucleus induction, gastric epithelial cells on days 3 and 4 after administration were fixed with 10% neutral-buffered formalin, stained with a combination of AO-DAPI, and analyzed under fluorescence microscopy. We also examined the induction of micronuclei in peripheral blood of these mice on days 2 and 3 after administration. Moreover, total RNA was extracted from gastric epithelial cells at 4h after administration, and p21 and plk2 expression was analyzed using a quantitative RT-PCR technique. 1) A significant increase of micronucleated cells was observed in the glandular stomach in mice treated with N-nitroso-N-methylurea (MNU) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) compared to mice treated with vehicle. 2) In peripheral blood, induction of micronuclei was observed in mice treated with MNU but not with MNNG. 3) p21 and plk2, which related to cell cycle arrest, were up-regulated in the glandular stomach in mice treated with MNU or MNNG compared to mice treated with vehicle. The present study showed that these assays using glandular stomach may help to evaluate the genotoxicity of chemicals after oral administration.