Acrylamide (AA), a proven rodent carcinogen, is found in a variety of commonly consumed human foods, which has raised public health concerns. AA is largely oxidized to the chemically reactive epoxide, glycidamide (GA), by cytochrome P450 2E1. The genotoxic effects of AA and GA have been extensively evaluated. However, the results in mammalian gene mutation tests were inconsistent, especially the genotoxic effects at the HPRT gene and TK gene. In this article, the relevant mutations induced by AA and GA on both gene loci in various test systems involving in vivo and in vitro tests are reviewed. It is confirmed that AA acts directly as a clastogen and produces weakly mutagenic effects at the HPRT gene probably by metabolic conversion of AA to GA. On the other hand, GA is a strong mutagen with high reactivity to DNA, inducing predominantly point mutations. The molecular mutation spectra of AA and GA at the HPRT and TK genes are also compared and summarized here, for better clarifying the mechanisms of mutation induced by these two compounds. These data would help to understand the mutagenicity of AA and its contribution to human cancers.
Many studies show that antigenotoxic ingredients are contained in our daily foods, including in a variety of mushrooms. Identification of antigenotoxic factors is expected to lead to the development of cancer-preventing agents. We previously demonstrated heat-unstable antimutagenic activity in a water-soluble extract of the mushroom Agrocybe cylindracea. In this study we show that antimutagenic components were precipitated in 30-40, 40-50 and 50-60% ammonium sulfate fractions of A. cylindracea extracts. The 30-40% and 40-50% precipitates appeared to contain the same substances and showed strong antimutagenic activity against 2-amino3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and N-nitrosodimethylamine in the Ames test, but not in the Drosophila in vivo DNA repair test. In contrast, the 50-60% precipitate showed antigenotoxic activity against MeIQx in Drosophila but little antigenotoxicity in bacteria. Thus, A. cylindracea contains at least two unique antimutagenic components that can be separated by ammonium sulfate precipitation. We attempted to purify the antimutagenic components contained in each ammonium sulfate fraction. The antimutagenic activity of the 30-50% ammonium sulfate fraction was detected in the flow-through fraction after application to a DEAE-Sepharose column. This fraction exhibited a single band of 27 kDa following electrophoretic analysis on a 15% SDS-polyacrylamide gel (SDS-PAGE). Sequence analysis of eight amino acids at the N-terminal of this protein indicated that this is a novel, previously unreported protein. The antimutagenic component(s) in the 50-60% ammonium sulfate fraction was eluted by 0.5 M NaCl from a DEAE-Sepharose column. As several bands were observed following SDS-PAGE analysis, we could not identify the active components in this fraction. The effects of the ammonium sulfate fraction on the activity of CYP enzymes that activate mutagens metabolically were examined: the 30-40% ammonium sulfate fraction showed strong inhibition of CYP1A, whereas the 50-60% fraction showed slight suppression of CYP1A. In conclusion, a 27-kDa protein from A. cylindracea may suppress mutation due to inhibition of the metabolism of indirect mutagens.
Transgenic rat gene-mutation assays can be used to assess genotoxicity of chemicals in target organs for carcinogenicity. Since gene mutations in transgenes are genetically neutral and thus accumulate along with treatment periods, the assays are suitable for genotoxicity risk assessment of chemicals using repeated-dose treatment methodologies. However, few studies have been conducted to examine the suitability of the assays in repeat-dose treatment protocols. In order to prove the utility of the transgenic rat assays, we treated gpt delta rats with aristolochic acid at 0.3 and 1 mg/kg by gavage daily for 28 days, and autopsied the rats 3 days after the final treatment, which is a protocol recommended by the International Workshop on Genotoxicity Testing (IWGT). Aristolochic acid exists in herbs and some other plants, and is carcinogenic in the kidney, bladder and stomach in rats. The mutant frequency (MF) in both the kidney and the liver increased significantly in a dose-dependent manner when the rats were treated with aristolochic acid. We concluded that the gpt delta rat assay is sensitive enough to detect gene mutations induced by aristolochic acid and also that the 28-day repeated-dose protocol is suitable for assessing genotoxicity of chemicals.
The transgenic rodent (TGR) assay has been widely used to study in vivo gene mutations by chemicals or radiation; however, an optimal protocol has not yet been established to assess unknown genotoxic potential. The International Workshop on Genotoxicity Testing (IWGT) strongly recommends a repeated-dose regimen for the TGR assay protocol for regulatory safety assessment as follows: a treatment period of 28 days and a sampling time of 3 days following the final treatment. In this study, TGR assays using F344 gpt delta transgenic rats were conducted at three laboratories to evaluate the validity of the IWGT protocol, as part of a collaborative study of the transgenic rat mutation assay. Male F344 gpt delta transgenic rats were orally treated with 2,4-diaminotoluene (2,4-DAT; hepatic carcinogen in rodents; 10 and 30 mg/kg/day) or 2,6-diaminotoluene (2,6-DAT; non-carcinogen in rodents; 60 mg/kg/day) once daily for 28 days. Rats were euthanized 3 days after the last dosing, and then mutant frequencies (MFs) of the gpt gene in the livers were studied. As a result, a significant increase in the MF was observed at 30 mg/kg in the 2,4-DAT-treated group, but not in the 2,6-DAT-treated group. These results were commonly observed among the three laboratories. In addition, the overall results from the three laboratories were in general agreement. These results indicate that 2,4-DAT induces gene mutation in the liver of gpt delta rats, but 2,6-DAT does not. These results also indicate that the F344 gpt delta transgenic rat mutation assay can distinguish differences in the in vivo mutagenic potential between a hepatic carcinogen and a non-carcinogen. Results from one laboratory showed more variability than those from the other two laboratories, and this appearance was due to the smaller number of colonies scored. Thus, these results demonstrate that the IWGT protocol for the TGR assays is valid, and show that consistent results are obtained among different laboratories.
This study was conducted to evaluate the effectiveness of a transgenic rat mutation assay using F344 gpt delta rats. We investigated the mutagenic potential in the lung of nickel subsulfide (Ni3S2), an insoluble fine-crystalline-metallic compound and a carcinogen in the rodent and human lung. Ni3S2 carcinogenicity has been proposed to act via both genotoxic and non-genotoxic mechanisms. Ni3S2 was intratracheally instilled into male gpt delta rats at doses of 0.5 and 1 mg/animal once a week for four weeks; these doses of Ni3S2 are high enough to induce inflammation in the lung. Following a period of 28 and 90 days after the first administration, the gpt mutant frequencies (MFs) in lung were determined in four independent laboratories, and Spi− selection for larger deletion mutations was done in one laboratory. The gpt MFs of the rats treated with Ni3S2 were not increased: all four laboratories obtained similar results with no statistical differences. The Spi− MFs were also not increased by exposure to Ni3S2. These results indicate that intratracheally instilled Ni3S2 is non-mutagenic in the lung of gpt delta transgenic rats; however, whether Ni3S2 is non-mutagenic in the lung or it induces mutations which are not detectable by transgenic rodent mutation assays requires further investigation.
The urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG) has been used as a biomarker of oxidative DNA damage in both the clinical and occupational setting. The urinary 8-OHdG in traffic policemen posted at busy traffic junctions were estimated along with the control population away from the busy traffic junctions those doing administrative job. A total of 105 urinary samples (60 samples of traffic policemen and 45 samples of control population) were collected for estimation of 8-OHdG and analyzed using enzyme linked immunosorbent assay (ELISA). The mean 8-OHdG was significantly higher (13.42±1.61) μg/g creatinine) than those of control group (9.34±1.36 μg/g creatinine) (p<0.05). The study showed that urinary 8-OHdG is associated with occupational exposure and other lifestyle factors.
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.
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