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A new in vivo gene mutation assay has been developed based on the phosphatidylinositol glycan anchor biosynthesis, Class A gene ( Pig-a in rodents) as an endogenous reporter. Using this Pig-a assay, the in vivo mutagenicity of a single dose of azathioprine (Aza) was investigated in red blood cells (RBC Pig-a assay) and reticulocytes (PIGRET) of rats. Eight-week old male rats were orally dosed once with Aza at 50, 100 and 200 mg/kg or ethylnitrosourea (ENU) at 10 and 40 mg/kg as a positive control. Because 4 out of 6 animals at 200 mg/kg of Aza died 3 days after the dosing, this dose group was excluded for analyses. The frequencies of Pig-a mutants in RBCs and reticulocytes (RET) were evaluated once a week for 4 weeks after the treatment. With a single exposure to ENU, the frequencies of Pig-a mutants in both RBCs and RETs increased in a time- and dose-dependent manner. In contrast, with Aza small effects that were not statistically significant were observed in rats at 21 and 14 days in the RBC Pig-a and PIGRET assays respectively. Based on the present results, the mutagenic potential of Aza is negligible after single oral administration in rats. [ABSTRACT FROM AUTHOR]

A new in vivo gene mutation assay has been developed based on the phosphatidylinositol glycan anchor biosynthesis, Class A gene ( Pig-a in rodents) as an endogenous reporter. Although a large number of chemicals have been evaluated in the rat Pig-a assay in 28-day repeat dose regimens, there was limited reporting of rat Pig-a assay after a single dose. A collaborative study by the Mammalian Mutagenicity Study group, which is a subgroup of the Japanese Environmental Mutagen Society, was conducted to verify the usefulness of the rat Pig-a assay after a single dose as a short-term genotoxicity test. As a part of this collaborative study, the in vivo mutagenicity of a single dose of pyrene (Pyr) was investigated in the red blood cell (RBC Pig-a assay) and in reticulocytes (PIGRET) of rats. Eight-week old male rats were orally dosed with Pyr at 500, 1000, and 2000 mg/kg or ethylnitrosourea (ENU) at 10 and 40 mg/kg as a positive control. The animals in each group were examined for Pig-a mutant frequencies (MF) except for animals in the 2000 mg/kg group because of mortality or severe toxicity. The Pig-a MF in RBCs and reticulocytes, as CD59 negative cells, were evaluated once a week for 4 weeks after the dosing. With a single exposure to ENU, the Pig-a MF in both RBCs and reticulocytes increased in a time- and dose-dependent manner. In contrast, no statistically significant effect was observed in rats dosed with Pyr at 500 and 1000 mg/kg. Therefore, Pyr was concluded to be negative in the RBC Pig-a assay and the PIGRET assay after a single oral administration in rats. The result was consistent with previously reported Pig-a assays with repeat dose regimens. [ABSTRACT FROM AUTHOR]

Formaldehyde (FA) is a very reactive compound that forms DNA adducts and DNA-protein crosslinks, which are known to contribute to FA-induced mutations and carcinogenesis. Post-translational modifications to histones have recently attracted attention due to their link with cancer. In the present study, we examined histone modifications following a treatment with FA. FA significantly phosphorylated histone H3 at serine 10 (H3S10), and at serine 28 (H3S28), the time-course of which was similar to the phosphorylation of H2AX at serine 139 (γ-H2AX), a marker of DNA double strand breaks. The temporal deacetylation of H3 was observed due to the reaction of FA with the lysine residues of histones. The phosphorylation mechanism was then analyzed by focusing on H3S10. The nuclear distribution of the phosphorylation of H3S10 and γ-H2AX did not overlap, and the phosphorylation of H3S10 could not be suppressed with an inhibitor of ATM/ATR, suggesting that the phosphorylation of H3S10 was independent of the DNA damage response. ERK and JNK in the MAPK pathways were phosphorylated by the treatment with FA, in which the JNK pathway was the main target for phosphorylation. The phosphorylation of H3S10 increased at the promoter regions of c-fos and c-jun , indicating a relationship between FA-induced tumor promotion activity and phosphorylation of H3S10. These results suggested that FA both initiates and promotes cancer, as judged by an analysis of histone modifications. [ABSTRACT FROM AUTHOR]

Formaldehyde (FA) is widely known to cause DNA damage. Recently, our study showed that FA can also inhibit a repair process of DNA damage, nucleotide excision repair (NER). DNA damage response (DDR) involving activation of phosphorylation pathways is important for the accuracy of the repair process, and the inhibition of the accurate repair would raise mutation rate, leading to cancer. We herein investigated whether FA influences phosphorylation of histone H2AX (γ-H2AX), an intermediate player of DDR signaling pathways. Human keratinocytes HaCaT were treated with FA and then exposed to UV known to generate clear γ-H2AX signal. UV-induced γ-H2AX was inhibited by FA in a dose-dependent manner. The repair of pyrimidine dimers was inhibited by FA, while the recruitments of γ-H2AX-related proteins, Mre11 and 53BP1, to damaged sites were also delayed. Mre11, Nbs-1, H2AX and ATM were not degraded after treatment with FA as opposed to NER-related protein, TFIIH. On the other hand, FA inhibited phosphorylation of ATM which acts upstream of γ-H2AX. These results suggest that FA can affect the repair of DNA damage via inhibition of the phosphorylation pathways of H2AX. Unlabelled Image • FA inhibited UV-induced γ-H2AX. • The repair of UV-generated DNA damage was inhibited by FA. • The recruitments of γ-H2AX-related proteins to damaged sites were delayed. • FA inhibited phosphorylation of ATM which acts upstream of γ-H2AX. • The multiple inhibition pathways of γ-H2AX may contribute to the FA carcinogenecity. [ABSTRACT FROM AUTHOR]

The reproducibility of the in vivo Pig-a gene mutation test system was assessed across 13 different Japanese laboratories. In each laboratory rats were exposed to the same dosing regimen of N -nitroso- N -ethylurea (ENU), and red blood cells (RBCs) and reticulocytes (RETs) were collected for mutant phenotypic analysis using flow cytometry. Mutant frequency dose response data were analysed using the PROAST benchmark dose (BMD) statistical package. Laboratory was used as a covariate during the analysis to allow all dose responses to be analysed at the same time, with conserved shape parameters. This approach has recently been shown to increase the precision of the BMD analysis, as well as providing a measure of equipotency. This measure of equipotency was used here to demonstrate a reasonable level of interlaboratory reproducibility. Increased reproducibility could have been achieved by increasing the number of cells scored, as this would reduce the number of zero values within the mutant frequency data. Overall, the interlaboratory trial was successful, and these findings support the transferability of the in vivo Pig-a gene mutation assay. [ABSTRACT FROM AUTHOR]