Search

Genotoxicity testing plays an important role in the safety assessment of pharmaceuticals, pesticides and chemical substances. Among the guidelines for various genotoxicity tests, the in vitro genotoxicity test battery comprises the bacterial Ames test and mammalian cell assays. Several chemicals exhibit conflicting results for the bacterial Ames test and mammalian cell genotoxicity studies, which may stem from the differences in DNA repair capacity or metabolism, between different cell types or species. For better understanding the mechanistic implications regarding conflict outcomes between different assay systems, it is necessary to develop in vitro genotoxicity testing approaches with higher specificity towards DNA-damaging reagents. We have recently established an improved thymidine kinase (TK) gene mutation assay (TK assay) i.e. deficient in DNA excision repair system using human lymphoblastoid TK6 cells lacking XRCC1 and XPA (XRCC1 −/−/ XPA −/−), the core factors of base excision repair (BER) and nucleotide excision repair (NER), respectively. This DNA repair-deficient TK6 cell line is expected to specifically evaluate the genotoxic potential of chemical substances based on the DNA damage. We focussed on four reagents, N -(1-naphthyl)ethylenediamine dihydrochloride (NEDA), p -phenylenediamine (PPD), auramine and malachite green (MG) as the Ames test-positive chemicals. In our assay, assessment using XRCC1 −/−/ XPA −/− cells revealed no statistically significant increase in the mutant frequencies after treatment with NEDA, PPD and MG, suggesting the chemicals to be non-genotoxic in humans. The observations were consistent with that of the follow-up in vivo studies. In contrast, the mutant frequency was markedly increased in XRCC1 −/−/ XPA −/− cells after treatment with auramine. The results suggest that auramine is the genotoxic reagent that preferentially induces DNA damages resolved by BER and/or NER in mammals. Taken together, BER/NER-deficient cell-based genotoxicity testing will contribute to elucidate the mechanism of genotoxicity and therefore play a pivotal role in the accurate safety assessment of chemical substances. [ABSTRACT FROM AUTHOR]

Mycotoxin fumonisin B1 (FB1) is a secondary metabolite that is produced by certain Fusarium species. Although numerous studies demonstrate toxic and carcinogenic effects of FB1, the underlying mechanisms have not been fully elucidated. In this study, we evaluated the epigenetic effects of FB1 for the first time using FLO assays, which detect epigenetic changes that affect the flocculation gene (FLO1) promoter activity in budding yeast. FLO assays showed increased reporter activities of the FLO1 promoter in the presence of 10 and 20 µM FB1. FB1 (20 µM) treatments also promoted flocculation. In subsequent in vitro methylation assays of a bacterial DNA methyltransferase (DNMT), FB1 treatments increased DNMT activities. Moreover, global DNA methylation was significantly increased in HEK293 cells treated with 100 µM FB1. Taken together, these results suggest that FB1 exposure leads to unique epigenetic alterations due to increased DNMT activities and demonstrate that FB1 may be an important risk factor for epigenetic dysfunction-associated human diseases including cancer. [ABSTRACT FROM AUTHOR]

The mycotoxin ochratoxin A (OTA) is considered to be a human carcinogen. However, the mode of its carcinogenetic action has not been elucidated. Recently, it has become evident that epigenetic changes influence the risk of developing cancer. Since it has been revealed that the yeast flocculation displayed by the strains transformed with human DNA methyltransferases (DNMT) can be regulated by epigenetic mechanisms, we examined the effect of OTA on the transcription level of FLO1, which mediates the flocculation phenotype. OTA but not a non-carcinogenetic mycotoxin deoxynivalenol (DON) inhibited the intensity of GFP fluorescence under the transcriptional regulation of FLO1 promoter in a dose-dependent manner. At the same time, OTA had no effect on the reporter activity under the control of modified FLO1 promoter with reduced CpG motifs. In addition, it was confirmed that the flocculation and FLO1 mRNA of DNMT gene-transformed yeast (DNMT yeast) were decreased by OTA. In vitro methylation assay using a bacterial DNMT revealed an inhibitory effect of OTA on the DNMT activity, and OTA treatment reduced the frequency of abnormally shaped nuclei which were often observed in DNMT yeast. These results suggest that the carcinogenicity of OTA may involve inhibition of DNMT-mediated epigenetic regulation. [ABSTRACT FROM AUTHOR]

We have previously reported that flocculation of a yeast co-transformed with the human DNA methyltransferase 1 (DNMT1) and DNMT3B genes was inhibited by DNMT inhibitors. It is well known that epigenetic mutagens can disturb nucleosome positioning via DNA methylation and/ or histone modification. In this study we first examined the effects of trichostatin A (TSA), a histone deacetylase inhibitor, on the flocculation level of yeast. TSA dose-dependently promoted the flocculation exhibited by the yeast transformed with the DNMT genes or empty vectors. Furthermore, TSA induced the expression of the flocculin-encoding gene FLO1. The anthracenederived alizarin, a natural madder root dye, has a potential for carcinogenesis promotion; however, the mode of action has not been elucidated. It is considered that epigenetic changes can promote cancer. Alizarin but not anthracene enhanced the flocculation level of the yeast. Similar to TSA, alizarin also upregulated FLO1 mRNA. Surprisingly, western blotting indicated that alizarin, but not anthracene, reduced the level of histone H3 in yeast, and alizarin-treated cells frequently displayed abnormally shaped nuclei. These findings suggest that alizarin uniquely influences nucleosome structure. Taken together with our previous findings, this study suggests that the DNMT genetransformed yeast strains are a useful tool for screening various classes of epigenetic mutagens. [ABSTRACT FROM AUTHOR]