We have already shown that the re-joining step of excision repair inhibits the formation of comet tails. In this study, we investigated how the formation of comet tails is supported by the incision step of excision repair. Three different human cells that are defective in nucleotide excision repair (NER), namely, XP3OSSV (derived from an XP group A patient), XPL3KA (derived from an XP group C patient), and CS2OSSV (derived from a Cockayne syndrome group A patient), and one NER wild-type human cell, TK6, were used. They were exposed to alkylating agents, bleomycin (BLM), or UVC, and then slides for the comet assay were prepared. Alkylating agents and BLM induced positive responses in the four studied cells. On the other hand, UVC induced positive responses in TK6, XPL3KA, and CS2OSSV cells, but not in XP3OSSV cells. UVC did not induce positive responses in XP3OSSV cells in the presence of DNA repair inhibitors (araC and hydroxyurea) or in their absence. Comet-positive response upon UVC irradiation was observed later in CS2OSSV than in TK6 and XPL3KA cells. It declined earlier in XPL3KA than in TK6 cells. On the basis of our results, the following features are suggested: 1. The incision step of NER is necessary to form comet tails. 2. If either TCR or GGR of NER acts, the comet tails can be formed.
SMD-502, a new vitamin D3 (VD3) analog, is an antipsoriatic drug candidate. The compound exhibited fewer side effects than known VD3 analogs in animal models, probably because the compound is rapidly converted into pharmacologically inactive form after permeating into systemic circulation from the application site on skin. This feature of the compound makes it difficult to assess genotoxic risk in vivo with the standard approach of bone marrow or peripheral blood micronucleus assays in rodents because of the low blood concentration level. To evaluate in vivo mutagenicity of the compound in the present study, mutant frequency (MF) in skin and liver of gpt delta transgenic mice was examined with percutaneous administration of SMD-502 for 28 days. In tissues collected 7 days after the end of administration, no significant increase in the MF was observed in either skin or liver. Additionally, when the compound was tested in a GDL1 cell line established from gpt delta mice, GDL1 cells exhibited no significant increase in MFs even under conditions in which they would be exposed to a much higher concentration of the compound than in the in vivo study. The results in this study further supported the consideration that SMD-502 has no mutagenic activity.
We investigated the antimutagenic activity of extract from mature and premature blackcurrant using the yeast loss of heterozygosity (LOH) system. Blackcurrant is a classical fruit that has long been used to make juice, jam, liqueur and sometimes medicines in Europe. Recently, its beneficial health effects, including anticarcinogenic and antioxidative activities, have been reported and associated with the functions of anthocyanins. However, it remains unclear how blackcurrant exhibits an anticarcinogenic effect. In this study, we demonstrated the evaluation of the antimutagenic activity of blackcurrant extract (BCE) using the yeast LOH system. Induction of mutations by several mutagens, namely, H2O2, MMS and UV, was significantly suppressed by the combined treatment of the two types of BCE. In conclusion, our findings clearly support the antimutagenic potential of blackcurrant, not only the mature fruit but also the premature fruit, as a useful food supplement.
We have reported the formation of 5-methylcytosine from cytosine in vitro, with methyl radicals generated from methionine sulfoxide (MetO). To confirm this reaction in vivo, MetO was added to the drinking water and administered to non-alcoholic steatohepatitis (NASH) mice, which develop hepatitis caused by endogenous oxidative stress. Histopathological examinations revealed incidences of hepatocellular carcinoma of 16.7% and 90% in the 0% and 3% MetO groups, respectively. Higher DNA methylation was detected in the promoter region of the p16 gene isolated from the livers of MetO-treated mice. The higher incidence of liver tumors may be due to the methyl radical-mediated formation of 5-methylcytosine in DNA, which triggers epigenetic changes.
17β-Estradiol (E2) is crucial for various physiological functions, such as in the development of the uterus and the mammary gland. However, prolonged exposure to E2 is a risk factor for breast cancer. E2 is metabolized to carcinogenic 4-hydroxyestradiol (4-OHE2) and non-carcinogenic 2-hydroxyestradiol (2-OHE2) by CYP1A1 and CYP1B1 in the breast tissue, respectively. These two catechol estrogens are converted to methylated metabolites by catechol-O-methyltransferase (COMT). 4-OHE2 has been reported to be further oxidized to quinone intermediates which react with purine bases in DNA to form depurinating adducts, which generate highly mutagenic apurinic (AP) sites. Recently, phosphorylation of histone H2AX (γ-H2AX) has emerged as a sensitive marker for not only DNA double-strand breaks but also various types of DNA damage. 4-OHE2-induced γ-H2AX in MCF-7 cells has never been reported yet. In this study, we investigated whether 4-OHE2 induces γ-H2AX in response to DNA damage in the presence or absence of Ro 41-0960, an inhibitor of COMT, in human breast cancer MCF-7 cells. AP sites and γ-H2AX were induced 1-2 h after treatment with 4-OHE2 and Ro 41-0960. The generation of intracellular reactive oxygen species (ROS) was also observed, as determined by 2′-7′-dichlorodihydrofluorescein diacetate fluorescence. By comparison, 2-OHE2 and Ro 41-0960 had no effect on AP sites, γ-H2AX or the generation of ROS. KU-55933, an inhibitor of ataxia telangiectasia mutated (ATM), decreased the formation of γ-H2AX in conjunction with 4-OHE2 and Ro 41-0960. These results demonstrate that 4-OHE2, in the presence of Ro 41-0960, induces ATM-dependent γ-H2AX in MCF-7 cells.
Degradation of four nucleosides, 2′-deoxyguanosine, 2′-deoxyadenosine, 2′-deoxycytidine, and thymidine, in potassium phosphate buffer of pH 7.4 by direct and alternating electric currents was monitored by HPLC. When a direct current of 3 V was applied to the nucleoside solution, all the nucleosides were consumed time-dependently. Results for voltage dependence showed that the nucleosides were decomposed above 0.8 V for 2′-deoxyguanosine, 1.5 V for 2′-deoxyadenosine, and 2.5 V for 2′-deoxycytidine and thymidine. Results of frequency dependence of alternating current at 3 V showed that increasing frequency caused decreasing consumption of all the nucleosides, and that no consumption was observed above 3,000 Hz. The results suggest that accidental ingestion of batteries or electrical shock by domestic power supply may induce alternation of nucleosides.
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