Study On Cytotoxicity, DNA Damage And Repair Induced By 1,4-benzoquinone In Vitro

Objective: 1,4-benzoquinone(BQ) is a yellow crystal-solid, which was an important row material in organic synthesie and was used in medical,chemical and dye industry widely. BQ is one of the main active metabolites of benzene.Some studies demonstrate clearly that BQ is the most potent metabolite in vitro. In recent years, some progresses have been made in the research on genotoxicity mechanism of BQ. Although BQ is a known leukemogen, the basis of its toxicity is not well defined.The aim of research is to examine the effect of BQ on cell viability,cell cycle and genetic damage, to explore possible genotoxicity mechanism from DNA damage and DNA repair in order to provide bases of effective protectional measures for occupational exposure benzene and BQ.Methods:In this study,the viability of V79 cells was measured by MTT method and the dose-effect relation could be decided;The cell cycle distribution of V79 cells treated with BQ were detected by flowcytometry;Alkli single cell gel electrophoresis (SCGE) was used to study the degree and type of DNA damage induced by BQ in V79 cells, and subsequent self-repair effect; DCFH-DA assay was used to study the content of reactive oxygen species(ROS) after V79 cells stained by the fluorescent probes , which had been treated for 2 hour by BQ; after co-cultured by BQ with various concentration of VitC , SCGE was used to study the DNA damage of V79 cells in order to observe the influence of VitC on BQ induced DNA damage and repair.Results: As showed in the MTT assay, V79 cell proliferation was inhibited obviously by BQ (treated 2 hours, from the concentration of 100μmol/L), and concentration-dependent relationship was show. Treated with BQ (25- 100μmol /L) for 24 h, the distribution of cell cycle was changed. Cells in S stage were increased, while the cells in G1 stage were decreased. The comet cells ratio elevated as the concentration increased in SCGE in the range of 12.5～100μmol/L showing significant statisitic differences (P < 0.05). All the analysis indexes including tail length, Olive tail moment and tail DNA % increased in a dose-dependent pattern.Levels of ROS induced by 50 and 100μmol/L p-BQ were significantly increased as evidenced by elevated fluorescence intensity (P<0.01). The comet cells ratio ,tail length , Olive tail moment elevated as the concentration increased in SCGE in the range of 12.5-100μmol/L showing significant statisitic differences (P<0.05). There was a linear correlation between the DNA damage and the ROS intensity(P<0.01). Combined treatment of vitamin C(40,200μmol/L)and BQ,the levels of the comet cells ratio ,tail length and Olive tail moment were significant lower than those of correspondent BQ alone treatment(P<0.05). However, 1000μmol/L vitamin C has an aggravation effect on the BQ induced DNA damage.Conclusion: The results of this study showed that:the first,conditions in vitro, low concentration BQ could inhibit proliferation of V79 cells and had a concentration-dependent mode; the second,the distribution of cell cycle could be altered by BQ in V79 cell, cells in S stage increased, while the cells in G1 stage decreased ; the third,DNA single strand breaks could induced by BQ and the damage could be self-repaired partly; the fourth,by flow cytometry and confocal laser scanning technology test results showed that, BQ at the concentrations of 50,100μmol/L induced enhanced ROS production. And it indicated that excess production of ROS may be a possible mechanism of the DNA damage induced by BQ; the fifth,Vitamin C at certain concentration(40,200μmol/L)has the antagonistic effects on oxidative stress and DNA damage induced by BQ.