Study On Cytotoxicity, DNA Damage Induced By Hydroquinone And ACF And Vc Repair Effect On DNA Damage In Vitro

Objective: Hydroquinone (HQ) is a crystal-solid, which was widely used in chemical and medical industry. HQ is one of the main active metabolites of benzene, a carcinogenic agent in long time test and can cause genetic damage in various experiment system. It also can increase the carcinogenesis effect of other chemical compound such as methyl-N-pentylnitrosamine and N-methyl-N-nitro-N- nitroguanidine. In recent years, some progresses have been made in the research on genotoxicity mechanism of HQ, but definite conclusion has not been made.The aim of research is to examine the effect of HQ on cell viability and genetic damage, to explore possible genotoxicity mechanism from DNA damage, DNA repair and apoptosis in order to provide bases of effective protectional measures for benzene and HQ exposed population group.Methods:In this study,the viability of V79 cells was measured by MTT method and the dose-effect relation could be decided;Alkli single cell gel electrophoresis (SCGE) was used to measure the degree and type of DNA damage caused by HQ in V79 cells, and subsequent self-repair effect; the AO/EB double labelling method was used to investigate V79 cells apoptosis induced by HQ. DCFH-DA assay was used to measure the content of reactive oxygen species(ROS) after V79 cells stained by the fluorescent probes , which had been treated for 0.5 or 1 hour by HQ; after co-cultured by HQ with various concentration of ACF or VitC , SCGE was used to study the DNA damage of V79 cells in order to observe the influence of ACF or VitC on HQ induced DNA damage and repair.Results: As showed in the MTT assay, V79 cell proliferation was inhibited obviously by HQ (treated 2 hours, from the concentration of 0.15mmol/L), and concentration-dependent relationship was show. The comet cells ratio was rise with the concentration increased in the five concentrations (0.015mmol/L-1.215mmol/L), which has significant statistic difference (P<0.01)。The four analysis indexs including tail-length,tail/head ratio, TM and CM were decreased with the repair time increased at the concentration of 0.405 mmol/L. The statistic differences between the repair time of 2h and 4h were significant statistic differences (P<0.01). As showed in the AO/EB double labelling method, apoptosis in V79 cell could been induced obviously by HQ (0.015mmol/L—1.215mmol/L,cultured 24 hours). Concentration- dependent relationship between HQ and apoptosis was shown.The ROS contents was increased with the concentration increased in the four concentrations (0.015mmol/L -0.405mmol/L), which have significant statistic differences(P<0.01) in the DCFH-DA assay. The ROS contents in the 0.5h group are higher than that of the 1h group, which also have significant statistic differences (P<0.01). As shown by the SCGE, comet cells ratio, TM and tail-length were decreased by 200μmol/L,20μmol/L vitamin C and 100mg/L ACF. The statistic difference was significant (P<0.05). However, 2000μmol/L vitamin C has an aggravation effect on the HQ induced DNA damage。Conclusion: V79 cell,s proliferation was inhibited significantly by HQ(from 0.15mmol/L). There was a concentration-dependent relationship between HQ concentration and cell viability.DNA damage, mainly SSB, may be induced by HQ(from 0.015mmol/L), and the damage can be self-repaired partly. In vitro, apoptosis can be induced, and ROS was generated by HQ in V79 cell, both of which have concentration-dependent relationship. The ROS is generated mainly within 0.5h.DNA damage caused by HQ in V79 cells can be reduced by middle and low concentration(200,20μmol/L) Vitamin C and middle concentration(100mg/L) ACF in vitro.