Study On Cytotoxicity, DNA Damage And Repair Induced By Phenol In Vitro

Objective: Phenol (PH) is a white crystal-solid, weak acid. It has been extensively used in organic synthesise,medical,agrochemical,chemical and dye industry. PH is one of the intermediate active metabolites of benzene. In recent years some studies demonstrate that PH can induce genotoxicity, so it can be an antileptic of genotoxicity. PH can do harm to the workers after long exposure, but the mechanism of its toxicity is not well defined.The aim of research is to study the effect of PH on cell viability,genetic damage and apoptosis, to explore possible genotoxicity mechanism from DNA damage and DNA repair in order to provide basis experimental data of effective measures for occupational exposure benzene and PH.Methods: (1) Chinese hamster lung fibroblast cells(V79 cells) were used as the system. Cytotoxicity of PH was determined by MTT Cell Proliferation Assay (MTT method) and the dose-effect relation could be decided; (2) Alkli single cell gel electrophoresis (SCGE) was used to study the degree and type of DNA damage induced by PH in V79 cells; (3) The apoptosis of V79 cells treated with PH were detected by flowcytometry; (4) 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 12 hours by PH; (5) After co-cultured by PH with various concentration of Vitamin C , SCGE was used to study the DNA damage of V79 cells in order to observe the influence of Vitamin C on PH induced DNA damage and repair and subsequent self-repair effect.Results: (1) As showed in the MTT assay, V79 cell proliferation was inhibited obviously by PH (treated 48 hours, from the concentration of 25mg/L), and concentration-dependent relationship was obvious. (2) The damage to DNA-strand cells significantly increased after exposure to 5mg/L PH for 12h. All the analysis indexes including Comet cells ratio,tail length,Olive tail moment and tail DNA % increased in a dose-dependent pattern.(3) Treated with PH (10～80 mg/L) for 12h, the apoptosis rate of V79 cells was increased significantly compared with the negative group. (4) Levels of ROS induced by 10mg/L PH 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 5～80mg/L (P <0.05). There was a linear correlation between the DNA damage and the ROS intensity( P <0.01),the apoptosis and the ROS intensity. (5) Combined treatment of Vitamin C(40μmol/L) and PH,the levels of the comet cells ratio ,tail length and Olive tail moment were significant lower than those of correspondent PH alone treatment( P <0.05). However, 1000μmol/L Vitamin C has an aggravation effect on the PH induced DNA damage.Conclusion: The results of this study showed that: (1) Conditions in vitro, low concentration PH could inhibit proliferation of V79 cells and had a concentration-dependent mode. (2) DNA single strand breaks could be induced by PH. (3) Compare with the negative group, the apoptosis rate of V79 were increased significantly in those groups that the concentration of Ph is above 5mg/L. (4) By flow cytometryt, results showed that, PH induced enhanced ROS production at the concentration of 10mg/L and more. That result indicated excess production of ROS may be a possible mechanism of the DNA damage induced by PH. (5) The DNA damage caused by PH could be self-repaired partly. Vitamin C at certain concentration (40μmol/L) has the antagonistic effects on oxidative stress and DNA damage induced by PH.