三氯乙酸染毒对肝L-02细胞的增殖作用及对DNA甲基化水平的影响

Short-term trichloracetic acid treatment induced the hepatic L-02 cells proliferation and DNA hypomethylation in human hepatic L-02 cells

目的 检测三氯乙烯（TCE）主要代谢产物三氯乙酸（TCA）对人肝L-02细胞染毒24h后的增殖作用，以及对DNA总体甲基化水平的影响，探索TCE对肝L02细胞的表型遗传毒性。方法选择0．1～0．9mmol／L浓度TCA作为合适的染毒剂量对肝L-02细胞染毒24h后分别进行下列试验：以CCK-8试剂盒观测细胞的生长曲线；以抗5-mC抗体进行免疫荧光检测细胞核中胞嘧啶甲基化总体水平（TCA0．9mmol／L染毒24h）；以流式细胞仪（FCM）检测其对细胞周期的影响及凋亡情况；提取细胞DNA进行琼脂糖电泳分析其对DNA的损伤作用。以上试验必要时设置5-氮杂胞苷（5-aza—dC5μmol／L）处理的L-02细胞为基因组DNA低甲基化的对照以及肝癌细胞作为细胞周期分析的对照。结果TCE在0．1～0．9mmol／L浓度下染毒24h后可以促进肝L-02细胞生长，并在0．9mmol／L浓度作用下24h可致肝L-02细胞DNA总体甲基化水平降低，荧光强度和正常细胞比较明显减弱；细胞周期分析发现TCA处理24h后再用正常培养基继续培养24h后可使细胞S＋G2期中细胞比例增加（与肝癌细胞接近）；DNA梯状电泳分析未发现DNA损伤性改变。结论TCA染毒24h可促进细胞生长，可诱导基因组DNA低甲基化，并造成细胞周期的改变，提示TCA对肝细胞的早期细胞毒性作用与表型遗传机制有关。

Objective To measure the effect of cell proliferation and explore the changes of gross DNA methylation status in L- 02 cells induced by the essential metabolite trichloracetic acid （TCA）of trichloroethylene （TCE） for short time（24h） and try to find out the possible epigenetic genetoxic within the process. Methods Measured the L-02 cells growth curve in different TCA concentrations （0.1 mmol/L, 0.3 mmol/L, 0.9 mmol/L） by CCK-8 kit; Immunofluorescence of anti-5- methylcytosine （5-mC） was used to detect the cells methylation levels of L-02 cells which treated with TCA（0.9mmol/L） for 24h, the trial used the 5-azadeoxycytidine （5-aza-dC, 5 μmol/L）as the hypomethylation control group; The flow cytometry（FCM） was employed to explore the cell cycle and apoptosis analysis. At last, the DNAs of each TCA treated group cells were extracted and processed agarose electrophoresis to detect the DNA injury. The liver cancer cell line HepG2 was employed as a control group in cell cycle analysis. Results Our study showed TCA induced the L-02 cells proliferation after short-term TCA（0.1- 0.9 mmol/L） treated for 24 h and decreased the DNA methylation levels for TCA（0.9 mmol/L） treated for 24 h. TCA increased the percentage of L-02 cells in S ＋ G2（M） phase for 24 h treatment with one day recovery. There was no DNA injury in the DNA ladder analysis. Conclusion All of above results indicate that TCA can induce the L-02 cells proliferation for short term treatment and the DNA hypomethylation may play a important role in the process of toxic action about TCA in hepatic L-02 ceils.