癌症高发区环境水样有机提取物诱导L-02细胞转化及其表观遗传学机制

目的:检测华中某癌症高发地区环境水样有机提取物诱导人肝细胞系L-02细胞转化的能力,并初步探讨其诱导细胞转化的表观遗传学机制。方法:分别采集研究地区河水、离河较近(约1 km)及离河较远(约20 km)的浅井水(约10 m),用固相萃取方法提取其中的有机物。用台盼蓝染色计数法和单细胞凝胶电泳试验检测受试物的细胞毒性和致DNA损伤效应,以受试物对L-02细胞进行长期染毒并利用软琼脂克隆形成试验和裸鼠皮下成瘤试验检测受试物诱导L-02细胞转化的能力。采用甲基化特异性PCR(methyrlation specific PCR,MSP)技术检测转化细胞中O~6-甲基鸟嘌呤-DNA甲基转移酶(O~6-methylguanine-DNA methyhransferase,MGMT)基因启动子区甲基化状况,并分别用实时荧光定量PCR和Western blot技术检测转化细胞MGMT mRNA和蛋白的表达水平。结果:河水、离河较近的井水、离河较远的井水的有机提取物处理L-02细胞24 h后,细胞存活率达70%的处理浓度分别为每毫升培养基30、150、200ml原水,且均含有致DNA损伤的物质。以此作为最高剂量对细胞进行长期染毒,12周以后各处理组细胞软琼脂克隆形成试验和裸鼠皮下成瘤试验均显示转化特性。MSP结果显示转化细胞中MGMT基因启动子区发生高甲基化,与DMSO溶剂对照组比较,转化细胞中MGMT mRNA和蛋白表达水平均下调(P0.05)。结论:研究地区环境水样有机提取物具有诱导L-02细胞转化的能力,MGMT基因启动子区的高甲基化可能参与有机物诱导的细胞转化过程。

Transformation and epigenetic changes of L-02 cells by organic extracts of water environment

OBJECTIVE:To assess the carcinogenicity of organic extracts of water environment and elucidate the possible epigenetic mechanism.METHODS:Surface water of X river and groundwater of Y and Z villages were collected,and solid phase extraction cartridges were usedto extract organic contaminants from the water samples.Trypan blue dye exclusion method was conducted to examine the cytotoxicity of the extracts,whilst the organic extracts-induced DNA breakage was measured using single cell gel electrophoresis assay.After long term treatment of the extracts,soft agar assay and tumor formation of immunodeficient mice were performed to test the carcinogenicity.Methylation-specific PCR (MSP) was employed to determine the methylation status of MGMT gene in both transformed cells and control L-02 cell line.Meanwhile,the mRNA and protein levels of MGMT were examined,using RT-q-PCR and Western blot assays. RESULTS:The organic extracts showed cytotoxicity against the L-02 cells and could induce DNA breakage.L-02 cells were malignantly transformed by the three organic extracts.Compared with the control cells,MGMT gene showed hypermethylation in promoter and it was down-regulated in transformed cells(P<0.05).CONCLUSION:The organic extracts of the water environment exhibited carcinogenicity and hypermethylation of MGMT promoter may be accountable.