六价铬暴露致细胞毒性及其对ROS产生和GPX4表达水平的影响

Effects of hexavalent chromium exposure on cytotoxicity and ROS production and expression of GPX4

目的探讨六价铬暴露对人肺腺癌上皮A549细胞和正常人支气管上皮16HBE细胞的细胞毒性及其活性氧(reactive oxygen species,ROS)含量和铁死亡标志物谷胱甘肽过氧化物酶-4(glutathione peroxidase 4,GPX4)表达水平的影响。方法选择重铬酸钾(K2Cr2O7)溶液,依据不同的处理浓度(0.2、0.8、1.6、3.2和4.8μmol/L)对A549和16HBE细胞进行处理,在不同的处理时间(0、1、12和24 h),采用CCK-8法检测A549细胞和16HBE细胞的增殖活力,荧光酶标仪检测ROS的含量,采用RT-PCR方法检测GPX4 mRNA的表达水平。结果与未处理的对照组相比,处理剂量≥1.6μmol/L时,A549细胞在处理12和24 h后均出现明显增殖抑制现象(均P<0.01),处理1 h后便对16HBE细胞增殖有显著的抑制作用(P<0.01),均存在时间-剂量效应关系。与16HBE细胞的表达水平相比,A549细胞中GPX4在mRNA水平显著高表达(P<0.05)。与未处理组相比,浓度为0.2、0.8、1.6、3.2、4.8μmol/L处理16HBE细胞1、12和24 h后均能引起细胞ROS含量增加(均P<0.01);而在A549细胞中,与未处理组相比,处理1 h后浓度为1.6、3.2、4.8μmol/L的处理组ROS含量增加(均P<0.01),当处理时间为12和24 h时浓度为0.2、0.8、1.6、3.2、4.8μmol/L均能引起细胞ROS含量增加。在16HBE细胞中,与未处理组相比,浓度为0.2、0.8、1.6、3.2、4.8μmol/L处理后的1、12和24 h,16HBE细胞GPX4 mRNA表达水平显著上调(均P<0.01);在A549细胞中,与未处理组相比,浓度为0.2、0.8、1.6、3.2、4.8μmol/L处理后的1和24 h,A549细胞GPX4 mRNA表达水平显著下调(均P<0.01),然而处理12 h后A549细胞GPX4 m RNA表达水平显著上调(P<0.01)。结论六价铬暴露均可引起A549和16HBE细胞的细胞毒性,存在时间-剂量效应关系,引起ROS含量增加和GPX4 mRNA表达水平发生改变。提示铁死亡标志物GPX4可能参与六价铬诱导肺癌发生发展的过程。

Objective To explore the effect of hexavalent chromium[Cr(VI)]exposure on the cytotoxicity,reactive oxygen species(ROS)levels and expression levels of glutathione peroxidase 4(GPX4),a major inhibitor of ferroptosis,in human lung adenocarcinoma(A549)cells and human normal bronchial epithelial(16HBE)cells.Methods A549 cells and 16HBE cells were cultured following standard protocols and exposed to different concentrations(0.2,0.8,1.6,3.2 and 4.8μmol/L)of potassium dichromate(K2Cr2O7)for various time points(0,1,12 and 24 h).The CCK-8 assay was performed to assess the proliferation activity of A549 cells and 16HBE cells,the ROS level was detected by fluorescent enzyme labeling instrument,and RT-PCR was used to detect the expression of GPX4mRNA.Results Compared with untreated control group,when the treatment dose was≥1.6μmol/L,the significant proliferation inhibition was observed at 12 and 24 h in A549 cells(all P<0.01),and the significant proliferation inhibition was observed at1 hour in 16HBE cells(P<0.01),and there were time-dose effect relationship.Compared with the expression level of GPX4mRNA in 16HBE cells,the expression level of GPX4 mRNA in A549 cells were increased significantly(P<0.05).After treatment with 0.2,0.8,1.6,3.2 and 4.8μmol/L at 1,12 and 24 h,the ROS productions in 16HBE cells were significantly increased as compared with those in the untreated control group(all P<0.01).In A549 cells,after treatment with 1.6,3.2 and 4.8μmol/L of potassium dichromate,the ROS productions were significantly increased at 1 h as compared with those in the untreated control group(P<0.01),and the ROS productions were increased after treatment with 0.2,0.8,1.6,3.2 and 4.8μmol/L at 12 and 24 h.In16HBE cells,after treatment with 0.2,0.8,1.6,3.2 and 4.8μmol/L,the mRNA expressions of GPX4 were significantly increased at1,12 and 24 h as compared with those in the untreated control group(all P<0.01).In A549 cells,after treatment with 0.2,0.8,1.6,3.2and 4.8μmol/L,the mRNA expressions of GPX4 were significantly decreased at 1 and 24 h as compared with those in the untreated control group(all P<0.01),while significantly increased at 12 h(all P<0.01).Conclusion The hexavalent chromium exposure could cause cytotoxic effect of A549 and 16HBE cells in a time-and-dose-dependent manner,and induce the increase of the ROS production and the change of expression levels of GPX4 mRNA,indicating an important role of GPX4 in the occurrence and development of lung cancer induced by hexavalent chromium.