活性氧在邻苯二甲酸单(2-乙基己基)酯所致心肌细胞DNA损伤中的作用

Effect of reactive oxidative species on MEHP-induced DNA damage in human cardiac cells

目的探讨邻苯二甲酸单(2-乙基己基)酯(MEHP)对人源心肌AC16细胞DNA的损伤作用及其可能机制。方法以人源心肌AC16细胞为研究对象,分为对照组,40、60、80、100和120μmol/L MEHP组。处理细胞24 h后采用DCFH-DA探针标记测定AC16细胞内活性氧(ROS)水平;单细胞凝胶电泳实验测定细胞DNA损伤情况;利用抗氧化剂N-乙酰-L-半胱氨酸(NAC)抑制AC16细胞内ROS生成,以观察ROS在MEHP所致AC16细胞DNA损伤中的作用。结果与对照组相比,AC16细胞经MEHP处理后,ROS水平随着MEHP浓度的增加而明显升高(P<0.05);对照组心肌细胞核呈圆形荧光团,强度均匀,边缘光滑,大小较一致,无明显拖尾。不同浓度MEHP处理后,各组心肌细胞出现彗星拖尾现象,且具有MEHP浓度依赖性。与对照组OTM值和TD值相比,MEHP组OTM值和TD值明显升高,且有统计学意义(P<0.05);预先给予NAC可明显降低MEHP所致的细胞ROS水平升高。与相应的MEHP组相比,NAC+MEHP组OTM值及TD值明显降低,且差异有统计学意义(P<0.05)。结论MEHP可引起人源心肌AC16细胞DNA损伤,机制可能与ROS生成有关。

Objective To investigate the role of reactive oxygen species(ROS)in MEHP-induced DNA damage of human cardiac AC16 cells.Methods The AC16 cells were treated with MEHP(40,60,80,100 and 120μmol/L)or solvent control for 24 h.After treatment,the reactive oxygen species(ROS)content was assessed by fluorescent probes DCFH-DA,while DNA damage was evaluated with comet assay.Furthermore,antioxidant N-acetylcysteine(NAC)was added to evaluate the effect of ROS in MEHP-induced DNA damage of AC16 cells.Results Compared to the control group,there was a concentration-dependent increase in ROS accumulation of AC16 cells upon MEHP treatment(P<0.05).In the control group,there showed yellow-round nuclei of AC16 cells with homogenous distribution and consistent size,the edges were smooth without comet tail.MEHP treatment induced significant DNA damage in AC16 cells with a dose-dependent manner,which displayed as the enlongated comet tail.The OTM values as well as percentage of tail DNA contents(the TD values)in MEHP-treatmented AC16 cells were markedly higher than that of the cells in the control group(P<0.05).Pre-treatment with NAC significantly attenuated the enhanced ROS content induced by MEHP.Besides,in the NAC addition groups,the OTM and TD values decreased significantly in the NAC+MEHP group compared with the MEHP-treatment group(P<0.05).Conclusion MEHP may induce DNA damage in AC16 cells,and oxidative stress might be involved in the underlying mechanisms.