重金属镉通过DNA氧化损伤途径诱导细胞中心体扩增

摘要

目的:研究氯化镉(CdCl2)对细胞中心体扩增的影响,及活性氧(ROS)和DNA损伤在CdCl2诱导细胞中心体扩增中的作用.方法:用不同浓度(0、10、20、40 μmol/L) CdCl2处理HCT116细胞48 h,MTT法检测细胞活性;用低浓度无毒性的CdCl2处理细胞48 h,免疫荧光实验观察细胞内中心体的扩增;用CdCl2(20 μmol/L)、CdCl2+N-乙酰半胱氨酸(NAC)处理细胞2h,活性氧检测试剂盒检测细胞内ROS水平的变化;用CdCl2(20 μmol/L)、CdCl2+NAC处理细胞6h,彗星电泳试剂盒检测细胞内DNA损伤水平的变化;用CdCl2(20 μmol/L)、CdCl2+NAC处理细胞48 h,免疫荧光观察细胞内中心体的扩增.结果:20 μmol/L或以下CdCl2处理细胞48 h不影响细胞活性;CdCl220 μmol/L或以下无毒性剂量CdCl2诱导细胞中心体发生扩增(P＜0.01),并呈剂量依赖效应;在20 μmol/L CdCl2处理下,细胞内ROS和DNA损伤水平均明显升高(P＜0.01),当有抗氧化剂NAC存在时,细胞内升高的ROS和DNA损伤水平均被明显抑制(P＜0.01);抗氧化剂NAC对CdCl2诱导的中心体扩增也有明显的抑制效果(P＜0.01).结论:氯化镉通过DNA氧化损伤途径诱导细胞中心体扩增.%Objective:To explore the effect of cadmium to centrosome as well as the role of reactive oxygen species(ROS) and DNA damage in the cadmium-induced centrosome amplification.Methods:The HCT116 colon cancer cells were treated with different concentration of CdCl2(0,10,20,40 μmol/L) to determine the sub-toxic doses of cadmium using MTT assay.The cells were treated with sub-toxic concentrations of CdCl2 for 48 h,and the amplification of intracellular centrosomes was detected by immunofluorescent staining under microscope.The cells were treated with CdCl2(20 μmol/L) and CdCl2+N-acetyl-L-cysteine(NAC) for 2 h,and the changes of intracellular ROS level were detected by a kit.The cells were treated with CdCl2(20 μmol/L) and CdCl2+NAC for 6 h,and the DNA damage level in cells was detected by comet assay kit.Results:CdCl2 did not reduce the cell viability at 20 μmol/L or below after treatment for 48 h.The non-toxic doses of CdCl2 increased the level of centrosomes amplification in a dose-dependent manner.Treatment with CdCl2(20 μmol/L) increased the intracellular ROS and DNA damage levels significantly(P＜0.01),both of which were inhibited in the presence of antioxidant NAC(P＜0.01).Antioxidant NAC also significantly inhibited the CdCl2-induced centrosome amplification(P＜0.01).Conclusion:Cadmium chloride induces cell centrosome amplification through oxidative DNA damage pathway.