OBJECTIVE To assess the DNA damage of copper 8-quinolinolate (CuQ) and to elucidate the plausible mechanisms.METHODS HepG2 cells were treated with CuQ0-4 μmol·L-1 for different time,DNA damage was measured by Comet assay.Catalase (CAT) activity,glutathione(GSH) level and thiobarbituric acid reactive substances (TBARS) were measured.NF-κB was examined using Western blotting.8-Hydroxydeoxyguanosine (8-OHdG) was measured by immunoperoxidase staining analysis.RESULTS CuQ 0.5 -4 μmol·L-1 caused significant increase of DNA migration in HepG2 cells.CuQ significantly decreased levels of GSH and activity of CAT in HepG2 cells (P ＜0.05).Moreover,CuQ significantly increased accumulation of the p65 subunit of NF-κB into nucleus,levels of lipid peroxidation product TBARS and the formation of 8-OHdG (P ＜0.05).The intracellular GSH level was modulated by pre-treatment with buthionine-(S,R)-sulfoximine (BSO),depletion of GSH in HepG2 cells pre-treated with BSO dramatically increased susceptibility of HepG2 cells to CuQ-induced DNA damage.CONCLUSION CuQ exerts DNA damage by oxidative stress and increases accumulation of p65 subunit of NF-κB into nucleus in HepG2 cells.u0000of GSH i%目的 评估8-羟基喹啉酮( CuQ)对HepG2细胞的DNA损伤作用并阐明其可能的作用机制.方法 CuQ 0 ～4 μmol·L-1处理HepG2细胞不同时间后,通过单细胞凝胶电泳实验检测细胞DNA损伤；分光光度法测定过氧化氢酶活性；苯二醛法测定细胞内谷胱甘肽(GSH)水平；硫代巴比妥酸反应物(TBARS)法检测细胞内脂质过氧化水平；Western印迹法检测NF-κB p65的变化；免疫组化方法检测细胞内8-羟基脱氧鸟苷(8-OHdG)的表达水平.结果 HepG2细胞与CuQ 0.5～4μmol· L-1作用1h后,DNA的迁移距离明显增加(P＜0.05),提示CuQ可引起DNA链断裂.CuQ能够造成细胞内GSH水平以及过氧化氢酶活性的降低.随着CuQ剂量的增加及染毒时间的延长,NF-κB由细胞浆逐渐转移至细胞核.CuQ还可以引起细胞内TBARS水平增高及8-OHdG表达水平的增强.采用GSH合成特异抑制剂DL-甲硫氨酸磺酰亚胺(BSO)预处理细胞,可明显增强CuQ对HepG2细胞DNA的损伤(P＜0.05).结论 CuQ可造成HepG2细胞氧化性DNA损伤,其作用机制与氧化应激及NF-κB p65在细胞核蓄积增高有关.
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