8-羟基喹啉酮对HepG2细胞氧化性DNA损伤的诱导作用(英文)

Inductive effect of copper 8-quinolinolate on oxidative DNA damage in HepG2 cells

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 0～4μmol.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1 h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。

OBJECTIVE To assess the DNA damage of copper 8-quinolinolate（CuQ） and to elucidate the plausible mechanisms.METHODS HepG2 cells were treated with CuQ 0-4 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（P0.05）.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（P0.05）.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.