大气可吸入颗粒物致HepG2细胞DNA损伤

目的 研究大气可吸入颗粒物 (PM10)致人肝细胞瘤细胞 (HepG2)DNA损伤及其可能机制.方法 单细胞凝胶电泳 (SCGE)试验检测细胞DNA链断裂;2',7'-二氢二氯荧光素 (DCFH)法测定细胞内活性氧水平;免疫组化方法测定8-羟脱氧鸟苷 (8-OHdG)在细胞内的表达水平;免疫细胞化学法检测细胞内核转录因子NF-κBp65蛋白表达水平.结果 大连市4个监测点PM10有机提取物致HepG2细胞DNA链断裂作用存在地点和季节差异;7.5～30μg/mLPM10有机提取物作用HepG2细胞1h后,尾DNA%增大,呈剂量依赖关系.HepG2细胞与7.5～30μg/mL的PM10有机提取物接触1h后,可引起细胞内ROS水平表达的明显增加;7.5～30μg/mL的PM10有机提取物作用于HepG2细胞3h后,细胞内8-OHdG水平的表达增强;HepG2细胞与30μg/mL的PM10有机提取物接触24h后,NF-κBp65蛋白表达水平明显增高.结论 PM10有机提取物可引起体外HepG2细胞DNA链断裂;DNA链断裂水平随不同季节和不同监测点而异;PM10引起DNA损伤的机制可能是细胞内ROS生成增多,8-OHdG表达增高及NF-κBp65蛋白表达水平升高而导致的氧化性DNA损伤.

Study on DNA damage of HepG2 cell induced by inhalable particulate matter

Objective To investigate DNA damage induced by inhalable particulate matter (PM10) and its mechanisms in HepG2 cells.Methods DNA strand breaks induced by extractable organic matter of PM10 was assessed by single cell gel electrophoresis (SCGE) assay.Intracellular generation of reactive oxygen species (ROS),expression of 8-hydroxydeoxyguanosine (8-OHdG) and protein expression of NF-κB p65 were estimated by 2,7-dichlorofluorescein (DCFH),immunocytochemistry staining and Western blot,respectively.Results Significant differences in percent of tail DNA induced by the extractable organic matter of PM10 were revealed between samples collected in summer and winter seasons and from different monitoring sites.The percent of tail DNA in HepG2 cells significantly increased in a dose-dependent manner after exposure to 7.5-30μg/mL extractable organic matter of PM10 for 1hr.After exposure to extractable organic matter of PM10 for 1hr,intracellular ROS increased significantly.The 8-OHdG was positive in the HepG2 cells treated with extractable organic matter of PM10.The protein expression of NF-κB p65 significantly increased in HepG2 cells after treatment with 30μg/mL extractable organic matter of PM10 for 24 hr.Conclusion The extractable organic matter of PM10 induces DNA strand breaks in HepG2 cells.Significant differences in percent of tail DNA relates to PM10 samples collected at different times and locations.The extractable organic matter of PM10 exerts DNA damage effects in HepG2 cells probably through oxidative DNA damage induced by intracellular ROS,increase of 8-OHdG formation and the expression of NF-κB p65.