低剂量纳米二氧化钛和乙酸铅联合诱导人胚肝细胞DNA损伤与修复作用

DNA Damage and OGG1 Expression Induced by a Combined Effect of Titanium Dioxide Nanoparticles and Lead Acetate in Human Hepatocytes

目的研究纳米二氧化钛(titanium dioxide nanoparticles,nano-TiO_2)与乙酸铅(PbAc)联合染毒对人胚肝细胞(L-02)的DNA损伤及修复作用。方法将L-02细胞分别以1μg/ml乙酸铅溶液和10、1、0.1、0.01、0.001μg/ml纳米二氧化钛溶液以及1μg/ml乙酸铅+0.001、0.01、0.1、1、10μg/ml纳米二氧化钛溶液处理24 h,同时,以1‰二甲基亚砜(dimethyl sulfoxide,DMSO)为阴性对照,30μmol/L H_2O_2为阳性对照。测定L-02细胞DNA的Olive尾矩、DNA加合物8-羟基脱氧鸟苷(8-OHdG)和DNA损伤修复酶8-羟基鸟苷DNA糖苷酶同源物1(OGG1)的水平。结果与阴性对照组比较,1μg/ml乙酸铅+1、10μg/ml纳米二氧化钛组L02细胞DNA的Olive尾矩和8-OHdG生成量明显升高,差异有统计学意义(P<0.05)。与1μg/ml乙酸铅单独染毒组比较,1μg/ml乙酸铅+10μg/ml纳米二氧化钛联合染毒组L02细胞DNA的Olive尾矩和8-OHdG生成量明显升高,差异有统计学意义(P<0.05)。与阴性对照组和1μg/ml乙酸铅单独染毒组比较,1μg/ml乙酸铅+0.001～1μg/ml纳米二氧化钛组L02细胞OGG1的表达水平均明显升高,差异有统计学意义(P<0.05)。随着乙酸铅的添加以及纳米二氧化钛染毒浓度的升高,L02细胞DNA的Olive尾矩和8-OhdG生成量均呈上升趋势,OGG1的表达水平呈下降趋势。乙酸铅和纳米二氧化钛对DNA损伤修复蛋白OGG1的表达存在协同作用,但对Olive尾矩水平和8-OHdG生成量不存在交互作用。结论低浓度乙酸铅和纳米二氧化钛联合暴露可使L02细胞DNA损伤增加,OGG1应激性增高以修复DNA损伤,但持续增加的DNA损伤会使OGG1水平下降。

Objective To observe the DNA damage and DNA repair protein expression induced by a joint ettect ot nano-TiO2 and PbAe in human hepatoeytes under non-UV. Methods L02 were exposed to nano-TiO2 in 10,1,0.1,0.01,0.001 μg/ml and 1 μg/ml PbAe treated alone and 10, 1,0.1,0.01,0.001 Ixg/ml mixed with 1 μg/ml PbAc for 24 h. A negative control（l%~ dimethyl sulfoxide,DMSO） and positive control （30 μmol/L H202） were included in each experiment. The DNA damage was determined by the comet assay. The generation of DNA adducts and the expressions of OGG1 were assessed by HPLC and western blotting assay, respectively. Results Compared with the negative control, OTM and 8-OHdG generation increased notably when treated with 10 and 1 Ixg/ml nano-TiO2 mixed with 1μg/ml PbAe （P〈0.05）. Compared with 1 μg/ml PbAc treated alone, 10 μg/ml nano-TiO2 mixed with 1 μg/ml PbAe induced significantly OTM and 8-OHdG generation increased （P〈0.05）. Compared with the negative control and 1μg/ml PbAe treated alone, 0.001 to 1μg/ml nano-TiO2 mixed with 1μg/ml PbAc induced significant increased in expression of OGG1 expression in L02 cells （P〈0.05）. There were uptrend in OTM and 8-OHdG generation but downtrend in OGG1 expression with added of PbAC and the increased of nano-TiO2 in the mixtures of nano-TiO2 and PbAc. Addition of nano-TiO2 with PbAC coordinated the expression OGG1 but not OTM and 8-OHdG generation in this study. Conclusion These findings indicate that low concentrations of nano-TiO2 and PbAc in combination induce DNA damage and 8-OHdG generation, while triggered OGG1 expression to repair the damaged DNA in L02 ceils in the absence of photoactivation. However,the increased DNA damage reduced the DNA damage repair capacity.