苯并(a)芘作用下的人支气管上皮细胞DNA损伤与XPF、XPG和ERCC1蛋白表达的关系

Association of DNA Damage and Expression of XPF,XPG and ERCC1 in 16HBE Exposed to Benzo[a]pyrene

目的探讨苯并(a)芘(Benzo[a]pyrene,B[a]P)作用下人支气管上皮细胞(16HBE)DNA损伤与着色性干皮病F、G蛋白[Xeroderma pigmentosum group F,G(XPF,XPG)]和切除修复交叉互补蛋白1(Excision repair cross-complementing 1,ERCC1)表达的关系。方法用B[a]P(0,1,2,4,8,16,32,64μmol/L)染毒16HBE细胞24h,用噻唑蓝(Methylthiazoletetrazolium,MTT)法检测B[a]P对细胞的毒作用。用Comet实验检测细胞DNA损伤并以Olive尾矩值(Olive Tail Moments,OTM)评价DNA损伤程度。用Western-blot检测XPF、XPG和ERCC1蛋白的表达水平。结果B[a]P浓度大于4μmol/L的各组细胞活性均显著性降低(P<0.05)。各染毒组细胞DNA损伤程度则明显增高(P<0.05)。在不同浓度B[a]P染毒细胞中目的蛋白的表达量有差异。在1μmol/L和2μmol/L B[a]P组细胞XPF、XPG表达增高,且2μmol/L组XPG水平与对照组相比,差异有统计学意义(P<0.05),但随染毒剂量增大(32,64μmol/L),XPF和XPG表达量明显降低(P<0.05)。ERCC1表达随染毒剂量的增高而逐渐增加,在16μmol/L达到峰值,随后逐步下降。回归分析显示XPF、XPG和ERCC1的表达与OTM值之间决定系数分别为0.691、0.745和0.642。结论B[a]P引起的XPF、XPG和ERCC1蛋白表达水平的变化与DNA损伤之间存在密切关系。

Objective To investigate the association of DNA damage and expression of xeroderma pigmentosum group F, G（XPF, XPG） and the expression of excision repair cross-complementing 1 （ERCC1） in human bronchial epithelia（16HBE） cells when exposing to benzo[a]pyrene（B[a]P）. Methods The 16HBE cells were exposed to B[a]P at concentrations of 0, 1, 2, 4, 8, 16, 32, 64 μmol/L for 24 hours. The viability of cells was determined by MTT assay. Comet assay was applied to determine the DNA damage. The levels of DNA damage were evaluated by the Olive Tail Moments（OTM）. The levels of protein expressions were detected by Western-blot. Results The viability of cells in the groups at concentrations of B[a]P〉4 μmol/L significantly decreased compared with the control （P〈0.05）. Furthermore, the dose-dependent increase in DNA damage was found in all of exposed groups. However, the targeted protein expressions were different in the exposed groups of different concentrations. XPF and XPG protein expressions increased in low concentrations of B[a] P（1 μmol/L and 2 μmol/L）. The levels of XPG in 2 μmol/L group increased significantly compared with the control group （P 〈0.05）. However, the levels of both XPF and XPG decreased significantly as the concentrations of B[a]P increased. The expression of ERCC1 increased as the exposure dosages gradually increased and reached to a peak at the concentration of 16 μmol/L B[a]P, and then descended slowly afterward. The regression analysis showed that the determination coefficients between OTM and protein levels of XPF, XPG and ERCC1 were 0. 691, 0. 745 and 0. 642, respectively. Conclusions A close correlation exists between the DNA damage and the changes of expression levels of XPF, XPG and ERCC1 in 16HBE cells when exposing to B[a]P.