双酚A与苯并[a]芘联合作用对三种乳腺上皮细胞的DNA损伤效应

DNA Damage Effect of Combination of Bisphenol A and Benzo[a]pyrene on Three Human Mammary Epithelial Cells

[目的]观察双酚A(BPA)与苯并[a]芘(BaP)联合作用对三种乳腺上皮细胞DNA损伤的影响。[方法]采用对DNA双链断裂标志pH2AX免疫荧光检测的方法,观察环境相关剂量水平(10-9,10-7mol/L)BPA、10-9mol/L17-β-雌二醇(雌激素对照)及其与10-6mol/L BaP联合作用对人乳腺上皮细胞(HMEC)、人永生化乳腺上皮细胞MCF-10A(CRL-10317)和人乳腺上皮肿瘤细胞MCF-7三种乳腺上皮细胞的DNA损伤效应(以二甲基亚砜作为溶剂对照),并应用实时荧光定量聚合酶链反应法检测三种细胞雌激素受体α和雌激素受体β的基因ESR1、ESR2mRNA水平的相对表达量。[结果]BPA对三种类型乳腺上皮细胞pH2AX阳性率均无明显影响。BaP可明显增加HMEC和MCF-7两种乳腺上皮细胞pH2AX阳性率:单独染毒时两种细胞pH2AX阳性率分别由(34.7±2.2)%和(3.1±0.3)%增至(63.6±1.2)%和(13.3±0.4)%;与10-7mol/L BPA、10-9mol/L 17-β-雌二醇分别联合作用可使MCF-7的pH2AX阳性率增至(15.1±0.2)%、(17.5±1.0)%,与单独染毒组相比,差异有统计学意义(P<0.05)。三种细胞内ESR1、ESR2基因表达有差异,其中ESR1基因表达差异明显,在MCF-7中的表达水平最高,为HMEC的1080倍、MCF-10A的5222倍。[结论]BPA与BaP联合作用与BaP单独作用相比,可引起MCF-7的DNA双链断裂损伤加剧,该作用可能与雌激素受体表达水平相关。

[ Objective ] To observe the DNA damage effect of Bisphenol A （BPA） in combination with benzo[α]pyrene （BaP） on three human epithelial cell lines, i.e. human mammary epithelial cells （HMEC）, MCF-10A, and MCF-7. [ Methods ] Immunofiuorescence was applied to observe the biomarker of DNA double-strand break in three human epithelial cells caused by a combination exposure to BPA （10^-9 mol/L, 10^-7 mol/L） and BaP （10^-6 mol/L）. The blank controls were administrated with dimethyl sulfoxide （DMSO） and the positive controls with 17-β-estradiol. Real-time polymerase chain reaction （PCR） was used to detect the relative mRNA expression levels of estrogen receptors （ESR1 and ESR2） in three cell lines. [ Results ] No notable differences in the pH2AX positive rates were detected between the BPA alone and the control groups. The pH2AX positive rates were increased remarkably after treatment of BaP to HMEC [ from （34.7 ± 2.2）% to （63.6 ± 1.2）% ] and MCF-7 [ from （3.1 ± 0.3）% to （13.3 ± 0.4）% ] cell lines. Compare with the BaP alone group, combination of 10^-7 mol/L BPA or 10^-9 mol/L 17-±-estradiol with BaP increased the pH2AX positive rate to （15.1 ± 0.2）% and （17.5 ± 1.0）%, respectively. The expression levels of estrogen receptor genes varied among three cell lines, and the expression level of ESR1 gene in MCF-7 cells were 1080 folds as high as in HMEC cells and 5 222 folds as in MCF-IOA cells. [ Conclusion ] Compare with the BaP alone exposure, the combination exposure to BPA and BaP can intensify the DNA double-strand break in MCF-7 cell line, and the effect may depend on the expression level of estrogen receptors.