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 Benzoapyrene on Three Human Mammary Epithelial Cells
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摘要:
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(10-9,10-7 mol/L) BPA、10-9 mol/L17-β-雌二醇(雌激素对照)及其与10-6 mol/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& #177;2.2)%和(3.1& #177;0.3)%增至(63.6& #177;1.2)%和(13.3& #177;0.4)%;与10-7 mol/L BPA、10-9 mol/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& #177;0.2)%、(17.5& #177;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1 080倍、MCF-10A的5 222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。 Abstract:Objective To observe the DNA damage effect of Bisphenol A (BPA) in combination with benzoapyrene (BaP) on three human epithelial cell lines, i.e. human mammary epithelial cells (HMEC), MCF-10A, and MCF-7. Methods Immunofluorescence 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 HMECfrom (34.7& #177;2.2)% to (63.6& #177;1.2)%and MCF-7from (3.1& #177;0.3)% to (13.3& #177;0.4)% cell lines. Compare with the BaP alone group, combination of 10-7 mol/L BPA or 10-9 mol/L 17-β-estradiol with BaP increased the pH2AX positive rate to (15.1& #177;0.2)% and (17.5& #177;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 1 080 folds as high as in HMEC cells and 5 222 folds as in MCF-10A 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.
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