摘要:
有机氯类农药为已确定的环境雌激素，易在体内蓄积产生慢性毒性危害人体健康。选择单一存在时无明显雌激素效应的残留剂量进行联合试验，观察联合作用后雌激素效应的增减。选取乳腺癌MCF-7细胞作为研究对象，对残留剂量下六氯苯(HCB)、β-六六六(β-BHC)和p,p’-滴滴涕(p,p’-DDT)联合后的雌激素效应机制进行了研究。首先，运用MTT法观察3种有机氯类农药残留剂量下单独和联合作用对MCF-7细胞生长的影响；然后，运用流式细胞仪测定有机氯类农药联合对MCF-7细胞周期的影响；之后，采用Western Blot法测定有机氯类农药联合后ERα、ERβ、ERK1/2、p-ERK1/2、Ki67、c-Myc和CyclinD1蛋白表达的变化。结果显示，β-BHC+p,p’-DDT组、HCB+β-BHC+p,p’-DDT组作用24 h后细胞增殖率为115.0%、120.1%，作用48 h后增殖率为121.2%、126.3%，其他联合组与对照相比未发生明显变化；β-BHC+p,p’-DDT组、HCB+β-BHC+p,p’-DDT组中MCF-7细胞的G1期细胞比例下降，S期细胞比例上升，细胞呈高增殖状态；β-BHC+p,p’-DDT组、HCB+β-BHC+p,p’-DDT组和雌二醇(E2)组作用48 h后，均降低ERα的蛋白表达，升高ERβ的蛋白表达，促进p-ERK1/2、Ki67、CyclinD1和c-Myc的蛋白表达。研究表明，在单一作用时不具有雌激素效应的残留剂量下，仅β-BHC+p,p’-DDT联合组和HCB+β-BHC+p,p’-DDT联合组具有明显雌激素效应，其效应机制为经雌激素受体途径，激活ERβ蛋白表达，抑制ERα蛋白表达，促进p-ERK1/2、Ki67、c-Myc和CyclinD1等相关蛋白的表达。
Abstract:
Organochlorine pesticides are established environmental estrogens that are prone to accumulate in the body and produce chronic toxicity that harms human health. In this experiment, the combination effect of residual dose with no obvious estrogenic effect in a single presence was observed. Breast cancer MCF-7 cells were selected to study the estrogenic mechanism of organochlorine pesticides including hexachlorobenzene (HCB), β-hexahexachlorocyclohexane (β-BHC) and p,p'-clofenotane (p,p'-DDT) at residual dose. Firstly, MTT assay was used to observe the effects of three residual organochlorine pesticides on the growth of MCF-7 cells. Flow cytometry was used to determine the effects of organochlorine pesticide combination on the cell cycle of MCF-7 cells. Western Blot method was used to determine the changes of ERα, ERβ, ERK1/2, p-ERK1/2, Ki67, c-Myc and CyclinD1 protein expression after the combination exposure of organochlorine pesticides. The results showed that the cell proliferation rate of β-BHC+p,p'-DDT, HCB+β-BHC+p,p'-DDT combined group was 115.0% and 120.1% after 24 h exposure. After 48 h exposure, the proliferation rate was 121.2% and 126.3%. However, no significant changes occurred in other combined groups, compared with the control. Flow cytometry analysis showed that the proportion of G1 phase cells decreased, the proportion of S phase cells increased, and the cell proliferation was high in the above two combined groups. Western Blot results showed that in the β-BHC+p,p'-DDT, HCB+β-BHC+p,p'-DDT combined group, and estradiol (E2) group, the expression of ERα protein was decreased, the expression of ERβ protein was increased, and the expressions of p-ERK1/2, Ki67, CyclinD1 and c-Myc protein were promoted. The study indicated that β-BHC+p,p'-DDT, HCB+β-BHC+p,p'-DDT combined group had significantly estrogenic effect, when no estrogenic effect was observed in their single presence at the same dose. Its mechanism was the estrogen receptor pathway, which is activating the ERβ protein expression, inhibiting the ERα protein expression, and promoting the expression of p-ERK1/2, Ki67, c-Myc, CyclinD1 and other related proteins.
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