从芳香烃受体入手探究呋喃丹的抗雌激素效应机制

Carbofuran, a carbamate pesticide, shows high toxicity to fish, birds and mammals. The use of carbofuran is widely in China, moreover its residues are frequently detected in vegetables and surface waters. Carbofuran is known to exert anti-estrogenic effects on females, but the exact mechanisms remains unclear. In the present project, using zebrafish (Danio rerio) and isolated cells, we will confirm the anti-estrogenic effects of carbofuran by measuring a variety of endpoints including fecundity, gonadosomatic index, ovarian morphology, mRNA and protein expression levels of vitellogenin (VTG). Then the fact that carbofuran interacts with the aromatic hydrocarbon receptor (AhR) is regarded as the molecular initiating event, we will further investigate the effects of carbofuran on the expression of estrogen receptors (ERs), the binding rates of 17β-estradiol (E2) to ERα as well as the competition of cofactors and E2-ERα to elucidate how carbofuran inhibit the function of E2 after activating the AhR pathway. Besides the effects of the expression of the steroidogenic enzymes involved in E2 production and the metabolism of E2 will be evaluated to clarify how carbofuran down-regulated the internal E2 levels following the activation of AhR by carbofuran. This research aims to ascertain the exact molecular initiating event of carbofuran and find out the underlying mechanisms of its anti-estrogenic effects. Our project will provide new thoughts for future studies focusing on anti-estrogenic mechanisms of other exogenous chemicals whose structures are similar to carbofuran.

呋喃丹是一种高毒的氨基甲酸酯类农药，目前在蔬菜及地表水中均频繁检出。呋喃丹对雌性生物体具有抗雌激素效应，然而其机制尚不明确。本项目以斑马鱼(Danio rerio)和离体细胞系为实验对象，从产卵量、性腺指数、卵巢形态结构、肝脏卵黄原蛋白(VTG)表达量等几个指标联合确定呋喃丹对雌性斑马鱼的抗雌激素效应；尔后以呋喃丹结合芳香烃受体(AhR)为分子起始事件，从呋喃丹对雌激素受体(ER)表达水平的影响、对17β-雌二醇(E2)与ERα结合率的影响、对共同辅助因子及E2-ERα的竞争四个方面，探究呋喃丹激活AhR通路后如何拮抗E2功能；再从呋喃丹对合成E2的类固醇生成酶表达水平的影响、对E2代谢的影响验证呋喃丹激活AhR通路后如何降低E2水平。以期明确呋喃丹在细胞内的真正分子起始事件，查明呋喃丹发挥抗雌激素效应的根本原因，为呋喃丹结构类似物的抗雌激素效应机制的研究提供新思路。

呋喃丹是一种高毒的氨基甲酸酯类农药，目前在蔬菜及地表水中均频繁检出，正逐渐成为威胁生态环境和人类健康的危险因子。本项目选用雌性斑马鱼和MCF-7细胞作为受试对象，结合在体和离体实验，首先从呋喃丹降低雌性斑马鱼产卵量、降低雌鱼GSI和卵母细胞成熟度、抑制E2对MCF-7细胞的增殖诱导作用及下调雌鱼肝脏和MCF-7细胞中诸多雌激素效应基因的转录四个不同的生物学层次综合阐明了呋喃丹的抗雌激素效应。接着我们通过添加AhR抑制剂ANF进一步证实了呋喃丹能够激活AhR通路，并以此为分子起始事件。在排除呋喃丹能够降低内源E2水平这条途径后，本项目从呋喃丹对雌激素受体的影响入手，证实了呋喃丹激活AhR通路后通过增强ERβ的表达和加速ERα的降解来拮抗E2的正常生理功能，从而揭示了呋喃丹发挥抗雌激素效应的根本原因。本项目的研究结果为评价外源化学物质的抗雌激素效应建立起了一个较为系统的指标体系，同时也将抗雌激素效应机制的探究向前推进至分子起始事件的层面，为今后呋喃丹结构类似物的抗雌激素效应机制研究及预测提供了新的参考。