环境雌激素对鱼类Kisspeptin/GnRH系统的神经毒性及作用机理研究

Bisphenol A and alkylphenols are environmental estrogen-like compounds being introduced into the aquatic ecosystems by human activities. Those chemicals have been confirmed by numbers of previous studies to have significant endocrine disrupting effects on aquatic organisms, especially on fish. Our previous studies have shown that those chemicals are also neurotoxic substances to fish. Upon exposure to those compounds, the expression of genes related to the development of nervous system in fish embryos has been changed, and cell apoptosis has been induced, and the expression levels of kisspeptin / GnRH system-related genes have been altered. According to those results, the present project is designed to evaluate the effects of bisphenol A and nonylphenol on kisspeptin / GnRH neural system in fish under individual and joint exposure to those chemicals at environmental relevant concentrations. The in vivo and in vitro neurotoxicity experiments will be set using the lived whole mount GnRH transgenic zebrafish embryos and the primary neural cells cultured from GnRH transgenic zebrafish embryos. The combined exposure with estrogen receptor antagonist and receptor knock-out strategies will be implemented. Using multicolor fluorescence labeling and confocal microscopy imaging methods, the project aims to explore the toxic response, signaling pathway and its role in the regulation mechanism of kisspeptin / GnRH system, the upstream regulatory neural system in the central nervous system modulating the response of Hypothalamic - Pituitary - Gonadal (HPG) axis in fish. The project is expected to clarify the action mechanisms of environmental estrogen-like chemicals regarding their endocrine disrupting effects and neurotoxicity on fish. Furthermore, these results should be of great scientific significance for breeding and population conservation for fish stocks.

双酚A和烷基酚类化合物是由人类活动引入水生生态系统的环境雌激素，已确认对水生生物特别是鱼类具有显著的内分泌干扰效应。申请人前期的研究发现该类物质对鱼类具有神经毒性，如影响胚胎神经发育相关基因的表达、诱导神经细胞凋亡、影响kisspeptin/GnRH系统基因的表达水平等。本项目拟在前期工作的基础上，以模式生物野生斑马鱼和绿色荧光标记的GnRH转基因斑马鱼为研究对象，以双酚A和壬基酚为目标物，设置环境相关浓度下单独暴露和联合暴露的在体和离体神经毒性实验，并通过拮抗剂共暴露和受体基因敲除等策略，结合多色荧光标记和共聚焦显微技术，从细胞和分子水平探索鱼类中枢神经系统在下丘脑-垂体-性腺轴上游的kisspeptin/GnRH调控系统对该类化合物的毒性响应及其作用途径和调控机理。本项目预期从根本上阐明环境雌激素类物质对鱼类内分泌干扰效应及神经毒性发生的根源，为保障鱼类种群繁育具有重要的科学意义。

环境内分泌干扰物对多种环境介质特别是水体的广泛污染是近年来全球关注的环境热点问题。双酚类环境雌激素污染物在各类水体中普遍能检测到，尽管浓度较低，但是其对水生生物产生低剂量长期的影响却不容忽视，对整个水生生态系统具有潜在的生态威胁。本项目以阐明环境雌激素内分泌干扰物质对鱼类的神经毒性以及其毒性在中枢神经系统层面的调控机理为目标，以环境雌激素类化合物对鱼类下丘脑–垂体–性腺（HPG）轴的作用途径出发，探索其上游kisspeptin/GnRH系统通过经典途径或旁路途径对HPG轴的调控作用通路。本项目按照申请书研究计划执行，已达到预期研究目标。项目研究发现双酚A（BPA）及其替代物双酚S（BPS）的暴露对斑马鱼胚胎发育阶段的神经内分泌系统具有相似的毒性效应，结果如下：（1）在环境浓度10和100μg/L BPA暴露下，斑马鱼三叉神经和下丘脑中GnRH3神经元数量出现显著性增多，神经内分泌系统相关基因（Kiss 1、Kiss 1r、GnRH3、LHβ、FSHβ、SV2c）的表达受到影响，暗示BPA会对斑马鱼胚胎发育阶段神经内分泌系统产生影响；（2）相同浓度的BPS能够显著诱导GnRH3神经元的发育和神经内分泌系统相关基因的表达水平，表明BPS有类似BPA的效应，能够显著干扰鱼类神经内分泌系统；（3）研究结果还表明，雌激素受体，甲状腺受体和芳香化酶通的抑制剂能够显著抑制BPA或BPS对内分泌系统相关基因包括Kiss 1、Kiss 1r、GnRH3、LHβ、FSHβ和ERa的诱导水平，表明BPA和BPS对鱼类的神经内分泌系统干扰作用与雌激素受体，甲状腺受体和芳香化酶三条通路之间存在密切的关系。此外，对比相同浓度BPS和BPA暴露对原代巨噬细胞的毒性效应发现，BPS和BPA都能够显著影响胞内自由基和免疫相关基因的表达水平，并且在诱导程度上，BPS和BPA有着非常类似的效应；但是不同于BPA的作用，BPS在诱导脂质过氧化和凋亡方面没有显著的效力；BPS可诱导erα和erβ2表达，而BPA仅诱导erα表达。最后，通过对野外采集的鱼类样品的分析检测，结合实验室暴露实验，本项目建立了鱼体中双酚和烷基酚类化合物的富集系数与水体中暴露浓度的相关性模型。本项目研究成果对于保护野生鱼类种群、保障养殖鱼类健康、维护水生生态系统平衡，都具有重要的科学意义。