ER和AhR通路介导的壬基酚对翡翠贻贝生殖内分泌干扰毒性研究

Nonylphenol is a typical environmental estrogen and persistent organic pollutant, and can affect the functions of reproductive, nervous and immune system of animals. Both AhR and ER are involved in xenobiotic metabolism and endocrine regulation of animals. Crosstalk has been observed between AhR and ER. AhR and ER may interact on each other through a number of different mechanisms due to existence of different contaminants. Nonylphenol may act as ligand of aryl hydrocarbon receptor (AhR) and estrogen receptor (ER), and modulates response genes of the two signaling pathways. According to the results of previous studies, we hypothesized that crosstalk of AhR and ER may affect the reproductive endocrine disruption effects of nonylphenol on marine invertebrates. How did AhR and ER signaling pathways involved in the toxic effects of nonylohenol was still unknown. The green mussel Perna viridis is commercially important seafood and is widely distributed in coastal waters of China. It has frequently been used as a biomonitor for contaminants in the marine environment. The aim of this project is to investigate the toxic effects and molecular mechanism of nonylphenol on the reproduction of P. viridis by using molecular biology, cytobiology and ecotoxicology technology. In this study, we are going to clone and analyze AhR, ER and cytochrome P450 (CYP) genes of P. viridis. The expression characteristics of AhR, ER, CYP and vitellogenin (Vtg) genes will be studied after nonylphenol challenge. AhR and ER gene of P. viridis will be silenced by RNAi technology to investigate crosstalk mechanism among AhR and ER in response to nonylphenol. Results of this study will shed light on understanding toxic effects of nonylphenol and provide important evidence to illustrate mechanisms on how nonylphenol affects marine invertebrate.

壬基酚是一种典型的环境雌激素和持久性有机污染物，可以对海洋生物的生殖发育产生影响，成为目前毒理学研究领域的热点。芳香烃受体（ER）和雌激素受体（AhR）通路在动物外源物质代谢和内分泌调节中发挥着重要作用，当有外源配体（如环境污染物）存在时，二者可交叉影响，进而影响动物生长、生殖和发育等一系列过程。研究表明，壬基酚可作为配体与ER和AhR结合，通过调节两条通路下游基因的表达，干扰动物的性腺发育和生殖内分泌。我们根据以往的研究结果，提出AhR和ER通路之间的相互作用可能介导了壬基酚对动物的生殖内分泌干扰这一假说。为验证上述观点，拟以海洋无脊椎动物——翡翠贻贝为研究对象，运用分子生物学、细胞生物学和生态毒理学技术手段，对翡翠贻贝芳香烃受体和雌激素受体通路相关基因进行克隆、表达分析和在壬基酚暴露下的功能验证，旨在从ER与AhR相互作用角度评价壬基酚对海洋无脊椎动物的毒性机制。

本项目以翡翠贻贝为研究对象，围绕“芳香烃受体通路和雌激素受体通路是否介导了壬基酚对翡翠贻贝的毒性”这一科学问题展开了相关的研究。应用分子生物学、转录组学和代谢组学等技术手段，分析了壬基酚胁迫对翡翠贻贝的毒性效应和毒性机制，并初步探讨了雌激素受体和芳香烃受体通路在壬基酚胁迫过程中的作用机制。研究主要从四个方面展开：（1）通过翡翠贻贝全长转录组的测定结合cDNA末端快速扩增技术，获得了翡翠贻贝雌激素受体基因（er）、芳香烃受体基因（ahr）、卵黄蛋白原基因（vtg）和细胞色素P450基因（cyp4），为后续实验的开展奠定了基础；（2）通过急性毒性实验的研究，确定了壬基酚对翡翠贻贝的96h LC50为2.75mg/L，进而结合壬基酚的环境浓度，确定了壬基酚亚急性胁迫实验浓度；（3）不同浓度壬基酚胁迫翡翠贻贝，发现壬基酚可以引起翡翠贻贝生殖细胞的释放，对翡翠贻贝性腺和鳃组织结构产生影响，高浓度壬基酚胁迫可以抑制翡翠贻贝芳香烃受体基因及其下游基因的表达，转录组和代谢组结果表明不同浓度壬基酚胁迫的作用机制存在差异，雄性翡翠贻贝较之雌性对壬基酚更为敏感；（4）开展了雌激素受体和芳香烃受体拮抗剂与壬基酚联合毒性研究，结合基因沉默技术，分析了雌激素受体通路和芳香烃受体通路基因表达模式变化。结果表明壬基酚胁迫不影响雌激素受体基因的表达，但可诱导卵黄蛋白原基因表达量及其蛋白含量，说明克隆得到的翡翠贻贝雌激素受体可能为非配体依赖型受体，同时可能有其他信号通路介导了壬基酚的毒性效应。与此同时，壬基酚对芳香烃受体及其通路下游基因有抑制作用，且未发现两个通路之间的相互作用。综上，本研究从分子、蛋白、组织和个体水平分析了壬基酚对翡翠贻贝的毒性效应，初步探讨了雌激素受体和芳香烃受体通路在壬基酚毒性机制中的作用，项目成果有助于更好的理解环境雌激素类污染物对海洋无脊椎动物的毒性机制。有关贝类雌激素受体及其通路相关基因功能的研究有待于进一步深入。