对壬基酚（4-NP）对菲律宾蛤仔的生殖内分泌干扰效应及其机理分析

Marine bivalves are an ecologically and economically important group of molluscs. Recently, wild population declining of marine bivalve species has received extensive attention. Gonadal neoplasia was also found to be more frequently appeared in marine bivalve which considered to be caused by endocrine disrupting chemicals (EDCs). In coastal environment, nonylphenol (NP) was often found to be the dominant EDC. However the reproductive toxicity of NP on molluscs was rarely studied. In the present study, by means of techniques including recombinant yeast assay, qRT-PCR and hybridization in situ etc., we will try to elaborate on the reproductive endocrine disrupting effects of 4-NP, the majority isomers of NP, on manila clam Ruditapes philippinarum. We will explore the mode of actions of 4-NP on steroids biosynthesis and estrogen receptor pathway of manila clams. The manila clams will be exposed to 4-NP during the gonad developing period. Histological changes of gonad, steroids levels, expression of reproductive function related genes and quality of gamete will be measured to elucidate whether imposex, gonadal development delay and sex ratio change will happen after 4-NP exposure. We will try to identify the potentially sensitive molecular targets of 4-NP in the reproductive endocrine system of manila clam, as well as to find out the causal relationship between the 4-NP induced adverse effects and the molecular mechanisms. The study may take a first attempt towards establishing methods for early warning evaluation of EDCs based on bivalves. The results may provide more ecotoxicological insights for resource protection of marine bivalves.

双壳贝类在海洋生态系统中具有重要地位，很多种类具有较高的经济价值。目前海产贝类的自然种群衰退以及贝类性腺瘤的发生率上升等问题引起了广泛关注，并推测与环境内分泌干扰物（EDCs）有关。壬基酚（NP）在很多地区沿海环境中被发现是含量最高的EDCs，然而迄今关于NP对贝类生殖内分泌干扰效应的研究非常有限。本课题以埋栖型经济贝类菲律宾蛤仔为研究对象，选择环境含量最高的4-NP为代表，运用重组酵母表达、qRT-PCR、LC-MS等技术，探索4-NP对其性激素合成途径、雌激素受体通路的作用模式，通过长期暴露实验分析4-NP对其性腺发育、激素调节、关键功能基因表达、性比变化以及配子质量的影响，解析4-NP对菲律宾蛤仔的生殖内分泌干扰机制，查明4-NP对其干扰作用的靶点，为开发基于双壳贝类的EDCs早期评价方法提供新的尝试。研究结果可为贝类资源保护和EDCs的海洋生态毒理评价提供理论依据与技术支撑。

本课题瞄准近海环境内分泌干扰物（EDCs）污染问题，以埋栖型经济贝类菲律宾蛤仔为研究对象，选择环境含量最高的对壬基酚（4-NP）为代表，利用重组酵母表达等分子生物学技术与生理、生化研究方法，探索了4-NP等EDCs对菲律宾蛤仔内分泌干扰效应机理，主要研究发现如下：利用RACE技术获得了雌激素受体（ER）和 雌激素相关受体（ERR）基因的cDNA全长，在此基础上利用酵母载体共转化技术构建了包含ER表达载体与雌激素受体响应元件（ERE）载体的重组酵母系统，并同时构建了长牡蛎、斑马鱼的ER/ERE重组酵母系统。利用两种贝类和斑马鱼的重组酵母系统，比较并评价了4种天然性激素（雌二醇、雌醇、孕酮、睾酮）和3种EDCs（4-NP、苯并[a]芘（BaP）、四溴双酚A（TBBPA））对ERs的诱导活性，发现这些化合物对贝类ER均具有诱导效应，并获得了这些化合物对3个物种ER转录激活活性影响的剂量-效应曲线。利用分子对接模拟初步探究了ER氨基酸残基与配体的相互作用，预测的配体结合腔与结合能大小顺序与重组酵母实验结果基本符合，从分子水平探究了EDCs对贝类雌激素受体的影响。以生殖内分泌干扰途径为研究背景，对比研究了E2和4-NP暴露对菲律宾蛤仔幼贝生长状况、性激素含量、性激素合成转运关键基因表达水平的影响，发现4-NP对性激素合成关键基因与性激素含量影响显著，并能显著诱导了幼体组织中卵黄蛋白原基因表达上调，说明4-NP从ER途径和性激素合成途径两方面造成内分泌干扰效应。综上，本研究查明了4-NP对菲律宾蛤仔生殖内分泌系统的作用靶点，解析了4-NP对菲律宾蛤仔的内分泌干扰机制，为贝类资源保护和EDCs的海洋生态毒理评价提供了理论依据与技术支撑。