微囊藻毒素对斑马鱼生殖内分泌的干扰效应及其机制研究

In recent studies, it has been found that microcystins (MCs), as a ubiquitous environmental pollutant, caused reproductive toxicity and disturbed the levels of sex hormone. However, there are very limited studies on the reproductive endocrine disruption of MCs. The specific mechanism of MCs reproductive endocrine disruption has been reported rarely. In light of the above, zebrafish were as experiment animal in this project. Through the exposure experiment of low dose of MC-LR, this project aims to 1) evaluate endocrine disrupting effects of MC-LR on the reproductive system in zebrafish of different developmental stages, 2) built the dose-effect relationship between the gonadal MC-LR concentration and endpoints of reproductive endocrine disruption based on the quantitative and qualitative analysis of gonadal MC-LR using HPLC-ESI-MS, 3) discuss the molecular mechanism for reproductive endocrine disruption of MC-LR on zebrafish from the views of HPGL axis pathway, sex hormone synthesis pathway and steroid receptors-mediated pathway using fluorescence quantitative PCR and Western blotting respectively, and further screen molecular targets and available sensitive biomarkers for reproductive endocrine disruption using proteomics technology and establish evaluation standards and models for risk evaluation of MC-LR on fish reproductive endocrine disruption. The results will try to provide scientific basis for evaluation of the potential harmful effects of MCs on human reproductive health and also for the risk management and health security of ecological environment to MCs pollution.

近来研究发现，微囊藻毒素（MCs）具有生殖毒性，造成性激素水平紊乱。然而，MCs相关的生殖内分泌干扰效应和机制的研究相对匮乏。针对这一问题，本项目以斑马鱼作为模型，MC-LR作为染毒物，通过低剂量暴露实验模拟野外染毒模式，评价MC-LR对不同发育阶段斑马鱼生殖内分泌系统的干扰效应；采用液质联用技术定性、定量分析染毒斑马鱼性腺中的MC-LR，建立MC-LR生殖内分泌干扰的剂量-效应关系；釆用实时荧光定量PCR和Western blot技术，分别从下丘脑-垂体-性腺-肝（HPGL）轴、性激素合成途径、类固醇受体介导途径系统的探讨MC-LR干扰性激素合成和作用的分子机制，并从蛋白组学水平挖掘MC-LR生殖内分泌干扰作用的靶蛋白，筛选可利用的敏感的生物标志物，建立MC-LR生殖内分泌干扰作用的评价标准和模型。研究开展对于评估MCs对水环境的危害及人类生殖健康的潜在威胁具有重要的理论和实际意义。

当前，湖泊富营养化和蓝藻水华频繁爆发致使微囊藻毒素（MCs）对水资源的污染越来越严重，由此产生的对水环境危害及人类健康威胁已成为人们关注的重大环境问题之一。大量研究表明，MCs不但是典型的肝毒素，还具有明显的生殖毒性，可对鱼类的繁殖和种群产生不利影响，然而其相关的生殖毒性机制尚未阐明。针对此，本研究以MC-LR作为染毒物，在MC-LR环境相关浓度范围内设置不同的浓度组，以斑马鱼为实验模型动物，分别从性腺指数，组织病理，激素含量，关键基因和蛋白的表达及组学分析研究多方面，深入系统的阐明了环境浓度的MC-LR长期反复暴露造成的生殖内分泌干扰机制，潜在的雌激素样作用机制及通过生殖毒性导致的跨代免疫毒性的机制。研究结果表明：MC-LR对鱼类生殖内分泌干扰的作用机制主要是通过干扰下丘脑-垂体-性腺轴上关键基因的表达，由此造成性腺病理损伤，性激素分泌紊乱，怀卵量的减小和精子活力降低，并且性腺中的芳香化酶CYP19a为其关键的作用因子；证实MC-LR是通过干扰类固醇激素的合成而引起雌激素样作用；揭示环境浓度的MC-LR造成的性腺损伤还会导致子代免疫受损，具有跨代的免疫毒性。研究的开展揭示水华污染可能是引发鱼类种群衰退和渔业资源减产的重要原因之一，同时也为全面评估蓝藻水华的危害影响及其生态负面效应提供了有力的参考依据。