新型有机卤代化合物调控维甲酸受体诱导鱼类胚胎发育毒性效应及分子机制

Emerging halogenated organic compounds are now causing increasing concerns due to their universal contamination in our daily life. Nevertheless, the related toxicological data are rather limited. Considering the irreversible hazardous effects of emerging endocrine disruptors on the wildlife and human beings, the efficient screening methods are highly required for these emerging chemicals of concern. The retinoic acid receptors (RARs) play important roles in the mediation of the endocrine system and the developmental biology in vertebrates, thus offering a very promising strategy for endocrine disrupting effect screening of emerging chemicals. In this project, new recombinant cell line models transfected with RAR isoforms will be developed to test RAR mediated endocrine disrupting effects of emerging halogenated organic chemicals, and their developmental toxicities will be studied using the embryos of the native fish species, the Chinese rare minnow (Gobiocypris rarus). The target chemicals in this project mainly include tetrabrominated bisphenol A (TBBPA) and its derivatives, per- and polyflurinated substances (PFASs), etc. The toxicological endpoints based on morphological, histopathological, and molecular biological levels, will be evaluated for RAR-mediated effects in fish embryo development. The crucial structural factors of the chemicals which regulate their binding affinities for RAR isoforms will be discussed, and the deformities induced by chemical exposure in fish embryonic ocular and cardiovascular development will be further studied. The research findings based on this project will offer solid technical support for the screening of emerging chemical-induced endocrine disrupting effects, and provide substantial new evidences on their toxicological profiles for the risk assessment of the related environmental exposure.

新型有机卤代化合物品种繁多、结构复杂，其环境污染已引起人们高度关注，然而这类化合物的毒理学效应研究刚刚起步，特别是其内分泌干扰效应的毒理学数据严重匮乏。维甲酸受体（RARs）介导的信号通路在机体生长发育过程中发挥了重要的作用，为新型污染物内分泌干扰效应评价提供了新的研究思路。本项目拟构建筛选具有不同亚型RARs调控活性的重组细胞离体分析技术，并基于中国特色的小型鱼类稀有鮈鲫模型，建立RARs调控胚胎发育的毒性评价体系，在离体与活体层面上研究新型有机卤代化合物对不同亚型RARs的激动或拮抗作用，探讨化合物暴露产生的鱼类胚胎发育毒性效应。着重分析污染物调控RARs活性的关键结构因子，以及其诱导鱼类胚胎眼与心血管发育畸变的作用靶点与致毒途径。本项目研究可为新型有机卤代化合物内分泌干扰效应筛选提供关键技术支持，也可为其环境暴露引发的生态风险与潜在人体健康效应评价提供重要科学依据。

新污染物种类繁多、对环境与人体健康的毒性数据欠缺，特别是其潜在的内分泌干扰效应值得关注。维甲酸受体是机体内分泌系统的重要靶点，探讨污染物干扰维甲酸受体信号通路产生的发育毒性是新污染物毒理学效应评价的重要基础。本项目通过构建维甲酸受体RAR alpha表达的质粒载体，采用人源细胞作为宿主，优化确立了RAR alpha活性筛选的细胞模型。研究同时结合酵母双杂交系统，筛选了75个化合物对RAR alpha的激动或拮抗活性。在确定典型污染物具有RAR alpha激动活性的基础上，利用斑马鱼胚胎模型探讨了化合物的发育毒性与神经毒理效应，并解析了其内在的构效关系。研究发现，基于MCF-7构建的RAR alpha表达载体pEF1α-RAR alpha-RFP和含有维甲酸反应元件（RARE）的荧光素酶报告基因载体pRARE-TA-Luc双转染细胞模型对RAR alpha激动剂或拮抗剂刺激响应灵敏度高、方法稳定性好，并可成功用于新型有机酚类化合物的内分泌干扰效应评价。大规模筛选新污染物的RAR alpha活性显示，大多数化合物具有受体拮抗活性，仅有少量化合物具有较弱的受体激活活性。针对具有RAR alpha激活活性的对羟基苯甲酸酯开展暴露研究显示，该类化合物可以引起斑马鱼胚胎发育异常，产生明显的形态学畸形现象与神经行为学改变，并且酯基碳链越长，毒性越强。以上研究成果，一方面为新污染物内分泌干扰效应筛选提供了重要技术支持，另一方面也为新污染物调控RAR alpha信号通路介导的毒理机制解析提供了科学思路。通过项目研究获得的化学品毒性数据，为其环境安全评价与管理标准拟定等提供重要依据。