微囊藻毒素的母代转移效应所引发的斑马鱼甲状腺毒性及其分子机制

With frequent occurrence of cyanobacterial blooms in recent years, cyanotoxins have become a great threat to aquatic ecosystem and public health. Microcystins (MC) are the most dangerous group of cyanotoxins, with microcystin-LR (MCLR) being one of the most abundant and toxic variants in blooms. Recently, endocrine-disrupting toxicity of MCLR has gotten more and more attention. Previous studies from our laboratory and others have shown that MCLR could alter gene expression in the hypothalamic-pituitary-thyroid (HPT) axis which might contribute to MCLR-induced thyroid hormone disruption, eventually causing growth retardation of fish. However, the molecular mechanism underlying this process has not yet been clarified. The studies of MCLR-induced thyroid toxicity at environmentally relevant concentrations are still limited. In our preliminary work, we established proteomic model of zebrafish larvae. Based on our previous work, the present study aims to investigate the molecular mechanism of thyroid toxicity induced by maternal transfer with chronic MC exposure at environmentally relevant concentrations, employing mophological observation, behavioral assessment, LC-MS, two-dimensional gel electrophoresis, real-time PCR and other relevant techniques. The present study was carried out in several levels including individual, tissue, protein and gene, the results of which will elucidate the molecular mechanism in thyroid disruption effect of MCLR from a new perspective. Our results will also be benefit to identify the potential biomarkers for MCLR induced thyroid toxicity and provide new ideas and theoretical references for preventing the hazards of MC.

世界各地蓝藻水华频繁暴发给水生态系统和人畜健康造成了严重危害。微囊藻毒素（Microcystins, MC）是危害最大的蓝藻毒素，其中以MCLR的毒性最强。近年来，MC的内分泌毒性受到越来越多的关注。国内外学者和申请人前期工作显示MC可通过下丘脑-垂体-甲状腺（HPT）轴干扰甲状腺激素分泌并引发甲状腺毒性来影响鱼类发育，但已有MC甲状腺毒性的研究多为描述性实验，关于机制的深入探讨和系统性研究不足，且缺乏长期低剂量染毒的研究。申请者开展了部分前期工作，建立了早期发育阶段斑马鱼蛋白组学研究模型。本项目拟在前期研究基础上采用慢性环境浓度染毒方式，运用形态学、行为学、液质联用、双向电泳、定量PCR等多种技术，从个体、组织、基因、蛋白等多个层面探讨MC在斑马鱼母代与子代间的转移效应以及毒性转移所引发的甲状腺毒性，筛选MC甲状腺毒性关键蛋白，拓展人们对MC诱发胚胎毒性的认知，为MC的防治提供理论依据。

世界各地蓝藻水华频繁暴发给水生态系统和人畜健康造成了严重危害。微囊藻毒素（Microcystins, MC）是危害最大的蓝藻毒素，其中以MCLR的毒性最强。近年来，MC的内分泌毒性受到越来越多的关注。国内外学者和申请人前期工作显示MC可通过下丘脑-垂体-甲状腺（HPT）轴干扰甲状腺激素分泌并引发甲状腺毒性来影响鱼类发育，但已有MC甲状腺毒性的研究多为描述性实验，关于机制的深入探讨和系统性研究不足，且缺乏长期低剂量染毒的研究。在本项目中，申请人将成年斑马鱼（F0）暴露于环境相关浓度的MCLR（1，5和25 μg/L）45天，然后将F1代胚胎置于不含有MCLR的培养液中孵化5天。研究发现，在F0代中，暴露于25 μg/L MCLR显著降低了雌鱼血清T4的水平，对T3的水平无影响；而子代（F1）T4和T3含量均显著降低，并伴随生长迟缓现象；F0与F1代斑马鱼HPT轴相关基因表达模式均发生改变。研究结果表明：（1）MCLR可通过影响母体对子代的甲状腺激素传递而影响子代胚胎的早期生长发育；（2）MCLR可通过在子代体内蓄积而引发子代HPT轴功能紊乱，继而影响子代生长发育。此外，在本项目中，申请人将斑马鱼胚胎暴露于环境相关浓度（0，0.3，3和30 μg/L）MCLR 90天，直到性成熟。组织切片结果显示，MCLR对大脑超微结构造成严重损伤，表现为有髓神经纤维的水肿和崩溃，内质网扩张和线粒体肿胀。此外，在此过程中斑马鱼游泳行为发生异常，提示MCLR可能引发神经毒性。利用实时定量PCR检测发现，斑马鱼脑中氨基酸g-氨基丁酸（GABA）和谷氨酸代谢途径相关基因的mRNA水平发生显著变化。综上可知，微囊藻毒素环境浓度慢性暴露可通过诱导斑马鱼GABA神经递质系统的改变来引发神经毒性。