典型微囊藻毒素对斑马鱼的联合胚胎发育毒性及机制研究

Microcystins, a family of the secondary metabolism toxin of algae blooms in eutrophic lake, have become a global concern for its harm to the environment and human health. However, so far, the research on combined toxicity of different kinds of MCs is still rare, and the combined toxicity effects and mechanism are largely unknown. In the present study, the single and combined toxicity of MC-LR and MC-RR to zebrafish (Daniorerio) embryos were investigated under different treatment conditions. The purpose of this investigation were to 1) evaluate the combined approach between MC-LR and MC-RR in vivo by mixture toxicity evaluation model, 2) find out the behaviors and characters of the combined developmental toxicity of MC-LR and MC-RR through the detection of pathological changes and physiological and biochemical indexes in experimental animals, 3) quantitative evaluate the absorption and metabolism rules of MC-LR and MC-RR in vivo by liquid chromatogram- mass spectrometry (LC-MS), 4) try to identify the potential sensitive biomarkers and material base involved in the process of toxicity damage through the comparison of differences in metabolite map under different treatment conditions, and explore the possible mechanism of the combined developmental toxicity of MC-LR and MC-RR from the perspective of metabolites. In conclusion, the present study would comprehensively assess the combined embryonic developmental toxicity of MC-LR and MC-RR in several levels including organism, tissue and molecule.

富营养化湖泊中蓝藻水华次生代谢产生的微囊藻毒素（MCs）对环境和人类健康的危害已成为全球关注的重大环境问题之一。然而，目前对多种MCs的联合毒理效应及机制缺乏深入的认识。本研究选择MC-LR和MC-RR作为典型模式化合物，研究不同暴露模式下其对斑马鱼的联合胚胎发育毒性。此研究的目的主要包括：1）通过复合毒性模型评估MC-LR和MC-RR的联合毒性及其作用方式；2）对染毒动物病理、生理生化指标的检测，确定MC-LR和MC-RR联合暴露后胚胎发育毒性的表现和特征，结合液质联用技术定量分析两种典型MCs在体内的吸收和代谢规律；3）比较不同染毒条件下代谢产物谱的差异响应，找到毒性损伤作用的敏感生物标志物，从机体代谢物质层面探讨MC-LR和MC-RR联合暴露的胚胎发育毒性的可能机制。本研究将对MC-LR和MC-RR联合暴露进入机体后在不同水平(个体、组织、分子）引发的胚胎发育毒性效应进行全面的评价。

本研究选取斑马鱼作为研究对象，探讨了MC-LR和MC-RR两种常见的微囊藻毒素异构体对胚胎发育毒性的联合作用方式，运用代谢组学手段，对染毒后斑马鱼仔鱼体内的差异代谢物进行了分析，运用分子生物学与传统胚胎发育毒性评价技术，对MC-LR和MC-RR进入机体后引发的联合胚胎发育毒性效应进行了评估，进一步探讨其诱发胚胎发育毒性的潜在致毒机制。取得的主要研究成果如下：.（1）在单一急性毒性和联合急性毒性试验的基础上，通过LC50和畸形率等指标测定，分别采用不同评价方法判断两种化合物的联合作用类型。不同评价方法对MC-LR和MC-RR的联合作用类型的判断结果一致，在暴露120h时两种异构体化合物表现为协同作用。.（2）MCs暴露可以在斑马鱼仔鱼体内迅速累积，并产生毒性作用。MC-LR和MC-RR联合暴露后斑马鱼早期生长发育受阻，孵化率、心率和体长明显降低，畸形率和死亡率显著升高。同时幼鱼出现尾部变形、脊柱弯曲和心包囊水肿等致畸现象。MCs联合暴露可以促进活性氧的产生，引起了斑马鱼仔鱼体内的氧化压力，并且这种影响程度在联合暴露组更为严重，同时抗氧化酶活性和GSH含量的显著变化是对抗氧化压力的应激反应，表明GSH途径在细胞抵抗MCs引起的机体毒性的重要作用。.（3）MCs暴露可以干扰斑马鱼氨基酸类、脂类等类物质的代谢，其毒性作用与脂肪酸、氨基酸、核酸等代谢通路密切相关。.（4）在斑马鱼早期发育阶段，下丘脑-垂体-甲状腺（HPT）轴和神经系统发育是MCs毒性作用的重要敏感靶标。MC-LR和MC-RR暴露的毒性机制包括：调节HPT轴上的重要基因的表达进而干扰甲状腺激素的合成、转化与分泌，从而影响斑马鱼胚胎和幼鱼的早期发育。干扰神经元发育，包括干扰神经细胞结构的形成、分化和再生，进而影响神经元正常的功能发挥；干扰兴奋性和抑制性神经递质之间的动态平衡，影响正常的神经传导。