斑马鱼胚胎暴露多环芳烃导致生命后期毒性效应及其表观遗传学机制

It is a new ecotoxicological task that early-life exposure to pollutants results in a persistent toxic effect throughout life in animals and the underlying mechanisms, which would raise a challenge to the risk assessment of pollutants. Based on our previous results, in this project, we will use zebrafish as experimental target to observe late-life toxic effects as well as transgenerational effects induced by embryonic exposure to polycyclic aromatic hydrocarbons (PAHs), and to research the underlying mechanisms via investigating the perturbation of DNA methylation and histone acetylation. The late-life toxic effects caused by embryonic exposure to benzo[a]pyrene and phenanthrene will be comprehensively examined and analyzed using histological, biochemical, genomics and molecular biological technology. Differentially expressed genes caused by embryonic PAH exposure will be investigated. The alteration of DNA methylation as well as histone modifications and their stability will be checked during the development and throughout life of the fish. The fluctuation in the methylation status of genes (including their promoters) related to the toxic effects (mainly concerning heart, reproduction and development) will be comprehended, the relation between the methylation status as well as histone acetylation and the expression of genes will be elucidated, and mechanisms underlying the late-life toxic effects induced by embryonic exposure to PAHs would be explored. The mechanisms underlying the transgenerational effects will be revealed via investigating the expression and epigenetic alterations of the imprinted genes, comparing the difference between the parental and maternal transgenerational effects, and analyzing their reasons. The results would provide critical biomolecular evidences to understand this important issue of toxicology, and indicate a novel idea and theoretical foundation for the risk assessment of contaminants.

生命早期暴露污染物产生长久的毒性效应及其机制是生态毒理学面临的新课题。本项目通过斑马鱼胚胎期暴露多环芳烃，采用组织学、生物化学、基因组学和分子生物学技术，全面地观察分析胚胎期暴露多环芳烃[苯并（a）芘和菲]对斑马鱼的后期毒性效应及传代效应；通过DNA甲基化和组蛋白修饰分析和理解这类效应的机制，通过基因组学技术了解多环芳烃诱导的差异表达基因，检测多环芳烃暴露对DNA甲基化和组蛋白乙酰化的影响以及在发育过程的稳定性；掌握与毒性效应（主要是心脏、生殖和发育毒性）相关基因甲基化和组蛋白乙酰化状态的变化，阐明基因甲基化和组蛋白乙酰化改变与基因表达的关系，揭示多环芳烃早期暴露产生持久性毒性效应的机制；通过印记基因的表达和表观遗传状态的变化，揭示传代效应的机制，比较分析雄性和雌性传代效应的差异和原因。研究成果将为理解这一毒理学的重要问题在分子机制上提供重要资料，为污染物的风险评估将提供新的思路和理论依据

生命早期暴露污染物产生长久的毒性效应及其机制是生态毒理学面临的新课题。本项目通过斑马鱼胚胎期暴露多环芳烃，观察分析胚胎期暴露多环芳烃[苯并（a）芘和菲]对斑马鱼的后期毒性效应及传代效应及其机制。.斑马鱼胚胎暴露菲或苯并（a）芘（0.05、0.5、5、50 nmol/L）96小时后，转移至清洁水中培养至成年。菲暴露组成年与正常成鱼进行交配所产胚胎的数量与受精率均下降；组织学检测结果显示，卵巢内成熟卵母细胞的相对数量减少，精巢内成熟精子的相对含量减少；性腺与肌肉样品中的睾酮与17β-雌二醇含量均下降。实时荧光定量PCR结果显示，脑-垂体-性腺轴的生殖相关的基因，如GnRH3、Fshβ、LHβ、LHγ、cyp19a1a、cyp19b、vtg1、vtg2、ar、esr1、esr2β的转录水平均下降。脑中GnRH3的启动子区域呈现高甲基化状态，这与其转录水平降低相吻合。而苯并（a）芘暴露后的成年雄鱼，除了脑-垂体-性腺轴的生殖相关的基因转录水平下调之外，精巢中配子发育相关的特异性基因转录水平下调， 其中ddx4, dnd1, nanos2的启动子甲基化水平的升高与它们的转录水平下调相关。这些途径可能是胚胎时期暴露导致成鱼生殖毒性的机制之一。.斑马鱼胚胎暴露菲（0.05、0.5、5、50 nmol/L）96小时后，在清洁水中培养至成年的鱼与正常未暴露的成鱼交配所产生的F1代仔鱼，颅面骨发育延迟，麦克氏软骨和下颌骨变短。母系后代比父系后代的影响更大。与颅面骨发育相关的基因runx2、fgf8、shh和ihh的转录水平下调。runx2、和shh的启动子甲基化水平在暴露的胚胎发生改变，并且遗传到F1代，这是颅面骨发育缺陷传代的原因。同时，观察到F1代心脏发育的缺陷，包括加快的心率、扩大的心脏和异常的收缩。母系仔鱼比父系仔鱼显示更大的心房与心室距离，而父系仔鱼比母系仔鱼显示更严重的心率不齐。与心脏发育相关的基因转录水平受到影响。这与它们的甲基化水平改变有关，相关性分析显示与组蛋白乙酰化水平也有关。.研究结果为理解生命早期暴露的后期效应在分子机制上提供重要资料，为污染物的风险评估将提供新的思路和理论依据。