丙烯酰胺对斑马鱼胚胎心脏发育毒性的分子毒理学效应及Notch信号通路激活机制

Acrylamide, a hazardous substance leading to environmental pollution and food safety issues, has recently raised toxicological concerns due to its cardiovascular developmental toxicity. However, related molecular toxicological effects and mechanisms of signaling pathway still remain poorly understood. Taking zebrafish as the animal model, the specific aims of this proposal are: (i) to elucidate the effects of acrylamide on morphological change, myocardial cell toxicity and oxidative damage during cardiac development of zebrafish embryos; (ii) to analyze the effects on spatiotemporal expression profiles of transcription factors for early cardiac development and genes for cardiomyocytes and atrioventricular functions and cardiac microstructure development; (iii) to demonstrate the mechanisms of cardiac developmental toxicity via interfering microRNA expression using combined techniques of overexpression, knock-down and target gene prediction; (iv) to observe the effects on Notch related target genes and downstream genes via activating Notch signaling, and reveal the mechanisms on acrylamide-induced endocardial cushions defect and atrioventricular valve hypertrophy; and (v) to mechanistically understand the interaction between Notch ligand and receptor domains using combined techniques of molecular cloning, co-immunoprecipitation and subcellular localization. Overall, we will systematically investigate the cardiac developmental toxicity of acrylamide based on toxicological effect, molecular mechanism and signaling pathway levels. The anticipated outcomes will be benefit for improving the evaluation system of developmental toxicity and toxicological effect of environmental contaminants, and promoting evidence- and traceability-based researches on national environmental pollution and food safety related risk factors.

丙烯酰胺作为集环境污染与食品安全问题于一身的有害物质，其心血管发育毒性成为毒理学最新关注的方向，但相关分子毒理学效应和信号通路机制知之甚少。本项目以斑马鱼为模式动物，旨在阐明丙烯酰胺对斑马鱼胚胎心脏发育的形态学改变、心肌细胞毒性和氧化损伤效应；分析其对心脏早期发育转录因子、心肌细胞与房室功能、心脏微观结构发育相关基因时空表达谱的影响，集过表达、敲减和靶基因预测于一体论证其通过干扰miRNA而致心脏发育毒性的机制；观察其激活Notch信号对靶基因和下游基因的影响，及导致心内膜垫缺陷及房室瓣膜肥大的作用机制，并集分子克隆、免疫共沉淀和亚细胞共定位技术于一体揭示其影响Notch配体-受体结构域互作的机制。总体上从毒理学效应、分子机制和信号通路三个层面系统研究丙烯酰胺的心脏发育毒性。预期成果为完善环境污染物发育毒性和毒理学效应评价体系奠定基础，并推动我国环境污染与食品安全危害因素的循证和溯源研究。

丙烯酰胺作为典型食品热加工污染物，已被证实以血红蛋白加合物的形式通过胎盘屏障进入胎儿体内，对胚胎造成发育毒性；而本课题组前期人群队列研究表明，丙烯酰胺的体内暴露水平与心血管疾病的发生率和死亡率呈显著正相关。由此，引发了丙烯酰胺对胚胎心脏发育毒性的思考探究。本项目以斑马鱼胚胎为模型，从毒理学效应、细胞生物学机制以及分子机制等层面系统研究丙烯酰胺急性暴露和亚慢性暴露后对心脏发育的影响及其致毒机理。研究结果表明，氧化应激可能是丙烯酰胺急性暴露的致毒机理，而亚慢性的丙烯酰胺暴露对斑马鱼的心脏形态、功能和心肌细胞生长及凋亡产生了不良的影响，从而确定心脏是丙烯酰胺发育毒性的重要靶器官。其次，结合毒理学效应、细胞生物学机制和分子机制等层面，探究了丙烯酰胺对心脏发育各个阶段的影响，建立时空表达图谱，全面系统地评估了丙烯酰胺心脏发育毒性作用，发现丙烯酰胺导致心脏早期发育的关键因子过度、异位表达，心肌细胞和心内膜细胞的数目减少，心脏房室通道的分化障碍，心内膜细胞向间质细胞的转变障碍，心肌和心内膜之间的物理间隙增大，最终影响房室瓣膜的发育和心脏成熟。最后，发现丙烯酰胺暴露干扰胚胎心脏发育过程中Notch信号通路，导致心室肌小梁增生和迁移受阻，揭示丙烯酰胺心脏发育毒性作用的靶通路，解释心室壁增厚而心脏功能下降的表型异常。相关研究成果为完善食品加工污染物发育毒性和毒理学效应评价体系提供科学依据，对于我国食品安全危害因素的循证和溯源研究具有重要意义。本项目执行期间共发表标注本基金资助的SCI论文8篇，其中IF5>5的论文6篇，参加国际会议并作口头报告3次，参加国内学术会议并作口头报告4次。项目负责人获得2018年中国毒理学会优秀青年科技奖。本项目共培养博士研究生2名，硕士研究生3名。