典型手性三唑类杀菌剂与人细胞色素CYP3A4酶相互作用的分子机制研究

The triazole fungicides were used quantitatively in China and their residues much exceeded the maximum residue limit,causing serious environmental health issues.The research abroad found the hepatocarcinogens properties of triazole fungicides and their risk management was further strengthened.However,the domestic research on toxic identification and evaluation of triazole fungicides is rare,especially there lacks the research on molecular mechanisms of action,showing the necessity and urgency to carry out relevant research.In this project,the QM/MM method together with fluorescence lifetime,cell proliferation assay and Western Blot analysis will be performed to investigate the interactions of eight typical triazole fungicides with human CYP3A4 enzyme.The focus of this project is to probe the stereoselective difference of enantiomers,to monitor conformational changes and activity change of human CYP3A4 enzyme at an atomic level of detail and to investigate the effect on CYP3A4 expression within HepG2 cell and on HepG2 cell proliferation.Our aim is to elucidate mechanism of toxic action of triazole fungicides,thus providing scientific data for risk assessment of environmental and human health hazards.

目前我国三唑类杀菌剂使用量大且残留超标严重，已引起严重的环境健康问题。国外已发现三唑类杀菌剂的致肝癌毒性，并加强了对三唑类杀菌剂的风险管理，而国内相关研究工作极少，尤其缺乏针对毒性作用分子机制的研究，这预示了开展相关研究工作的必要性和紧迫性。本项目将采用QM/MM计算方法并结合荧光寿命、细胞增殖、蛋白质印迹等实验研究8种典型手性三唑类杀菌剂与人CYP3A4酶的动态相互作用。在对映体水平上考察对映体的立体选择性差异;在分子水平上考察对人CYP3A4酶构象变化、酶活性的影响；在细胞水平上考察对HepG2细胞内CYP3A4酶表达的影响和促人肝癌HepG2细胞体外增殖效应；以期诠释手性三唑类杀菌剂毒性作用的分子机制，为环境安全和人体健康风险评估提供重要的科学数据支持。

当前三唑类杀菌剂的大量使用导致其在多种水果蔬菜中残留超标，其暴露对人体健康造成潜在的危害。本项目研究了多种手性农药的对映体拆分方法及绝对化学构型归属，考察了三唑类杀菌剂对人CYP3A4酶活性、HepG2细胞内CYP3A4 mRNA水平和蛋白表达的影响。采用荧光光谱学和拉伸动力学、分子动力学模拟等理论方法研究了与CYP3A4分子交互作用的机制。构建了多种手性农药拆分方法；建立了基于圆二色谱和含时密度泛函(Time-dependent density functional theory, TDDFT)方法的手性污染物绝对构型表征方法；发现了三唑类杀菌剂对人CYP3A4酶抑制活性的对映体选择性差异；从构效关系角度利用分子动力学模拟方法揭示了三唑类杀菌剂对映体与CYP3A4酶结合模式和结合能量的不同，表征了三唑类杀菌剂诱导的CYP3A4酶构象变化，诠释了对映体选择性差异机制；明晰了典型三唑类杀菌剂苯醚甲环唑通过ROS介导的线粒体途径和内质网应激相关通路诱导HepG2细胞凋亡的通路；证实了典型三唑类农药CYP3A4酶抑制活性与其雄激素干扰效应的高度相关性。这为当前三唑类杀菌剂及相关农药的风险评价及毒性作用机制研究提供了借鉴。相关研究成果已发表SCI收录论文19篇（其中一篇进入ESI 1%），国际会议学术报告3次，国内会议学术报告5次。