吡咯里西啶生物碱致母、胎肝毒性差异及其代谢活化机制

Developmental toxicity of traditional Chinese medicine (TCM) and the underlying mechanisms are important issues in TCM Chinese safety study. Previously, the applicant has demonstrated pyrrolizidine alkaloids (PAs), a group of compounds widely existing in TCM, induced developmental toxicities in rats. Our preliminary experimental results and literature review also indicate fetus is much more vulnerable than mother to PAs-induced hepatotoxicity. Based on the understanding that cytochrome P450 (CYP) 3A is the major enzyme in activating PAs to toxic metabolites and the expression of CYP3A isoforms is significantly different in the livers of mother and fetus, we hypothesize that liver damage induced by PAs is the result of metabolic activation of PAs in situ, and that the differential hepatotoxicity by PAs in mother and fetus could be attributed to higher capacities of CYP3A isoforms specifically expressed in fetal liver. By using two representative PAs (retrorsine and monocrotaline), this project will further confirm the difference in hepatotoxicity by PAs between mother and fetus in vivo. We will then determine the role of specific human maternal and fetal hepatic CYP3A isoforms that mediate diverse metabolic injury of PAs, by several complementary experimental systems including baculovirus expression system, Flp-In CHO cell and transgenic zebrafish. Moreover, we will identify the key amino acid(s) affecting metabolic capacity of maternal and fetal CYP isoforms for PAs. This mechanistic study will be conducted by docking PAs into the active site of homology-based model of CYP3A7 and site-directed mutagenesis. Results from our proposed project will provide important information on the safety evaluation of PAs-containing TCM and novel mechanistic approaches in treatment and prevention of PAs-induced developmental toxicity.

研究传统中药的发育毒性及其机制是我国中药安全性研究亟需解决的问题。申请人前期证实中药单体成分-吡咯里西啶生物碱（PAs）导致大鼠发育毒性，并存在原位代谢损伤机制；结合文献在预实验基础上，发现孕期PAs暴露对母肝无明显毒性，但胎肝损伤严重；体外代谢显示，大鼠母、胎肝微粒体有PAs代谢活化差异，提出PAs引起母、胎肝毒性的差异很可能源于两者表达的不同CYP3A亚型代谢活化PAs能力的差异。本项目以两种PAs（倒千里光碱和野百合碱）为代表，从整体实验确证PAs的母、胎肝毒性差异；在亚细胞、细胞和转基因动物水平确认母、胎肝特异性CYP3A亚型介导了PAs的代谢损伤差异；通过基于CYP3A7同源模建结构的定点突变，确定影响两酶亚型代谢活化PAs差异的关键氨基酸。本项目从PAs代谢活化差异角度，探究中药成分对胎儿的特异性发育毒性及其发生机制，为解析中药发育毒性提供新的研究思路和手段。

中药单体成分吡咯里西啶生物碱（PAs）以其肝毒性引起了广泛关注，PAs本身无毒，需经过细胞色素P450 3A（CYP3A）代谢生成脱氢吡咯代谢物发挥毒性。本室前期研究证实其具有发育毒性，文献和本室预实验结果提示PAs对母胎毒性有差异。本项目证实了PAs对母胎（子）的毒性差异现象，并从代谢角度证实其发生机制。以倒千里光碱（RTS）和野百合碱（MCT）作为PAs 的代表和研究工具，建立了大鼠发育毒性的动物模型，证实PAs几乎可完全通过胎盘屏障进入胎仔体内，而PAs在胎仔的原位代谢损伤是引起发育毒性的主要原因；子代幼鼠对PAs毒性的敏感性远高于母鼠，PAs暴露对母肝无明显毒性，但胎肝和哺乳期子代肝脏损伤严重；体外代谢显示，出生后仔鼠肝微粒体对两种PAs尤其是RTS的代谢远远高于母肝，证实PAs引起母、子肝毒性的差异很可能源于两者表达的CYP3A亚型不同。而胎鼠体外和体内PAs代谢结果均显示弱于母鼠，胎盘出现了明显损伤，提示胎盘损伤、胎肝原位代谢损伤和未发育成熟胎肝对PAs毒性更敏感可能共同造成了PAs对胎鼠肝毒性更显著。但人母子特异性CYP3A亚型对PAs代谢没有明显差别。此外我们发现PAs对胎仔的发育毒性具有性别差异，雄激素和孕烷X受体交互作用可能是造成雄性CYP3A表达降低的主要原因。根据流行病学调查和动物实验提示，妊娠期肝内胆汁淤积母亲与子代成年后代谢综合征发病显著相关，很可能源于宫内的糖脂代谢改变。本研究发现PAs能导致妊娠期母鼠血胆汁酸水平升高，造成母鼠高血脂和胎仔血脂异常，主要原因为胎盘对脂质转运增加和胎肝脂代谢的编程改变。本项目结果深入阐明了PAs的发育毒性机制，提出母血PAs作为胎儿暴露生物标志物的可能性；从代谢角度揭示了PAs毒性的母胎（子）差异和性别差异及其机制；同时提示孕期PAs暴露除了能造成发育迟缓、肝毒性等近期发育毒性效应，还可能具有致子代成年后代谢综合征的远期毒性效应。以上结果为加深对PAs毒性作用的认识，探究PAs所致胎源性疾病提供了实验数据，并有助于早期诊断和预防PAs所致的发育毒性。