吡咯里西啶生物碱反应性代谢产物形成DNA-DNA＆DNA-Protein交联结合研究

Pyrrolizidine alkaloids (PAs) are widespread, and many of them present in traditional Chinese medicine (TCM). Exposure to PAs can lead to carcinogenesis. PAs have become a potential public health issue to humans. PAs are considered to be non-toxic themselves, and they are metabolized by cytochromes P450 to electrophilic species that are responsible for the carcinogenesis. However, the detailed mechanisms of PA-induced carcinogenesis remain unknown. The hypothesis of the study is that PAs are biotransformed to reactive metabolites which conjugate DNA/proteins to form DNA-DNA and DNA-Protein cross-links, are thought to interfere with chromatin folding, DNA replication, transcription, and repair, potentially contributing to the process of cell cancer. To probe the hypothesis, the following studies will be performed, including 1) Explore the structure of DNA-DNA and DNA-protein cross-links formed by reaction of reactive metabolites of PAs with DNA/Protein in vitro and in vivo; 2) Identification of the proteins that are reacted with reactive metabolites to form DNA-Protein cross-links. This application in respect of chemical research of toxicology allows us to elucidate the molecular mechanisms of carcinogenesis induced by PAs and to provide scientific foundation for TCM safety.

吡咯里西啶生物碱（PAs）是一类分布广泛的生物碱，存在于许多常用中药。PAs具有致癌性，暴露于PAs能导致多种多样的肿瘤，是一大类威胁人类健康的有害物质。一般认为PAs本身无毒，进入体内后被P450酶代谢成的反应性代谢产物才是真正的致癌物质，但具体致癌机理尚未清楚。我们提出假说：PAs在肝脏被代谢成具化学活性的代谢产物，进而与DNA和蛋白质发生相互作用生成DNA-DNA和DNA-Protein交联产物，阻碍DNA的复制和翻译等功能，从而产生致癌作用。为验证该假说，本项目将1）研究PAs反应性代谢产物在体外和体内与DNA/Protein相互作用形成DNA-DNA和DNA-protein交联产物结构信息; 2）鉴定与PAs致癌性相关的形成DNA-Protein交联产物的蛋白质。本项目从毒理化学角度，阐述PAs致癌的分子机制，为中药安全使用提供理论基础。

吡咯里西啶生物碱（Pyrrolizidine alkaloids，PAs）存在于多种常用中药。PAs引起的急性肝损伤临床时有报道，已有多起中毒死亡案例。本课题的主要内容是研究PAs的反应性代谢产物与生物分子DNA和蛋白质相互作用形成的DNA-DNA交联结合物和DNA-Protein交联结合物，剖析PAs致肝毒性的作用机制。DNA-DNA交联结合物的研究结果显示：在体外与体内实验中，均检测到PAs反应性代谢产物与2'-脱氧鸟苷形成的双分子DNA-DNA交联结合物。DNA-Protein交联结合物的研究结果显示：在体外实验中，脱氢野百合碱（Dehydro-monocrotaline）与小分子半胱氨酸/赖氨酸和2'-脱氧鸟苷/2'-脱氧腺苷能发生反应形成千里光次碱的C7位为氨基酸，千里光次碱的C9位为脱氧核苷的交联结合物；进一步实验中，采用模型蛋白（BSA）替换小分子半胱氨酸/赖氨酸进行反应，采用模型DNA（ctDNA）替换小分子2'-脱氧鸟苷/2'-脱氧腺进行反应，这种交联结合还能发生，主要是与蛋白质上的半胱氨酸和DNA上的2'-脱氧鸟苷发生反应形成DNA-Protein交联结合物；并且，使用脱氢野百合碱与Nα-乙酰-半胱氨酸-O-甲酯（NACOMe，替代半胱氨酸）和2'-脱氧鸟苷反应合成、制备、纯化得到了四种结合物，采用核磁共振波谱法和电子圆二色谱法确定了四种结合物的结构；在体内实验中，给予小鼠野百合碱（monocrotaline）和倒千里光碱（retrorsine），也检测到PAs反应性代谢物与DNA和蛋白质发生交联结合的产物，其结构为：7(R)-Cys-DHP-1-dG。这些研究进展使得我们对PAs致毒机制有了更深入的了解。