Mechanisms underlying activation and detoxification of aristolochic acids in human hepatic and renal cells

马兜铃酸(AA)，世界各地用于药用的马兜铃属植物的主要成分， 是强效致癌物质和肾毒素。重要的是，再生障碍性贫血的独特突变特征是 记录在上尿路癌、膀胱癌、肾细胞癌、肝细胞癌和 肝内胆管细胞癌。据估计，在中国和其他亚洲国家，草药在那里 是最广泛使用的，1亿人面临患AA相关癌症和/或慢性肾脏的风险 疾病。在美国和欧洲，含有AA的草本补充剂通过互联网和 继续使用，尽管有相反的警告。此外，在巴尔干国家，马兜铃属植物 富含AA的农田、毒害土壤和农作物。考虑到上述所有情况，迫切需要 了解AA的生物转化途径，以通过设计新的化学物质来减少人体暴露 控制参与AA新陈代谢的酶的活性的试剂。对生物多样性途径的有限认识 AA的生物转化，被目前这一研究领域关于Aa的作用的冲突所放大 硫基转移酶和硝基还原酶在诱导AA毒性中的作用，阻止了这种策略的发展。 这项建议建立在我们在早期研究中获得的两个重要发现的基础上。使用一个完整的人类“肝脏-- 在“芯片上肾脏”系统中，我们报告了AA的激活不仅发生在肾脏，也发生在肝脏。我们也 发现新的还原酶可能对AA的代谢和毒性起重要作用。因此，这一行动的目标是 研究是为了评估新型还原酶在AA代谢和毒性中的作用，并解决一个争议 磺基转移酶和硝基还原酶参与人体肝和肾中AA的生物激活。 为了实现这些目标，我们在人类肝脏中采用了一种有针对性的CRISPR/Cas9基因组编辑方法 HepG2和肾HK-2细胞株在可能涉及的基因中产生双等位移码突变 在AA的代谢中。工程细胞株将根据它们对AA的敏感性进行评估，并与 各自的亲代细胞。质谱学和DNA后标记技术将被应用于定量 AA的主要代谢物及其DNA加合物。表达相应野生型和 催化失活蛋白将被用来转化敲除细胞系，以验证参与 AA毒性中的特殊酶功能。为了支持在培养细胞中的发现，重组人 蛋白质和细胞裂解产物AA和N-羟基马兜铃内酰胺，已知的AA代谢物，将被研究。 这项研究的成功完成将建立参与AA生物转化的新基因。这 信息将使临床科学家了解旨在减少遗传毒性和细胞毒性的疗法的设计 暴露，并将有助于确定有患再生障碍性贫血相关疾病风险的个人。鉴于全球范围内 暴露在AA中，这项研究对全球公共卫生具有重大影响。最后，产生的细胞系 然后，我们的研究将可用于研究其他人类致癌物质、毒素和药物。

Aristolochic acids (AA), principal components of Aristolochia plants used worldwide for medicinal purposes, are potent carcinogens and nephrotoxins. Importantly, a unique mutational signature for AA has been documented in upper urothelial tract cancer, bladder cancer, renal cell carcinoma, hepatocellular carcinoma and intrahepatic cholangiocarcinoma. It is estimated that in China and other Asian countries, where herbal remedies are most widely used, 100 million people are at risk of developing AA-related cancers and/or chronic renal disease. In the US and Europe, herbal supplements containing AA are marketed through the Internet and continue to be used despite warnings to the contrary. Furthermore, in Balkan countries, Aristolochia plants are abundant in farming fields, poisoning soil and crops with AA. Considering all the above, there is an urgent need to understand biotransformation pathways of AA in order to reduce human exposure by devising novel chemical agents that control the activity of enzymes involved in AA metabolism. The limited knowledge of pathways for biotransformation of AA, amplified by the current conflict in this area of research regarding the role of sulfotransferases and nitroreductases in inducing AA toxicities, prevents the development of such strategies. This proposal builds on two important findings we obtained in earlier studies. Using an integrated human “liver- kidney-on-a-chip” system, we reported that activation of AA occurs in the liver as well as in the kidney. We also found that novel reductases might be important for AA metabolism and toxicity. Thus, the objective of this research is to evaluate the role of novel reductases in AA metabolism and toxicity and to resolve a controversy over the involvement of sulfotransferases and nitroreductases in bioactivation of AA in human liver and kidney. To achieve these goals, we employ a targeted CRISPR/CAS9 genome editing approach in human hepatic HepG2 and renal HK-2 cell lines to generate double-allelic, frame-shifting mutations in genes putatively involved in metabolism of AA. Engineered cell lines will be evaluated in terms of their sensitivity to AA and compared with respective parental cells. Mass spectrometric and DNA postlabelling techniques will be applied to quantify the major metabolites of AA and their DNA adducts, respectively. Plasmids expressing corresponding wild-type and catalytically inactive proteins will be used to transform knock-out cell lines in order to verify the involvement of particular enzymatic function in AA toxicities. To support findings in cultured cells, activities of recombinant proteins and cell lysates toward AA and N-hydroxyaristolactams, known metabolites of AA, will be studied. Successful completion of this research will establish novel genes involved in the biotransformaton of AA. This information will inform clinical scientists on design of therapeutics geared to reduce genotoxic and cytotoxic exposure, and will aid in defining individuals at risk of developing AA-related diseases. Given the worldwide exposure to AA, this research has major implications for global public health. Finally, the cell lines generated in our studies will then be available for use in investigations of other human carcinogens, toxins and drugs.