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

马兜铃酸（Aristolochic Acids，AA）是马兜铃属植物的主要成分， 是强致癌物和肾毒素。重要的是，AA的独特突变特征已经被发现。 在上尿路上皮癌、膀胱癌、肾细胞癌、肝细胞癌和 肝内胆管癌据估计，在中国和其他亚洲国家， 使用最广泛的是，1亿人有患AA相关癌症和/或慢性肾脏疾病的风险。 疾病在美国和欧洲，含有AA的草药补充剂通过互联网销售， 尽管有相反的警告，但仍继续使用。此外，在巴尔干半岛的国家，马兜铃属植物是 大量存在于农田中，用AA毒害土壤和作物。考虑到上述情况，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类暴露 控制参与AA代谢的酶活性的试剂。对疾病途径的有限了解 AA的生物转化，放大了目前的冲突，在这方面的研究的作用， 磺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。 这一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。利用一个完整的人类“肝脏- 在“芯片上的肾”系统中，我们报道了AA的激活发生在肝脏以及肾脏中。我们也 发现新的还原酶可能对AA代谢和毒性很重要。因此，这一目标 研究的目的是评估新的还原酶在AA代谢和毒性中的作用，并解决一个有争议的问题。 磺基转移酶和硝基还原酶参与人肝脏和肾脏中AA的生物活化。 为了实现这些目标，我们在人类肝脏中采用了靶向CRISPR/CAS9基因组编辑方法。 HepG 2和肾HK-2细胞系在涉及pupil的基因中产生双等位基因移码突变 在AA代谢中的作用。工程化细胞系将根据其对AA的敏感性进行评估，并与 各自的亲本细胞。质谱和DNA后标记技术将被应用于定量 AA及其DNA加合物的主要代谢产物。表达相应野生型和 将使用无催化活性的蛋白质转化敲除细胞系，以验证 在AA毒性中的特殊酶功能。为了支持在培养细胞中的发现， 将研究蛋白质和细胞裂解物对AA和N-羟基马兜铃内酰胺（AA的已知代谢物）的作用。 本研究的成功完成将为建立AA生物转化相关的新基因奠定基础。这 这些信息将为临床科学家提供设计治疗方法的信息， 暴露，并将有助于确定有发生AA相关疾病风险的个体。鉴于全球 暴露于AA，这项研究对全球公共卫生具有重大意义。最后，在细胞中产生的细胞系 我们的研究将可用于其他人类致癌物，毒素和药物的研究。

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.