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

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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.