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.
