Project title: The Role of CYP1A Enzymes in Aristolohic Acid Toxicity and Mutagenicity: Clues to Mechanism and Disease
Completed in: 2010
Exposure to aristolochic acid (AA) has been indetified as the cause of aristolochic acid nehropathy (AAN) and a putative risk factor for endemic (Balkan) nephropathy (EN). Historically, AA-related research to date has focused on the activation of AA to mutagenic and nephrotoxic metabolites, but the processes by which AA is detoxified are also relevant to disease risk. Although originally classified as AA activation enzymes. CYPs 1A1 and 1A2 have recently been proposed instead as AA detoxification enzymes because of their ability to metabolize AA1, the predominant toxic form of AA, to AA1a, a less toxic metabolite. In vivo experiments have demonstrated that hepatic clearance of AA1 via the presence/induction of CYP1A enzymes may be relevant to the detoxification of AA1 due to AA1a production and reduced amount of AA1 reaching the kidney. To delineate the roles of human CYP1A enzymes in AA activation and detoxification, a series of in vitro and in vivo experiments were developed and conducted. The first of these employed a modified Ames assay to examine the role of CYP enzymes, including CYP1A2, in attenuating AA-induced mutagenicity. In a second set of experiments, the propensity of human liver and kidney microsomes to form AA1-DNA adducts was assessed using microsome incubations, which contained AA1, calf thymus DNA, and NADPH. The liver microsomes, which varied by as much as 60-fold in their CYP1A2 activity, were also tested for their ability to catalyze the production of AA1a. Lastly, an in vivo study was conducted in which C57BL/6J, Cyp1a1/1a2(-/-)_hCYP1A1/1A2(-), and hCYP1A1_1A2_Cyp1a1/1a2(-/-)-Ahrb1 transgenic mice were treated with AA1 to investigate the similarities and differences between mouse and human CYP1A enzymes with regard to AA1 detoxification. A subset of these mice were also pre-treated with 3-methylcholanthrene, which allowed for additional comparisons of CYP1A induction between the wild-type and humanized mice. Collectively, these experiments demonstrate the capability of humanized mice. Collectively, these experiments demonstrate the capability of human CYP1A enzymes to detoxify AA1. Given these findings, individuals with low CYP1A expression or activity may be at increased risk for AA-related diseases, a theory which may be further investigated using epidemiologic methods.