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Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, has been associated with the development of urothelial cancer in humans. Understanding which human enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual's susceptibility to this plant carcinogen. Using the 32 P postlabeling assay, we examined the ability of microsomal samples from 8 human livers and from 1 human kidney to activate AAI, the major component of the plant extract AA, to metabolites forming adducts in DNA. Microsomes of both organs generated DNA adduct patterns reproducing those found in renal tissues from humans exposed to AA. 7-(deoxyadenosin-N 6 -yl)aristolactam I, 7-(deoxyguanosin-N 2 -yl)aristolactam I and 7-(deoxyadenosin-N 6 -yl)aristolactam II were identified as AA-DNA adducts formed from AAI by all human hepatic and renal microsomes. To define the role of human microsomal enzymes in the activation of AAI, we investigated the modulation of AAI-DNA adduct formation by cofactors and selective inhibitors of microsomal reductases, cytochrome P450 (CYP) enzymes, NADPH:CYP reductase and NADH:cytochrome b 5 reductase. We also determined whether the activities of CYP and NADPH:CYP reductase in different human hepatic microsomal samples correlated with the levels of AAI-DNA adducts formed by the same microsomal samples. On the basis of these studies, we attribute most of the activation of AAI in human hepatic microsomes to CYP1A2. In contrast to human hepatic microsomes, in human renal microsomes NADPH:CYP reductase is more effective in AAI activation. In addition, prostaglandin H synthase is another enzyme activating AAI in renal microsomes. The results demonstrate for the first time the potential of microsomal enzymes in human liver and kidney to activate AAI by nitroreduction.

shifts oxidation of the anticancer drug ellipticine by cytochromes P450 1A1 and 1A2 from its detoxication to activation, thereby modulating its pharmacological efficacy. Biochemical Pharmacology, Elsevier, 2011, 82 (6) This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Page 1 of 45A c c e p t e d M a n u s c r i p A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 3 ABSTRACTEllipticine is a pro-drug, whose activation is dependent on its oxidation by cytochromes P450 (

Aristolochic acid I (AAI) is a plant alkaloid causing aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. AAI is detoxified by cytochrome P450 (CYP)-mediated O-demethylation to 8-hydroxyaristolochic acid I (aristolochic acid Ia, AAIa). We previously investigated the efficiencies of human and rat CYPs in the presence of two other components of the mixed-functions-oxidase system, NADPH:CYP oxidoreductase and cytochrome b5, to oxidize AAI. Human and rat CYP1A are the major enzymes oxidizing AAI. Other CYPs such as CYP2C, 3A4, 2D6, 2E1, and 1B1, also form AAIa, but with much lower efficiency than CYP1A. Based on velocities of AAIa formation by examined CYPs and their expression levels in human and rat livers, here we determined the contributions of individual CYPs to AAI oxidation in these organs. Human CYP1A2 followed by CYP2C9, 3A4 and 1A1 were the major enzymes contributing to AAI oxidation in human liver, while CYP2C and 1A were most important in rat liver. We employed flexible in silico docking methods to explain the differences in AAI oxidation in the liver by human CYP1A1, 1A2, 2C9, and 3A4, the enzymes that all O-demethylate AAI, but with different effectiveness. We found that the binding orientations of the methoxy group of AAI in binding centers of the CYP enzymes and the energies of AAI binding to the CYP active sites dictate the efficiency of AAI oxidation. Our results indicate that utilization of experimental and theoretical methods is an appropriate study design to examine the CYP-catalyzed reaction mechanisms of AAI oxidation and contributions of human hepatic CYPs to this metabolism.