Human enzymes involved in the metabolic activation of carcinogenic aristolochic acids: Evidence for reductive activation by cytochromes P450 1A1 and 1A2

Aristolochic acid (AA), a naturally occurring nephrotoxin and rodent carcin ogen, has recently been associated with the development of urothelial cance r in humans. Determining the capability of humans to metabolize AA and unde rstanding, which human enzymes are involved in AA activation is important i n the assessment of individual susceptibility. Using the nuclease P1-enhanc ed version of the P-32-postlabeling assay, we compared the ability of human , minipig and rat hepatic microsomal samples to activate AA to metabolites forming DNA adducts. Human microsomes generated AA-DNA adduct profiles repr oducing those found in renal tissues from humans exposed to AA. Identical p atterns of AA-DNA adducts were also observed when AA was activated by minip ig and rat microsomes. Therefore, microsomes of both animals are suitable i n vitro systems mimicking the enzymatic activation of AA in humans. To defi ne the role of specific P450 enzymes and NADPH:P450 reductase in the activa tion of AA by human microsomes we investigated the modulation of AA-DNA add uct formation by specific inducers or selective inhibitors of P450s and cof actors or inhibitors of NADPH:P450 reductase. The inducer of P450 1A1/2, be ta -naphthoflavone, significantly stimulated the levels of AA-DNA adducts f ormed by rat microsomes, but inducers of P450 2B1/2 and 2E1 had no such eff ect. Furthermore, only inhibitors of the P450 1A subfamily (alpha -naphthof lavone, furafylline) significantly decreased the amount of adducts formed b y microsomes from humans, minipigs and rats. a-Lipoic acid, an inhibitor of NADPH:P450 reductase, inhibited adduct formation too, but to a lower exten t. On the basis of these results, we attribute most of the microsomal activ ation of AA to P450 1A1 and 1A2, although a role of NADPH:P450 reductase ca nnot be ruled out. With purified enzymes (recombinant P450 1A1/2 and NADPH: P450 reductase) and microsomes from baculovirus transfected insect cells ex pressing recombinant human P450 1A1/2 and NADPH: P450 reductase, the partic ipation of these enzymes in the formation of AA-DNA adducts was confirmed. These results are the first report on the activation of AA by human enzymes and clearly demonstrate the role of P450 1A1, 1A2, and NADPH:P450 reductas e in catalyzing the reductive activation of AA.