The mechanism of stimulation of NADPH oxidation during the mechanism-based inactivation of cytochrome P450 2B1 by N-methylcarbazole: redox cycling and DNA scission.

The oxidation rate of NADPH is markedly stimulated during the mechanism-based inactivation of cytochrome P450 2B1 by N-methylcarbazole (NMC) in a reconstituted system consisting of NADPH-cytochrome P450 reductase, cytochrome P450 and phospholipid. The stimulation of NADPH oxidation in this system is due to 1-hydroxy-N-methylcarbazole (1-OH-NMC), one of the major metabolites of NMC. The 1-OH-NMC is further metabolized in an NADPH-dependent manner by the reconstituted system or by purified NADPH-cytochrome P450 reductase to give a more polar metabolite which has been isolated by HPLC. The conversion of 1-OH-NMC to this product was inhibited by superoxide dismutase (SOD), and incubation of the 1-OH-NMC with hypoxanthine-xanthine oxidase resulted in the formation of the same product, suggesting that the superoxide anion was involved in the metabolism of 1-OH-NMC by the reductase. Redox cycling activity during the metabolism of 1-OH-NMC by reductase has been demonstrated. The oxidation of NADPH by the reductase in the presence of 35 microM 1-OH-NMC was enhanced approximately 23-fold [240 nmol of NADPH oxidized/(min.nmol of reductase)] relative to control levels in the presence of 500 microM NMC [10.5 nmol/(min.nmol of reductase)]. 1-OH-NMC (35 microM) caused a 40-fold increase in the rate of formation of superoxide during its metabolism by reductase. The rapid rates of NADPH oxidation and superoxide formation were inhibited by the addition of reduced glutathione (GSH) to the reaction mixture. Neither SOD nor GSH inhibited the reductase activity directly.(ABSTRACT TRUNCATED AT 250 WORDS)