Role of human liver P450s and cytochrome b5 in the reductive metabolism of 3′-azido-3′-deoxythymidine (AZT) to 3′-amino-3′-deoxythymidine

Our laboratory has shown that human liver microsomes metabolize the anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) via a P450-type reductive reaction to a toxic metabolite 3'-amino-3'-deoxythymidine. (AMT). In the present study, we examined the role of specific human P450s and other microsomal enzymes in AZT reduction. Under anaerobic conditions in the presence of NADPH, human liver microsomes converted AZT to AMT with kinetics indicative of two enzymatic components, one with a low K-m, (58-74 mu M) and V-max (107-142 pmol AMT formed/min/mg protein) and the other with a high K-m (4.33-5.88 mM) and V-max (1804-2607 pmol AMT formed/min/mg). Involvement of a specific P450 enzyme in AZT reduction was not detected by using human P450 substrates and inhibitors. Antibodies to human CYP2E1, CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2A6 were also without effect on this reaction. NADH was as effective as NADPH in promoting microsomal AZT reduction, raising the possibility of cytochrome b(5) (b(5)) involvement. Indeed, AZT reduction among six human liver samples correlated strongly with microsomal b(5) content (r(2) = 0.96) as well as with aggregate P450 content (r(2) = 0.97). Upon reconstitution, human liver b(5) plus NADH:b(5) reductase and CYP2C9 plus NADPH:P450 reductase were both effective catalysts of AZT reduction, which was also supported when CYP2A6 or CYP2E1 was substituted for CYP2C9. Kinetic analysis revealed an AZT K-m of 54 mu M and V-max of 301 pmol/min for b(5) plus NADH:b(5) reductase and an AZT K-m of 103 mu M and V-max of 397 pmol/min for CYP2C9 plus NADPH:P450 reductase. Our results indicate that AZT reduction to AMT by human liver microsomes involves both b(5) and P450 enzymes plus their corresponding reductases. The capacity of these proteins and b(5) to reduce AZT may be a function of their heme prothestic groups. (C) 1998 Elsevier Science Inc.