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 Km (58-74 microM) and Vmax (107-142 pmol AMT formed/min/mg protein) and the other with a high Km (4.33-5.88 mM) and Vmax (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 b5 (b5) involvement. Indeed, AZT reduction among six human liver samples correlated strongly with microsomal b5 content (r2 = 0.96) as well as with aggregate P450 content (r2 = 0.97). Upon reconstitution, human liver b5 plus NADH:b5 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 Km of 54 microM and Vmax of 301 pmol/min for b5 plus NADH:b5 reductase and an AZT Km of 103 microM and Vmax of 397 pmol/min for CYP2C9 plus NADPH:P450 reductase. Our results indicate that AZT reduction to AMT by human liver microsomes involves both b5 and P450 enzymes plus their corresponding reductases. The capacity of these proteins and b5 to reduce AZT may be a function of their heme prothestic groups.
Pan-Zhou
X R
XR
Department of Pharmacology and Toxicology, Center for AIDS Research, University of Alabama at Birmingham, 35294-0019, USA.
Cretton-Scott
E
E
Zhou
X J
XJ
Yang
M X
MX
Lasker
J M
JM
Sommadossi
J P
JP
D003577:Cytochrome P-450 Enzyme System / Q000502:physiology*; D015786:Cytochromes b5 / Q000502:physiology*; D015224:Dideoxynucleosides / Q000378:metabolism*; D006801:Humans; D016014:Linear Models; D008862:Microsomes, Liver / Q000201:enzymology*; D010084:Oxidation-Reduction; D015215:Zidovudine / Q000378:metabolism*
D015224:Dideoxynucleosides; D015215:Zidovudine; C019252:3'-aminothymidine; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System