REDUCTION OF 3'-AZIDO-2',3'-DIDEOXYNUCLEOSIDES TO THEIR 3'-AMINO METABOLITE IS MEDIATED BY CYTOCHROME-P-450 AND NADPH-CYTOCHROME P-450 REDUCTASE IN RAT-LIVER MICROSOMES

Using in vitro liver systems, we previously demonstrated that 3'-azido -3'-deoxythymidine (AZT) is reduced to a highly toxic metabolite, 3'-a mino-3'-deoxythymidine (AMT) through a NADPH-dependent system. This pa thway also occurs for other 3'-azido-2',3'-dideoxynucleosides (3'-azid o ddNs), indicating that reduction to a 3'-amino metabolite is a gener al catabolic route of this class of compounds. This study was undertak en to understand the enzymatic reaction responsible for this catabolic pathway. Rat liver microsomes were exposed to 1 mM [H-3]AZT or 1 MM [ H-3]AzddU, and incubated under various conditions. Reduction to the 3' -amino derivative was enhanced 5-fold by the addition of NADPH. When F AD or FMN was combined with NADPH, AMT and AMddU formation was enhance d 2-fold. Addition of equimolar FAD and FMN enhanced azido reducing ac tivity by 3-fold and 5-fold when compared with NADPH alone for AZT and AzddU, respectively, Exposure to carbon monoxide inhibited 3'-amino f ormation approximately 60%, consistent with involvement of cytochrome P-450 (P-450). This inhibitory effect was not detected in the presence of combined flavin and NADPH; in control incubations that contained t hese cofactors but no microsomes, AMT or AMddU formation was not obser ved. This suggests that a flavoprotein, possibly NADPH-cytochrome P-45 0 reductase (P-450 reductase), is also involved in azido reduction. Pr eincubation with various P-450 ligands resulted in variable inhibition ; reduction of AZT and AzddU was decreased approximately 20-80%. In ad dition to these observations, preincubation with polyclonal antibodies to rat P-450 IIB1 and to rat P-450 reductase resulted in 33-50% and 2 5%, respectively, inhibition of AMT formation. Treatment of rats with phenobarbital led to a 3-fold enhancement of AMT and AMddU formation i n the treated microsomes. These results suggest that both the P-450 re ductase and P-450 are involved in the reduction of AzddNs. The P-450 s ubfamily IIB is probably responsible for this reaction in rats.