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
Em. Cretton et Jp. Sommadossi, 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, Drug metabolism and disposition, 21(5), 1993, pp. 946-950
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.