Background: Recently, we showed that, in addition to cytochrome P-4502E1 (CYP2E1), CYP1A2 and CYP3A4 also contribute to the microsomal ethanol oxidizing system (MEOS). When MEOS activity is measured, sodium azide commonly is used to block the contaminating catalase. However, although CYP2E1 is considered insensitive to azide, its effect on the other P-450s is unknown. Therefore, the aim of the present study was to determine the effect of azide on human recombinant and hepatic CYP2E1, CYP1A2, and CYP3A4.
Methods And Results: Concentrations of sodium azide as low as 0.1 mM markedly inhibited the specific ethanol oxidation (mean +/- SEM) by recombinant CYP1A2 and CYP3A4 expressed in HepG2 cells (to 16 +/- 1% and 22 +/- 2% of control without azide, respectively; p < 0.01). By contrast, the specific activity of CYP2E1 was only slightly (and not significantly) inhibited at this azide concentration (to 79 +/- 12% of control). Similarly, in human liver microsomes (n = 6), 0.1 mM azide strongly inhibited CYP1A2-dependent (to 25 +/- 2%) and CYP3A4-dependent (to 15 +/- 2%) ethanol oxidation, whereas CYP2E1 was inhibited only at 10 mM azide (to 60 +/- 10%). Azide also strongly affected the apparent kinetic values of all three isoenzymes. Furthermore, azide inhibited the specific monooxygenase activities, both by recombinant and microsomal P-450s. CYP2E1-specific p-nitrophenol hydroxylation was the most sensitive to azide, whereas CYP1A2-dependent 7-methoxyresorufin O-dealkylation was only slightly inhibited. Judging from its effect on p-nitrophenol hydroxylation by human liver microsomes, the inhibition of azide was competitive (Ki 0.09 mM).
Conclusions: Sodium azide at a concentration as low as 0.1 mM inhibited ethanol oxidation by CYP1A2 and CYP3A4. With CYP2E1, although oxidation of 50 mM ethanol was not inhibited by 0.1 mM azide, higher azide concentrations were inhibitory and 0.1 mM azide seemed to affect the kinetics of ethanol oxidation by CYP2E1. Therefore, azide should be avoided when measuring the MEOS activity because it may lead to underestimation, especially of CYP1A2- and CYP3A4-dependent ethanol oxidation.
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