Received for publication July 15, 2004.
Revised September 14, 2004.
Accepted for publication September 14, 2004.

Short-Term Inhibitory Effects of Nitric Oxide on Cytochrome P450-Mediated drug Metabolism: Time Dependency and Reversibility Profiles in Isolated Perfused Rat Livers

Abstract

Nitric oxide (NO) is implicated as a mediator in the decreased catalytic activities of P450 enzymes during inflammation or infection. Here, we examined the time course and the reversibility of the NO effect on P450s using isolated perfused rat livers. Livers were perfused at a constant rate with the NO donor sodium nitroprusside (SNP) for 0.5 or 1 h, followed by washout periods of 0-2.5 h. At the end of perfusion, microsomes were prepared and analyzed for P450 activities and other metabolic markers. Whereas 0.5 h of NO exposure caused an irreversible decline (~30%) in total P450 content, a greater decline after 1 h of NO (~55%) was mostly (~30%) reversible, a pattern identical to that observed for the microsomal heme content. NO exposure also caused an enzyme-selective and time-dependent decline in P450 activities. Whereas the pattern of decline and reversibility of activities were qualitatively similar for CYP3A2, 2C11, 2E1, and 1A1/2, they differed for 2B1/2 and 2D1 in that the decline in the activity was delayed (1 h) for 2B1/2 and not observed for 2D1. This may be attributed to the accessibility of heme or cysteine thiolate and/or the presence/reactivity of critical cysteinyl amino acid residues in various P450 enzymes. Additionally, for most enzymes, the activity showed a biphasic decline, one within 1 h of SNP perfusion and another after 2 h of washout. This was associated with an identical biphasic decline in the microsomal free thiols, presumably due to the rapid and slow reaction of NO and peroxynitrite, respectively, with critical P450 thiols. The short-term effects of NO on P450 are time-dependent, enzyme-selective, with both reversible and irreversible mechanisms.

Key words: CYP inhibition, cytochrome P450, cytochrome P450 isoforms, isolated perfused liver, kinetics, liver microsomes

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