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

Abstract

ABSTRACT:Nitric oxide (NO) is implicated as a mediator in the decreased catalytic activities of cytochrome P450 (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 to 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 (ϳ30%) in total P450 content, a greater decline after 1 h of NO (ϳ55%) was mostly (ϳ30%) 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 and enzyme-selective, with both reversible and irreversible mechanisms.

Enzyme-Selective Effects of Nitric Oxide on Affinity and Maximum Velocity of Various Rat Cytochromes P450

“…Supporting the above findings, very recently (Vuppugalla and Mehvar, 2004a), we showed the inhibitory effects of NO on P450 to be rapid, concentration-dependent, and enzyme-selective in an isolated perfused rat liver (IPRL) model. Furthermore, we also conducted time course studies to assess the mechanisms responsible for the NO-mediated P450 inhibition and demonstrated that the short-term inhibitory effects of NO are time-dependent, consisting of both reversible and irreversible components (Vuppugalla and Mehvar, 2004b).One of the novel findings of our IPRL studies (Vuppugalla and Mehvar, 2004a,b) was the apparent lack of effect of NO donors on CYP2D1 enzyme, along with different degrees and time courses of inhibition or reversal of activities for other P450 enzymes. These studies were carried out only with a single substrate concentration.…”

Selective Effects of Nitric Oxide on the Disposition of Chlorzoxazone and Dextromethorphan in Isolated Perfused Rat Livers

“…The NO-induced decrease in glucuronide formation (Fig. 5) may be because reduction in the activity of UDPGT resulted from an interaction of NO with the critical thiol-containing amino acid residues of the enzyme, similar to that reported between the P450 apoprotein and NO (Minamiyama et al, 1997;Vuppugalla and Mehvar, 2004b). In addition to the decrease in the formation of DOR and HOM glucuronides (Fig.…”

“…The differential effects of NO on the V max values of these enzymes may be because of differences between the two enzymes in the accessibility of heme and/or cysteine thiolate residues to NO (Gergel et al, 1997). Additionally, because NO reacts with thiol groups of amino acid residues in the apoprotein (Minamiyama et al, 1997;Vuppugalla and Mehvar, 2004b), it may affect the binding of substrates to these enzymes and therefore impact their K m values, selectively. This is because the degree of involvement of the thiolcontaining cysteine residues in the substrate binding may be different for various P450 enzymes (Vuppugalla and Mehvar, 2005).…”

“…Additionally, we also showed that the effects of NO are time-dependent, consisting of both reversible and irreversible components (Vuppugalla and Mehvar, 2004b). Further studies (Vuppugalla and Mehvar, 2005) indicated that the inhibitory effects of NO on P450 enzymes are mediated through selective alterations in the V max and/or K m of various enzymes.…”

“…However, all of these studies were conducted using microsomal preparations obtained from livers exposed to NO donors. Because during the preparation of microsomes some of the rapidly reversible interactions of NO with heme, such as the formation of nitrosyl-heme complexes, may be lost (Wink et al, 1993;Gergel et al, 1997;Vuppugalla and Mehvar, 2004b), our observations in microsomal preparation may not be directly extrapolated to intact liver or animals. Therefore, it is not clear whether the lack of effect of NO on CYP2D1 observed in our microsomal preparations Mehvar, 2004a,b, 2005) is indeed real or a result of the experimental model used.…”