Hepatic Disposition and Effects of Nitric Oxide Donors: Rapid and Concentration-Dependent Reduction in the Cytochrome P450-Mediated Drug Metabolism in Isolated Perfused Rat Livers

Abstract

Various mechanisms, including high levels of cytokines and nitric oxide (NO), have been proposed as mediators for inflammation-induced cytochrome 450 down-regulation. However, the contribution of each of these mediators to the observed effects is controversial. We used an isolated perfused rat liver (IPRL) model to test the direct effects of NO donors on CYP450 down-regulation in the absence of cytokines or other confounding in vivo factors. Our hypothesis was that NO rapidly and concentration-dependently decreases CYP450 activities in IPRL. Livers were perfused (60 min) with 50 to 500 M sodium nitroprusside (SNP) or 100 to 500 M isosorbide dinitrate (ISDN) as NO donors, and the perfusate and biliary disposition of SNP, ISDN, and generated nitrate/nitrite (NO x ) were determined. Additionally, at the end of perfusion, catalytic activities and protein levels of various cytochrome isoenzymes were measured. Both SNP and ISDN exhibited linear hepatic disposition with extraction ratios of ϳ0.30 and 0.50, respectively. Furthermore, although in small amounts, both NO donors and NO x were found in the bile. Except for CYP2D1, the catalytic activities of all the studied isoenzymes were substantially (up to 85%) decreased by both NO donors. However, the apoprotein levels of isoenzymes remained largely unchanged. Additionally, the inhibitory effects of NO donors were concentration-dependent, with the concentrations of SNP producing one-half of maximum inhibition being in the order of 2C11 Ͼ 2B1/2 Ͼ 2E1 ϭ 3A2 Ͼ 1A1/2. These studies indicate that the effects of NO on the down-regulation of cytochrome 450 catalytic activity are rapid, concentration-dependent, and isoenzyme-selective.

Roles of nitric oxide in inflammatory downregulation of human cytochromes P450

“…NO can bind to P450s and cause inhibition by reversible or irreversible pathways [14,15], and therefore NO may be one cause of the enzyme inhibition observed in inflammatory states [16,17]. Stimulation of NO synthesis may also be involved in the down-regulation of hepatic CYP mRNAs or proteins by inflammation, because these effects could be blocked or attenuated by NOS inhibitors in either in vivo or cell culture models [18][19][20].…”

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

“…The role of cytokines in this down-regulation is relatively well established; they are known to inhibit drug metabolism by acting at the level of gene transcription (Ghezzi et al, 1986;Warren et al, 1999). However, the role of NO is still subject to controversy (Sewer and Morgan, 1998), although it has been proposed that NO acts at both transcriptional and post-translational levels (Khatsenko et al, 1993;Wink et al, 1993;Minamiyama et al, 1997).Very recently, we reported that the effects of NO on P450 are rapid, concentration-dependent, and enzyme-selective (Vuppugalla and Mehvar, 2004a). Additionally, we also showed that the effects of NO are time-dependent, consisting of both reversible and irreversible components (Vuppugalla and Mehvar, 2004b).…”

“…Very recently, we reported that the effects of NO on P450 are rapid, concentration-dependent, and enzyme-selective (Vuppugalla and Mehvar, 2004a). Additionally, we also showed that the effects of NO are time-dependent, consisting of both reversible and irreversible components (Vuppugalla and Mehvar, 2004b).…”

“…The previous studies (Vuppugalla and Mehvar, 2004a), which utilized the same model used here (single-pass IPRL), indicated that at equal inlet concentrations of Ն400 M, nitrite/nitrate concentrations in the outlet perfusate samples were Ն2-fold higher for ISDN than that for SNP. In contrast, the amount of nitrite/nitrate found in the bile of SNP-perfused IPRL was 5-fold higher than that after perfusion of the livers with ISDN.…”

“…Finally, a 200-l aliquot of each sample was injected onto the HPLC system. The HPLC method used for the analysis of the drug and metabolites was based on the modifications (Vuppugalla and Mehvar, 2004a) made to a previously reported method (Yu and Haining, 2001). The levels of unconjugated drug and metabolites were also measured using the same procedure, without the ␤-glucuronidase incubation step.…”