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

Abstract

ABSTRACT:The rapid and direct effects of nitric oxide (NO) donors sodium nitroprusside (SNP) and isosorbide dinitrate (ISDN) on the hepatic and biliary disposition of chlorzoxazone (CZX), a marker of CYP2E1, and dextromethorphan (DEM), a marker of CYP2D1, were studied in a single-pass isolated perfused rat liver model. Livers (n ‫؍‬ 30) were perfused with constant concentrations of NO donors (0-120 min) in addition to infusion of CZX or DEM (60-120 min), and periodical outlet and bile samples were collected. Both ISDN and SNP significantly reduced (30 and 60%, respectively) the hepatic extraction ratio of CZX and decreased (50 and 70%, respectively) the recovery of the CYP2E1-mediated metabolite, 6-hydroxychlorzoxazone, in the outlet perfusate and bile. As for DEM, both NO donors increased (up to 3.5-fold) the recovery of the CYP2D1-mediated metabolite dextrorphan (DOR) in the outlet perfusate. However, this was associated with a simultaneous decrease (50-75%) in the excretion of the metabolite into the bile, thus resulting in no change in the overall recovery of DOR as a result of NO donor treatment. The decrease in the biliary excretion of DOR was caused by NO-induced simultaneous reductions in both the conjugation of DOR and biliary clearance of DOR conjugate. Additionally, both SNP and ISDN significantly reduced the metabolism of DEM to 3-hydroxymorphinan, which is mostly regulated by CYP3A2. These studies in an intact liver model confirm the selectivity of the inhibitory effects of NO donors on cytochrome P450 enzymes, which was recently reported in microsomal studies, and expand these inhibitory effects to conjugation pathways.Cytochrome P450 (P450) enzymes are a major class of hemecontaining proteins that participate in the biotransformation of xenobiotics. Studies have shown that the ability of the liver to metabolize drugs is compromised in the presence of infectious diseases or disease states associated with inflammation (Morgan, 1997;Renton, 2001Renton, , 2004Riddick et al., 2004;Ling and Jamali, 2005). Large quantities of cytokines and nitric oxide (NO) released during inflammation are implicated as the major mediators for the observed down-regulation of P450 activities and enzyme levels (Morgan, 1997). 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). Further studies (Vuppugalla and Mehvar, 2005) ind...

Divergent Effects of Nitric Oxide Donors on the Biliary Efflux Transporters in Isolated Perfused Rat Livers: Nitric Oxide-Independent Inhibition of ABC Transporters by Sodium Nitroprusside

“…These data suggest that although ISDN-500 does not affect the Mrp2-mediated biliary transport of RH-Glu (no change in its CL bile ), it may affect the formation of the metabolite (reduced recovery). Indeed, our own previous work [8] showed that SNP and ISDN at a concentration of 400 M significantly inhibited the glucuronidation of dextrorphan and 3-hydroxymorphinan, two metabolites of dextromethorphan, in an IPRL similar to that used here. Collectively, our data using RH-123 and its glucuronidated metabolite suggest that whereas SNP-400 reduces the biliary transport of both RH-123 (P-gp) and RH-Glu (Mrp2), neither process is affected by ISDN.…”

“…Elevated levels of this important bioregulator are observed in a number of hepatic disorders, such as inflammation, cirrhosis, and chronic exposure to cholesterol and alcohol [2][3][4]. Earlier works from our laboratory [5][6][7][8] and by others [2,[9][10][11][12] have shown inhibition of the expression and activities of many isoenzymes of the hepatic drug metabolizing enzymes cytochrome P450 (P450) in the presence of increased concentrations of NO. Thus, diseases that cause an upregulation of NO production, such as septic shock, have been shown to alter drug metabolism [13][14][15].…”

Effects of Normothermic Hepatic Ischemia–Reperfusion Injury on the In Vivo, Isolated Perfused Liver, and Microsomal Disposition of Chlorzoxazone, a Cytochrome P450 2E1 Probe, in Rats

“…The procedures for the isolation and perfusion of the livers were similar to those reported before. 19,22,23 In brief, after opening the abdominal wall, bile duct, portal vein (inlet), and suprahepatic vena cava (outlet) were cannulated. The livers were then transferred to a water jacketed (37 • C), all glass perfusion system (Radnoti Glass Technology Inc., Monrovia, California) and perfused in a singlepass manner at a constant flow rate of 30 mL/min [∼3-4 mL/(min g) liver].…”

Effects of Hypoxia Due to Isovolemic Hemodilution on an Ex Vivo Normothermic Perfused Liver Model

“…A different experimental approach, utilized from the 19th century onwards, consists of the use of ex vivo perfused models for the study of liver physiology [5,6], metabolism [7], and drug toxicity [7][8][9][10][11]. Ex vivo perfusion maintains normal physiological parameters, histology, and hepatocyte function for relatively prolonged periods, allowing analysis of the organ's responses to biochemical and hormonal variations [12][13][14].…”