The effects of early and late administration of inhibitors of inducible nitric oxide synthase in a thioacetamide-induced model of acute hepatic failure in the rat

Abstract

Background/Aims: Nitric oxide (NO) is a pivotal mediator of inflammation. Its role in acute hepatic failure (AHF) is controversial. We investigated the role of NO, and the hypothesis that inhibition of inducible NO synthase (iNOS) activity would improve outcome in liver failure in rats, using the iNOS inhibitors l-NAME and aminoguanidine (AMG).

Methods: AHF was induced by two intraperitoneal injections of thioacetamide (TAA). Seven groups (n=10) were studied. Group I: TAA alone. Groups II, III and IV were additionally pre-treated with the NO precursor l-arginine (300 mg/kg i.p.), or iNOS inhibitors AMG (100 mg/kg s.c.), or N^G-nitro-l-arginine methyl ester (l-NAME) (100 mg/kg s.c.) for 5 days, respectively. Groups V, VI and VII received l-arginine, AMG or l-NAME commencing immediately after TAA administration. Clinical and biochemical parameters were assessed serially, and mortality investigated in further similar cohorts for each regime.

Results: AMG, pre-treatment but not post-treatment, significantly improved outcome including mortality (10 vs. 70%, P<0.005). The less selective iNOS inhibitor l-NAME was not beneficial. Arginine pre-and post-treatment, and iNOS inhibition post-treatment, worsened clinical parameters of TAA-induced liver failure.

Conclusions: Administration of the iNOS inhibitor AMG prior to insult reduces the severity of damage and improves mortality.

Introduction

Nitric oxide (NO) is a highly reactive oxidant. In the liver it is produced both by parenchymal and non-parenchymal cells. Over-production of NO in the liver from l-arginine via inducible nitric oxide synthase (iNOS) [1], [2], [3] has been implicated as an important part of the cascade of events that takes place in the pathogenesis of septic shock and in various forms of hepatic injury, inflammation and acute hepatic failure (AHF) [4], [5]. Supplementation of the NO precursor l-arginine has been shown to exacerbate damage in models of inflammation and injury [6].

The precise mechanisms underlying the actions of NO are controversial and as yet are unknown. NO has been implicated in the haemodynamic changes in the systemic circulation of patients with both AHF and chronic liver disease [7]. It is as an important contributor to haemodynamic changes in the brain and in the pathogenesis of cerebral oedema associated with AHF [8], [9]. In vitro and in vivo studies have also shown NO to down-regulate cytochrome P450 and suppress liver protein and DNA synthesis, and to induce apoptosis and necrosis, all of which may contribute to liver failure [10], [11]. NO also inhibits catalase activity, suggesting that it may alter the detoxification of cytotoxic free radicals, and reacts with superoxide anions to form peroxynitrite, which can react with sulphydryl residues in cell membranes leading to lipid peroxidation, and with DNA leading to cytotoxicity [2].

As modulators of NO production have become available, it is important to assess their potential utility in AHF. We utilized the thioacetamide (TAA) model of AHF in the rat. Thioacetamide is metabolized to toxic metabolites thioacetamide sulfine (sulphoxide) and sulfene (sulphone) [12], [13]. Damage involves generation of free radicals and superoxide anions causing lipid peroxidation [14]. Cellular consequences include interruption of cellular calcium homeostasis, disruption of membranes of the endoplasmic reticulum and failure of amino acid incorporation into liver proteins. Hepatic necrosis [10], [15] and apoptosis [16] result.

Thioacetamide causes hepatic failure in rats using protocols of varying dose and timing [10], [17], [18]. Reproducibility of the model is affected by several factors, such as intra-species variation, age, diet, and also concomitant supportive therapy. We characterized the clinical, biochemical, metabolic and histological pattern of thioacetamide-induced hepatic failure in the Wistar rat, to create a model of substantial severity (∼70% mortality), which fulfils the criteria of reproducibility, reversibility and provision of a therapeutic window, suggested by Terblanche et al. [19] for animal models of AHF.

Previous reports illustrate contradictory findings following NO inhibition in experimental AHF, in part reflecting choice of iNOS inhibitor, doses of hepatotoxin and precise experimental design [6], [10], [20]. We report here the effect of two inhibitors of NO synthesis, l-NAME (N^G-nitro-l-arginine methyl ester), a relatively non-specific inhibitor of NO synthesis, and AMG, a more selective inhibitor of iNOS [21], on the course of thioacetamide-induced AHF in rats. We also investigated the effect of preloading animals with l-arginine, which by providing the substrate for iNOS should exaggerate the consequences of NO generation. Our results indicate that NO is involved in the pathogenesis of thioacetamide induced liver damage and AHF in rats, and selective iNOS inhibition minimizes the severity of TAA-induced damage induced subsequently.