diethyl-maleate and Endotoxemia

The inducible nitric oxide synthase (iNOS) promoter contains nuclear factor kappaB (NF-kappaB) binding sites. NF-kappaB activation is determined, in part, by the intracellular redox status. The aim of this study was to determine the importance of the cellular glutathione status in relation to NF-kappaB activation and iNOS expression in hepatocytes in vivo and in vitro. For in vivo experiments, rats were injected with endotoxin and sacrificed 6 hours later. Glutathione was depleted by diethylmaleate. For in vitro experiments, cultured hepatocytes from untreated rats were exposed to a cytokine mixture. Glutathione levels were depleted by diethylmaleate and restored by N-acetylcysteine. iNOS expression was assessed by Western blot, reverse transcription polymerase chain reaction, nitric oxide (NO) metabolites, and immunohistochemistry. NF-kappaB binding was assessed by electrophoretic mobility shift assay. Endotoxin-induced iNOS expression in rat liver was prominent in hepatocytes, Kupffer cells, and inflammatory cells, in particular neutrophils. Glutathione depletion prevented iNOS induction in hepatocytes, but not in inflammatory cells. iNOS protein levels were in accordance with iNOS messenger RNA and NO metabolites in plasma. Glutathione depletion did not affect neutrophil infiltration. Cytokines strongly induced iNOS in cultured hepatocytes. Induction was prevented by glutathione depletion and could be restored by addition of N-acetylcysteine. NF-kappaB binding correlated with iNOS induction. In conclusion, in this study we show that iNOS induction in hepatocytes in vivo and in vitro is dependent on the intracellular glutathione status and correlates with NF-kappaB binding. Glutathione-depletion has no effect on the expression of iNOS in inflammatory cells, nor on neutrophil infiltration.

Topics: Acetylcysteine; Animals; Binding Sites; Blotting, Western; Endotoxemia; Gene Expression; Glutathione; Immunohistochemistry; Kupffer Cells; Liver; Male; Maleates; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Promoter Regions, Genetic; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction

Manipulation of the intracellular redox state has been shown to alter cell activation pathways with resultant changes in cellular function. Previous studies have suggested that thiol oxidation, using the glutathione-depleting agent diethyl maleate (DEM), was able to inhibit endothelial cell activation. We hypothesized that this agent might exert beneficial effects following endotoxemia in the rat, a model in which transendothelial migration of neutrophils is central to the development of hepatocellular injury. Sprague-Dawley rats treated intraperitoneally with lipopolysaccharide (LPS) (200 microg/kg) plus D-galactosamine (GalN) (600 mg/kg) developed hepatocellular necrosis, as evidenced by liver enzyme release and morphological changes. Pretreatment with DEM abrogated this injury in a dose-dependent fashion. Histology revealed reduced neutrophil accumulation in both the parenchyma and sinusoids, consistent with reduced neutrophil sequestration and transendothelial migration. This effect appeared to be related to the ability of DEM to prevent LPS-induced up-regulation of both vascular cell adhesion molecule-1 (VCAM-1) mRNA and intercellular adhesion molecule-1 (ICAM-1) mRNA in the liver, as well as reducing tumor necrosis factor (TNF) mRNA expression. In addition, DEM prevented hepatocyte apoptosis following LPS treatment. The effect was reproduced when TNF was used as an inflammatory stimulus, suggesting a direct protective effect on the hepatocyte. Taken together, these studies show that redox manipulation through thiol oxidation may represent a novel approach to preventing liver necrosis and apoptosis in inflammatory conditions.

Topics: Alanine Transaminase; Animals; Apoptosis; Endotoxemia; Glutathione; Intercellular Adhesion Molecule-1; Liver; Male; Maleates; Necrosis; NF-kappa B; Oxidation-Reduction; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1