Esther Ardite

Selective glutathione depletion of mitochondria by ethanol sensitizes hepatocytes to tumor necrosis factor

BACKGROUND & AIMS Tumor necrosis factor (TNF)-alpha induces cell injury by generating oxidative stress from mitochondria. The purpose of this study was to determine the effect of ethanol on the sensitization of hepatocytes to TNF-alpha. METHODS Cultured hepatocytes from ethanol-fed (ethanol hepatocytes) or pair-fed (control hepatocytes) rats were exposed to TNF-alpha, and the extent of oxidative stress, gene expression, and viability were evaluated. RESULTS Ethanol hepatocytes, which develop a selective deficiency of mitochondrial glutathione (mGSH), showed marked susceptibility to TNF-alpha. The susceptibility to TNF-alpha, manifested as necrosis rather than apoptosis, was accompanied by a progressive increase in hydrogen peroxide that correlated inversely with cell survival. Nuclear factor kappaB activation by TNF-alpha was significantly greater in ethanol hepatocytes than in control hepatocytes, an effect paralleled by the expression of cytokine-induced neutrophil chemoattractant. Similar sensitization of normal hepatocytes to TNF-alpha was obtained by depleting the mitochondrial pool of GSH with 3-hydroxyl-4-pentenoate. Restoration of mGSH by S-adenosyl-L-methionine or by GSH-ethyl ester prevented the increased susceptibility of ethanol hepatocytes to TNF-alpha. CONCLUSIONS These results indicate that mGSH controls the fate of hepatocytes in response to TNF-alpha. Its depletion caused by alcohol consumption amplifies the power of TNF-alpha to generate reactive oxygen species, compromising mitochondrial and cellular functions that culminate in cell death.

VCAM-1 and ICAM-1 mediate leukocyte-endothelial cell adhesion in rat experimental colitis

BACKGROUND & AIMS The molecular mechanisms responsible for leukocyte recruitment in experimental colitis are poorly understood. The aims of this study were to measure expression of endothelial intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) and to determine their role in leukocyte recruitment in experimental colitis. METHODS Rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis and control rats were studied 1, 7, or 21 days after treatment. ICAM-1 and VCAM-1 expressions were measured by the double radiolabeled antibody technique. Leukocyte-endothelial cell interactions were determined in colonic venules by fluorescence intravital microscopy. Therapeutic effects of treatment with anti-VCAM-1 antibodies were also assessed. RESULTS Colonic endothelial ICAM-1 was constitutively expressed and did not increase in colitic animals. In contrast, constitutive expression of VCAM-1 was low but markedly increased (6-fold) 1 and 7 days after induction of colitis. Increased colonic expression of VCAM-1 paralleled macroscopic damage score, myeloperoxidase activity, and increased leukocyte adhesion in colonic venules. The latter was significantly decreased by immunoneutralization of ICAM-1 and completely abrogated by immunoneutralization of VCAM-1. Long-term administration of anti-VCAM-1 antibody resulted in significant attenuation of colitis. CONCLUSIONS Induction of colitis in rats by TNBS is followed by up-regulation of endothelial VCAM-1. VCAM-1 and constitutive ICAM-1 are major determinants of leukocyte recruitment to the inflamed intestine.

Replenishment of Glutathione Levels Improves Mucosal Function in Experimental Acute Colitis

Because reactive oxygen species (ROS) have been implicated as mediators of inflammatory bowel disease (IBD), the purpose of the present work was to determine the functional role of mucosal GSH in the trinitrobenzenesulfonic acid in 50% ethanol (TNBS+ethanol)-induced colitis in rats. Mucosal samples were taken to evaluate the temporal relationship between the extent of injury, the levels of glutathione (GSH) during acute colitis induced by TNBS+ethanol, and the effect of N-acetylcysteine (NAC) administration. In vitro assays revealed the interaction of TNBS with GSH leading to the almost instantaneous disappearance of GSH, while the reductive metabolism of TNBS by GSSG reductase generated ROS. Mucosal samples from TNBS+ethanol-treated rats indicated a direct correlation between GSH depletion and injury detected as soon as 30 minutes after TNBS+ethanol administration that persisted 24 hours post treatment. Although, short term depletion of mucosal GSH per se by diethylmaleate did not result in mucosal injury, the oral administration of NAC (40 mm) 4 hours after TNBS+ethanol treatment increased GSH stores (2–fold), decreasing the extent of mucosal injury (60–70%) examined at 24 hours post treatment. However, an equimolar dose of dithiothreitol failed to increase GSH levels and protect mucosa from TNBS+ethanol-induced injury. Interestingly, GSH levels in TNBS+ethanol-treated rats recovered by 1–2 weeks, an effect that was accounted for by an increase of γ-glutamylcysteine synthetase (γ-GCS) activity due to an induction of γ-GCS-heavy subunit chain mRNA. Thus, TNBS promotes two independent mechanisms of injury, GSH depletion and ROS generation, both being required for the manifestation of mucosal injury as GSH limitation renders intestine susceptible to the TNBS-induced ROS overgeneration. Accordingly, in vivo administration of NAC attenuates the acute colitis through increased mucosal GSH levels, suggesting that GSH precursors may be of relevance in the acute relapse of IBD.

Glutathione Depletion Impairs Myogenic Differentiation of Murine Skeletal Muscle C2C12 Cells through Sustained NF-κB Activation

Skeletal muscle differentation is a complex process regulated at multiple levels. This study addressed the effect of glutathione (GSH) depletion on the transition of murine skeletal muscle C2C12 myoblasts into myocytes induced by growth factor inactivation. Cellular GSH levels increased within 24 hours on myogenic stimulation of myoblasts due to enhanced GSH synthetic rate accounted for by stimulated glutamate-L-cysteine ligase (also known as gamma-glutamylcysteine synthetase) activity. In contrast, the synthesis rate of GSH using gamma-glutamylcysteine and glutamate as precursors, which reflects the activity of the GSH synthetase, did not change during differentiation. The stimulation of GSH stores preceded the myogenic differentiation of C2C12 myoblasts monitored by expression of muscle-specific genes, creatine kinase (CK), myosin heavy chain (MyHC), and MyoD. The pattern of DNA binding activity of NF-kappaB and AP-1 in differentiating cells was similar both displaying an activation peak at 24 hours after myogenic stimulation. Depletion of cellular GSH levels 24 hours after stimulation of differentiation abrogated myogenesis as reflected by lower CK activity, MyHC levels, MyoD expression, and myotubes formation, effects that were reversible on GSH replenishment by GSH ethyl ester (GHSEE). Moreover, GSH depletion led to sustained activation of NF-kappaB, while GSHEE prevented it. Furthermore, inhibition of NF-kappaB activation restored myogenesis despite GSH depletion. Thus, GSH contributes to the formation of myotubes from satellite myoblasts by ensuring inactivation of NF-kappaB, and hence maintaining optimal GSH levels may be beneficial in restoring muscle mass in chronic inflammatory disorders.