Jason C. Lambert

A Critical Involvement of Oxidative Stress in Acute Alcohol-Induced Hepatic TNF-α Production

Tumor necrosis factor-α (TNF-α) production is a critical factor in the pathogenesis of alcoholic liver injury. Both oxidative stress and endotoxin have been implicated in the process of alcohol-induced TNF-α production. However, a cause-and-effect relationship between these factors has not been fully defined. The present study was undertaken to determine the mediators of acute alcohol-induced TNF-α production using a mouse model of acute alcohol hepatotoxicity. Alcohol administration via gavage at a dose of 6 g/kg to 129/Sv mice induced hepatic TNF-α production in Kupffer cells as demonstrated by measuring protein levels, immunohistochemical localization, and mRNA expression. Alcohol intoxication caused liver injury in association with increases in plasma endotoxin and hepatic lipid peroxidation. Treatment with an endotoxin neutralizing protein significantly suppressed alcohol-induced elevation of plasma endotoxin, hepatic lipid peroxidation, and inhibited TNF-α production. Treatment with antioxidants, N-acetyl-l-cysteine, or dimethylsulfoxide, failed to attenuate plasma endotoxin elevation, but significantly inhibited alcohol-induced hepatic lipid peroxidation, TNF-α production and steatosis. All treatments prevented alcohol-induced necrotic cell death in the liver. This study thus systemically dissected the relationship among plasma endotoxin elevation, hepatic oxidative stress, and TNF-α production following acute alcohol administration, and the results demonstrate that oxidative stress mediates endotoxin-induced hepatic TNF-α production in acute alcohol intoxication.

Metallothionein-Independent Zinc Protection from Alcoholic Liver Injury

Previous studies using metallothionein (MT)-overexpressing transgenic mice have demonstrated that MT protects the liver from oxidative injury induced by alcohol. The mechanism of action of MT is unknown. Because MT primarily binds to zinc under physiological conditions and releases zinc under oxidative stress and zinc is an antioxidant element, it is likely that zinc mediates the protective action of MT. The present study was undertaken to determine the distinct role of zinc in hepatic protection from alcoholic injury. MT I/II-knockout (MT-KO) mice along with their wild-type controls were treated with three gastric doses of ethanol at 5 g/kg at 12-hour intervals. Zinc sulfate was injected intraperitoneally in a dosage of 5 mg/kg/day for 3 days before ethanol treatment. MT concentrations in MT-KO mice were very low and zinc concentrations in MT-KO mice were lower than in wild-type mice. Zinc treatment significantly elevated hepatic MT concentrations only in wild-type mice and increased zinc concentrations in both MT-KO and wild-type mice. Ethanol treatment caused degenerative morphological changes and necrotic appearance in the livers of MT-KO mice. Microvesicular steatosis was the only ethanol-induced change in the liver of wild-type mice. Ethanol treatment decreased hepatic glutathione concentrations and increased hepatic lipid peroxidation, and the concentrations of lipid peroxide products in the wild-type mice were lower than in the MT-KO mice. All of these alcohol-induced toxic responses were significantly suppressed by zinc treatment in both MT-KO and wild-type mouse livers. These results demonstrate that zinc, independent of MT, plays an important role in protection from alcoholic liver injury. However, MT is required to maintain high levels of zinc in the liver, suggesting that the protective action of MT in the liver is likely mediated by zinc.

Preservation of Intestinal Structural Integrity by Zinc Is Independent of Metallothionein in Alcohol-Intoxicated Mice

Intestinal-derived endotoxins are importantly involved in alcohol-induced liver injury. Disruption of intestinal barrier function and endotoxemia are common features associated with liver inflammation and injury due to acute ethanol exposure. Zinc has been shown to inhibit acute alcohol-induced liver injury. This study was designed to determine the inhibitory effect of zinc on alcohol-induced endotoxemia and whether the inhibition is mediated by metallothionein (MT) or is independent of MT. MT knockout (MT-KO) mice were administered three oral doses of zinc sulfate (2.5 mg zinc ion/kg body weight) every 12 hours before being administered a single dose of ethanol (6 g/kg body weight) by gavage. Ethanol administration caused liver injury as determined by increased serum transaminases, parenchymal fat accumulation, necrotic foci, and an elevation of tumor necrosis factor (TNF-alpha). Increased plasma endotoxin levels were detected in ethanol-treated animals whose small intestinal structural integrity was compromised as determined by microscopic examination. Zinc supplementation significantly inhibited acute ethanol-induced liver injury and suppressed hepatic TNF-alpha production in association with decreased circulating endotoxin levels and a significant protection of small intestine structure. As expected, MT levels remained undetectable in the MT-KO mice under the zinc treatment. These results thus demonstrate that zinc preservation of intestinal structural integrity is associated with suppression of endotoxemia and liver injury induced by acute exposure to ethanol and the zinc protection is independent of MT.

Suppression of fas-mediated signaling pathway is involved in zinc inhibition of ethanol-induced liver apoptosis

Apoptosis is critically involved in hepatic pathogenesis induced by acute alcohol exposure. This study was undertaken to test the hypothesis that zinc interferes with an important Fas ligand-mediated pathway in the liver, leading to the inhibition of ethanol-induced apoptosis. Male 129/SvPCJ mice were injected subcutaneously with ZnSO4 (5 mg of Zn ion/kg) in 12-hr intervals for 24 hr before intragastric administration of ethanol (5 g/kg) in 12-hr intervals for 36 hr. Ethanol-induced apoptosis in the liver was detected by a terminal deoxynucleotidyl transferase nick-end labeling assay and was further confirmed by electron microscopy. The number of apoptotic cells in the livers pretreated with zinc was significantly decreased, being only 15% of that found in the animals treated with ethanol only. Characteristic apoptotic morphological changes observed by electron microscopy were also inhibited by zinc. Importantly, zinc inhibited ethanol-induced activation of caspase-3, the primary executioner protease responsible for alcohol-induced liver apoptosis, and caspase-8 as determined by enzymatic assay. Immunohistochemical analysis revealed that zinc inhibited ethanol-induced endogenous Fas ligand activation, which is a key component in signaling pathways leading to hepatic caspase-8 and subsequent caspase-3 activation and apoptosis. These results demonstrate that zinc is a potent inhibitor of acute ethanol-induced liver apoptosis, and this effect occurs primarily through zinc interference with Fas ligand pathway and the suppression of caspase-3.

Roles of Kupffer Cells in Alcoholic Liver Disease

This article summarizes presentations at the 2004 International Society for Biomedical Research on Alcoholism held in Heidelberg/Mannheim, Germany. The organizers and chairpersons were Yoshiyuki Takei and Gavin E Arteel. The presentations were on 1) Kupffer cell-derived mediators involved in alcoholic liver disease: reactive oxygen and nitrogen species, by Gavin E. Arteel; 2) Kupffer cell-derived mediators in alcoholic liver disease: tumor necrosis factor-a, by Laura E. Nagy; 3) metformin prevents acute alcohol-induced fat accumulation in mouse liver, by Ina Bergheim; 4) gender difference in alcoholic liver disease, by Nobuyuki Enomoto; and 5) Kupffer cells as a therapeutic target of alcoholic liver disease, by Yoshiyuki Takei.

925 A critical involvement of oxidative stress in acute alcohol-induced hepatic TNF-alpha production

Tumor necrosis factor-alpha (TNF-alpha) production is a critical factor in the pathogenesis of alcoholic liver injury. Both oxidative stress and endotoxin have been implicated in the process of alcohol-induced TNF-alpha production. However, a cause-and-effect relationship between these factors has not been fully defined. The present study was undertaken to determine the mediators of acute alcohol-induced TNF-alpha production using a mouse model of acute alcohol hepatotoxicity. Alcohol administration via gavage at a dose of 6 g/kg to 129/Sv mice induced hepatic TNF-alpha production in Kupffer cells as demonstrated by measuring protein levels, immunohistochemical localization, and mRNA expression. Alcohol intoxication caused liver injury in association with increases in plasma endotoxin and hepatic lipid peroxidation. Treatment with an endotoxin neutralizing protein significantly suppressed alcohol-induced elevation of plasma endotoxin, hepatic lipid peroxidation, and inhibited TNF-alpha production. Treatment with antioxidants, N-ACETYL-L-CYSTEINE, or dimethylsulfoxide, failed to attenuate plasma endotoxin elevation, but significantly inhibited alcohol-induced hepatic lipid peroxidation, TNF-alpha production and steatosis. All treatments prevented alcohol-induced necrotic cell death in the liver. This study thus systemically dissected the relationship among plasma endotoxin elevation, hepatic oxidative stress, and TNF-alpha production following acute alcohol administration, and the results demonstrate that oxidative stress mediates endotoxin-induced hepatic TNF-alpha production in acute alcohol intoxication.