Mitochondria are thought to play a major role in hepatic oxidative stress associated with alcohol-induced liver injury. Thus, the hypothesis that delivery of the mitochondrial isoform of superoxide dismutase (Mn-SOD) via recombinant adenovirus would reduce alcohol-induced liver injury was tested. Rats were given recombinant adenovirus containing Mn-SOD (Ad.SOD2) or ␤-galactosidase (Ad.lacZ) and then fed alcohol enterally for 4 weeks. Mn-SOD expression and activity of Ad.SOD2 in liver mitochondria of infected animals was increased nearly 3-fold compared with Ad.lacZ-infected controls. Mitochondrial glutathione levels in Ad.lacZinfected animals were decreased after 4 weeks of chronic ethanol, as expected, but were unchanged in Ad.SOD2-infected animals. Alanine aminotransferase was elevated significantly by ethanol, an effect that was prevented by Ad.SOD2. Moreover, pathology (e.g. the sum of steatosis, inflammation, and necrosis) was elevated dramatically by ethanol in Ad.lacZ-treated rats. This effect was also blunted in animals infected with Ad.SOD2. Neutrophil infiltration was increased about 3-fold in livers from both Ad.lacZ-and Ad.SOD2-infected rats by ethanol treatment. Moreover, ESR-detectable free radical adducts in bile were increased about 8-fold by ethanol. Using 13 C-labeled ethanol, it was determined that nearly 60% of total adducts were due to the ␣-hydroxyethyl radical adduct. This increase in radical formation was blocked completely by Ad.SOD2 infection. Furthermore, apoptosis of hepatocytes was increased about 5-fold by ethanol, an effect also blocked by Ad.SOD2. Interestingly, tumor necrosis factor-␣ mRNA was elevated to the same extent in both Ad.lacZ-and Ad.SOD2-infected animals follows ethanol exposure. These data suggest that hepatocyte mitochondrial oxidative stress is involved in alcohol-induced liver damage and likely follows Kupffer cell activation, cytokine production, and neutrophil infiltration. These results also support the hypothesis that mitochondrial oxidant production is a critical factor in parenchymal cell death caused by alcohol.Alcoholic liver disease results from dose-and time-dependent exposure to alcohol (1), but precise mechanisms of pathology are still largely unknown. Endotoxin and Kupffer cells have been implicated in the mechanism of early alcoholinduced liver injury using the enteral feeding model of Tsukamoto-French (2). For example, endotoxin derived from the gut activates Kupffer cells in the liver (3). In support of this idea, gut sterilization with nonabsorbable antibiotics or inactivation of Kupffer cells by gadolinium chloride (GdCl 3 ) prevents alcohol-induced liver injury in this model (4, 5). Furthermore, Kupffer cells, which release effectors and cytokines, are a major source of TNF␣ 1 in the liver (6). Indeed, TNF␣ messenger RNA in liver increased after 4 weeks of treatment with ethanol (7). Moreover, early alcohol-induced liver injury was attenuated by anti-TNF␣ antibodies and largely prevented in TNF receptor 1 knockout mice (8, 9). Thus, it is clear t...

It has been reported that ischemic preconditioning of the heart or brain has a possible relevance to heat shock protein (HSP). It is still unknown, however, whether HSP induced by means of ischemic preconditioning of the liver is a direct factor in the acquisition of tolerance to succeeding ischemia-reperfusion injury. In the present study we used ischemic preconditioning of the liver to verify the effects of induced HSP72 in the liver on the subsequent longer warm ischemia and reperfusion. Rats preconditioned with short-term (15-minute) ischemia were compared with rats preconditioned by heat exposure or with control rats. After a 48-hour recovery from the sublethal stress for preconditioning, all rats were exposed to longer (30-minute) warm ischemia and reperfusion. Forty-eight hours after ischemic preconditioning, HSP72 was clearly induced in the liver, as well as in the liver preconditioned with heat shock, but not in the kidney or heart. This ischemic preconditioning also attenuated the liver damage in the subsequent ischemia-reperfusion injury, improving the restoration of hepatic function during reperfusion and resulting in higher postischemic rat survival. According to the proposed model of tolerance acquisition for ischemia-reperfusion injury by stress preconditioning, these observations support the speculation that the induced HSP72 plays some beneficial role in this protection mechanism.

L-2-oxothiazolidine-4-carboxylic acid (OTC) is a cysteine prodrug that maintains glutathione in tissues. Here, its effect on alcohol-induced liver injury in an enteral alcohol feeding model was investigated. Male Wistar rats were given control high-fat or ethanol containing diets enterally for 4 weeks. Treated rats received 500 mg/kg/d of dietary OTC. Ethanol delivery, weight gain, and the cyclic pattern of ethanol in the urine were not different between the OTC-ethanol and ethanol groups. After 4 weeks, serum aspartate transaminase (AST), necrosis and inflammation were elevated significantly by ethanol compared with appropriate high-fat controls, effects blocked by OTC. Moreover, ethanol elevated hepatic tumor necrosis factor ␣ (TNF-␣) messenger RNA (mRNA) and the nuclear transcription factor nuclear factor B (NFB) 2-3 fold. NFB in isolated Kupffer cells was also increased by ethanol. These effects were all blocked by OTC treatment. Additionally, superoxide production was higher in Kupffer cells isolated from ethanol-treated rats, an effect blunted by OTC. OTC also increased circulating glutathione (GSH) levels about 2-fold; however, GSH levels were not affected by ethanol or OTC in livers from the groups studied. Surprisingly, GSH was elevated by ethanol and OTC treatment in isolated Kupffer cells about 2-fold. Moreover, GSH (Ki-10 mol/L) and cysteinylglycine, but not oxidized glutathione (GSSG) or OTC, blunted the LPS-induced increase in calcium in isolated Kupffer cells, possibly by activating a glycine-gated chloride channel due to their structural similarity with glycine. Collectively, it is concluded that GSH is protective, in part, by increasing circulating GSH, which blunts activation of Kupffer cells via the glycine-gated chloride channel. (HEPATOLOGY 2000;31:391-398.)Kupffer cells release numerous mediators that participate in metabolic regulation, inflammation, and immune function. 1,2 Recent work from this laboratory has shown a central role for Kupffer cells in mechanisms of alcohol-induced liver injury. For example, when Kupffer cells were destroyed using gadolinium chloride (GdCl 3 ), injury caused by chronic alcohol administration in the Tsukamoto-French enteral alcohol feeding model was blocked. 3 Moreover, Kupffer cells are activated by bacterial endotoxin, and elimination of endotoxin with antibiotics 4 or Lactobacillus feeding 5 also diminished injury to the liver caused by alcohol. This concept was further strengthened by the observation that an antibody to tumor necrosis factor ␣ (TNF-␣) and a mouse lacking the TNF-receptor 1 6 exhibited diminished injury due to chronic ethanol 7 (for review see Thurman 8 ).Using the same enteral feeding model, Hirano et al. 9 showed that ethanol decreased hepatic glutathione (GSH) levels. Decreased glutathione occurs in many disease states including ischemia-reperfusion associated with heart disease. 10 Interestingly, they also showed that ethanol had a more pronounced effect on mitochondrial GSH content, and suggested that it could play an important role...

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