Chronic alcohol administration increases gut-derived endotoxin in the portal blood, which activates Kupffer cells and causes liver injury. Mice (C3H/HeJ) with mutations in toll-like receptor 4 (TLR4) are hyporesponsive to endotoxin. To test the hypothesis that TLR4 is involved in early alcohol-induced liver injury, the long-term intragastric ethanol feeding protocol developed by Tsukamoto and French for rats was adapted to mice. Animals with nonfunctional TLR4 and wild-type mice (C3H/HeOuJ) were compared. Two-month-old female mice were fed a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin as control continuously for 4 weeks. There was no difference in mean urine alcohol concentrations between the groups. Dietary alcohol significantly increased liver-to-body weight ratios and serum alanine transaminase (ALT) levels in wild-type mice (109 ؎ 18 U/L) over high-fat controls (40 ؎ 3 U/L), effects that were blunted significantly in mice with a mutation of TLR4 (55 ؎ 9 U/L). While no significant pathologic changes were observed in high-fat controls, dietary ethanol caused steatosis, mild inflammation, and focal necrosis in wild-type animals (pathology score ‫؍‬ 5.2 ؎ 1.2). These pathologic changes were significantly lower in TLR4-deficient mice fed ethanol (score ‫؍‬ 2.0 ؎ 1.3). Endotoxin levels in the portal vein were increased significantly after 4 weeks in both groups fed ethanol. Moreover, ethanol increased tumor necrosis factor ␣ (TNF-␣) mRNA expression in wildtype, but not in TLR4-deficient, mice. These data are consistent with the hypothesis that Kupffer cell activation by endotoxin via TLR4 is involved in early alcohol-induced liver injury. (HEPATOLOGY 2001;34:101-108.)Bacterial lipopolysaccharide (LPS; endotoxin), an abundant and essential component of the outer membrane of gramnegative bacteria, provokes a generalized proinflammatory response in the infected host that sometimes leads to septic shock and multiple organ failure. 1,2 LPS also causes liver injury in many experimental models. 3,4 Chronic alcohol administration increases gut-derived endotoxin in the portal circulation, thereby activating Kupffer cells to produce several proinflammatory cytokines such as tumor necrosis factor ␣ (TNF-␣) and interleukin (IL)-1. 4,5 Indeed, intestinal sterilization with antibiotics (polymixin B and neomycin) 6 and reduction of gram-negative bacteria with lactobacillus feeding 7 prevented alcohol-induced liver injury, indicating that increased endotoxin levels in the portal circulation are involved in the development of early alcohol-induced liver injury.More than 30 years ago, mice of the C3H/HeJ strain were found to have a defective response to bacterial endotoxin. 8 Recent work showed that a mutation at the Lps locus is responsible for this effect and that C3H/HeJ mice have a missense mutation in the third exon of the toll-like receptor 4 (TLR4) gene encoded by Lps. 9 TLR4, a transmembrane protein with a cytoplasmic domain that bears homology to the IL-1 receptor, is expressed in monocytes and m...

Activation of Kupffer cells by gut-derived endotoxin is associated with alcohol-induced liver injury. Recently, it was shown that CD14-deficient mice are more resistant to endotoxin-induced shock than wild-type controls. Therefore, this study was designed to investigate the role of CD14 receptors in early alcohol-induced liver injury using CD14 knockout and wild-type BALB/c mice in a model of enteral ethanol delivery. Animals were given a high-fat liquid diet continuously with ethanol or isocaloric maltose-dextrin as control for 4 wk. The liver to body weight ratio in wild-type mice (5.8 ± 0.3%) was increased significantly by ethanol (7.3 ± 0.2%) but was not altered by ethanol in CD14-deficient mice. Ethanol elevated serum alanine aminotransferase levels nearly 3-fold in wild-type mice, but not in CD14-deficient mice. Wild-type and knockout mice given the control high-fat diet had normal liver histology, whereas ethanol caused severe liver injury (steatosis, inflammation, and necrosis; pathology score = 3.8 ± 0.4). In contrast, CD14-deficient mice given ethanol showed minimal hepatic changes (score = 1.6 ± 0.3, p < 0.05). Additionally, NF-κB, TGF-β, and TNF-α were increased significantly in wild-type mice fed ethanol but not in the CD14 knockout. Thus, chronic ethanol feeding caused more severe liver injury in wild-type than CD14 knockouts, supporting the hypothesis that endotoxin acting via CD14 plays a major role in the development of early alcohol-induced liver injury.

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...

The oxidant source in alcohol-induced liver disease remains unclear. NADPH oxidase (mainly in liver Kupffer cells and infiltrating neutrophils) could be a potential free radical source. We aimed to determine if NADPH oxidase inhibitor diphenyleneiodonium sulfate (DPI) affects nuclear factor-kappaB (NF-kappaB) activation, liver tumor necrosis factor-alpha (TNF-alpha) mRNA expression, and early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-16 g. kg(-1). day(-1)) continuously for up to 4 wk, using the Tsukamoto-French intragastric enteral feeding protocol. DPI or saline vehicle was administered by subcutaneous injection for 4 wk. Mean urine ethanol concentrations were similar between the ethanol- and ethanol plus DPI-treated groups. Enteral ethanol feeding caused severe fat accumulation, mild inflammation, and necrosis in the liver (pathology score, 4.3 +/- 0.3). In contrast, DPI significantly blunted these changes (pathology score, 0.8 +/- 0.4). Enteral ethanol administration for 4 wk also significantly increased free radical adduct formation, NF-kappaB activity, and TNF-alpha expression in the liver. DPI almost completely blunted these parameters. These results indicate that DPI prevents early alcohol-induced liver injury, most likely by inhibiting free radical formation via NADPH oxidase, thereby preventing NF-kappaB activation and TNF-alpha mRNA expression in the liver.