Microarray gene analysis of the liver in a rat model of chronic, voluntary alcohol intake.

The mechanisms underlying alcoholic liver disease are not fully understood. It has been established that alcohol interferes with transcriptional and translational regulatory steps of cell function. To understand such an effect, assessment of alcohol-induced changes in the simultaneous expression of a large number of genes may prove very useful. The purpose of the current study was to test a large number of genes ( approximately 8700) for possible changes in expression induced by alcohol alone or in addition to treatment with lipopolysaccharide (LPS), a putative mediator of alcohol effects on the liver. Male rats were fed an alcohol-containing liquid diet (Lieber-DeCarli) for 14-15 weeks, injected with Escherichia coli LPS (0.8 mg x kg(-1)), and killed 24 h later. Blood samples were taken for determination of plasma liver enzyme activity, and liver samples were obtained for histologic evaluation and total RNA extraction. Total RNA was analyzed for gene expression (Rat Toxicology U34 Array; Affymetrix, Santa Clara, CA). Of 8740 genes on the microchip, 2259 were expressed in the liver. Seven hundred ninety-eight genes underwent significant changes induced by either alcohol or LPS, but listed in this article are only those that significantly increased or decreased expression twofold or more. The genes were assigned to functional groups and reviewed. Gene changes were discussed from two viewpoints: relevance to established hypotheses of alcohol and LPS mechanisms of action and revealing of novel mechanisms of alcohol-induced liver injury. Application of DNA microarray technology to the study of alcohol-induced liver injury generated novel theoretical and experimental approaches to alcohol-induced liver injury.

Alcohol, but not lipopolysaccharide-induced liver apoptosis involves changes in intracellular compartmentalization of apoptotic regulators.

BACKGROUND: While alcohol-induced augmentation of liver apoptosis has been demonstrated in humans and laboratory animals, the underlying mechanisms are not fully elucidated. This study addresses the question whether alcohol and bacterial lipopolysaccharide (LPS), a putative mediator of alcohol effects on the liver, induce augmentation of liver apoptosis by intrinsic or extrinsic signaling pathways. This information may prove important for future design of therapies for alcoholic liver disease. METHODS: Male rats were fed either an alcohol-containing liquid diet or an isocaloric, control diet for 15-16 weeks. At the end of feeding period, the rats were treated with LPS (0.8 mg.kg-1 body weight) or sterile saline and killed 3 and 24 hr later. The liver and blood were sampled for histology and biochemical assays. Hepatocytes were isolated by collagenase perfusion and fractionated to yield mitochondria and cytoplasm. The propensity of mitochondria to undergo permeability transition in the presence of a Ca2+ overload was determined along with distribution of various apoptotic regulators (AIF, Smac2, Bax, cytochrome c, Bcl-XL, Bfl-1, and caspase-2) between mitochondria and cytoplasmic fractions. RESULTS: Increased liver apoptosis in alcohol-treated rats was associated with translocation of several apoptotic regulators between mitochondria and cytoplasm in a manner suggesting that alcohol induces augmentation of apoptosis by recruiting intrinsic apoptotic signals. LPS treatment of rats counteracted alcohol-induced changes in intracellular compartmentalization of apoptotic regulators despite an increased rate of apoptosis. LPS may, therefore, recruit extrinsic apoptotic signals, such as proinflammatory cytokines. CONCLUSIONS: Hepatocytes are to be able to mount an apoptotic response to both intrinsic and extrinsic signals. Alcohol increases liver apoptosis predominantly through an intrinsic signaling pathway while LPS recruits extrinsic signaling pathways.

Epidermal growth factor protects the liver against alcohol-induced injury and sensitization to bacterial lipopolysaccharide.

BACKGROUND: Whereas the role of proinflammatory cytokines in the pathogenesis of alcoholic liver disease has been at the forefront of investigation, a possible role for anti-inflammatory cytokines in this disease has received little attention. This study investigated (1) the hepatic protective effect of an anti-inflammatory cytokine, epidermal growth factor (EGF), against deleterious effects of alcohol and sensitization to bacterial lipopolysaccharide (LPS), and (2) the possible mechanisms that underlie such protection. METHODS: Male C57BL/6 mice were fed a Lieber-DeCarli liquid diet that contained alcohol or an isocaloric replacement for 6 weeks. The animals then were treated daily with human EGF for 7 days (5 microg/mouse), after which they were injected with either LPS (1 mg/kg of body weight) or vehicle and killed 8 hr later. Blood and liver were analyzed for plasma aminotransferase activity, liver histology, liver apoptotic nuclei, mRNA of several cytokines (tumor necrosis factor [TNF]-alpha, interleukin [IL]-1beta, IL-6, and IL-10), apoptotic ligands (TRAIL), cytokine receptors (TNFRp55), pro- and antiapoptotic regulators/adaptors (Fas receptor, FasL, FADD, TRADD, RIP, Bak, Bax, Bcl-X, Bcl-2 and Bcl-w), and caspase-8. RESULTS: Alcohol increased plasma aminotransferase activity and sensitized the liver to the effects of LPS, such as polymorphonuclear infiltration, occurrence of necrotic foci and microabscesses, and increased apoptosis. These changes were associated with elevated mRNA expression of proapoptotic regulators/adaptors. EGF either counteracted or markedly blunted most of these effects. EGF did not affect liver mRNA expression of TNF-alpha, IL-1beta, IL-6, and IL-10, which suggested that these cytokines were not involved in EGF protective effect. EGF protection was mediated by down-regulation of apoptosis through suppression of proapoptotic gene expression. CONCLUSIONS: EGF protects the liver against both alcohol-induced liver damage and liver sensitization to bacterial LPS through down-regulation of apoptosis.

A role for interleukin-10 in alcohol-induced liver sensitization to bacterial lipopolysaccharide.

BACKGROUND: Proinflammatory cytokines play an important role in alcohol-induced liver injury. The role of anti-inflammatory cytokines in the initiation and progression of alcoholic liver disease has received little attention. This study tested the hypothesis that an imbalance exists between pro- and anti-inflammatory cytokines in the liver during chronic exposure to alcohol. Alcohol exposure results in predominantly proinflammatory cytokine secretion and liver injury. METHODS: IL-10 knock-out and their C57BL/6J counterpart wild-type mice were fed alcohol in drinking water for 7 weeks. At the end of alcohol feeding, Gram-negative bacterial lipopolysaccharide (LPS) was administered, and the animals were killed after 3 and 8 hr. Liver histology, plasma alanine aminotransferase and aspartate aminotransferase activity, tumor necrosis factor-alpha, interleukin (IL)-1beta and IL-10 levels, and liver cytokine messenger RNA levels were measured. RESULTS: Alcohol feeding and LPS treatment did not change plasma enzyme activity levels in wild-type mice. In the IL-10 knock-out mice, LPS alone increased aspartate aminotransferase and alanine aminotransferase enzyme activity, and this was potentiated by alcohol. Alcohol induced liver steatosis in both wild-type and knock-out mice. LPS markedly enhanced the histological effects further, especially in the knock-out mice, with the emergence of focal necrosis, polymorphonuclear infiltration, and microabscesses in the liver. Plasma tumor necrosis factor-alpha and IL-1beta levels were not affected by alcohol alone. Proinflammatory cytokine levels were increased by LPS and further enhanced by alcohol treatment, particularly in the IL-10 knock-out mice. IL-10 plasma levels in the wild-type animals were down-regulated by alcohol. Changes in liver cytokine messenger RNA paralleled those seen in plasma cytokine levels. CONCLUSIONS: Alcohol-induced liver sensitization to LPS in wild-type mice may involve down-regulation of IL-10. This anti-inflammatory cytokine, known for its hepatoprotective effects, is secreted simultaneously with proinflammatory cytokines. IL-10 may also limit alcohol-induced liver damage by counteracting the effects of proinflammatory cytokines.