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Background Our previous work showed that binge drinking in the rat induced hepatic steatosis which correlated with reduced expression of AMP-activated protein kinase (AMPK). In this study, we used the rat model to investigate the role of adiponectin (Adip), sirtuin 1 (SIRT1), AMPK, and lipin 1 (LIP 1) signaling, a central controlling pathway of lipid metabolism in hepatic steatosis. Methods The serum Adip and tumor necrosis factor-alpha (TNF-α) as well as liver Adip receptors (AdipoR1 and AdipoR2) SIRT1, AMPK, phosphorylated AMPK (p-AMPK), sterol regulatory element-binding proteins (SREBPs), acetyl-CoA carboxylase (ACC), LIP 1, lipocalin-2 (LCN2), and serum amyloid A1 were assessed in the rat model where 16 weeks of gavaged alcohol were administered. Results In this model of ethanol (EtOH) administration, hepatic steatosis, necrosis, as well as inflammation were increased over the 16-week period. The level of TNF-α in the serum was increased while the Adip content decreased significantly, and there was an inverse relationship between the content of TNF-α and Adip. The mRNA and protein expression of AdipoR2, SIRT1, and AMPK was suppressed by EtOH in the rats' hepatic tissue. Additionally, EtOH significantly decreased p-AMPK by 90% over the 16-week period. In parallel, there was a 2.53- and 1.82-fold increase of lipogenic genes SREBP1c and ACC, and a 3.22- and 4.12-fold increase of LIP 1 and LIP 1 β mRNA expression, respectively, in the hepatic tissue of the rats. Conclusions Our present observations demonstrate that the impaired Adip-SIRT1-AMPK signaling pathway contributes, at least in part, to the development of alcoholic fatty liver disease in EtOH binge rats.

Cyclooxygenase-1 (COX-1) is the constitutive form of the COX enzyme family, which produces bioactive lipids called prostanoids. Although the role of COX-2 in liver diseases has been studied, little is known about the function of COX-1 in liver injury. We aimed to investigate the role and mechanism of COX-1 in acute liver injury. Carbon tetrachloride (CCl(4)) was administered to induce acute liver injury in wild-type or COX-1-deficient mice. Both genetic (partially or completely) deletion of COX-1 expression and pharmacological inhibition of COX-1 activity in mice exacerbated acute liver injury induced by CCl(4), revealing the (1) histopathological changes and increased serum levels of aminotransferases; (2) oxidative stress in the liver partly through the action of cytochrome P450 2E1-dependent pathway; (3) enhanced inflammatory and chemoattractive responses with increased number of activated macrophages; and (4) increased apoptosis through both intrinsic and extrinsic apoptotic pathways. These pathological changes were partly through the modulation of transcription factor-dependent pathways (eg, NF-κB and C/EBP-α). Pre-treatment with prostaglandin E2 (PGE(2)) or 5-lipoxygenase (5-LO) inhibitor in homozygous COX-1 knockout mice significantly ameliorated CCl(4)-induced hepatic injury. In addition, level of hepato-protective molecules (eg, OSM and OSMR) and associated liver regeneration pathway were significantly inhibited by the deficiency of COX-1 but restored by the addition of PGE(2) or the inhibition of 5-LO. Furthermore, the alternative arachidonic acid metabolism pathway of 5-LO, which induced additional inflammation in the liver, was activated in response to the deficiency of COX-1. In conclusion, basal expression of COX-1 is essential for the protection of liver against chemical-induced hepatotoxicity and required for hepatic homeostatic maintenance.

Background Alcoholic liver disease (ALD) continues to be a major cause of morbidity worldwide. The exact mechanisms for ALD pathogenesis are not fully understood. There is currently no known available drug for ALD. Previous studies have suggested that ethanol (EtOH)-induced hepatic insulin resistance, through the inhibition of adenosine monophosphate-activated protein kinase (AMPK) and the expression of adiponectin as well as downstream enzymes, contribute to the development of ALD. This study was to determine the effects of EtOH on AMPK activity as well as the protective effect of metformin. Methods Forty male Wistar rats weighing 200 ± 20 g were randomized into 4 groups (n = 10) as follows: A = control group—rats received rodent chow; B = control + metformin group—rats received metformin (200 mg/kg/d intragastrically [IG]) at 21:00; C = EtOH group—rats were gavaged with alcohol of gradually increasing concentrations (30 to 60%, 5 to 9 g/kg/d) twice a day (9:00 and 16:00); D = EtOH + metformin group—rats received the same amount of EtOH as the rats in group C, and in addition received metformin (200 mg/kg/d IG) at 21:00. After 16 weeks, blood and liver samples were collected for further study. Results Chronic EtOH consumption led to liver injury both histologically and biochemically accompanied by insulin resistance, reduced AMPK activity, and dysregulation of downstream enzymes. Decreased levels of circulating adiponectin and decreased expression of proliferator-activated receptor gamma coactivator-1α (PGC-1α) and peroxisome proliferator-activated receptors-α (PPAR-α) in the hepatic tissue were observed. Treatment with metformin attenuated the severity of liver injury, restored AMPK activity and normalized the expression of acetyl-CoA carboxylase and fatty acid synthase. In addition, metformin also increased the circulating adiponectin and liver adiponectin receptor 2 expression. Furthermore, PGC-1α and PPAR-α activities were also restored. Conclusions EtOH exposure induces hepatic insulin resistance. Metformin improved insulin resistance and reversed liver injury through the activation of AMPK and normalized adiponectin signaling making metformin a promising drug for the treatment of ALD.

To investigate the protective mechanisms of an 85 % pure extract of (−) epigallocatechin gallate (EGCG) in the development of fibrosis, oxidative stress and inflammation in a recently developed dietary-induced animal model of non-alcoholic fatty liver disease (NAFLD). Female Sprague–Dawley rats were fed with either normal rat diet or high-fat diet for 8 weeks to develop NAFLD. For both treatments, rats were treated with or without EGCG (50 mg/kg, i.p. injection, 3 times per week). At the end, blood and liver tissue samples were obtained for histology, molecular, and biochemical analyses. Non-alcoholic fatty liver disease (NAFLD) rats showed significant amount of fatty infiltration, necrosis, fibrosis, and inflammation. This was accompanied by a significant expressional increase in markers for fibrosis, oxidative stress, and inflammation. TGF/SMAD, PI3 K/Akt/FoxO1, and NF-κB pathways were also activated. Treatment with EGCG improved hepatic histology (decreased number of fatty score, necrosis, and inflammatory foci), reduced liver injury (from ~0.5 to ~0.3 of ALT/AST ratio), attenuated hepatic changes including fibrosis (reduction in Sirius Red and synaptophysin-positive stain) with down-regulation in the expressions of key pathological oxidative (e.g. nitrotyrosine formation) and pro-inflammatory markers (e.g. iNOS, COX-2, and TNF-α). EGCG treatment also counteracted the activity of TGF/SMAD, PI3 K/Akt/FoxO1, and NF-κB pathways. Treatment with EGCG did not affect the healthy rats. Epigallocatechin gallate (EGCG) reduced the severity of liver injury in an experimental model of NAFLD associated with lower concentration of pro-fibrogenic, oxidative stress, and pro-inflammatory mediators partly through modulating the activities of TGF/SMAD, PI3 K/Akt/FoxO1, and NF-κB pathways. Therefore, green tea polyphenols and EGCG are useful supplements in the prevention of NAFLD.

Background To investigate whether “binge” and escalating alcohol exposure in the rat influences the development of pathological liver injury. Methods Time courses for the formation of eicosanoids by cyclooxygenase (COX), oxidative stress and nitrosative stress production, expression of hypoxia-inducible factor 1 (HIF-1), cytokines, hepatic tissue necroinflammation, and fibrosis were assessed in rats during 16 weeks of daily alcohol gavage. Results In this model of binge and escalating levels of alcohol, hepatic steatosis, necrosis, and inflammation as well as fibrosis were increased over the 16-week period. The levels of COX-2, oxidative stress, nitrosative stress, HIF-1, proinflammatory mediators (tumor necrosis factor-α, interleukin 1β [IL-1β], IL-6), and procollagen-I were increased over the 16-week period. The content of IL-10 in rat serum increased at the end of 4 and 8 weeks but decreased thereafter and was significantly decreased at 12 and 16 weeks. Conclusions A rat model of alcoholic liver disease (ALD) with long-term binge and escalating ethanol exposure was developed. Our data support the hypothesis that enhanced eicosanoid production by COX, oxidative stress and nitrosative stress, HIF-1, and the imbalance between pro- and anti-inflammatory cytokines plays an important role in the pathogenesis of ALD.

Thromboxane levels are increased in rats fed ethanol (EtOH), whereas thromboxane inhibitors reduce alcoholic liver injury. The aim of this study is to determine whether thromboxane inhibitors could attenuate the already established alcoholic liver injury.Rats were fed EtOH and liquid diet for 6 weeks by intragastric infusion to induce liver injury after which EtOH was continued for 2 more weeks, and the rats were treated with either a thromboxane synthase inhibitor (TXSI) or a thromboxane receptor antagonist (TXRA). Liver pathology, lipid peroxidation, nuclear factor-kappa-B (NF-κB) activity, tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), and transforming growth factor-beta1 (TGF-β(1) ) were evaluated.Administration of fish oil and EtOH caused fatty liver, necrosis, inflammation and fibrosis accompanied by increased in lipid peroxidation, NF-κB activity, and expression of TNF-α, COX-2, and TGF-β(1) . Treatment with the thromboxane inhibitors ameliorated a certain level of the pathological and biochemical abnormalities. In particular, TXSI in addition to reducing necrosis, inflammation and fibrosis also decrease the severity of fatty liver.Thromboxane inhibitors attenuated the alcoholic liver injury, inflammation and fibrotic changes despite continued EtOH administration. Inhibition of the production of thromboxane by thromboxane inhibitor and receptor antagonists may be a useful treatment strategy in clinical alcoholic liver disease.

Scope Lycium barbarum polysaccharide (LBP) is a water fraction of wolfberry, which has been demonstrated to possess a hepatoprotective effect in several liver disease models. However, the anti-alcoholic liver disease (anti-ALD) mechanism of LBP has not been investigated thoroughly. Its protective effects on both male and femal mice are investigated in the current study. Methods and results A chronic ethanol-fed ALD in vivo model is applied to study the effect of LBP in both male and female mice. It is observed that ethanol causes more severe liver injury in female than male mice, and the ameliorative effects of LBP are also more significant in female mice, which are impaired after complete bilateral oophorectomy. The hepatic SCD1 expression is found to be positively correlated with the severity of the liver damage and the main mediator of LBP inducer of protection. The AMPK-CPT pathway is also activated by LBP to rebalance the dysregulated lipid metabolism during ALD development. By using concurrent sodium palmitate and an ethanol-induced in vitro cell damage model in AML-12 cell line, it is characterized that LBP directly interacts with ERα instead of ERβ to activate the SCD1-AMPK-CPT pathway. Conclusions LBP is an effective and safe hepatoprotective agent against ALD primarily through the SCD1-AMPK-CPT pathway after ERα agonist.

The anti-inflammatory and antioxidant effects of epigallocatechin-3-gallate (EGCG) are considered important forces in attenuate liver injury and fibrosis. The aim of the study was to investigate the effect of EGCG on the expression of fibrogenic factors and whether EGCG attenuates the severity of oxidative stress and inflammatory response in chronic liver injury. Mice were administered with CCl(4) together with or without EGCG for 8 weeks (n=6-8 per group). Histopathological and biochemical analyses were carried out. The mRNA expression levels of TNF-alpha, COX-2, iNOS, alpha-smooth muscle actin (alpha-SMA), transforming growth factor (TGF-beta(1)), pro-collagen-I, matrix metalloproteinases (MMP-2, -9) and their inhibitors (TIMP-1, -2) were determined by RT-PCR. The collagen deposited in the liver was detected by Sirius Red staining. The formation of nitrotyrosine was measured as a marker of oxidative stress. The activity level of NF-kappaB and the expression level of C/EBP were also assessed. Chronic CCl(4) treatment caused liver injury, oxidative stress and nitrosative stress, and collagen accumulation in the liver. The expression levels of pro-inflammatory and pro-fibrotic mediators and the activity of NF-kappaB were increased. Treatment with EGCG significantly reduced liver injury, oxidative stress and the inflammatory response. EGCG also significantly reduced the formation of collagen in the liver, the expression of alpha-SMA and all of the assayed pro-fibrogenic markers except TIMP-2 and MMP-9. EGCG significantly attenuated the severity of CCl(4)-induced liver injury and the progression of liver fibrosis. The protective effect of EGCG may in part be a consequence of the reduction in oxidative stress and the pro-inflammatory response.

Background: Ethanol metabolism increases production of reactive oxygen species, including superoxide (O ·― 2 ) in the liver, resulting in significant oxidative stress, which causes cellular damage. Superoxide dismutase (SOD) is an antioxidant enzyme that converts superoxide to less toxic intermediates, preventing O ·― 2 accumulation. Because the absence of SOD would confer less resistance to oxidative stress, we determined whether damage to hepatic proteolytic systems was greater in SOD ―/― than in SOD +/+ mice after chronic ethanol feeding. Methods: Female wild-type (SOD +/+ ) and Cu/Zn-SOD knockout (SOD ―/― ) mice were pair-fed ethanol and control liquid diets for 24 days, after which liver injury was assessed. Results: Ethanol-fed SOD ―/― mice had 4-fold higher blood ethanol, 2.8-fold higher alanine aminotransferase levels, 20% higher liver weight, a 1.4-fold rise in hepatic protein levels, and 35 to 70% higher levels of lipid peroxides than corresponding wild-type mice. While wild-type mice exhibited fatty liver after ethanol administration, SOD ―/― mice showed no evidence of ethanol-induced steatosis, although triglyceride levels were elevated in both groups of knockout mice. Ethanol administration caused no significant change in proteasome activity, but caused lysosomal leakage in livers of SOD ―/― mice but not in wild-type mice. Alcohol dehydrogenase activity was reduced by 50 to 60% in ethanol-fed SOD ―/― mice compared with all other groups. Additionally, while ethanol administration induced cytochrome P450 2E1 (CYP2E1) activity in wild-type mice, it caused no such induction in SOD ―/― mice. Unexpectedly, ethanol feeding significantly elevated total and mitochondrial levels of glutathione in SOD knockout mice compared with wild-type mice. Conclusion: Ethanol-fed SOD ―/― mice exhibited lower alcohol dehydrogenase activity and lack of CYP2E1 inducibility, thereby causing decreased ethanol metabolism compared with wild-type mice. These and other atypical responses to ethanol, including the absence of ethanol-induced steatosis and enhanced glutathione levels, appear to be linked to enhanced oxidative stress due to lack of antioxidant enzyme capacity.

The asialoglycoprotein (ASGP) receptor is an abundant hepatocyte-specific receptor involved in receptor-mediated endocytosis. This receptor's abundance and function is decreased by chronic ethanol administration prior to the appearance of pathology such as necrosis or inflammation. Hence, this study aimed to determine if ASGP receptor function is required to protect against liver injury by utilizing a knockout mouse model lacking functional ASGP receptor in the setting of carbon tetrachloride (CCl(4)) hepatotoxicity. Briefly, ASGP receptor-deficient (RD) mice and wild-type (WT) mice were injected with 1ml/kg body weight of CCl(4). In the subsequent week, mice were monitored for liver damage and pathology (aspartate transaminase (AST), alanine transaminase (ALT) and light microscopy). The consequences of CCl(4) injection were examined by measuring alpha-smooth muscle actin (alpha-SMA) deposition, contents of malondialdehyde and the percentage of apoptotic hepatocytes. After CCl(4) injection, RD mice showed increased liver pathology together with significantly increased activities of AST and ALT compared to that in WT mice. There were also significantly more apoptotic bodies, lipid peroxidation and deposition of alpha-SMA in RD mice versus WT mice following CCl(4) injection. Since these two mouse strains only differ in whether or not they have the ASGP receptor, it can be concluded that proper ASGP receptor function exerted a protective effect against CCl(4) toxicity. Thus, receptor-mediated endocytosis by the ASGP receptor could represent a novel molecular mechanism that is responsible for subsequent liver health or injury.

Reduced expression of endothelial nitric oxide synthase (eNOS) in chronic liver disease can reduce hepatic perfusion and accelerate fibrosis. The relationship between eNOS expression and liver fibrogenesis remains unclear. We investigated whether L-arginine attenuated chronic liver fibrosis through eNOS expression. Chronic liver injury was induced by administration of carbon tetrachloride (CCl(4)) to mice for 8 weeks. 5-Methylisothiourea hemisulphate (SMT), an iNOS inhibitor, or L-arginine, a NOS substrate were injected subcutaneously. CCl(4)-induced hepatotoxicity, oxidative stress and accumulation of collagen were detected in the liver. The expression levels of inducible NOS (iNOS) and nuclear factor kappa-B (NF-kappaB) activity in the liver after CCl(4) treatment were increased but eNOS expression and activator protein-1 (AP-1) activity were decreased. Both SMT and L-arginine effectively reduced CCl(4) induced oxidative stress and collagen formation, but L-arginine showed a significantly greater suppression of collagen formation, iNOS expression and NF-kappaB activity. L-arginine also restored the level of eNOS and AP-1 activity. L-arginine was more effective than SMT in suppressing liver fibrosis. L-arginine might improve NO production which facilitates hepatic blood flow and thus retards liver fibrogenesis. Our results showed that the reduced eNOS expression in CCl(4)-treated mice was reversed by L-arginine. Furthermore, L-arginine also reversed the reduced AP-1 activity, an eNOS promoter.

BACKGROUND: The intragastric (IG) ethanol infusion model results in fatty liver, necrosis, inflammation and fibrosis. This model was utilized to study the pathogenesis of alcoholic liver disease (ALD). Disadvantages of the IG model include maintenance of the animals and equipment expense. To develop a voluntary feeding model for ALD, we took advantage of two important observations in the IG model: (i) female rats demonstrate greater severity of alcohol-induced liver injury than males and (ii) rats fed fish oil as a source of fatty acids develop more severe alcoholic liver injury than rats fed other fatty acids with ethanol. METHODS: Female Wistar rats (205 to 220 g) were fed for 8 weeks a diet containing 8% ethanol, fish oil (30% of calories), protein, and dextrose. Pair-fed controls (FD) received dextrose in amounts isocaloric to ethanol. The following measurements were made: liver pathology [fatty liver (0 to 4), necrosis, inflammation and fibrosis by Sirius Red], endotoxin and alanine aminotransferase (ALT) in plasma, urine ethanol, lipid peroxidation, nuclear factor kappa-B (NF-kappaB) and mRNA levels for tumor necrosis factor-alpha (TNF-alpha), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). Protein levels for iNOS and nitrotyrosine were evaluated by immunohistochemistry and Western Blot analysis. Liver proteasome and cytochrome P450 2E1 activity and protein levels of asialoglycoprotein receptor (ASGPR) were also evaluated. In addition, mRNA levels of fibrogenic markers were assessed. RESULTS: All animals lost weight for the initial 2 to 3 weeks but then gained weight until killing at 8 weeks. There was, however, a significant difference (p < 0.05) in weight between the ethanol-fed (Etoh) and (FD) groups at the end of the experiment. The mean urine ethanol levels ranged between 190 and 240 mg/dl. The severity of pathological changes was greater (p < 0.01) in Etoh vs. FD: fatty liver, 3.0 +/- 1.2 vs. 1.2 +/- 0.4; necrosis (foci/mm(2)), 3.9 +/- 2.3 vs. 0.4 +/- 0.3; inflammation (cells/mm(2)), 19.0 +/- 6.3 vs. 1.8 +/- 0.6. Centrilobular collagen deposition (% area), assessed by Sirius Red staining, was greater in Etoh vs. FD. Levels of endotoxin, ALT, CYP2E1 and lipid peroxidation markers were also higher (p < 0.01) in Etoh vs. FD. Levels of NF-kappaB and mRNA of pro-inflammatory mediators (TNF-alpha, COX-2, iNOS) and procollagen-I were increased (p < 0.05) in ethanol-fed rats. Immunohistochemical analysis showed more intense staining for both iNOS and nitrotyrosine in the centrilobular areas in the Etoh vs. FD groups. The greater area of positive staining for iNOS and nitrotyrosine in Etoh vs. FD was confirmed by Western Blot analysis. An increase in the expression of mRNA for profibrogenic genes (p < 0.05) was seen in ethanol-fed rats. CONCLUSIONS: A voluntary feeding regimen consisting of fish oil and ethanol in female rats is technically less demanding yet produces pathological and biochemical changes similar to those observed with the IG model. Pathological changes include fatty liver, necrosis and inflammation. Increased NF-kappaB and mRNA and protein levels of the pro-inflammatory mediators TNF-alpha, COX-2 and iNOS, coincided with the presence of necroinflammatory changes. The voluntary feeding regimen is proposed as an alternative to the IG model in the study of alcoholic liver injury.

Background: l-Buthionine (S,R) sulfoximine (BSO) is an inhibitor of glutathione biosynthesis and has been used as an effective means of depleting glutathione from cells and tissues. Here we investigated whether treatment with BSO enhanced ethanol-induced liver injury in mice. Methods: Female C57Bl/6 mice were pair fed with control and ethanol-containing liquid diets in which ethanol was 29.2% of total calories. During the final 7 days of pair feeding, groups of control-fed and ethanol-fed mice were given 0, 5 or 7.6 mM BSO in the liquid diets. Results: Compared with controls, ethanol given alone decreased total liver glutathione. This effect was exacerbated in mice given ethanol with 7.6 mM BSO, causing a 72% decline in hepatic glutathione. While ethanol alone caused no decrease in mitochondrial glutathione, inclusion of 7.6 mM BSO caused a 2-fold decline compared with untreated controls. l-Buthionine (S,R) sulfoximine did not affect ethanol consumption, but serum ethanol levels in BSO-treated mice were nearly 6-fold lower than in mice given ethanol alone. The latter decline in serum ethanol was associated with a significant elevation in the specific activities of cytochrome P450 2E1 and alcohol dehydrogenase in livers of BSO-treated animals. Ethanol consumption caused a 3.5-fold elevation in serum alanine aminotransferase levels but the enzyme fell to control levels when BSO was included in the diet. l-Buthionine (S,R) sulfoximine administration also attenuated ethanol-induced steatosis, prevented the leakage of lysosomal cathepsins into the cytosol, and prevented the ethanol-elicited decline in proteasome activity. Conclusions: l-Buthionine (S,R) sulfoximine, administered with ethanol, significantly depleted hepatic glutathione, compared with controls. However, despite the decrease in hepatic antioxidant levels, liver injury by ethanol was alleviated, due, in part, to a BSO-elicited acceleration of ethanol metabolism.

Background: Nonalcoholic fatty liver disease (NAFLD) is a common hepatic condition that may progress to end-stage liver disease. High-fat diets in animals reproduce many of the features found in nonalcoholic steatohepatitis. Objective: To understand how various dietary or genetic factors influence the development of fatty liver and consequently NAFLD, we performed microarray-based expression profiling of genes, induced by fish oil and dextrose diet, a putative mediator of alcohol-like effects on the liver of the female rat. Design: Male and age-matched female rats were fed fish oil and dextrose for 4 weeks. Hepatic RNA from each sample was extracted and used for microarray analysis. Results: A large number of genes underwent significant changes in the female liver as compared to male controls. In the female rat liver, biological theme analysis demonstrated a shift in the transcriptional program which included upregulation of genes involved in lipid metabolism, chaperone activity, mitochondrial and oxidoreductase activity combined with downregulation of genes involved in nucleic acid metabolism. The differential expression of genes of interest identified by microarray technique was validated by real-time reverse transcription-polymerase chain reaction. Ingenuity computational pathway analysis tools were used to identify specific regulatory networks of genes operative in promoting liver injury. Conclusions: The use of networks stated above allowed us to identify genes involved in cell death, apoptosis, peroxisome proliferator-activated receptor alpha-regulated lipid metabolism and mitogen-activated protein kinase signaling pathways.

The aims of the present review are to summarize and to discuss the role of hypoxia-inducible factor-1 (HIF-1) and the expression and functions of vasoactive substances in chronic hypoxemia with specific focus in the liver and the carotid body. Vascular remodelling and vasoactive substances play important functional roles in the adaptive response to chronic hypoxemia for the maintenance of oxygen homeostasis in all systems in man. HIF-1 regulates the gene expression of vasoactive substances such as vascular endothelial growth factor (VEGF), endothelin-1 (ET-1) and enzymes for producing nitric oxide (NO). Recent studies have shown the effect of chronic hypoxia on the expression of HIF-1alpha and HIF-1-target genes in multiple organ systems including the liver and the carotid body. Results are consistent with increases in the hematocrit levels, pulmonary arterial pressure and right heart mass developed during chronic hypoxia. In addition, the carotid body is also hyperplastic and increases in organ mass with increased levels of HIF-1alpha and the vasoactive substances. These molecules increase the mitotic activity and modulate the excitability of the chemoreceptor. Intriguingly, the liver morphology, serum alanine aminotransferase and 8-isoprostane levels are within normal range in chronic hypoxia, suggesting the absence of significant oxidative stress. Yet, the HIF-1alpha is upregulated and the mRNA and protein levels of VEGF, ET-1, inducible and constitutive NO synthases are elevated in the liver during chronic hypoxia. In conclusion, the adaptive response to long-term hypoxemia involves compensatory mechanisms mediated by expressing significant levels of HIF-1alpha and vasoactive substances regulated by HIF-1.

Background: Macrophage migration inhibitory factor (MIF) has emerged to be a pivotal cytokine in immune-mediated diseases. Patients and methods: To investigate the role of MIF in chronic hepatitis B infection, we studied two groups of hepatitis B surface antigen positive patients: group 1 (immune tolerant, n=16) and group 2 (immune clearance, n=16). Serum level of MIF was measured by enzyme-linked immunosorbent assay and intrahepatic expression of MIF, macrophage and T-cell localisation were detected by double immunohistochemistry. Results: An increased serum MIF correlated significantly with increased serum alanine aminotransferase activity (r=0.73, P

Background: Recently, considerable attention has been focused on dietary and medicinal phytochemicals that inhibit, reverse, or retard diseases caused by oxidative and inflammatory processes. Green tea polyphenols have both antioxidant and antiinflammatory properties. Objective: We examined the effects of green tea polyphenols in carbon tetrachloride– treated mice, a model of liver injury in which oxidant stress and cytokine production are intimately linked. We tested the effect of a pure form of epigallocatechin gallate (EGCG), the major polyphenol in green tea, in mice treated with carbon tetrachloride. Design: Eight-week-old ICR mice were administered 20 L/CCl4 kg dissolved in olive oil. Two different doses of EGCG, 50 and 75 mg/kg, were tested. Control mice were treated with saline and olive oil. We analyzed liver histopathology, lipid peroxidation, and messenger RNA and protein concentrations of inducible nitric oxide synthase. Additionally, nitric oxide– generated radicals were assessed by electron paramagnetic resonance spectroscopy, and protein concentrations were measured by immunohistochemistry and Western blot analysis. Results: Carbon tetrachloride administration caused an intense degree of liver necrosis associated with increases in lipid peroxidation, inducible nitric oxide synthase messenger RNA and protein, nitrotyrosine, and nitric oxide radicals. EGCG administration led to a dose-dependent decrease in all of the histologic and biochemical variables of liver injury observed in the carbon tetrachloride–treated mice. Conclusions: Green tea polyphenols reduce the severity of liver injury in association with lower concentrations of lipid peroxidation and proinflammatory nitric oxide– generated mediators. Green tea polyphenols can be a useful supplement in the treatment of liver disease and should be considered for liver conditions in which proinflammatory and oxidant stress responses are dominant. Am J Clin Nutr 2004;80:742–51.

Multiple lines of evidence suggest a role for endogenous lipopolysaccharides in toxin-induced liver injury. Toll-like receptor 4 has recently been implicated as a cell surface receptor important for lipopolysaccharide responsiveness. In these experiments, we sought to determine the role of toll-like receptor 4 in acute liver injury by carbon tetrachloride by utilizing the naturally occurring toll-like receptor 4 mutant and wild-type mice strains. Mice were injected with either carbon tetrachloride or the carrier. Serum transaminase levels peaked at 24 hr after carbon tetrachloride administration for both wild-type and mutant mice, with no significant histological difference in initial liver injury between the two groups. However, an overall decrease in hepatocyte proliferation was found in the mutant mice. Examination of the liver tissue revealed significant decreases in intrahepatic expressions of proinflammatory mediators. In conclusion, our results suggest that toll-like receptor 4 is important in the hepatic regenerative response to CCl4 liver injury via its role in modulating the inflammatory response to hepatic injury.

This article represents the proceedings of a symposium at the 2002 ISBRA/RSA meeting in San Francisco, USA. The organizer and chair was H. Ishii and co-chair was A.A. Nanji. The presentations were (1) Introduction: Apoptosis in alcoholic liver disease, by A. A. Nanji; (2) Mitochondria, oxidative stress and apoptosis in alcoholic liver disease, by M. Adachi; (3) Regulation of cell death by mitochondrial glutathione, by J.C. Fernandez-Checa; (4) Toxicity of ethanol in HepG2 cells that express CYP2E1, by A.I. Cederbaum; (5) Is alcohol-enhanced liver apoptosis a pathogenic factor in alcoholic liver disease? by I.V. Deaciuc.

Induction of NF-κB-mediated gene expression has been implicated in the pathogenesis of alcoholic liver disease (ALD). Curcumin, a phenolic antioxidant, inhibits the activation of NF-κB. We determined whether treatment with curcumin would prevent experimental ALD and elucidated the underlying mechanism. Four groups of rats (6 rats/group) were treated by intragastric infusion for 4 wk. One group received fish oil plus ethanol (FE); a second group received fish oil plus dextrose (FD). The third and fourth groups received FE or FD supplemented with 75 mg · kg−1 · day−1of curcumin. Liver samples were analyzed for histopathology, lipid peroxidation, NF-κB binding, TNF-α, IL-12, monocyte chemotactic protein-1, macrophage inflammatory protein-2, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and nitrotyrosine. Rats fed FE developed fatty liver, necrosis, and inflammation, which was accompanied by activation of NF-κB and the induction of cytokines, chemokines, COX-2, iNOS, and nitrotyrosine formation. Treatment with curcumin prevented both the pathological and biochemical changes induced by alcohol. Because endotoxin and the Kupffer cell are implicated in the pathogenesis of ALD, we investigated whether curcumin suppressed the stimulatory effects of endotoxin in isolated Kupffer cells. Curcumin blocked endotoxin-mediated activation of NF-κB and suppressed the expression of cytokines, chemokines, COX-2, and iNOS in Kupffer cells. Thus curcumin prevents experimental ALD, in part by suppressing induction of NF-κB-dependent genes.

The article summarizes the proceedings of a symposium on recent advances in research on the pathogenesis of alcoholic liver disease at the 2001 RSA meeting in Montreal, Canada. The chairs were Amin A. Nanji and Samuel W. French. The presentations were (1) Role of inflammatory mediators in alcoholic liver injury by Amin A. Nanji, (2) Role of endotoxin, lipopolysaccharide binding protein, CD14 and Toll receptors in alcoholic liver injury by Grace Su, (3) Fatty acid ethyl esters: toxicity, metabolism and markers of ethanol intake by Michael Laposata, and (4) Cyclic changes in gene expression when rats are fed alcohol at a constant rate by Samuel W. French.

Alcoholic liver injury is more severe and rapidly developing in women than men. To evaluate the reason(s) for these gender-related differences, we determined whether pathogenic mechanisms important in alcoholic liver injury in male rats were further upregulated in female rats. Male and age-matched female rats (7/group) were fed ethanol and a diet containing fish oil for 4 wk by intragastric infusion. Dextrose isocalorically replaced ethanol in control rats. We analyzed liver histopathology, lipid peroxidation, cytochrome P-450 (CYP)2E1 activity, nonheme iron, endotoxin, nuclear factor-kappa B (NF-kappa B) activation, and mRNA levels of cyclooxygenase-1 (COX-1) and COX-2, tumor necrosis factor-alpha (TNF-alpha), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2). Alcohol-induced liver injury was more severe in female vs. male rats. Female rats had higher endotoxin, lipid peroxidation, and nonheme iron levels and increased NF-kappa B activation and upregulation of the chemokines MCP-1 and MIP-2. CYP2E1 activity and TNF-alpha and COX-2 levels were similar in male and female rats. Remarkably, female rats fed fish oil and dextrose also showed necrosis and inflammation. Our findings in ethanol-fed rats suggest that increased endotoxemia and lipid peroxidation in females stimulate NF-kappa B activation and chemokine production, enhancing liver injury. TNF-alpha and COX-2 upregulation are probably important in causing liver injury but do not explain gender-related differences.

Chronic ethanol ingestion in rats and humans results in significant alterations in sex steroid levels and expression of sex hormone-dependent phenotype. In this study, we used the intragastric feeding model in male rats to determine hepatic sex hormone receptor activity under circumstances of chronic ethanol exposure and differing degrees of liver injury induced by type of dietary fat. Pathological analysis and quantitation of hepatic androgen receptor (AR) and estrogen receptor (ER) activity, serum sex hormones, and sex hormone-responsive protein and mRNA expression were performed. The activity of the physiologically relevant nuclear form of both AR and ER was significantly decreased with ethanol and correlated inversely with the severity of liver injury. Serum testosterone levels, as well as expression of an androgen-dependent hepatic mRNA, were decreased by ethanol and progressive liver injury. Serum estradiol increased with liver injury. We postulate that these changes in receptor activity may be due to the oxidative stress, reduced cellular energy, and/or altered cytokine milieu known to occur in this model.

Background: Fatty acid ethyl esters (FAEE) are nonoxidative ethanol metabolites that have been shown to be long term markers of ethanol intake and have been implicated as mediators of ethanol-induced cell injury. Previous studies have indicated that the fatty acid composition of the FAEE found in the plasma of human subjects after ethanol ingestion is predominantly ethyl palmitate and ethyl oleate. This raised the possibility that there is some selectivity toward the fatty acid used for FAEE to be exported from the liver into the blood. Methods: To address the hypothesis that the fatty acid composition of FAEE secreted from organs, such as the liver and pancreas, differs from the fatty acid composition of FAEE in the organs, this study was performed using rats that received ethanol by intra-arterial infusion. Results: It was found that the fatty acids in FAEE differed significantly in plasma versus liver, bile versus liver, and pancreatic secretions versus pancreas. Conclusions: These results indicate that organs selectively export certain FAEE species.

Inflammatory stimuli and lipid peroxidation activate nuclear factor kappa B (NF-kappaB) and upregulate proinflammatory cytokines and chemokines. The present study evaluated the relationship between pathological liver injury, endotoxemia, lipid peroxidation, and NF-kappaB activation and imbalance between pro- and anti-inflammatory cytokines. Rats (5 per group) were fed ethanol and a diet containing saturated fat, palm oil, corn oil, or fish oil by intragastric infusion. Dextrose isocalorically replaced ethanol in control rats. Pathological analysis was performed and measurements of endotoxin were taken, lipid peroxidation, NF-kappaB, and messenger RNA (mRNA) levels of proinflammatory cytokines (tumor necrosis factor-alpha [TNFalpha], interleukin-1 beta [IL-1beta], interferon-gamma, [IFN-gamma], and IL-12), C-C chemokines (regulated upon activation, normal T cell expressed and secreted [RANTES], monocyte chemotactic protein [MCP]-1, macrophage inflammatory protein [MIP]-1alpha), C-X-C chemokines (cytokine induced neutrophil chemoattractant (CINC), MIP-2, IP-10, and epithelial neutrophil activating protein [ENA]-78), and anti-inflammatory cytokines (IL-10, IL-4, and IL-13). Activation of NF-kappaB and increased expression of proinflammatory cytokines C-C and C-X-C chemokines was seen in the rats exhibiting necroinflammatory injury (fish oil-ethanol [FE] and corn oil-ethanol[CE]). These groups also had the highest levels of endotoxin and lipid peroxidation. Levels of IL-10 and IL-4 mRNA were lower in the group exhibiting inflammatory liver injury. Thus, activation of NF-kappaB occurs in the presence of proinflammatory stimuli and results in increased expression of proinflammatory cytokines and chemokines. The Kupffer cell is probably the major cell type showing activation of NF-kappaB although the contribution of endothelial cells and hepatocytes cannot be excluded. Downregulation of anti-inflammatory cytokines may additionally exacerbate liver injury.

In this study, we examined the role of fibrogenic cytokines in alcohol-induced fibrosis. In particular, we examined the production of a novel fibrogenic cytokine, fibrosin, among others, by fibroblasts in response to ethanol in vitro; we also studied the production of fibrosin in an animal model of alcohol-induced liver injury. This model system utilizes the intragastric feeding rat model in which rats are fed different dietary fats and ethanol or dextrose. Our study showed that physiologic concentrations of ethanol directly induced proliferation of fibroblasts in vitro and also stimulated the production of cytokines. In particular, fibrosin, the novel fibrogenic cytokine, was produced. Other cytokines such as TGFbeta, IL-6, and TNFalpha were also induced. Also, exposure of fibroblasts to interleukin-1beta, interleukin-6, and tumor necrosis factor alpha induced production of fibrosin. In the fish oil-ethanol-fed rats which showed fibrotic lesions in the liver, fibrosin mRNA as well as protein was expressed. Fibrosin was not detected in control rats not exhibiting fibrosis. These studies show that ethanol can directly stimulate fibroblast proliferation and production of fibrogenic cytokines. It is likely that fibrosin, which may be derived from inflammatory cells, contributes to alcohol-induced hepatic fibrosis in vivo.

Essentially all patients with significant alcoholic liver disease (ALD) have clinical manifestations of malnutrition. 101 Work by Lieber and others, however, has demonstrated that ALD can occur despite adequate nutrition. 85,86 This suggests that the liver injury of ALD is attributable to direct toxic effects of alcohol and its metabolites or the consequences of alcohol metabolism. Major research efforts into these areas have uncovered several potential mechanisms that may play a role in the liver injury of ALD. In all likelihood, several mechanisms operate simultaneously to initiate, potentiate, or perpetuate liver injury once it is initiated in clinical ALD. In addition, the initiating events or factors in alcoholic liver injury and the reasons why some individuals are predisposed to ALD are not known. Although interesting data recently were reported on the mechanisms of liver fibrosis in ALD, alcoholic hepatitis (AH) and acute liver injury are the foci of our discussion because most investigators agree that AH is an intermediate but potentially reversible stage of ALD. It is hoped that further knowledge concerning the pathogenesis of AH will allow the development of more effective treatments for this potentially lethal disease. The concepts of liver injury caused by alcohol-related oxidative stress (lipid peroxidation), immune response (cytokines), fatty acid metabolites, acetaldehyde production (protein–acetaldehyde adduct formation), mitochondrial dysfunction, and synergism of viral liver injuries, have been proposed. 81,145 These are the areas that have received major investigative attention over the past decade and are reviewed in this article.

Increased hepatic oxidative stress with ethanol administration is hypothesized to be caused either by enhanced pro-oxidant production or decreased levels of antioxidants or both. We used the intragastric feeding rat model to assess the relationship between hepatic antioxidant enzymes and pathological liver injury in animals fed different dietary fats. Male Wistar rats (5 per group) were fed ethanol with either medium-chain triglycerides (MCTE), palm oil (PE), corn oil (CE), or fish oil (FE). Control animals were fed isocaloric amounts of dextrose instead of ethanol with the same diets. The following were evaluated in each group: liver pathology, lipid peroxidation, manganese superoxide dismutase (MnSOD) levels, copper-zinc SOD (CuZnSOD) levels, glutathione peroxidase (GPX) levels, and catalase (CAT) levels. All enzymes were evaluated using activity assays and immunoblots. Rats fed FE showed the most severe pathology (fatty liver, necrosis, and inflammation), those fed CE showed moderate changes, those fed PE showed fatty liver only, and those fed MCTE were normal. Parameters indicative of lipid peroxidation (conjugated dienes and thiobarbituric acid–reactive substances) were also greater in rat livers from animals fed the diets high in polyunsaturated fatty acids (CE and FE). CuZnSOD, GPX, and CAT activities showed an inverse correlation (r = −.92, P < .01) with severity of pathological injury, with the lowest levels for both enzymes found in FE-fed rats. Decreased enzyme activity in CE- and FE-fed rats was accompanied by similar decreases in immunoreactive protein. Ethanol administration did not cause significant decreases in enzyme activity in groups that showed no necroinflammatory changes (MCTE and PE). MnSOD activity showed no significant change in any ethanol-fed group. Our results show that decreases in CuZnSOD, GPX, and CAT occur in rats showing pathological liver injury and also having the highest levels of lipid peroxidation. These results suggest that feeding dietary substrates that enhance lipid peroxidation can exacerbate both ethanol-induced oxidative damage as well as necroinflammatory changes. The decrease in activity of antioxidant enzymes observed in animals fed diets high in polyunsaturated fatty acids and ethanol could possibly increase the susceptibility to oxidative damage and further contribute to ethanol-induced liver injury.

We investigated the potential of dietary saturated fatty acids to decrease endotoxemia and suppress expression of cyclooxygenase 2 (Cox-2) and tumor necrosis factor alpha (TNF-alpha) in established alcohol-induced liver injury. Six groups (five rats/group) of male Wistar rats were studied. Rats in group 1 were fed a fish oil-ethanol diet for 6 weeks. Rats in groups 2, 3, and 4 were fed fish oil and ethanol for 6 weeks. Ethanol administration was stopped at this time, and the rats were switched to isocaloric diets containing dextrose with fish oil (group 2), palm oil (group 3), or medium-chain triglycerides (group 4) as the source of fat for an additional 2 weeks. Rats in groups 5 and 6 were fed fish oil-ethanol and fish oil-dextrose, respectively, for 8 weeks. Liver samples were analyzed for histopathology, lipid peroxidation, and levels of messenger RNA (mRNA) for Cox-2 and TNF-alpha. Concentrations of endotoxin were determined in plasma. The most severe inflammation and fibrosis were detected in groups 1 and 5, as were the highest levels of endotoxin, lipid peroxidation, and mRNA for Cox-2 and TNF-alpha. After ethanol was discontinued, there was minimal histological improvement in group 2 but near normalization of the histology, including regression of fibrosis, in groups 3 and 4. Histological improvement was associated with decreased levels of endotoxin, lipid peroxidation, and reduced expression of Cox-2 and TNF-alpha. The data indicate that a diet enriched in saturated fatty acids (groups 3 and 4) effectively reverses alcohol-induced liver injury, including fibrosis. The therapeutic effects of saturated fatty acids may be explained, at least in part, by reduced endotoxemia and lipid peroxidation, which in turn result in decreased levels of TNF-alpha and Cox-2.

BACKGROUND & AIMS: Thromboxane levels correlate with severity of liver injury in rats given alcohol. The aim of this study was to evaluate the effect of thromboxane inhibitors on pathological changes in experimental alcoholic liver disease. METHODS: Male Wistar rats were given a liquid diet and ethanol intragastrically for 1 month. The thromboxane inhibitors tested were a thromboxane receptor antagonist (TXRA) and a thromboxane synthase inhibitor (TXSI). Pathological changes, liver and plasma thromboxane levels, 6-ketoprostaglandin F1 alpha levels, lipid peroxidation, and messenger RNA levels for tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF) beta were evaluated. RESULTS: Treatment with thromboxane inhibitors prevented necrosis and inflammation. In the TXSI-treated group, fatty liver was also decreased. Ethanol administration led to a 3-4-fold increase in liver thromboxane levels; a reduction in thromboxane levels and lipid peroxidation was seen in the TXSI group. In all treatment groups, TNF-alpha and TGF-beta messenger RNA levels were decreased. CONCLUSIONS: The prevention of necroinflammatory changes in thromboxane- treated groups is related to a decrease in TNF-alpha levels. Inhibition of TGF-beta expression may also prevent fibrosis in ethanol-treated rats. (Gastroenterology 1997 Jan;112(1):200-7)

We used the intragastric feeding rat model for alcoholic liver disease to evaluate the relationship between morphologic and functional indicators of endothelial cell dysfunction.Twelve groups of rats (4-5 rats/group) were fed the following diets: saturated fat and dextrose (SD), saturated fat and ethanol (SE), corn oil and dextrose (CD), corn oil and ethanol (CE). Four of the 12 groups were sacrificed at 2 weeks, four groups at 4 weeks and remaining four groups at 8 weeks. The following were evaluated at sacrifice: pathologic changes in the liver, endothelial cell proliferation using a monoclonal antibody to proliferating cell nuclear antigen, factor VIII-related antigen staining of endothelial cells in liver, plasma endotoxin, hyaluronan and prostaglandin F2 alpha.Only CE rats at 4 and 8 weeks showed pathologic changes. The plasma levels of HA were significantly higher in the CE groups compared to the other groups at all time intervals studied. In the CE rats, a significant correlation was obtained between plasma endotoxin and hyaluronan (r = 0.84, p0.01). Endotoxin levels also correlated significantly with the number of G1/S arrested hepatic sinusoidal endothelial cell (r = 0.61, p0.05). A role for prostaglandin F2 alpha, in causing endothelial dysfunction, was suggested by a significant correlation between plasma hyaluronan and prostaglandin F2 alpha levels (r = 0.95, p0.01). Positive factor VIII related antigen staining of hepatic endothelial cells was seen in rats with high plasma hyaluronan levels.We propose that endotoxin, mediating part of its effect through prostaglandin F2 alpha, plays a role in hepatic sinusoidal endothelial cell G1/S arrest. This morphologic change, associated with increased plasma hyaluronan levels, precedes capillarization in this model of alcoholic liver injury.

The ability of a cell to withstand oxidative stress has been hypothesized to be related to its ploidy status. We used the intragastric feeding rat model for alcoholic liver disease to evaluate the relationship between severity of liver injury, antioxidant mRNA levels, and DNA ploidy of liver cells. Rats were fed ethanol with different dietary fats (saturated fat, corn oil, and fish oil); pair-fed control animals received isocaloric amounts of dextrose. All animals were euthanized at 1 month and had evaluation of pathologic changes in the liver, DNA content by flow cytometry, and mRNA levels for catalase and glutathione peroxidase. The fish oil-ethanol group exhibited the most severe pathology, the corn oil-ethanol group had intermediate pathologic changes, and no pathologic changes were seen in the saturated fat-ethanol and dextrose-fed controls. Flow cytometric analysis of propidium iodide-stained nuclei revealed that saturated fat-dextrose and corn oil-dextrose groups had about 65% of cells with (diploid) G1 DNA content and about 30% of cells with tetraploid (4C) nuclei. The fish oil-dextrose had a significantly higher (p0.001) number of 4C cells (67.4 +/- 2.1%) compared to the other two dextrose-fed groups. In the animals showing pathologic liver injury, there was a higher percentage of cells with hypertetraploid nuclei. The highest percentage of these hypertetraploid cells was seen in the fish oil-ethanol group. Catalase and glutathione peroxidase mRNA levels correlated significantly with polyploidy. A significant correlation was seen between the number of cells in the greater than G2 + M phase and glutathione peroxidase mRNA levels (r = 0.91, p0.01) and catalase mRNA. The different slopes of correlation analysis between catalase mRNA and dietary fats show that the degree of saturation of fatty acids may influence catalase mRNA expression in cells with different ploidy states. We propose that polyploidization of liver cell nuclei may serve as a defense mechanism against ethanol-induced hepatotoxicity. This defense mechanism may also, in part, account for the antiregenerative effect of ethanol on hepatocytes.

To evaluate the relationship between heat-shock gene expression and the severity of pathologic liver injury and lipid peroxidation in experimental alcoholic liver disease, we used the intragastric-feeding rat model. Six groups of male Wistar rats weighing between 225 and 250 g were fed liquid diets containing different dietary fats (saturated fat, corn oil, and fish oil) and ethanol or dextrose for 1 month. Pathologic injury, mRNA and protein levels of HSP70, microsomal conjugated dienes, and hydrogen peroxide were evaluated at sacrifice. Fish oil-ethanol fed rats developed the most severe injury, while the saturated fat-ethanol group showed no liver injury. The highest levels of HSP70 mRNA were seen in the fish oil-ethanol group. There was no difference in HSP70 protein levels between the groups. The levels of HSP70 mRNA correlated significantly with microsomal conjugated dienes (r = 0.87, P < 0.01) and hydrogen peroxide (r = 0.90, P < 0.01). Increased centrilobular HSP70 expression was seen in rats showing liver injury. The close relationship seen between oxidant stress and HSP70 mRNA but not protein suggest that the binding of HSP70 protein to other damaged proteins reduces free HSP70 protein leading to increased HSP70 expression. HSP70 expression may also be a cellular adaptive response to ethanol-induced oxidative stress.

Background & Aims: Regulation of blood flow and oxygen supply are important pathogenetic factors in alcoholic liver disease. Because nitric oxide may have an important role, its effects on alcoholic liver injury were investigated. Methods: Rats were fed ethanol intragastrically with either saturated fat or corn oil. Spontaneous production of NO by liver nonparenchymal cells was compared in the two dietary groups. Two additional groups of rats fed corn oil and ethanol were treated with either an NO inhibitor (l-NAME) or supplemented with l-arginine. Liver pathology and plasma NO production were evaluated. Results: In the corn oil and ethanol group, a progressive decrease in liver nonparenchymal cell NO production and increased plasma NO levels were associated with liver injury. Reduced nicotinamide adenine dinucleotide phosphate diaphorase staining showed increased centrilobular staining of hepatocytes in the corn oil and ethanol group and l-NAME-treated group. Moreover, l-NAME increased the severity, whereas l-arginine supplementation completely prevented liver injury. In the saturated fat and ethanol group, in which there was no liver injury, the levels of NO 2 − in nonparenchymal supernatant were 5–10-fold higher than in the corn oil and ethanol group. Conclusions: Decreased NO production by nonparenchymal cells may contribute to liver injury in ethanol-fed rats, and the compensatory increase in hepatocyte NO production may contribute to centrilobular liver injury.

We carried out a study to relate the effect of the type of dietary fat and ethanol on antioxidant enzyme mRNA levels in liver in the intragastric feeding rat model. Different types of dietary fat were administered [saturated fat (SE), corn oil (CE) and fish oil (FE)] with ethanol to induce varying severities of liver injury. Ethanol-fed rats were pair-fed with dextrose-fed controls that received isocaloric amounts of dextrose. All animals were killed at 1 month and the following studies were carried out: evaluation of severity of pathologic liver injury, mRNA quantitation for catalase, glutathione peroxidase (GPx), and manganese superoxide dismutase (MnSOD), microsomal conjugated dienes, and hydrogen peroxide. SE animals had no liver injury, FE animals had severe liver injury, and CE animals had moderate liver injury. Ethanol induced GPx mRNA in all dietary groups, with the highest levels seen in the FE group. The pattern of catalase mRNA induction was similar to that of GPx mRNA. In contrast, MnSOD mRNA was decreased compared to controls in animals that developed pathologic liver injury, i.e., CE and FE groups. A positive correlation was seen between conjugated diene levels and GPx mRNA (r = 0.88, P < 0.01) and catalase mRNA. The similar slopes for the relationship between conjugated dienes and catalase in the fish oil and non-fish oil groups indicate that the same degree of lipid peroxidation increases catalase mRNA to a greater degree in fish oil-fed rats. A positive correlation was also seen between catalase mRNA and H2O2 (r = 0.95, P < 0.001).

Capillarization of the hepatic sinusoid occurs in alcoholic liver disease. Because endothelial cell proliferation is relevant to capillarization, we used the intragastric feeding rat model to evaluate the relationship between pathological liver injury and endothelial cell proliferation.Male Wistar rats (225-250 g) were fed liquid diets containing corn oil and ethanol for periods ranging between 1 week and 2 months. At the time the rats were killed, the severity of pathological injury and endothelial cell proliferation using anti-rat proliferating cell nuclear antigen/antibody was evaluated.Two distinctly different populations of proliferating sinusoidal lining cells were identified; one population showed relatively weak granular staining consistent with cells arrested at the G1/S boundary. The other population of cells showed strong granular staining of the nucleoplasm and nucleoli (cells in mid to late S phase). A strong correlation (r = 0.85; P0.01) was obtained between pathological severity and G1/S-arrested endothelial cells. There was no correlation between cells in S phase.The presence of an increased number of G1/S-arrested endothelial cells in animals with severe pathological change suggests that stimuli are present for both endothelial cell proliferation and G1/arrest. The identification of these stimuli could lead to a better understanding of the pathogenesis of alcoholic liver disease.

The rat intragastric feeding model for alcoholic liver disease was used to study the effect of different diets on the fatty acid composition of liver microsomes. Rats were fed corn oil and ethanol (CE), saturated fat and ethanol (SF+E) or corn oil and dextrose (CD) for either 2 or 4 weeks. Rats were also fed saturated and dextrose (SF+D) for 4 weeks. In comparison with the CD diet, lower levels of arachidonic acid were detected in rats fed the CE, SF+E, and SF+D diets. However, the diet-induced changes in levels of arachidonic acid varied as a function of length of feeding. In rats fed the CE diet, we detected a significant decrease in the level of arachidonic acid compared with CD animals. Conversely, in rats fed the SF+E diet, the level of arachidonic acid increased compared with the SF+D group. In addition, a significant correlation was noted between levels of oleic acid and arachidonic acid in both corn oil (r = -0.85, p < 0.01) and saturated fat (r = -0.76, p < 0.05) groups. However, the changes in levels of arachidonic acid and oleic acid were in opposite directions in the two groups. Levels of docosahexaenoic acid decreased between the 2 and 4 weeks in animals maintained on the CE diet. Levels of stearic acid increased between 2 and 4 weeks in rats fed the SF+E diet. The lowest level of linoleic acid was detected in the SF+D and SF+E groups, but levels of linoleic acid remained constant in all groups throughout the study.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of chronic ethanol intake on the levels of α-tocopherol and γ-tocopherol in serum and liver of both vitamin E-deficient and normal rats were studied. An intragastric feeding rat model was used. Both normal and vitamin E-deficient animals were fed a liquid diet and ethanol for 1 month. In pair-fed animals, dextrose was isocalorically replaced by ethanol. The blood ethanol level in the ethanol-fed animals was between 150 and 250 mg/dL. Liver peroxidation was determined by measuring thiobarbituric acid reactive substances (TBARS). Plasma alanine aminotransferase (ALT) was increased by 3-fold in vitamin E-deficient ethanol-fed rats compared with normal ethanol-fed rats. Plasma α- and γ-tocopherol were decreased in the normal ethanol-fed rats by 22.3 and 65%. respectively (P < 0.01). Liver α- and γ-tocopherol were also decreased by 51.7 and 76%, respectively (P < 0.01). Vitamin E-deficient animals had significantly lower mean plasma α-tocopherol (5670 vs 530 ng/mL, P < 0.01), and ethanol feeding did not decrease the levels any further. However, ethanol feeding decreased liver α-and γ-tocopherol by 58.5 and 56.5% (P < 0.01), respectively, beyond the already low levels observed in this group. There was an inverse correlation between liver TBARS and liver α-tocopherol (r = − 0.59, P < 0.05) and γ-tocopherol (r = −0.65, P < 0.02). Also of significance is that ethanol feeding decreased the plasma and liver γ-tocopherol more than the α--tocopherol in both normal and vitamin E-deficient animals. In conclusion, ethanol feeding markedly decreased both α- and γ-tocopherol in livers of normal and vitamin E-deficient rats, but it only decreased plasma levels of tocopherols in normal rats. The higher ALT in vitamin E-deficient animals and the inverse correlation between TBARS and α- and γ-tocopherol suggest that enhanced lipid peroxidation is associated with greater severity of liver injury induced by ethanol in vitamin E-deficient rats.

We evaluated the expression of interleukin-1 alpha, interleukin-1 beta, tumor necrosis factor-alpha and transforming growth factor-beta mRNAs in the intragastric-feeding rat model of alcoholic liver disease. Rats were fed different diets for periods of 2 or 4 wk. Animals fed saturated fat and ethanol and the corn oil-dextrose control group had no liver injury, whereas animals fed corn oil and ethanol showed pathologic changes. RNA was extracted from the livers at the time of killing, reverse-transcribed and amplified; polymerase chain reaction products were subjected to electrophoresis on agarose gel. Interleukin-1 alpha mRNA was present in all groups at 2 and 4 wk; interleukin-1 beta and transforming growth factor-beta mRNAs were present in all groups at 4 wk. Tumor necrosis factor-alpha mRNA was absent in all groups at 2 wk but was present in the corn oil-ethanol group only at 4 wk. Because pathological liver injury was evident in the corn oil-ethanol group by 4 wk, the presence of tumor necrosis factor-alpha mRNA at this time suggests a pathogenetic role for tumor necrosis factor-alpha in alcohol-induced liver injury.

We investigated the association between vitamin E, lipid peroxidation and eicosanoid production in experimental alcoholic liver injury. We used the intragastric feeding rat model in which animals were fed corn oil and ethanol (CO+E) and corn oil and dextrose (CO+D) for 2 and 4 week periods. At sacrifice, we measured plasma levels of alpha-tocopherol, 8-isoprostane, thromboxane B2 (TXB2) and 6-ketoprostaglandin F1 alpha (6-KetoPGF1 alpha). Animals fed CO+E had significantly lower concentrations of alpha-tocopherol and higher concentrations of 8 isoprostane at both 2 and 4 weeks. a significant inverse correlation was seen between alpha-tocopherol concentrations and the TXB2: PGF1 alpha ratio (r = 0.72, p0.01). A positive correlation was seen between the TXB2: PGF1 alpha ratio and 8 isoprostane levels (r = 0.84, p0.001). These results suggest that vitamin E depletion and enhanced lipid peroxidation may affect eicosanoid metabolism in experimental alcoholic liver disease in such a way so as to increase the thromboxane to prostacyclin ration.

We used the intragastric feeding rat model for alcohol liver disease to investigate the relationship between endothelin and pathologic liver injury. Rats were fed the following diets for periods of 1, 2 and 4 weeks: corn oil plus ethanol (CE), corn oil plus dextrose (CD) and saturated fat plus ethanol (SE). Plasma endothelin levels were significantly higher in the CE group compared to the other groups at all time periods studied. The CE animals, also, developed pathologic liver injury which is in contrast to the SE and CD animals which showed no pathologic changes. We propose that endothelin, because of its powerful vasoconstrictive effects, leads to a reduction in hepatic blood flow which is important in the pathogenesis of alcoholic liver disease.

Kupffer cells play a major role in alcoholic liver disease. Oxidative stress and endotoxin are major mediators of the inflammatory process in alcoholic hepatitis. Recent evidence supports the suggestion that endotoxin-induced signal transduction begins with CD14-mediated activation of Toll-receptor 4 and subsequent activation of nuclear factor-kappa B (NF-kappa B) binding activity. Free radicals from reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in Kupffer cells also activate NF-kappa B binding activity. Inflammatory cytokines and cyclooxygenase-2 are up-regulated in response to binding of NF-kappa B. A combined role for tumor necrosis factor-alpha (TNF-alpha) and cyclooxygenase-2 is important in the pathogenesis of alcoholic hepatitis.

We investigated the protective effects of two non-steroid anti-inflammatory drugs, indomethacin (COX-1 and COX-2 inhibitors) and nimesulide (specific COX-2 inhibitor) on the hepatic injury induced by lipopolysaccharide in d-galactosamine sensitized (Gal/LPS) mice. ICR male mice were injected with a single dose of Gal/LPS with or without pre-treatment of 3mg/kg indomethacin or 30 mg/kg nimesulide (single i.p. injection). Sixteen hours later, blood and liver tissues of mice were collected for histological, molecular, and biochemical analyses. Our results showed marked reduction of hepatic necrosis, serum ALT, and tissue TBARS levels in both indomethacin- and nimesulide-pre-treated mice when compared with Gal/LPS-treated mice. Western blot and RT-PCR analysis showed decreased levels of iNOS mRNA, iNOS protein, and nitrotyrosine formation in both COX inhibitor pre-treated groups when compared with Gal/LPS-treated group. There was an inverse relationship between COX-1 and COX-2 expressions, as well as between COX-2 and C/EBP-α expressions in COX inhibitors groups, Gal/LPS and control groups. COX inhibitors reduced the expression of TNF-α mRNA and the activity of NF-κB which were elevated by Gal/LPS treatment. We conclude that COX inhibitors protected the liver from Gal/LPS-induced hepatotoxicity. COX inhibitors could be considered as potential agents in the prevention of acute liver failure and sepsis.

The intragastric tube feeding model is ideal for the study of the role of dietary factors and the effect of drugs on experimental alcoholic liver disease (ALD), since the model allows us to study the effect of a single variable in the diet on the pathology of liver where the blood alcohol level (BAL) is maintained over 150 mg%. By varying the dietary fatty acid composition we showed that the pathology was worsened by increasing linoleic acid or polyunsaturated fatty acids (PUFAs) in the diet where cytochrome P4502E1 (CYP2E1) was increased posttranslationally by high BAL. Concomitant with the increase in CYP2E1 there was evidence for an increase in lipid peroxidation (LP) by microsomes. Protein adducts of the products of LP were increased in the blood. Isoniazid (INH) enhanced this process and the pathology of ALD when INH was fed at therapeutic levels with ethanol. Preliminary studies show that diallyl sulfide, which inhibits and destroys liver CYP2E1 selectively, also modified the pathologic effects of ethanol. Thus we postulate that CYP2E1 induction plays a central role in the pathogenesis of ALD.

Based on our previous observations that linoleic acid is an important dietary requirement for the development of liver injury in the intragastric feeding rat model for alcoholic liver disease, we postulated that the conversion of linoleic acid to arachidonic acid and various eicosanoids could be important in the pathogenesis of liver injury. We showed that liver nonparenchymal cell production of thromboxane B2 and leukotriene B4 was higher in rats fed corn oil and ethanol (liver injury model) than in animals fed saturated fat and ethanol (no liver injury). In contrast, prostaglandin E2 levels were lower in the former group. The best correlate of pathologic changes was the plasma level of thromboxane B2. The importance of thromboxanes in alcoholic liver injury is further suggested by the fact that inhibition of thromboxane production is accompanied by amelioration of liver injury.

We evaluated the role of changes in microsomal phospholipases (A and C) and arachidonic acid in the intragastric rat feeding model. The experimental animals (male Wistar rats), divided into 4-5 rats/group, were fed the following diets: corn oil and ethanol and corn oil plus dextrose. One set of groups was killed after 2 weeks of feeding, and the second set was killed after 1 month. For each animal, microsomal analysis of cytochrome P-450 2E1 (CYP 2E1) and fatty acids was done. Fourteen animals had analyses of phospholipase C (PLC) and phospholipase A (PLA), and 10 animals had measurements of conjugated dienes. A significant correlation was obtained between the level of CYP 2E1 and the decrease in arachidonic acid (AA) from baseline levels (r = 0.69, p < 0.01). The decrease in AA also correlated with the increase in conjugated dienes (r = 0.70, p < 0.05). PLA and PLC activities were both significantly increased in the corn oil and ethanol groups. The activity of PLC correlated with the decline in AA (r = 0.69, p < 0.01). The correlations noted between the decrease in microsomal AA and CYP 2E1 induction and conjugated diene formation suggest that these processes may be interlinked especially in regard to generation of lipid peroxides that may play a role in alcoholic liver injury.

We studied the effects of long-term ethanol ingestion and dietary fat on Ito cell activation morphometrically in rats. Sixteen pairs of Wistar male rats were divided into two groups, one fed tallow and the other fed corn oil as the source of dietary fat. Each group of rats were pair-fed a nutritional adequate liquid diet containing either corn oil (CF) or tallow (TF) as fat as well as protein and carbohydrate. Half of each group received ethanol, the rest were pair-fed isocaloric amounts of dextrore via an implanted gastric tube for up to 5 months. Morphometric analysis of the change in fat and rough endoplasmic reticulum (RER) of Ito cells was performed on electron micrographs obtained from monthly biopsies including baseline. Ito cell activation was assessed by a decrease in the ratio of fat/RER in Ito cells. The ratio of fat/RER in Ito cells of alcoholic rats fed the CF diet (CFA) gradually decreased. The ratio war found to be lower than in the pair-fed control rats (CFC) at 5 months of feeding. CFA 1.74 ± 0.57, vs. 7.46 ± 2.05, respectlvely, p < 0.05, mean ± se). Ito cell fat also significantly decreased at 5 months of feeding (p < 0.05). The fat/ RER ratio In CFA significantly decreased only subsequent to the development of fatty change, necrosis, and inflammation followed by fibrosis of the liver. In contrast, the TFA rats did not show a significant decrease in the fat/RER ratio in the Ito cells throughout the study, while TFC rats showed an increase in the fat/RER ratio. Minimal pathological changes were observed In the livers of CFC, TFA, and TFC rats. These results indicate that activation of Ito cells at a signifiant level occurred only late in the course of feeding alcohol after moderate to severe abnormalities in liver hlstology had developed, although activation may have begun at an earlier time of ethanol feeding. The results Indicate that dietary fatty acid composition may be an important factor in the pathogenesis of ethanol-induced Ito cII activation.

The variety of animal models used in the study of alcoholic liver disease reflects the formidable task of developing a model that replicates the human disease. We show that oral feeding of fatty acids derived from fish oil and ethanol induces fatty liver, necrosis, inflammation, and fibrosis. Together with the study of oxidative and nitrosative stress markers, cytokines, proteasome function, and protein studies, this model has provided an inexpensive and technically simple method of establishing pathological alcoholic liver injury.