Differential contributions of C3, C5, and decay-accelerating factor to ethanol-induced fatty liver in mice

BACKGROUND AND AIMS: The complement pathway is an important component of the innate and adaptive immune response. Here we tested the hypothesis that activation of complement is required for development of ethanol-induced fatty liver. METHODS: Wild-type mice and mice lacking the third (C3) or fifth (C5) components of the complement activation pathway, as well as mice lacking decay-accelerating factor (CD55/DAF), a complement regulatory protein, were fed Lieber-DeCarli ethanol-containing diets for 6 weeks or pair-fed control diets. RESULTS: Ethanol feeding to wild-type mice increased C3a in plasma. Wild-type and C5-/- mice fed the ethanol diet developed hepatic steatosis characterized by microvesicular and macrovesicular lipid accumulation and increased triglyceride content. C3-/- mice did not develop steatosis, while CD55/DAF-/- mice accumulated even more hepatic triglyceride after ethanol feeding than wild-type mice. Levels of serum alanine aminotransferase and hepatic tumor necrosis factor alpha, indicators of hepatocyte injury and inflammation, respectively, were increased in wild-type and CD55/DAF-/- mice but not in C5-/- mice after ethanol feeding. In contrast to the protective effect of C3-/- against ethanol-induced steatosis, levels of both alanine aminotransferase and tumor necrosis factor alpha were increased in C3-/- mice after ethanol feeding. CONCLUSIONS: Here we have identified several elements of the complement system as important contributors to ethanol-induced fatty liver. C3 contributed primarily to the accumulation of triglyceride in the liver, whereas C5 was involved in inflammation and injury to hepatocytes. Further, the absence of CD55/DAF exacerbated these responses, suggesting that CD55/DAF serves as a barrier to ethanol-induced fatty liver.     

L-Buthionine (S,R) sulfoximine depletes hepatic glutathione but protects against ethanol-induced 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.     

内质网分子伴侣糖调节蛋白94在大鼠酒精性肝损伤中的高表达和意义

目的检测酒精性肝损伤大鼠肝组织内质网分子伴侣糖调节蛋白94的表达情况,探讨酒精性肝损伤的发病机制.方法24只SD雌性大鼠分成对照组和模型组.模型组大鼠给予乙醇加鱼油灌胃配合高脂饮食8周,建立酒精性肝损伤模型,应用半定量逆转录聚合酶链反应(RT-PCR)法和免疫组化法检测大鼠肝组织内质网分子伴侣糖调节蛋白94 mRNA和蛋白水平.结果与正常组比较,模型组大鼠肝组织糖调节蛋白94 mRNA和蛋白表达明显增强(P＜0.01).结论酒精性肝损伤大鼠存在糖调节蛋白94高表达,这可能是该病的发病机制之一.     

β-catenin is essential for ethanol metabolism and protection against alcohol-mediated liver steatosis in mice

The liver plays a central role in ethanol metabolism, and oxidative stress is implicated in alcohol-mediated liver injury. β-Catenin regulates hepatic metabolic zonation and adaptive response to oxidative stress. We hypothesized that β-catenin regulates the hepatic response to ethanol ingestion. Female liver-specific β-catenin knockout (KO) mice and wild-type (WT) littermates were fed the Lieber-Decarli liquid diet (5% ethanol) in a pairwise fashion. Liver histology, biochemistry, and gene-expression studies were performed. Plasma alcohol and ammonia levels were measured using standard assays. Ethanol-fed (EtOH) KO mice exhibited systemic toxicity and early mortality. KO mice exhibited severe macrovesicular steatosis and 5 to 6-fold higher serum alanine aminotransferase and aspartate aminotransferase levels. KO mice had a modest increase in hepatic oxidative stress, lower expression of mitochondrial superoxide dismutase (SOD2), and lower citrate synthase activity, the first step in the tricarboxylic acid cycle. N-Acetylcysteine did not prevent ethanol-induced mortality in KO mice. In WT livers, β-catenin was found to coprecipitate with forkhead box O3, the upstream regulator of SOD2. Hepatic alcohol dehydrogenase and aldehyde dehydrogenase activities and expression were lower in KO mice. Hepatic cytochrome P450 2E1 protein levels were up-regulated in EtOH WT mice, but were nearly undetectable in KO mice. These changes in ethanol-metabolizing enzymes were associated with 30-fold higher blood alcohol levels in KO mice. CONCLUSION: β-Catenin is essential for hepatic ethanol metabolism and plays a protective role in alcohol-mediated liver steatosis. Our results strongly suggest that integration of these functions by β-catenin is critical for adaptation to ethanol ingestion in vivo.     

Immunohistochemical characterization of hepatic malondialdehyde and 4-hydroxynonenal modified proteins during early stages of ethanol-induced liver injury

BACKGROUND: Chronic ethanol consumption is associated with hepatic lipid peroxidation and the deposition or retention of aldehyde-adducted proteins postulated to be involved in alcohol-induced liver injury. The purpose of this study was to characterize hepatocellular formation of aldehyde-protein adducts during early stages of alcohol-induced liver injury. METHODS: Female Sprague Dawley(R) rats were subjected to the intragastric administration of a low-carbohydrate/high-fat total enteral nutrition diet or a total enteral nutrition diet containing ethanol for a period of 36 days. Indexes of hepatic responses to ethanol were evaluated in terms of changes in plasma alanine aminotransferase activity, hepatic histopathologic analysis, and induction of cytochrome P-4502E1 (CYP2E1). Immunohistochemical methods were used to detect hepatic proteins modified with malondialdehyde (MDA) or 4-hydroxynonenal (4-HNE) for subsequent quantitative image analysis. RESULTS: After 36 days of treatment, rats receiving the alcohol-containing diet displayed hepatic histopathologies characterized by marked micro- and macrosteatosis associated with only minor inflammation and necrosis. Alcohol administration resulted in a 3-fold elevation of plasma alanine aminotransferase activity and 3-fold increases (p < 0.01) in hepatic CYP2E1 apoprotein and activity. Quantitative immunohistochemical analysis revealed significant (p < 0.01) 5-fold increases in MDA- and 4-HNE modified proteins in liver sections prepared from rats treated with alcohol. The MDA- or 4-HNE modified proteins were contained in hepatocytes displaying intact morphology and were colocalized primarily with microvesicular deposits of lipid. Aldehyde-modified proteins were not prevalent in parenchymal or nonparenchymal cells associated with foci of necrosis or inflammation. CONCLUSIONS: These results suggest that alcohol-induced lipid peroxidation is an early event during alcohol-mediated liver injury and may be a sensitizing event resulting in the production of bioactive aldehydes that have the potential to initiate or propagate ensuing proinflammatory or profibrogenic cellular events.     

Xanthine Oxidase Status in Ethanol-Intoxicated Rat Liver

The status of xanthine oxidase in ethanol-induced liver injury has been investigated in the rat, by acute and chronic ethanol treatments. A 38% increase of the enzyme O-form was observed after repeated ethanol administration. Chronic intoxication caused a significant decrease of total xanthine oxidase activity after both prolonged ethanol feeding and life span ethanol ingestion. The intermediate D/O-form of xanthine oxidase (that can act either as an oxidase or as a dehydrogenase, being able to react with O2 as well as with NAD+ as electron acceptor) increased 5.5-fold after prolonged ethanol feeding.     

茶多酚治疗慢性酒精性肝损伤的实验研究

目的 建立酒精性肝病大鼠模型,观察茶多酚对酒精性肝病大鼠血清氨基转移酶活性和肝脏病理变化的影响,探讨茶多酚对酒精性肝损伤的防治作用。 方法 SD大鼠分成3组:酒精组(酒精7g·kg-1·d-1灌胃)、茶多酚组(酒精7g·kg-1·d-1 茶多酚0.25g·kg-1·d-1灌胃)和对照组(等渗盐水灌胃)。各组分别于4周末、12周末和24周末处死大鼠留取肝脏标本,用于HE染色和Masson染色。 结果 酒精组大鼠血清氧基转移酶水平较对照组升高,茶多酚组大鼠与酒精组相比,其值有明显降低,差异有统计学意义(P<0.05)。HE染色显示酒精组大鼠肝细胞浆出现不同程度的脂肪变性,小叶各带可见不同程度的点、灶状或片状坏死,24周大鼠可见桥接坏死。Masson三色染色可见24周大鼠汇管区边缘有绿染胶原纤维包绕增生,肝窦中可见绿染胶原纤维分布。茶多酚组肝脂肪变和炎症程度轻于酒精组,未发现桥接坏死。 结论 茶多酚对酒精性肝损伤具有一定的保护作用。     

Large-scale gene profiling of the liver in a mouse model of chronic, intragastric ethanol infusion

BACKGROUND/AIMS: The mechanisms underlying alcohol-induced liver injury are not fully elucidated. An approach in this direction would consist of an all-inclusive assessment of gene expression in the liver. The purpose of this study was to perform a comprehensive analysis of gene expression in the livers of mice treated with ethanol by means of intragastric infusion. METHODS: An ethanol- or glucose-enriched liquid diet was fed to animals for 4 weeks via a long-term gastrostomy catheter. The animals were killed and plasma alanine:2-oxoglutarate aminotransferase (ALT) assay, liver histology and total RNA analysis by microarray gene technology were performed. RESULTS: Alcohol increased ALT, induced steatosis, necrosis and inflammation. A total of 12,423 genes were analyzed for expression out of which 4867 were expressed by the liver. Alcohol repressed expression of 11 genes, induced expression of 13 genes, and up- or down-regulated expression of 44 and 42 genes >2-fold, respectively. Gene expression analysis identified several genes that have not previously been tested for alcohol effects. CONCLUSIONS: This study: (i) expands the knowledge of mechanism(s) of action of ethanol; (ii) indicates novel pathways of ethanol action on the liver, and (iii) illustrates the utility of microarray gene analysis in hepatology research.     

A dual effect of N-acetylcysteine on acute ethanol-induced liver damage in mice.

Reactive oxygen species (ROS) have been associated with acute ethanol-induced liver damage. N-acetylcysteine (NAC) is a glutathione (GSH) precursor and direct antioxidant. In this study, we investigated the effects of NAC on acute ethanol-induced liver damage. Female ICR mice were administered by gavage with a single dose of ethanol (6g/kg). NAC was administered in two different modes. In mode A, mice were injected with different doses of NAC at 30min before ethanol. In mode B, mice were injected with different doses of NAC at 4h after ethanol. Acute ethanol-induced liver damage was estimated by measuring serum alanine aminotransferase (ALT) activity and histopathological changes. Result showed that a single dose of ethanol (6g/kg) caused a significant increase in serum ALT activity, followed by microvesicular steatosis and necrosis in mouse liver. Pretreatment with NAC significantly protected against acute ethanol-induced liver damage in a dose-independent manner. Correspondingly, pretreatment with NAC significantly attenuated acute ethanol-induced lipid peroxidation and GSH depletion and inhibited hepatic TNF-alpha mRNA expression. By contrast, post-treatment with NAC aggravated ethanol-induced hepatic lipid peroxidation and worsened acute ethanol-induced liver damage in a dose-dependent manner. Taken together, NAC has a dual effect on acute ethanol-induced liver damage. Pretreatment with NAC prevent from acute ethanol-induced liver damage via counteracting ethanol-induced oxidative stress. When administered after ethanol, NAC might behave as a pro-oxidant and aggravate acute ethanol-induced liver damage.     

Acute Ethanol Intoxication Inhibits Neutrophil β2-Integrin Expression in Rats During Endotoxemia

The effects of acute ethanol intoxication on neutrophil [polymorphonuclear leukocyte (PMN)] adhesion molecule expression and certain other functional properties during endotoxemia were studied in rats to elucidate the mechanisms underlying the immunosuppressive effects of ethanol. Acute ethanol intoxication was induced by an intraperitoneal injection of 20% ethanol at a dose of 5.5 g of ethanol/kg. Control animals received an intraperitoneal injection of saline. Thirty minutes after intraperitoneal injection, animals were given a 90-min intravenous infusion of Escherichia coli endotoxin (total dose of 112.5 μg/rat in 2.5 ml of saline) or saline. Certain rats received granulocyte colony-stimulating factor (G-CSF; 50 μg/kg in 5% dextrose, subcutaneous injection twice daily) or vehicle pretreatment for 2 days before intravenous endotoxin infusion. Endotoxemia significantly upregulated CD11b/c and CD18 expression on PMNs when compared with those of saline-infused rats. Acute ethanol intoxication inhibited this endotoxin-induced upregulation of CD11b/c and CD18 expression on PMNs. Ethanol intoxication also suppressed the phagocytic activities of PMNs in saline-infused rats, but this suppression failed to reach statistical significance in endotoxin-infused rats. Hydrogen peroxide generation by PMNs in saline- or endotoxin-infused rats was not affected by ethanol intoxication. Histological examination showed extensive PMN sequestration in the liver after endotoxin infusion, and ethanol intoxication significantly attenuated this hepatic sequestration of PMNs. G-CSF pretreatment enhanced neutrophil phagocytosis, CD11b/c and CD18 expression in endotoxin-infused rats, and prevented the ethanol-induced inhibition of neutrophil CD18 expression and phagocytosis. The impairment of β₂-integrin expression on PMNs may be one mechanism underlying ethanol-induced defects of neutrophil delivery into tissue sites of infection. G-CSF may be of benefit to the infected alcoholic host by enhancing leukocyte defense functions.     

The Effect of Betaine in Reversing Alcoholic Steatosis

The feeding of ethanol to experimental animals results in fatty infiltration of the liver. Recent findings have shown that ethanol-induced steatosis is accompanied by a lowering in hepatic S-adenosylmethionine (SAM) levels. It is known that SAM provides substrates for reduced glutathione formation and offers the cell protection from toxic metabolic oxidants. A recent study in this laboratory demonstrated that dietary supplementation with betaine generated increased SAM in the liver and protected against ethanol-induced steatosis. The present study not only showed that betaine supplementation to rats protects the liver from alcoholic steatosis, but also demonstrated that once steatosis is established, treatment with betaine partially reversed the steatosis after cessation of ethanol feeding. Furthermore, this study indicated that betaine supplementation to the diet had the capacity to attenuate steatosis despite the continued feeding of ethanol.     

Deposition of Cellular Fibronectin Increases before Stellate Cell Activation in Rat Liver during Ethanol Feeding

Cellular fibronectin (cFN)—a structural extracellular matrix protein—facilitates cell adhesion, migration, and differentiation during organ development; wound healing; tissue regeneration; and fibrogenic processes. cFN is deposited early in various fibrotic diseases and seems to function as a template for deposition of other extracellular matrix proteins, such as collagen type I and laminin, in the injured area. We have compared the relative changes in cFN levels with other pathogenic markers of alcoholic liver injury over time of ethanol feeding in the rat. Male Wistar rats were allowed free access to a liquid diet containing 36% of total energy as ethanol or pair-fed an isocaloric control diet for 4, 8, and 12 weeks. Serum alanine arnino-transferase activity and total liver lipid were increased in ethanol-fed animals, compared with pair-fed controls after 4,8, and 12 weeks of feeding. Liver lipid content was higher in ethanol-fed rats as early as 4 weeks and was further increased by 12 weeks of feeding. Total fibronectin and cFN protein quantity was greater in liver from ethanol-fed rats after 8 and 12 weeks (fibronectin: 2.3-fold and 2.6-fold; cFN: 4.3-fold and 2.6-fold higher than pair-fed at 8 and 12 weeks, respectively). α-Smooth muscle actin, an indicator of hepatic stellate cell activation, was increased in the liver of ethanol-fed rats after 12 weeks of feeding (344% higher compared with pair-fed), with no differences observed at any earlier time points. In summary, increases in hepatic immunoreactive cFN content were observed subsequent to increased liver lipid concentration, but before hepatic stellate cell activation in rats fed the ethanol-based diet. These data suggest that deposition of cFN in the liver during long-term ethanol consumption may represent an early response to injury similar to that observed in other models of liver injury and wound healing.     

Decreased proteasome activity is associated with increased severity of liver pathology and oxidative stress in experimental alcoholic liver disease

BACKGROUND: Because of its role in degrading the bulk of intracellular proteins and eliminating damaged proteins, the proteasome is important in maintaining cell viability. Previously, we showed a 35-40% decrease in proteasome peptidase activity when ethanol was administered to rats by intragastric infusion. We hypothesized that this reduction was caused by ethanol-elicited oxidative stress, the degree of which varies depending on the method of ethanol administration. This study examined the relationship of proteasome activity and content with ethanol-induced oxidative stress and the degree of liver injury. METHODS: Rats were given ethanol or isocaloric dextrose-containing liquid diets by intragastric infusion for 1 month. The diets contained medium-chain triglycerides (MCT), palm oil (PO), corn oil (CO), or fish oil (FO) as the principal source of fat. RESULTS: Rats given ethanol and MCT exhibited no significant liver pathology, whereas cumulative pathology scores in ethanol-fed rats given PO, CO, or FO were 2.5, 5.4 and 7.0, respectively, indicating that ethanol and FO caused the greatest liver damage. The severity of liver pathology in the last three groups of animals correlated with levels of lipid peroxides and serum 8-isoprostanes. Alpha smooth muscle actin, an indicator of stellate cell activation, was increased relative to controls in the livers of all ethanol-fed rats except FO-fed animals, in which both control and ethanol-fed rats had similar levels of this protein. In livers of CO and FO ethanol-fed rats, proteasome chymotrypsin-like activity was decreased by 55-60%, but there was no quantitative alteration in 20S proteasome subunit content. In contrast, ethanol affected neither proteasome activity nor its content in MCT- and PO-treated animals. CONCLUSIONS: Our findings indicate that the severity of liver injury and ethanol-induced oxidative stress is associated with a reduction in proteasome catalysis.     

Granulocyte Colony-Stimulating Factor Prevents Ethanol-Induced Impairment in Host Defense in Septic Rats

Ethanol is a potent immunosuppressive agent that impairs neutrophil effector function. The purpose of this study was to determine whether granulocyte colony-stimulating factor (G-CSF), a cytokine that increases neutrophil number and functional activity, could prevent the ethanol-induced impairment of antibacterial host defense. Rats were injected with human recombinant G-CSF for 2 days. Eight hr after the last injection of G-CSF, animals were infused with ethanol (or saline) for 1 hr before the subcutaneous injection of live Escherichia coli . The infusion of alcohol was continued after the bacterial challenge and produced blood alcohol levels of 275–300 mg/dl. In control animals, the injection of E. coll resulted in a marked leukopenia. There was an influx of leukocytes into the subcutaneous space where the bacteria were injected, and neutrophil accumulation in tissues adjacent to the focus of infection (i.e., dorsal skin and muscle). Based on myeloperoxidase activity, there was no detectable accumulation of neutrophils in other soft tissues. In acutely intoxicated rats, leukocyte migration to the inflammatory site was impaired, and the number of viable bacteria isolated from the subcutaneous pocket was markedly increased. G-CSF prevented the sepsis-induced leukopenia, increased the influx of neutrophils in to the infection site, reduced the number of bacteria in the subcutaneous lavage fluid, and decreased the incidence of bacteremia in ethanol-treated rats when compared with rats not receiving G-CSF. These results demonstrate that G-CSF is a potent immunomodulator that stimulates neutrophil recruitment selectively to the site of infection and that can be used to ameliorate the ethanol-induced impairment in bacterial host defense.     

Liver Microsomal Fatty Acid Composition in Ethanol-Fed Rats: Effect of Different Dietary Fats and Relationship to Liver Injury

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. Histological evaluation indicated that ethanol-induced liver injury was limited to rats fed the diet containing corn oil for 4 weeks. Thus, diet-dependent differences in liver microsomal fatty acid composition may help to explain why ethanol-induced liver injury occurs in rats fed corn oil, but not saturated fat.     

Gender Differences in Phagocytic Responses in the Blood and Liver, and the Generation of Cytokine-Induced Neutrophil Chemoattractant in the Liver of Acutely Ethanol-Intoxicated Rats

Phagocytosis by circulating and liver-recruited polymorphonuclear leukocytes (PMNs) and Kupffer cells was studied in acutely ethanol-intoxicated, age-matched male and female rats. Acute ethanol intoxication in female rats is associated with a more effective phagocytic PMN response in the circulating blood, but with lower phagocytic activities by liver-recruited PMNs and Kupffer cells than in their male counterparts. Endotoxin [lipopolysaccharide (LPS)] treatment (consisting of a Wmin intravenous infusion of a nonlethal dose) of acutely ethanol-intoxicated male and female rats results in enhanced phagocytic responses in liver-sequestered PMNs and Kupffer cells, but not in circulating PMNs. However, the LPS challenge elicits a lesser phagocytic response in liver PMNs and Kupffer cells of female rats than in males. Significant gender differences exist in the extent of hepatic PMN infiltration in ethanol plus LPS-treated rats, which is paralleled by very similar difterences in CDll b/c adhesion molecule expression in circulating PMNs and cytokine-induced neutrophil chemoattractant generation by hepatocytes and Kupffer cells. Taken together, these data indicate a smaller phagocytic response to fight infection in the liver of acutely alcohol-intoxicated female rats, but also a mechanism to afford some protection against neu-trophil-associated tissue injury.     

Dietary Betaine Promotes Generation of Hepatic S-Adenosylmethionine and Protects the Liver from Ethanol-Induced Fatty Infiltration

Previous studies have shown that ethanol feeding to rats alters methionine metabolism by decreasing the activity of methionine synthetase. This is the enzyme that converts homocysteine in the presence of vitamin B₁₂ and N⁵-methyltetrahydrofolate to methionine. The action of the ethanol results in an increase in the hepatic level of the substrate N⁵-methyltetrahydrofolate but as an adaptive mechanism, betaine homocysteine methyltransferase, is induced in order to maintain hepatic S-adenosylmethionine at normal levels. Continued ethanol feeding, beyond 2 months, however, produces depressed levels of hepatic S-adenosylmethionine. Because betaine homocysteine methyltransferase is induced in the livers of ethanolfed rats, this study was conducted to determine what effect the feeding of betaine, a substrate of betaine homocysteine methyltransferase, has on methionine metabolism in control and ethanol-fed animals. Control and ethanol-fed rats were given both betainelacking and betaine-containing liquid diets for 4 weeks, and parameters of methionine metabolism were measured. These measurements demonstrated that betaine administration doubled the hepatic levels of S-adenosylmethionine in control animals and increased by 4-fold the levels of hepatic S-adenosylmethionine in the ethanol-fed rats. The ethanol-induced infiltration of triglycerides in the liver was also reduced by the feeding of betaine to the ethanol-fed animals. These results indicate that betaine administration has the capacity to elevate hepatic S-adenosylmethionine and to prevent the ethanol-induced fatty liver.     

Tezosentan, an endothelin receptor antagonist, limits liver injury in endotoxin challenged cirrhotic rats

BACKGROUND/AIMS: Lipopolysaccharide (LPS) induces liver injury which is associated with upregulated endothelin (ET)-1 production. The aim of this study was to investigate the effects of tezosentan, a non-selective ETA and ETB receptor antagonist, in LPS challenged rats with cirrhosis. METHODS: Rats with cirrhosis received LPS and then tezosentan or placebo one hour later. Four hours after LPS administration, rats were killed to measure serum transaminase activity and plasma tumour necrosis factor alpha (TNF-alpha) levels. Hepatic inducible nitric oxide synthase (iNOS), myeloperoxidase (MPO), a marker of neutrophil infiltration, and cyclooxygenase (COX)-2 expression were also measured. RESULTS: LPS administration significantly decreased arterial pressure and significantly increased plasma endothelin levels. Following LPS and tezosentan administration, serum aspartate aminotransferase and alanine aminotransferase activities were similar to those in the control group while they were increased by more than 700% with LPS alone. Plasma TNF-alpha levels were significantly lower in rats receiving LPS and tezosentan (182 (38) pg/ml) compared with those receiving LPS alone (821 (212) pg/ml). Tezosentan significantly decreased hepatic MPO activity and hepatic neutrophils but had no effect on LPS induced iNOS or COX-2. Survival rate was significantly higher in rats receiving LPS plus tezosentan (80%) than in rats receiving LPS alone (50%). CONCLUSION: In LPS challenged cirrhotic rats, tezosentan administration prevents LPS induced liver injury by decreasing intrahepatic neutrophil infiltration. In addition, tezosentan increases survival in these rats.     

Involvement of tumor necrosis factor in endotoxin-triggered neutrophil adherence to sinusoidal endothelial cells of mouse liver and its modulation in acute phase

Tumor necrosis factor (TNF) has been shown to mediate lipopolysaccharide-induced neutrophil adhesion to liver sinusoidal endothelium in vivo. Female NMRI mice received either 5 micrograms lipopolysaccharide (R595) per animal alone (model A) or together with 116 mumol D-galactosamine (model B). One hour after injection, TNF activity in the serum was detectable to an equal extent in both models. Neutrophils in the liver, which had been identified by chloroacetate esterase staining of liver sections and quantitated by light microscopy, started to increase at 1 h and were elevated 10-fold above baseline at 6 h after application in (A) and (B). If 0.5 micrograms TNF instead of lipopolysaccharide was injected alone (model C) or together with D-galactosamine (model D), neutrophil influx into the liver was comparable to that observed in (A) or (B). Alanine aminotransferase activity in the serum was nearly normal in (A) and (C) 6 h after injection, while it reached levels up to 50-fold above baseline in models (B) and (D). This reflects the well-known D-galactosamine sensitization against lipopolysaccharide or TNF. Furthermore, degranulation of a large number of intrasinusoidal neutrophils could be observed 9 h after lipopolysaccharide-galactosamine injection. The administration of 116 mumol D-galactosamine per animal alone led neither to a measurable TNF activity in the serum nor to an increase in alanine aminotransferase activity or number of liver neutrophils. If the animals had received 50 microliter turpentine subcutaneously 24 h prior to lipopolysaccharide, TNF or D-galactosamine injection, the induced acute-phase reaction suppressed the increase of liver neutrophils in all models. Acute-phase reaction also prevented neutrophil degranulation and the rise of alanine aminotransferase in (B) to a great extent, while serum TNF activity was only minimally affected. It is concluded that TNF mediates neutrophil adhesion to the sinusoidal endothelium in vivo and that acute-phase reactants prevent lipopolysaccharide- or TNF-induced neutrophil influx into the liver.