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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.

Previously, we have shown that an increased expression level of iNOS but a reduction in the expression of eNOS is associated with increased oxidative stress markers in CCl₄-induced experimental liver fibrosis. The present study aimed to investigate the effect of L-arginine and 5-methylisothiourea hemisulfate (SMT) in the expression of profibrogenic factors in chronic liver injury. ICR mice were treated with CCl₄ with or without treatment of L-arginine, an NO donor, or SMT, an iNOS inhibitor. The expression of matrix metalloptroteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), α-smooth muscle actin (α-SMA), tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2) were investigated by RT-PCR. The activity of the MMP-2 and MMP-9 were measured by zymography. Our results showed that CCl₄-treated mice showed significant up-regulation of expression of pro-fibrogenic factors, TNF-α and COX-2. Treatment with L-arginine or SMT showed a significant reduction in CCl₄-induced expression of these pro-fibrogenic factors, TNF-α and COX-2. In conclusion, both SMT and L-arginine effectively attenuated the progression of CCl₄-induced liver fibrosis. SMT suppresses iNOS mediated NO production. However, L-arginine augments NO production. The similar effect of the two drugs on liver fibrosis indicates that there may be two distinct pathways of NOS mediated fibrogenesis in chronic liver injury by iNOS and eNOS. Our results suggest that eNOS-mediated liver fibrogenesis may play a more important role than that of iNOS in chronic liver injury. Taken together, these results support the contention that NO plays an active role in the progression of liver fibrosis and hepatocellular damage.

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.

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.

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.

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.

Background and aims.Treatment with acetaldehyde dehydrogenase inhibitors leads to increased liver acetaldehyde levels and prevents hepatic inflammation and necrosis in ethanol-fed rats. This is accompanied by IκBα preservation and decreased activation of nuclear factor (NF)-κB. The presentin vitrostudy was aimed to clarify whether acetaldehyde has an effect on degradation of IκBα and activation of NF-κB in LPS-stimulated rat Kupffer cells.Methods.Kupffer cells were isolated from male Sprague–Dawley rats and preincubated with various concentrations of acetaldehyde (25–100 μM). Thereafter the cells were stimulated with LPS, and cytosolic and nuclear fractions were prepared. IκBα and p65 proteins and activation of NF-κB were evaluated.Results.In LPS-stimulated rat Kupffer cells, acetaldehyde diminished proteolytic degradation of IκBα, inhibited nuclear translocation of cytosolic p65 protein, and, accordingly, markedly decreased NF-κB activation.Conclusions.Acetaldehyde is clearly involved in the stabilization of IκBα protein and suppression of NF-κB activation in rat Kupffer cells. Acetaldehyde may form an adduct with IκBα, thus making the protein less susceptible to degradation.

Background: Women exhibit greater liver damage than men after chronic alcohol consumption. Similar findings are reported in animal models. Here, we determined whether differential liver injury occurred in male and female rats after feeding these animals liquid diets containing either ethanol or isocaloric dextrose with fish oil as the sole source of lipid. Methods: Control and ethanol liquid diets containing fish oil were pair-fed to male and female rats for 8 weeks. Liver damage was evaluated by triglyceride accumulation, lipid peroxide formation, serum transaminases, histological evaluation, and the activities of selected lysosomal and hepatoprotective enzymes. Results: Fatty liver was detected after ethanol feeding in both genders, but in female rats, triglyceride levels were 60% higher, lipid peroxides were 2-fold higher, and inflammatory cells were more evident than in males. A 2-fold elevation of cathepsin B in hepatic cytosol fractions, indicating lysosomal leakage, was detected in ethanol-fed female rats but no such elevation was observed in males. The basal activity of the hepatoprotective enzyme, betaine-homocysteine methyltransferase was 4-fold higher in livers of control male rats than females, and the enzyme activity was further elevated in ethanol-fed male rats but not in females. Conclusions: Thus, female rats given ethanol in a diet containing fish oil exhibited more severe liver damage than males. We propose that this difference results, in part, from a greater tendency by females to accumulate hepatic fat, thereby enhancing the potential for oxidative stress, which in turn leads to hepatic inflammation. In addition, our findings indicate that female rats have a higher susceptibility to liver damage because of a reduced capacity for hepatoprotection.

Alcoholic liver disease (ALD) is an increasingly recognized condition that may progress to end-stage liver disease. In addition to alcohol consumption, genetic factors, dietary fatty acids, gender and viral infection potentiate the severity of alcoholic liver injury. In humans, significant gender differences in susceptibility to ALD are observed. In the intragastric infusion rat model of ALD, female rats developed more severe liver injury than males. To understand the effect of gender on the development of more severe ALD in female rats, we performed a microarray based expression profiling of genes in rats fed with fish oil and ethanol diet. A large number of genes showed significant changes in female livers compared to males. The upregulated genes in female liver were involved in proteosome endopeptidase activity, catalytic activity, lipid metabolism, alcohol metabolism, mitochondrial and oxidoreductase activity. The downregulated genes were involved in oxidoreductase activity, chaperone activity, and electron transport activity in the female liver as demonstrated by biological theme analysis. Ingenuity computational pathway analysis tools were used to identify specific regulatory networks of genes operative in promoting liver injury. These networks allowed us to identify a large cluster of genes involved in lipid metabolism, development, cellular growth and proliferation, apoptosis, carcinogenesis and various signaling pathways. Genes listed in this article that were significantly increased or decreased (expression two fold or more) were assigned to pathological functional groups and reviewed for relevance to establish hypotheses of potential mechanisms involved in ALD in female liver injury.

Expression of hepatocyte growth factor (HGF) and its receptor, c-met is up-regulated in various forms of liver injury. This study evaluated the relationship between HGF and c-met expression and pathological changes in experimental alcoholic liver disease. Rats (5 per group) were fed ethanol and a diet containing saturated fat corn oil or fish oil by intragastric infusion. Dextrose isocalorically replaced ethanol in controls. In a second set of experiments, Kupffer cells, endothelial cells and hepatocytes were isolated from rats in each group. Pathological evaluation and analysis of HGF and c-met expression were performed in liver and the different cell types. Increased expression of HGF and c-met expression was detected in the liver of rats showing necroinflammatory changes. The Kupffer and endothelial cells were primarily responsible for the increase in HGF, c-met expression was seen only in hepatocytes. Thus, up-regulation of HGF and c-met occurred in the presence of the necrosis and inflammation suggesting that HGF may be acting to protect against liver injury or accelerate the regenerative process.

The use of animal models has contributed to greater understanding of how alcoholic liver disease (ALD) develops, and of how the severity of liver injury is influenced by factors other than alcohol, such as nutrition, oxygen deprivation (as occurs with sleep apnea or smoking), and gene regulation. This article focuses on the use of one animal model in particular, the intragastric feeding model in rats. This model allows scientists to rigorously control an animal’s consumption of both alcohol and dietary nutrients and is providing important information on the mechanisms of injury of alcoholic liver disease.

As occurs in people, nonalcoholic fatty liver disease (NAFLD) is associated strongly with obesity, diabetes, and dyslipidemia in experimental animals. There are many animal models that have been used to investigate the pathogenesis of nonalcoholic fatty liver disease. Most of this work has used mice or rats that are fed diets high in fat or carbohydrates, or mice that exhibit a genetic deficiency of a satiety factor such as leptin, 5-adenosylmethionine,or enzyme deficiencies in fatty acid oxidation. The purpose of this article is to update information regarding animal models in the pathogenesis of NAFLD.

Both dietary fatty acids and alcohol play an important role in the pathogenesis of alcoholic liver disease. Findings of studies in rats show a steatogenic role for dietary fat. A role for polyunsaturated fatty acids in alcoholic liver disease is supported by results of studies, which show that pathologic changes occur only in rats fed ethanol with polyunsaturated fatty acids. The mechanisms through which the fatty acids promote alcoholic liver disease include enhanced oxidative stress, production of endotoxin, and increased expression of proinflammatory cytokines.

Alcoholic liver disease is associated with a state of hepatic fatty acid overload. We examined the effect of ethanol and different types of dietary fat on the expression of mRNA for liver fatty acid binding protein (L-FABP), peroxisome proliferator-activated receptor-alpha (PPARalpha), and peroxisomal fatty acyl CoA oxidase (FACO). Four groups of rats (n = 5) were fed intragastrically, a liquid diet with or without ethanol (10-16 g/kg/day) for 4 weeks. Pair-fed controls received isocaloric amounts of dextrose. The source of fat was either corn oil or fish oil. Ethanolfed rats developed fatty liver, necrosis, and inflammation; the changes were more severe in the fish oil-ethanol (FE) rats. PPARalpha mRNA levels were not different between groups, although there was a trend toward increased levels in ethanol-fed rats. We calculated L-FABP/PPARalpha and FACO/PPARalpha ratios as a measure of FACO and L-FABP up-regulation relative to PPARalpha expression. Both FACO/PPARalpha and L-FABP/PPARalpha ratios were significantly decreased in FE rats. However, only L-FABP/PPARalpha was decreased in corn oil plus ethanol rats. Also, the level of L-FABP/mRNA correlated inversely with the degree of fatty liver in ethanol-fed rats. Since expression of PPARalpha response genes was impaired in ethanol-fed rats, we determined whether activation of PPARalpha would normalize the PPARalpha response and prevent the pathological changes in ethanol-fed rats. Treatment with clofibrate, a PPARalpha-activating ligand, led to a marked decrease in fatty liver and complete abrogation of necroinflammatory changes in FE rats. Also, nuclear factor kappaB activation and up-regulation of tumor necrosis factor-alpha and cyclooxygenase-2 was also abolished in clofibrate-treated rats. We conclude that adaptive gene regulation of FACO and L-FABP by PPARalpha is impaired in ethanol-fed rats and that treatment with clofibrate, a PPARalpha ligand, prevents alcohol-induced pathological liver injury, possibly by reversing the above changes.

It has been demonstrated that the oral administration of ethanol (Lieber-DeCarli liquid diet) to rats results in a decreased expression and content of the asialoglycoprotein receptor (ASGP-R) in the resultant fatty liver. In the present study, we wanted to determine whether the extent of impaired receptor content was correlated with the severity of liver pathology by using the intragastric feeding model. When ASGP-R protein and mRNA levels were measured in animals infused with ethanol or dextrose in the presence of fish oil (FO) or medium-chain triglyceride as the source of fat, more significant impairments to the ASGP-R were observed in the FO-ethanol group compared with the medium-chain triglyceride-ethanol group. Furthermore, only the FO-ethanol group showed pathological liver changes. These results demonstrate that a correlation exists between the progression of alcohol-associated liver injury, as defined by the severity of liver pathology, and an ethanol-induced decline in ASGP-R content.

The effect of chronic ethanol intake, with and without an iron chelator, on the activity of rat membrane ATPases was investigated. Using the intragastric feeding model, male Wistar rats (250 g) were fed a liquid diet and ethanol for 1 month. In control pair-fed animals, ethanol was isocalorically replaced by dextrose. In addition to the above groups, two groups of animals (dextrose or ethanol-fed) also received an oral iron chelator (1,2-dimethyl-3-hydroxypyrid-4-one, L 1 ) (25 mg/kg/day for 30 days). The blood ethanol levels were maintained between 150 and 300 mg/dL. Red cells were washed immediately with ice-cold saline, membranes were prepared, and ATPases were measured. The mean Ca 24+ pump ATPase in animals fed ethanol was lower than in dextrose-fed controls. In contrast, Na + /K + pump ATPase was enhanced following chronic ethanol treatment. The addition of 1 1 ) to the diet prevented the changes in both the Ca 2+ -ATPase and Na + /K + -ATPase in ethanol-fed rats. Although the exact mechanism for the prevention of changes in ATPase activity by L 1 is unknown, it is not a result of non-specific interaction between the chelator and membranes. Incubation of purified membranes with different concentrations of L 1 for 60 min at 37° had no effect on the activity of the ATPase. In conclusion, chronic intake of ethanol specifically inhibited Ca 2+ pump ATPase and enhanced Na + /K + -ATPase in rat red blood cell membranes. The iron chelator, L 1 , corrected both of these ethanol-induced changes.

Macrophage migration inhibitory factory (MIF) regulates macrophage accumulation at sites of injury and can promote the inflammatory response. We studied MIF expression in the intragastric feeding rat model for alcoholic liver injury. Male and age-matched female rats were fed ethanol or dextrose with fish oil. Two groups of male rats were fed medium-chain triglycerides with ethanol or dextrose. Analysis of liver histopathology, lipid peroxidation, endotoxin, mRNA, and immunohistochemistry for MIF, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were carried out. Male and female rats fed fish oil and ethanol showed necroinflammatory liver injury and had the highest expression of MIF, TNF-alpha, and IFN-gamma in the liver. Decreased levels of MIF protein were seen in rats with higher endotoxin levels, suggesting that preformed MIF is released into the circulation. MIF is an important mediator of the inflammatory response in alcoholic liver disease and a potential therapeutic target.

Endotoxemia and oxidative stress activate nuclear factor kappa B (NF-kappaB) in alcoholic liver injury. In alcohol-fed rats, activation of NF-kappaB is associated with the development of necro-inflammatory changes in the liver. Whether activation of NF-kappaB occurs prior to development of liver injury is unknown. We determined whether activation of NF-kappaB preceded histopathological liver changes. Male Wistar rats were fed a liquid diet containing ethanol by continuous infusion through permanently implanted gastric tubes. Radical intermediates detected by spin trapping were measured in bile prior to killing. After 2 weeks of treatment, samples of liver tissue were obtained for histopathological examination, for evaluation of NF-kappaB, and determination of messenger RNA levels of cytokines, chemokines and cyclo-oxygenase-2. No pathological changes in liver were seen after 2 weeks of intragastric feeding. However, activation of NF-kappaB was seen in the livers from ethanol-fed rats. In addition, elevated mRNA levels of hepatic pro-inflammatory cytokines (TNF-alpha and IL12), chemokines MIPIalpha and MIP-2) and cyclo-oxygenase-2 were seen in association with activation of NF-kappaB and increased levels of free radicals and endotoxin. Thus, activation of NF-kappaB, associated with elevated mRNA levels of pro-inflammatory stimuli, precedes the histopathological liver changes in experimental alcoholic liver disease in rats.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Carol A. Casey and Amin Nanji. The presentations were (1) Mechanisms of apoptosis in alcoholic liver disease, by Amin A. Nanji; (2) Impaired receptor-mediated endocytosis: Its role in alcoholic apoptosis, by Carol A. Casey; (3) Toxicity of ethanol in HepG2 cells that express CYP2E1, by Arthur I. Cederbaum; (4) Mitochondrial regulation of ethanol-induced hepatocyte apoptosis, by M. Adachi; and (5) Apoptosis in alcoholic hepatitis, by T. Takahashi.

Apoptosis occurs in both clinical and experimental alcoholic liver disease. The mechanisms involved in alcohol-induced apoptosis of liver cells are not completely understood. Induction of cytochrome P450 2E1, the alcohol-inducible cytochrome P450, is one of the proposed mechanisms. Exposure of Hep G2 cells expressing cytochrome P450 2E1 to arachidonic acid leads to increased lipid peroxidation and apoptosis. Increased levels of iron in the liver also promote lipid peroxidation and are associated with increased numbers of apoptotic hepatocytes. Tumor necrosis factor (TNF) acting through its receptors can induce apoptosis in hepatocytes. Increased levels of tumor necrosis factor and its receptors have been described in alcoholic liver disease. The liver is also CD95 receptor positive and in liver tissue from patients with alcoholic hepatitis, the CD95 ligand is expressed at high levels in hepatocytes. Cytotoxic T lymphocytes could, through the CD95 receptor-ligand interaction, promote apoptosis.

Based on studies that show a role for the low-density lipoprotein (LDL)-receptor in arachidonic acid delivery and eicosanoid synthesis in macrophages, the present study investigated the effect of cholesterol supplementation on pathological changes and thromboxane (TX) synthesis in alcoholic liver injury. Male Wistar rats were intragastrically fed ethanol with either corn oil or fish oil for 1 month. Control rats received isocaloric amounts of dextrose instead of ethanol. An additional group of rats fed either ethanol or dextrose with fish oil or corn oil were supplemented with 1% cholesterol. At the time of killing, all rats had the following evaluated: liver histopathology, lipid peroxidation, liver and plasma thromboxane levels, plasma endotoxin and messenger RNA (mRNA) levels of LDL-receptor, tumor necrosis factor alpha (TNF-alpha), cyclooxygenase (Cox)-1 and -2, and transforming growth factor beta (TGF-beta). Rats fed ethanol with either fish oil or corn oil developed fatty liver, necrosis, inflammation, and central vein collagen deposition. Cholesterol supplementation enhanced the degree of fibrosis but prevented necrosis and inflammation. These alterations in pathological changes by cholesterol were accompanied by absent TNF-alpha and Cox-2 mRNAs, decreased thromboxane levels, decreased lipid peroxidation, and increased TGF-beta mRNA. Cholesterol enrichment of the diet thus decreases proinflammatory components, but enhances fibrosis in ethanol-fed rats. (Hepatology 1997 Jul;26(1):90-7)

measurements of lipid peroxidation, tumor necrosis factor (TNF)-a, COX-1 and COX-2 messenger RNA that stimulate expression of COX-2, we hypothesized (mRNA), endotoxin, and liver and plasma thromboxane that COX-2 would be induced in experimental ALD. In were performed. Results: Increased expression of COX- the rat intragastric feeding model of ALD, we and others 2 mRNA was detected in the livers of rats showing have shown that an inverse relationship exists between necroinflammatory changes. The Kupffer cell was the the state of saturation of lipids in diet and the severity cell primarily responsible for the increase in COX-2 of alcohol-induced liver injury. 13-17 Thus, rats fed etha- mRNA level. Increased expression of COX-2 was associ- nol and diets enriched in saturated fatty acids 14 are pro- ated with increased levels of endotoxin, TNF-a mRNA, tected from liver injury, whereas diets enriched in poly- lipid peroxidation, and synthesis of thromboxane. COX- unsaturated fatty acids 15-17 promote alcoholic liver 1 mRNA was decreased in Kupffer cells in rats with the injury. Thus, the rat intragastric feeding model is useful most severe liver injury. Conclusions: Up-regulation of to elucidate the pathogenesis of ALD. In the current COX-2 in alcoholic liver injury occurred in the presence study, we used this model of liver injury to study the of proinflammatory stimuli and resulted in increased synthesis of inflammatory and vasoactive eicosanoids. relationship between liver pathology and COX expres- Down-regulation of COX-1 may result in decreased syn- sion and to attempt to identify the factors responsible thesis of cytoprotective eicosanoids and additionally for induction of COX-2 in ALD. exacerbate liver injury. Our results show that necrosis and inflammation in ALD are associated with increased expression of COX- 2 and higher levels of plasma endotoxin, liver TNF-a

We evaluated the role of endothelial cell (EC) proliferation in experimental alcoholic liver disease (ALD) using different dietary models of ALD. Rats were divided into treatment groups to receive ethanol with either corn oil (CO + E), fish oil (FO + E), saturated fat (SF + E), and corn oil with a novel quinone derivative (E3330) and cimetidine. All ethanol-fed rats in the different groups were pair-fed with dextrose replacing the ethanol-derived calories. All ethanol-fed groups and their controls were fed for periods ranging from 1 to 4 weeks. For each animal, immunocytochemical staining for PCNA was performed on paraffin-embedded tissue sections and the number of endothelial cells staining for PCNA per 10 high-power fields (HPF) was determined. Rats fed CO + E and FO + E developed pathological changes, whereas none of the histologic features of ALD were seen in SF + E rats. The severity of injury was reduced in the quinone and cimetidine-treated groups. A higher rate of EC proliferation (6–30×) was seen in the SF + E group (no liver injury) than in the CO + E and FO + E groups (pathologic changes present). The difference was evident by 1 week, which is well before pathologic changes can be recognized (usually 4 weeks). An increase in EC proliferation was seen in the E3330 and cimetidine-treated groups. Our study indicates that the proliferative response of EC in ethanol-fed rats may be a factor in the progression to liver injury. Suppression of ethanol-induced EC proliferation in CO + E and FO + E groups occurred prior to development of liver injury; a lack of suppression of EC proliferation is associated with absence (SF + E and CO + E + cimetidine) or reduction in severity (CO + E + E3330) of liver injury.

Background & Aims: Lipid peroxidation may be important in the pathogenesis of alcoholic liver injury. The purpose of this study was to determine whether a saturated fatty acid-based therapy (palm oil) could decrease lipid peroxidation and alcoholic liver injury during ethanol withdrawal. Methods: Three groups of male Wistar rats (5 rats/group) were studied. Rats in group 1 were fed a fish oil-ethanol diet for 6 weeks; rats in groups 2 and 3 were fed a fish oil-ethanol diet for 6 weeks before treatment with fish oil-dextrose (group 2) or palm oil-dextrose (group 3) for 2 weeks. Liver samples were analyzed for histopathology, lipid peroxidation, fatty acid composition, cytochrome P450 2E1 activity, and tocopherol levels. Results: By 6 weeks, all rats had developed fatty liver, inflammation, and necrosis. Group 2 showed minimal histological improvement, whereas group 3 showed near normalization of the histology. The improvement in group 3 was associated with decreased lipid peroxidation and P450 2E1 activity. Higher levels of ω-3 fatty acids were detected in group 2 than group 3. Tocopherol levels were similar among the groups. Conclusions: A diet enriched in saturated but not unsaturated fatty acids reversed alcoholic liver injury. This effect may be explained by down-regulation of lipid peroxidation.

We used the intragastric feeding rat model to investigate the relationship between severity of alcoholic liver injury, apoptosis, bcl-2 protein expression, and lipid peroxidation. Rats were fed ethanol with different dietary fats (saturated fat, corn oil, and fish oil) for a 1-month period. Apoptosis was evaluated using an immunohistochemical method, and flow cytometry. Bcl-2 protein concentrations in liver were evaluated by Western blot analysis and lipid peroxidation by measurement of conjugated dienes. Pathological changes (fatty liver, necrosis, and inflammation) were present in corn oil-ethanol and fish oil-ethanol groups only. The highest number of apoptotic cells were seen in the group of rats exhibiting liver injury. The fish oil-ethanol-fed group had the highest concentrations of bcl-2 protein; this protein was localized in the bile duct epithelial and inflammatory cells. A significant correlation was seen between bcl-2 protein assessed densitometrically and the number of inflammatory cells/mm2 (r = 0.78, p < 0.02) and conjugated diene levels (r = 0.82, p < 0.01). Increased numbers of apoptotic cells were seen in rats developing ethanol-induced pathological liver injury. Increased bcl-2 protein concentration are associated with the presence of inflammatory cells and lipid peroxidation.

We used the intragastric feeding rat model for alcoholic liver disease to evaluate the relationship among intercellular adhesion molecule-1 (ICAM-1) expression, tumor necrosis factor-alpha (TNF-alpha), plasma endotoxin, and inflammatory changes in the liver. Rats were fed different dietary fats (saturated fat, corn oil, and fish oil) with ethanol; control rats were fed isocaloric amounts of dextrose instead of ethanol. At sacrifice the following were evaluated: liver pathologic changes, TNF-alpha mRNA by reverse transcription-PCR, plasma endotoxin, and ICAM-1 by immunohistochemistry and immunoblot analysis. Upregulation of ICAM-1 in endothelial lining cells in central and portal veins was observed in rats showing evidence of pathologic changes. Rats fed fish oil and ethanol, which exhibited the most severe inflammation, also showed hepatocyte ICAM-1 staining. The presence of ICAM-1 staining, in general, correlated with the level of TNF-alpha mRNA expression and plasma endotoxin levels. Upregulation of ICAM-1 in rats fed ethanol may contribute to the inflammatory changes seen in this model. The association between ICAM-1 upregulation and endotoxin and TNF-alpha mRNA suggests a role for these mediators in the inflammatory process in alcoholic liver injury.

The influence of dietary fat and alcohol on hepatic microsomal levels of cytochromes P-450 2E1, 2B, and 4A; phospholipases A and C; and UDP-glucuronosyltransferase was studied in the intragastric feeding rat model for alcoholic liver injury. Eight groups of animals were evaluated. Control and ethanol fed rats received either saturated fat or corn oil and were killed after 2 weeks and 1 month of feeding. All animals were pair-fed by continuous infusion of liquid diet through permanently implanted gastric cannulas. Alcoholic liver injury developed only in the corn oil-ethanol-fed groups and was manifest by 1 month. Livers were subjected to the following analyses: pathologic evaluation of liver injury; levels of cytochromes P-450 2E1, 2B, and 4A protein and mRNA; aniline hydroxylase activity; and phospholipase A and C and UDP-glucuronosyltransferase activities. Ethanol-induced increases in cytochromes P-450 2E1 and 2B protein determined by Western blotting were greatest in the corn oil-ethanol-fed group, which developed pathologic changes in the liver. Cytochromes P-450 2E1 and 2B1 mRNA levels were unaffected, suggesting that posttranscriptional mechanisms are responsible for the increase in the corresponding P-450 proteins. In contrast, cytochrome P-450 4A levels were higher in the saturated fat-ethanol groups compared with the corn oil-ethanol groups. Phospholipase A and phospholipase C levels were higher in the corn oil-ethanol groups compared with pair-fed dextrose controls and the saturated fat-ethanol groups. UDP-glucuronosyltransferase levels declined with time in the ethanol-fed groups. These observations are discussed in the context of a model whereby the induction of phospholipases A and C and cytochromes P-450 2E1 and 2B1 in corn oil-ethanol-fed rats provide arachidonic acid substrate and induce lipid peroxidation, respectively. These changes may account for the more severe pathologic changes that develop in corn oil-ethanol-fed animals compared with animals fed saturated fat and ethanol.

Chronic ethanol intake impairs several parameters of immune function. Since there is evidence that cytokine production by immune cells may contribute to the immunosuppressive effect of ethanol, we examined interleukin 1 (IL1) production by liver non-parenchymal cells (NPC) in ethanol-fed rats. Male Wistar rats (225-250 g) were fed by continuous intragastric infusion. The source of fat was either saturated fat or polyunsaturated fat. In addition, the effect of a quinone compound on IL1 production was assessed. Animals were fed for various periods: 1 week, 2 weeks, 1 month and 2 months. NPC were isolated and stimulated by lipopolysaccharide. IL1 production by NPC and the ratio of stimulated to unstimulated (S:U) IL1 production were evaluated in the different groups and related to the presence of liver injury. As expected, animals fed corn oil and ethanol (CO+E) developed pathologic liver injury, whereas animals fed saturated fat and ethanol (SF+E) had no liver injury. A progressive decrease in the S:U IL1 ratio was seen in the CO+E group over the 8-week period. The ratio in the SF+E group was higher. The quinone compound reversed the suppressive effect of ethanol on IL1 production. In summary, ethanol-induced suppression of IL1 production was modulated by diet and the presence of liver injury. This suppression of IL1 production was reversed by a quinone compound; the exact mechanism for the reversal of this inhibition is unknown.

We have previously shown a relationship between plasma endotoxin levels and severity of alcoholic liver injury in the intragastric feeding rat model. We attempted to reduce both circulating endotoxin and liver injury in this model by administering a lactobacillus strain (species GG) which survives for prolonged periods in the gastrointestinal tract. Male Wistar rats were fed ethanol and liquid diet containing corn oil (CO+E). Another group of animals (CO+E+L) received the diet containing ethanol plus a daily bolus of lactobacilli GG concentrate (10(10) CFU). All animals were sacrificed after one month. All animals had plasma endotoxin measurements and evaluation of severity of pathologic changes in the liver. The weight gain and blood alcohol levels were similar in both groups. The mean +/- SE of the pathology score was significantly higher (3.4 +/- 0.85) in the CO+E group compared to the CO+E+L group (0.5 +/- 0.3, P < 0.01). The virtual absence of pathologic changes in the latter group was accompanied by significantly lower endotoxin levels (8.4 +/- 2.9 pg/ml in CO+E+L group vs 48.3 +/- 7.8 pg/ml in CO+E group, P < 0.01). Feeding of strains of lactobacilli that survive in the gastrointestinal tract reduces endotoxemia and alcohol-induced liver injury in the rat. Lactobacillus species GG provides a potential nontoxic form of therapy for both endotoxemia and alcoholic liver disease.

We evaluated whether fish oil or vitamin E administration affected ethanol-induced changes in membrane ATPases. Male Wistar rats (225–250 g) were fed, through a gastric tube a liquid diet containing fish oil (25% of calories) and ethanol for one month. Another group of animals was given supplemental vitamin E (300 u/kg). In the pair-fed control animals, ethanol-derived calories were replaced with dextrose. The blood ethanol levels were maintained between 150 and 350 mg/dL. At sacrifice, the red cells were immediately washed with ice-cold saline, membranes were prepared and ATPases measured. These was no difference in the Na + K + ATPase, Ca 2+ ATPase and Mg 2+ ATPase activities between the fish oil-dextrose and corn oil-dextrose groups. A decrease in Ca 2+ ATPase and an increase in Na + K + ATPase was seen with ethanol feeding; these change are similar to those seen in corn oil-ethanol fed rats. In contrast, Vitamin E administration prevented the ethanol-induced changes in ATPases. This observation provides support for the role of lipid peroxidation in alcohol-induced changes in cell membrane ATPase activities.

We used the intragastric feeding rat model for alcoholic liver disease to investigate the relationship between prostacyclin and liver injury. Rats were fed the following diets for periods ranging from 1 to 8 weeks: corn oil plus ethanol (CO+E), corn oil plus dextrose (CO+D), saturated fat plus ethanol (SF+E) and saturated fat plus dextrose (SF+D). Prostacyclin production (assessed by 6-ketoprostaglandin F1 alpha) by liver non-parenchymal cells decreased steadily over the 8 week period in animals fed CO+E (liver injury present) whereas in animals fed SF+E (no liver injury) there was no change in prostacyclin production. Plasma levels of 6-ketoprostaglandin F1 alpha were also significantly lower in the CO+E group compared to the other groups studied. We propose that decreased prostacyclin production by liver non-parenchymal cells may contribute to the hepatotoxic effect of ethanol.

The present study evaluated the role of endotoxin, prostaglandin E 2 (PGE 2 ), leukotriene B 4 (LTB 4 ) and thromboxane B 2 in alcoholic liver injury. Animals (4–5 per group per time period) were fed ethanol and either saturated fat (SE group) or corn oil (CE group) and sacrificed at various time intervals: 1, 2, and 4 weeks. Non parenchymal cells (NPC) were isolated and the spontaneous and LPS-stimulated production of eicosanoids was evaluated. In addition, severity of pathology, plasma levels of endotoxin, TXB 2 , LTB 4 and PGE 2 were compared in the two groups. The severity of fatty, liver, necrosis and inflammation was significantly greater in the CE group compared to the SE group after 1 month. Plasma endotoxin levels were significantly higher in the CE group vs. SE group at all time periods studied. PGE 2 levels in NPCS and plasma were in general higher in the SE group. LTB 4 levels in NPCS declined with time in the SE group; in contrast the levels rose in the CE group. There was no difference in LTB 4 levels in plasma between the two groups. TXB 2 levels in plasma and NPCS were significantly higher in the CE group at all times. The pattern of LPS-stimulated eicosanoid production by NPC in the two groups (SE and CE) suggested the development of endotoxin tolerance in the CE group. However, an additional modulatory effect of ethanol was also suggested by our findings.

To the Editor: Studies of the metastatic behavior of cancer suggest that one of the factors influencing the site at which tumor cells lodge is the presence of a special “soil” that favors the survival and growth of these tumor cells. A variety of hormonal and growth factors have been shown to influence the expansion of a metastatic colony. One such factor is evidence that tumor cells and platelets interact to promote metastasis1. Tumor cells can activate platelets that undergo the release reaction and make available as yet unidentified mediators that promote tumor growth. In some models, the degree . . .

Arachidonic acid concentrations in liver are decreased in response to ethanol administration. In addition, the oxygenated products of arachidonic acid metabolites could affect the severity of alcoholic liver injury. Selective utilization of arachidonic acid by the cytochrome P-450 system could, in part, account for the decrease in arachidonic acid. To evaluate this pathway further, male Wistar rats were fed different dietary fats: medium chain triglycerides, palm oil, and corn oil or fish oil with either ethanol or isocaloric amounts of dextrose. Histopathology, cytochrome P-4502E1 (CYP2E1) and cytochrome P-4504A (CYP4A), and omega- and (omega-1)-hydroxylation products of lauric and arachidonic acids were evaluated. Ethanol induction of CYP2E1 was related to the concentration of polyunsaturated fatty acids in the diet; induction of CYP4A by ethanol was seen in all groups. The highest levels of 11-hydroxy-lauric acid and 19-hydroxyarachidonic acid (omega-1) were seen in rats fed ethanol with palm oil and corn oil. Highly significant correlations were seen between the (omega-1)-hydroxylation products and CYP2E1 activity. No correlation was seen between the omega-hydroxylation products and CYP2E1 activity. In contrast, the levels of omega-hydroxylation products correlated with CYP4A. The overall results showed a significant increase in (omega-1)-hydroxylation products in rats fed diets containing significant amounts of linoleic acid (i.e., palm oil and corn oil).

The authors evaluated the quality and reliability of four desktop analyzers in the outpatient clinic. Twenty-seven nontechnologists (NTs) participated in the study. These included nurses, physicians, and medical students. The instruments and tests evaluated were as follows: Reflotron (glucose, cholesterol, triglycerides, gamma-glutamyltransferase and urea); Seralyzer (creatinine, glucose, potassium, aspartate aminotransferase, and hemoglobin); Vision (glucose, urea, cholesterol, triglycerides, alkaline phosphatase, and uric acid); and DT60 (sodium, potassium, glucose, amylase, uric acid, bilirubin, and creatinine). For precision studies, low and high control material was used, and method comparison was done with methods in routine use in the laboratory. The range of coefficients of variation (CVs) for the analyzers with NTs was as follows: Reflotron: CV, 2.4-7.9%; Seralyzer CV, 1.4-18.7%; Vision: CV, 1.5-2.7%; DT60: CV, 2.5-46.8. The percentage results that is different by greater than 10% between the NTs and trained technologists was related to the complexicity of procedure for each analyzer and was the lowest for the Vision analyzer and greatest for the Seralyzer.

This article reviews male and female endocrine physiology and investigates male hypogonadism, amenorrhea, and male and female infertility.

A case of chlorambucil overdose is presented. The clinical manifestations were acute renal failure and seizures. We are not aware of this combination of clinical features being previously reported with chlorambucil overdose.

We evaluated whether arterial blood samples for pH and blood gas analysis need to be transported on ice. We found that although the changes in pH, pCO2 and pO2 were greater in samples kept at room temperature versus those kept on ice, the difference was probably not of clinical significance until the period of time after arterial puncture exceeded 20 minutes. We recommend that arterial blood samples do not need to be kept on ice if the analysis for pH and gases is performed within 20 minutes of blood being drawn.

We report a case of autoimmune hemolytic anemia, in a 28-year-old man with a warm-reacting IgG antibody with anti-Sdx activity. The unusual characteristics of the patient included the drama