Effect of Chronic Ethanol Administration on Protein Catabolism in Rat Liver

Hepatic protein catabolism was measured in rats which were pair-fed a liquid diet containing either ethanol or isocaloric maltose-dextrin (control diet). Within 12 days after initiation of pair feeding, the level of total hepatic protein in ethanol-fed rats was 26% higher than that in pair-fed control rats. During this time interval, the catabolic rates of both short-lived [3H]puromycin-labeled proteins and long-lived native [14C]bicarbonate-labeled proteins were measured in the two groups of animals. The degradation rate of short-lived [3H]puromycinyl proteins and peptides was the same in ethanol-fed and pair-fed control rats. However, the overall catabolic rate of long-lived proteins in rats fed the ethanol liquid diet for 2-10 days was 37-40% lower than that in pair-fed controls. This difference in protein turnover was not a general phenomenon, since the time-dependent decay of [14C]proteins in the hepatic microsome fraction of ethanol-fed rats was 33% slower than that in pair-fed controls, but the apparent rate of cytosolic protein catabolism was the same in both groups of animals. The differences in protein turnover did not reflect quantitative changes in lysosomal proteases since the activities of four hepatic lysosomal cathepsins were unaffected by alcohol administration. When rats were subjected to longer periods of pair feeding (16-25 days), the difference in overall hepatic protein catabolism between ethanol-fed rats and their pair-fed controls was considerably attenuated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ethanol Administration Alters the Proteolytic Activity of Hepatic Lysosomes

Protein accumulation in liver cells contributes to alcohol-induced hepatomegaly and is the result of an ethanol-elicited deceleration of protein catabolism (Alcohol Clin Exp Res 1349, 1989). Because lysosomes are active in the degradation of most hepatic proteins, the present studies were conducted to determine whether ethanol administration altered the proteolytic activities of partially purified hepatic lysosomes. Rats were fed liquid diets containing either ethanol (36% of calories) or isocaloric maltodextrin for periods of 2–34 days. Prior to death, all animals were injected with [³H]leucine to label hepatic proteins. Rats subjected to even brief periods of ethanol feeding (2–8 days) exhibited significant hepatomegaly and hepatic protein accumulation compared with pair-fed control animals. Crude liver homogenates and isolated lysosomal-mitochondrial and cytosolic subfractions were incubated at 37°C, and the acid-soluble radioactivity generated during incubation was measured as an index of proteolysis. At neutral pH, in vitro protein breakdown in incubated liver homogenates and subcellular fractions from control and ethanol-fed rats did not differ significantly. The extent of protein hydrolysis increased when samples were incubated at pH 5.5, which approximates the pH optimum for catalysis by lysosomal acid proteases. Under the latter conditions, partially purified lysosomes from control animals had 2-fold higher levels of proteolysis than corresponding fractions from ethanol-fed rats. The difference in proteolytic capacity appeared to be related to a lower latency and a higher degree of fragility of lysosomes from ethanol-fed rats at the acidic pH. The results suggest that ethanol-induced alterations in lysosomal membranes may be partially responsible for their altered capacities for protein hydrolysis. Such changes may result from ethanol-related alterations in lipid metabolism that may affect lysosome biogenesis or the maturation of lysosomes from autophagic vacuoles.     

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.     

Effects of chronic ethanol administration on hepatic glycoprotein secretion in the rat

The effects of chronic ethanol feeding on protein and glycoprotein synthesis and secretion were studied in rat liver slices. Liver slices from rats fed ethanol for 4-5 wk showed a decreased ability to incorporate [14C]glucosamine into medium trichloracetic acid-precipitable proteins when compared to the pair-fed controls; however, the labeling of hepatocellular glycoproteins was unaffected by chronic ethanol treatment. Immunoprecipitation of radiolabeled secretory (serum) glycoproteins with antiserum against rat serum proteins showed a similar marked inhibition in the appearance of glucosamine-labeled proteins in the medium of slices from ethanol-fed rats. Minimal effects, however, were noted in the labeling of intracellular secretory glycoproteins. Protein synthesis, as determined by measuring [14C]leucine incorporation into medium and liver proteins, was decreased in liver slices from ethanol-fed rats as compared to the pair-fed controls. This was the case for both total proteins as well as immunoprecipitable secretory proteins, although the labeling of secretory proteins retained in the liver slices was reduced to a lesser extent than total radiolabeled hepatic proteins. When the terminal sugar, [14C]fucose, was employed as a precursor in order to more closely focus on the final steps of hepatic glycoprotein secretion, liver slices obtained from chronic ethanol-fed rats exhibited impaired secretion of fucose-labeled proteins into the medium. When ethanol (5 or 10 mM) was added to the incubation medium containing liver slices from the ethanol-fed rats, the alterations in protein and glycoprotein synthesis and secretion caused by the chronic ethanol treatment were further potentiated. The results of this study indicate that liver slices prepared from chronic ethanol-fed rats exhibit both impaired synthesis and secretion of proteins and glycoproteins, and these defects are further potentiated by acute ethanol administration.     

Effects of ethanol feeding and withdrawal on plasma glutathione elimination in the rat

Chronic ethanol feeding increases hepatic turnover and sinusoidal efflux of glutathione in rats. The present study was performed to determine whether the observed increase in glutathione efflux was due to increased extrahepatic requirements for glutathione. The concentration and disposition of plasma glutathione were determined in rats fed liquid diets containing 36% of calories as ethanol or pair-fed an isocaloric mixture with carbohydrate replacing ethanol calories for 5 to 8 weeks. The half-life and plasma clearance of [35S]glutathione were found to be similar in ethanol-fed and control rats and in rats withdrawn 24 hr from ethanol. Uptakes of the sulfur moiety of [35S]glutathione by kidney, jejunal mucosa, liver, lung, spleen, muscle and heart were also unchanged by ethanol feeding. The plasma glutathione concentration was significantly higher in ethanol-withdrawn rats 22.30 +/- 3.06 nmoles per ml (p less than 0.05) compared to pair-fed controls (13.51 +/- 2.04), while rats continuing to drink ethanol had intermediate levels (16.96 +/- 2.22). Plasma cysteine levels were slightly, but not significantly, higher in ethanol-fed rats. These findings suggest that increased sinusoidal efflux of glutathione in ethanol-fed rats is due to a direct effect of ethanol on hepatic glutathione transport and not due to an alteration in extrahepatic disposition of glutathione. In order to characterize further the effects of ethanol feeding on glutathione-dependent detoxification, activities of glutathione S-transferase, glutathione reductase and gamma-glutamyltransferase were determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for enhanced secretory function of hepatic macrophages after long-term ethanol feeding in rats.

Rats were pair-fed nutritionally adequate liquid diets, containing ethanol as 36% of energy or an isocaloric amount of carbohydrate for 4-6 weeks. Ruffle formation of hepatic macrophages in the periportal area observed with a transmission electron microscope (which reflects their extent in activation) was more remarkable in ethanol-fed rats than in control rats. The ability of hepatic macrophages to produce superoxide anions assessed in situ by formazan deposition after liver perfusion with nitro-blue tetrazolium and phorbol myristate acetate was enhanced after such ethanol feeding. A similar result was seen 24 h after withdrawal of ethanol feeding. These findings suggest that long-term ethanol consumption may activate hepatic macrophages in secretory function.     

Evidence for enhanced secretory function of hepatic macrophages after long-term ethanol feeding in rats

ABSTRACT— Rats were pair-fed nutritionally adequate liquid diets, containing ethanol as 36% of energy or an isocaloric amount of carbohydrate for 4–6 weeks. Ruffle formation of hepatic macrophages in the periportal area observed with a transmission electron microscope (which reflects their extent in activation) was more remarkable in ethanol-fed rats than in control rats. The ability of hepatic macrophages to produce superoxide anions assessed in situ by formazan deposition after liver perfusion with nitro-blue tetrazolium and phorbol myristate acetate was enhanced after such ethanol feeding. A similar result was seen 24 h after withdrawal of ethanol feeding. These findings suggest that long-term ethanol consumption may activate hepatic macrophages in secretory function.     

Ethanol-Associated Alterations in the Kinetics of Putrescine Uptake and Metabolism by the Regenerating Liver

Biosynthesis of the polyamines, putrescine, spermidine, and spermine is required for DNA synthesis and liver regeneration after partial hepatectomy. We have previously reported that chronic ethanol consumption impairs polyamine synthesis and significantly retards liver regeneration after partial hepatectomy. In those studies, supplementation with putrescine restored hepatic DNA synthesis in ethanol-fed rats but exerted no effect in pair-fed controls. These differences in the response to putrescine treatment may have resulted from ethanol-associated differences in hepatic uptake, release, or metabolism of putrescine. To resolve these issues and define more completely how putrescine treatment affects DNA synthesis, we now assess the kinetics of putrescine uptake and metabolism after intraperitoneal or intravenous injection of radiolabeled putrescine (1.2 mmol/kg, specific activity 1 microCi/mmol) into rats fed 36% ethanol diets or isocaloric, nonethanol diets for 6 weeks prior to partial hepatectomy. After putrescine treatment, hepatic putrescine concentrations were greater in ethanol-fed rats than controls. Differences in post-treatment hepatic putrescine levels between ethanol and pair-fed groups could not be explained by differences in the rates of hepatic putrescine uptake or excretion into bile; residual de novo synthesis of putrescine from ornithine or metabolism of hepatic putrescine to its polyamine products, spermidine and spermine. Indeed, supplemental putrescine was not appreciably converted to spermidine or spermine in either ethanol or control rats. Hence, these latter polyamines are unlikely to be responsible for the treatment-associated improvement in DNA synthesis that has been noted in ethanol-fed rats. This suggests that putrescine itself acts to restore hepatic DNA synthesis in ethanol-fed rats.     

Chronic ethanol consumption alters the glutathione/glutathione peroxidase-1 system and protein oxidation status in rat liver

BACKGROUND: Alcohol-induced liver damage is associated with oxidative stress, which might be linked to disturbances in liver antioxidant defense mechanisms. The effect of chronic ethanol consumption on the mitochondrial and cytosolic glutathione/glutathione peroxidase-1 (GSHPx-1) system and oxidative modification of proteins was therefore studied in the rat. METHODS: Male Sprague-Dawley rats were fed liquid diets that provided 36% total calories as ethanol for at least 31 days. Pair-fed controls received isocaloric diets with ethanol calories substituted with maltose-dextrins. Mitochondrial and cytosolic fractions were prepared from livers and assayed for GSHPx-1 and glutathione reductase activities and total and oxidized concentrations of glutathione. Catalase activity was measured in the postmitochondrial supernatant. Levels of GSHPx-1, lactate dehydrogenase, and the beta subunit of the F1 portion of the ATP synthase protein were determined by western blot analysis. Concentrations of mitochondrial and cytosolic protein carbonyls were measured to assess ethanol-induced oxidation of proteins. RESULTS: Chronic ethanol consumption significantly decreased cytosolic and mitochondrial GSHPx-1 activities by 40% and 30%, respectively. Levels of GSHPx-1 protein in cytosol were unaffected by ethanol feeding, whereas there was a small decrease in GSHPx-1 protein levels in mitochondria isolated from ethanol-fed rats. Glutathione reductase activities were increased in both intracellular compartments and catalase activity was increased as a consequence of ethanol exposure. Cytosolic total glutathione was mildly decreased, whereas ethanol feeding increased mitochondrial levels of total glutathione. Chronic ethanol feeding significantly increased both cytosolic and mitochondrial concentrations of protein carbonyls by 30% and 60%, respectively. CONCLUSIONS: This study demonstrates that chronic ethanol-induced alterations in the glutathione/GSHPx-1 antioxidant system might promote oxidative modification of liver proteins, namely those of the mitochondrion, which could contribute to the adverse effects of ethanol on the liver.     

Hepatic thyroid hormone levels following chronic alcohol consumption: direct experimental evidence in rats against the existence of a hyperthyroid hepatic state

To study the effect of chronic alcohol consumption on hepatic levels of thyroid hormones, female Sprague-Dawley rats (n = 24) were pair-fed nutritionally adequate liquid diets containing either ethanol (36% of total calories) or isocaloric carbohydrates for 21 days. Compared to controls, chronic alcohol consumption failed to result in a significant change of hepatic thyroid hormone levels (thyroxine: 14.7 +/- 1.81 ng per gm of liver wet weight vs. 15.0 +/- 1.59; triiodothyronine: 2.60 +/- 0.16 ng per gm of liver wet weight vs. 2.66 +/- 0.18). Similar results were obtained when the hepatic levels of thyroid hormones were expressed per total liver, per gram of liver protein or per 100 gm of body weight. Moreover, prolonged alcohol ingestion led to a significant reduction of serum total thyroxine by 31.6% (p less than 0.001), free thyroxine by 38.9% (p less than 0.02), total triiodothyronine by 40.2% (p less than 0.001) and free triiodothyronine by 56.1% (p less than 0.001) when compared to their pair-fed controls, whereas thyroid-stimulating hormone levels remained virtually unchanged. These data, therefore, clearly show that chronic alcohol consumption is incapable of creating a hyperthyroid hepatic state in rats, and limit the rationale for antithyroid treatment in patients with alcoholic liver disease.     

Hepatic Adenosine in Rats Fed Ethanol: Effect of Acute Hyperoxia or Hypoxia

The level of adenosine was measured in monthly biopsied livers from rats fed ethanol and a high fat/low protein diet in order to test a hypothesis that hepatic adenosine is increased due to enhanced breakdown of adenine nucleotides in which ATP and total adenylate pool were decreased by chronic ethanol feeding. The ethanol-fed rats showed a significantly higher average level of adenosine compared to the pair-fed controls. When investigated monthly, however, adenosine in ethanol-fed rats increased only after the decrease in ATP had stabilized and AMP remained unchanged, indicating that these changes were not temporarily related. The average percentage of change in adenosine after acute hyperoxia or hypoxia were variable both in ethanol-fed and pair-fed rats. There was a tendency for a positive correlation between the percentage of change of adenosine and AMP after hyperoxia regardless of ethanol feeding. A negative correlation between the percentage of change of adenosine and energy charge, and a positive correlation between the percentage of change of adenosine and AMP were seen after hypoxia regardless of ethanol feeding. Adenosine levels changed rapidly in response to changes in systemic of pO2 in both the ethanol-fed and control rats, indicating that the liver maintained its normal response to the changes in energy state. The results indicate that chronic ethanol feeding does increase the level of adenosine in the liver and that this level remains responsive to acute changes in pO2.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Chronic ethanol consumption alters rat liver plasma membranes and potentiates release of alkaline phosphatase

The aim of this study was to investigate possible mechanisms involved in the elevation of serum alkaline phosphatase activity in alcoholics. Male Sprague-Dawley rats were pair-fed nutritionally adequate liquid diets containing ethanol as 36% of energy or an isocaloric amount of carbohydrate for 4-5 wk. Serum alkaline phosphatase activity was increased moderately but significantly. Hepatocytes isolated from ethanol-fed animals exhibited pronounced morphologic alterations of their plasma membranes by scanning electron microscopy and a reduced content of alkaline phosphatase despite an increase in total liver alkaline phosphatase content. Chronic ethanol feeding also potentiated the release of alkaline phosphatase from the cells during incubation with 50 mM ethanol. Furthermore, chronic ethanol feeding resulted in reduced recovery of alkaline phosphatase in hepatic plasma membranes isolated by sucrose gradient centrifugation but did not affect the recoveries of other plasma membrane markers (5'-nucleotidase and Na+,K+-adenosine triphosphatase) nor the subcellular distribution of alkaline phosphatase in the nuclear, mitochondrial, microsomal, and cytosolic fractions. These findings suggest that the increased serum alkaline phosphatase levels observed in response to chronic ethanol feeding may be due, at least in part, to increased lability of this plasma membrane enzyme.     

Ethanol-induced changes in hepatic free radical defense mechanisms and fatty-acid composition in the miniature pig

In the miniature pig, ethanol consumption has been reported to induce alterations in hepatic antioxidant defense capacity, which could result in increased risk of peroxidative damage. However, ethanol may also induce changes in membrane fatty acid composition, which could reduce the risk of peroxidative damage. This study examined lipid peroxidation, antioxidant defense and fatty acid composition in livers from miniature pigs fed ethanol in diets containing 12% of their calories as fat for 20 mo. After 12 and 20 mo of feeding, ethanol-fed pigs had higher hepatic manganese-superoxide dismutase activity, lower hepatic copper concentrations and low hepatic copper-zinc-superoxide dismutase and glutathione peroxidase activities compared with controls. Lipid peroxidation as assessed by thiobarbituric acid reacting substance assay was lower in liver homogenate and mitochondrial and microsomal fractions from ethanol-fed pigs than in controls. The percentage contribution of highly unsaturated fatty acids to total fatty acids in liver homogenates (after 12 mo of feeding) and microsome fractions (after 20 mo of feeding) was lower in the ethanol-fed pigs than in the controls, resulting in a lower peroxidizability index. Ethanol-fed pigs had minimal or no hepatic damage as assessed by histological methods. We suggest that the relative resistance of microsomes to lipid peroxidation is due to the lower peroxidizability index in the ethanol-fed pigs and may account in part for the absence of significant histopathological findings after 20 mo of ethanol feeding.     

Lysosomal leakage and lack of adaptation of hepatoprotective enzyme contribute to enhanced susceptibility to ethanol-induced liver injury in female rats

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.     

Effect of chronic ethanol ingestion on hepatic and intestinal acyl coenzyme a:cholesterol acyltransferase and 3-hydroxy-3-methylglutaryl coenzyme a reductase in the rat

Two groups of rats were pair-fed diets in which 36% of the calories were provided by either ethanol or dextrimaltose. After 60 days on these liquid diets, rats fed ethanol were significantly smaller than control rats fed dextrimaltose. Serum cholesterol levels in ethanol-fed animals were 20% higher than control rats. Cholestasis was not observed histologically, and serum alkaline phosphatase and bilirubin levels were the same in both groups. The livers of animals ingesting ethanol accumulated triglycerides and cholesterol. The increase in cholesterol was due to an increase in cholesteryl esters. The cholesterol content of liver microsomes, however, was unchanged by ethanol feeding. A small increase in unesterified cholesterol was observed in intestinal microsomes prepared from animals receiving ethanol. Microsomal fatty acids in liver and intestine were unchanged by the ethanol diet. Chronic ingestion of ethanol in these animals failed to change acyl coenzyme A:cholesterol acyltransferase or 3-hydroxy-3-methylglutaryl-coenzyme A reductase activities in the intestine. In contrast, the activities of acyl coenzyme A:cholesterol acyltransferase and 3-hydroxy-3-methylglutaryl-coenzyme A reductase were significantly increased in the livers of rats receiving ethanol. Thus, the chronic ingestion of ethanol caused a marked accumulation of hepatic cholesteryl esters. This was associated with a significant increase in the activities of enzymes that control the rates of both cholesterol synthesis and cholesterol esterification in the liver. These observed changes in enzyme activities may contribute to the lipid accumulation which occurs in these livers. Chronic ethanol consumption did not alter cholesterol metabolism in the intestine.     

Development of Tolerance to Inhibitory Effect of Ethanol on Endothelium-dependent Vascular Relaxation in Ethanol-fed Rats

Rats were maintained on liquid diets containing ethanol (35% of total calories) or an equicaloric volume of sucrose instead of ethanol for 10 wk. Vascular strips of isolated rat aortas were mounted in organ chambers to record isometric tension. Ethanol in vitro inhibited the endothelium-dependent relaxation responses to acetylcholine and ATP in both pair-fed control and ethanol-fed rats. The inhibitory effect of ethanol was greater in the pair-fed rats. In addition, the magnitudes of these relaxation responses in the absence of ethanol in vitro in pair-fed rats were similar to those in the presence of ethanol in ethanol-fed rats. In the absence of ethanol in vitro, the relaxations in response to acetylcholine and ATP in the ethanol-fed rats were greater than in the pair-fed rats. These results suggest that chronic ethanol consumption can induce tolerance to ethanol-induced inhibition of endothelium-dependent relaxation responses to acetylcholine and ATP, and that the relaxations can become adapted to the presence of plasma levels of ethanol, which may inhibit the relaxation in vivo. The augmented relaxation in the ethanol-fed rats may result from the mechanism causing tolerance to the inhibitory effect of ethanol.     

Depletion of liver and esophageal epithelium vitamin A after chronic moderate ethanol consumption in rats: inverse relation to zinc nutriture

This study was designed to determine whether chronic moderate ethanol ingestion alters the levels of vitamin A of liver and esophageal epithelium and if this is dependent on zinc nutriture. Forty male Sprague-Dawley 4-week-old rats were divided into five groups: zinc-deficient (0.9 ppm), ethanol-fed; zinc-deficient; zinc-adequate (25 ppm); zinc-adequate (25 ppm), ethanol-fed; and zinc-supplemented (50 ppm), ethanol-fed. All rats received liquid Lieber-DeCarli diet containing 4,000 IU per liter of vitamin A for 5 weeks. Zinc-deficient, ethanol-fed rats and zinc-adequate, ethanol-fed rats and zinc-supplemented, ethanol-fed rats received 15.5% of the caloric intake as ethanol while zinc-deficient and zinc-adequate rats received isocaloric amounts of maltose dextrin. All groups were pair-fed to zinc-deficient, ethanol-fed rats. In addition, a group of eight rats designated as weight-restricted controls were fed a diet similar to the one given to zinc-adequate rats but in the amount to obtain a final weight as in the zinc-deficient group. After 35 days, the liver histology was normal in all rats, and no fat accumulation was noted. Hepatic vitamin A concentration was significantly decreased in zinc-adequate, ethanol-fed rats (41 +/- 10 micrograms per gm) and further in zinc-supplemented, ethanol-fed rats (12 +/- 5 micrograms per gm) as compared to controls (137 +/- 49). A highly significant negative correlation between serum zinc and liver vitamin A was found in ethanol-fed animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatic mitochondrial glutathione depletion and progression of experimental alcoholic liver disease in rats.

Long-term ethanol feeding has been shown to selectively reduce hepatic mitochondrial glutathione content by impairing mitochondrial uptake of this thiol. In this study, we assessed the role of this defect in evolution of alcoholic liver disease by examining the mitochondrial glutathione pool and lipid peroxidation during progression of experimental alcoholic liver disease to centrilobular liver necrosis and fibrosis. Male Wistar rats were intragastrically infused with a high-fat diet plus ethanol for 3, 6 or 16 wk (the duration that resulted in induction of liver steatosis, necrosis and fibrosis, respectively). During this feeding period, the cytosolic pool of glutathione remained unchanged in the ethanol-fed animals compared with that in pair-fed controls. In contrast, the mitochondrial pool of glutathione selectively and progressively decreased in rats infused with ethanol for 3, 6 or 16 wk, by 39%, 61% and 85%, respectively. Renal mitochondrial glutathione level remained unaffected throughout the experiment. Serum ALT levels increased significantly in the ethanol-fed rats at 6 wk and remained elevated at 16 wk. In the mitochondria with severely depleted glutathione levels at 16 wk, enhanced lipid peroxidation was evidenced by increased malondialdehyde levels. Thus a progressive and selective depletion of mitochondrial glutathione is demonstrated in the liver in this experimental model of alcoholic liver disease and associated with mitochondrial lipid peroxidation and progression of liver damage.