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 ethanol-fed 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 betaine-lacking 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.     

Wünderlich syndrome caused by metastatic gastric sarcoma in the kidney. Report of a case

Two of the most important biochemical hepatic pathways in the liver are those that synthesize methionine and S-adenosylmethionine (SAM) through the methylation of homocysteine. This article reviews some recent findings in this laboratory, which demonstrate that ethanol feeding to rats impairs one of these pathways involving the enzyme methionine synthetase (MS), but by way of compensation increases the activity of the enzyme betaine:homocysteine methyl transferase (BHMT), which catalyzes the second pathway in methionine and SAM biosynthesis. It has been shown that despite the inhibition of MS, the enhanced BHMT pathway utilizes hepatic betaine pools to maintain levels of SAM. Subsequent to the above findings, it has been shown that minimal supplemental dietary betaine at the 0.5% level generates SAM twofold in control animals and fivefold in ethanol-fed rats. Concomitant with the betaine-generated SAM, ethanol-induced hepatic fatty infiltration was ameliorated. In view of the fact that SAM has already been used successfully in the treatment of human maladies, including liver dysfunction, betaine, shown to protect against the early stages of alcoholic liver injury as well as being a SAM generator, may become a promising therapeutic agent and a possible alternative to expensive SAM in the treatment of liver disease and other human maladies.     

Altered T-lymphocyte responsiveness to polyclonal cell activators is responsible for liver cell necrosis in alcohol-fed rats

The role of T-cell activation in alcoholic liver disease was investigated in rats fed alcohol and subsequently exposed to concanavalin A (Con A). Following Con A injection (20 mg/kg body weight), greater increases in liver-to-body weight ratio and ALT levels were observed at 12 and 24 hr in rats fed ethanol, compared with control rats fed sucrose. Furthermore, increases in serum interleukin-6 and tumor necrosis factor-alpha levels were noted in ethanol-fed rats, with maximal levels detected at 4 hr declining thereafter, but remaining above control levels at 24 hr. Analysis of T-cell subpopulations showed an increased percentage of CD4+, CD5+, and CD8+ T cells in blood from all groups, but not in liver perfusate. In contrast, a significant increase in the percentage of activated CD25+ T cells was detected in both blood and liver perfusate from rats fed ethanol even 24 hr after Con A injection. When CD4+ and CD8+ T cells from liver perfusate were cultured in the absence or presence of Con A, an increase in interleukin-6 and tumor necrosis factor-alpha production in supernatants was observed in ethanol-fed rats. In cultures stimulated with Con A, a 2- to 8-fold increase in cytokine production was detected, with intrahepatic CD4+ T cells being the major source. Immunohistological analysis revealed infiltration of CD4+ T cells around portal vein and central vein areas associated with fatty liver and severe hepatic necrosis. The results suggest that alcohol consumption induced a dysregulated T-cell population that mediated hepatic necrosis following polyclonal activation with Con A.     

Transport of reduced glutathione in hepatic mitochondria and mitoplasts from ethanol-treated rats: effect of membrane physical properties and S-adenosyl-L-methionine

Ethanol intake depletes the mitochondrial pool of reduced glutathione (GSH) by impairing the transport of GSH from cytosol into mitochondria. S-Adenosyl-L-methionine (SAM) supplementation of ethanol-fed rats restores the mitochondrial pool of GSH. The purpose of the current study was to determine the effect of ethanol feeding on the kinetic parameters of mitochondrial GSH transport, the fluidity of mitochondria, and the effect of SAM on these changes. Male Sprague-Dawley rats were fed ethanol-liquid diet for 4 weeks supplemented with either SAM or N-acetylcysteine (NAC). SAM-supplementation of ethanol-fed rats restored the mitochondrial GSH pool but NAC administration did not. Kinetic studies of GSH transport in isolated mitochondria revealed two saturable, adenosine triphosphate (ATP)-stimulated components that were affected significantly by chronic ethanol feeding: lowering Vmax (0.22 and 1.6 in ethanol case vs. 0.44 and 2.7 nmol/15 sec/mg protein in controls) for both low and high affinity components with the latter showing an increased Km (15.5 vs. 8.9, mmol/L in ethanol vs. control). Mitochondria from SAM-supplemented ethanol-fed rats showed kinetic features of GSH transport similar to control mitochondria. Determination of membrane fluidity revealed an increased order parameter in ethanol compared with control mitochondria, which was restricted to the polar head groups of the bilayer and was prevented by SAM but not NAC supplementation of ethanol-fed rats. The changes elicited in mitochondria by ethanol were confined to the inner membrane; mitoplasts from ethanol-fed rats showed features similar to those of intact mitochondria such as impaired transport of GSH and increased order parameter. A different mitochondrial transporter, adenosine diphosphate (ADP)/ATP translocator, was unaffected by ethanol feeding. Furthermore, fluidization of mitochondria or mitoplasts from ethanol-fed rats by treatment with a fatty acid derivative restored their ability to transport GSH to control levels. Thus, ethanol-induced impaired transport of GSH into mitochondria is selective, mediated by decreased fluidity of the mitochondrial inner membrane, and prevented by SAM treatment.     

Upper airway muscle activity and upper airway resistance in young adults during sleep

Ethanol consumption slows down the rate of hepatic protein catabolism. The present study was conducted to determine whether ethanol consumption, given by voluntary (pair) feeding or by intragastric administration, affected the peptidase activities of the proteasome in rat liver. Rats were pair-fed liquid diets containing either ethanol or isocaloric maltose-dextrin. A separate group of animals was intragastrically infused continuously with similar liquid diets containing either ethanol or isocaloric dextrose. Crude liver homogenates and their cytosolic fractions were assayed for their chymotrypsin-like (Cht-L), trypsin-like (T-L), and peptidyl-glutamyl-peptide hydrolase (PGPH) activities, using specific fluorogenic peptides as substrates. Voluntary ethanol feeding did not affect the three peptidase activities of the proteasome. However, intragastric ethanol administration caused a 35% to 40% decline in the Cht-L and the T-L activities, but did not significantly change the PGPH activity. The lower peptidase activities in cytosol samples from intragastrically ethanol-fed rats were not restored to control levels by overnight dialysis, nor by the inclusion of low levels of sodium dodecyl sulfate (SDS) or of 0.5 mmol/L adenosine triphosphate (ATP) in the proteasome assay mixture. Immunoblot analyses using anti-rat liver proteaseome exhibited equal levels of immunoreactive proteasome subunits in livers of control and ethanol-fed rats. Similar results were obtained when blots were probed with antibody made specifically against the proteasome subunit, LMP-7. The results indicate that intragastric, but not voluntary, ethanol consumption differentially affects the separate catalytic activities of the proteasome without affecting its steady-state levels. Such changes may be related to the degree of ethanol-induced oxidative stress.     

Effects of ethanol administration on components of the ubiquitin proteolytic pathway in rat liver

Hepatic protein accumulation during ethanol administration may result partly from an ethanol-elicited decline in hepatic protein degradation, which we have previously shown. We conducted the current studies to examine the effects of ethanol administration on the levels of hepatic ubiquitin, an 8.5-kd protein which is an important mediator of extralysosomal protein catabolism. Rats were pair-fed liquid diets containing either ethanol (36% of calories) or isocaloric maltose-dextrin for 1 to 5 weeks. Ubiquitin was immunochemically quantified by competitive enzyme-linked immunosorbent assay (ELISA) in crude cytosol fractions from whole liver and in 12,000g supernatants of hepatocyte lysates. Ubiquitin levels in hepatic cytosol fractions of ethanol-fed rats exceeded those of controls by about 30%. Isolated hepatocytes from ethanol-fed animals also showed a 40% to 75% elevation of ubiquitin above that in cells of pair-fed controls and this difference exceeded the relative rise in hepatocellular protein. In hepatocyte lysates subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, we detected monomeric ubiquitin and higher molecular mass ubiquitin-protein conjugates. However, the immunoblot analyses revealed no quantitative changes in the level of either free or conjugated ubiquitin. The ubiquitin conjugating activity of crude and diethyl aminoethyl-fractionated liver cytosols of ethanol-fed rats had equal capacities to those from controls in catalyzing the formation of ubiquitin-protein conjugates. Our findings indicate that chronic ethanol consumption increased the level of immunoreactive ubiquitin in rat liver. This may have resulted from enhanced ubiquitin production because of an ethanol-elicited stress response and/or decreased catabolism of ubiquitin and its conjugates. Our findings also provide no indication that the ethanol-elicited reduction in hepatic proteolysis is because of a ubiquitin-mediated mechanisms.     

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 13:49, 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 degrees 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.(ABSTRACT TRUNCATED AT 250 WORDS)     

Filgrastim restores interleukin-2 production in blood from patients with advanced human immunodeficiency virus infection

BACKGROUND & AIMS: Malondialdehyde and acetaldehyde react together with proteins and form hybrid protein conjugates designated as MAA adducts, which have been detected in livers of ethanol-fed rats. The aim of this study was to examine the immune response to MAA adducts and other aldehyde adducts during long-term ethanol exposure. METHODS: Rats were pair-fed for 7 months with a liquid diet containing either ethanol or isocaloric carbohydrate. Circulating antibody titers against MAA adducts and acetaldehyde adducts were measured and characterized in these animals. RESULTS: A significant increase in antibody titers against MAA-adducted proteins was observed in the ethanol-fed animals. Competitive inhibitions of antibody binding indicated that the circulating antibodies against MAA-modified proteins in the ethanol-fed rats recognized mainly a specific, chemically defined MAA epitope. Antibody titers to reduced and nonreduced acetaldehyde adducts were very low, and no significant differences were observed between ethanol-fed and control animals. Significant plasma immunoreactivity to not only MAA-adducted but also unmodified rat liver proteins (cytosol, microsomes, and especially plasma membrane) were also observed in the ethanol-fed rats. CONCLUSIONS: Long-term ethanol feeding generates circulating antibodies not only against MAA epitopes but possibly also against unmodified, native (self) protein epitopes, suggesting that MAA adducts could trigger harmful autoimmune responses.     

Iron increases ethanol toxicity in rat liver

Clinical evidence indicates that patients with iron overload are more susceptible to liver cell damage from alcohol than persons with normal iron stores. Iron may act as a co-factor to catalyze the lipid peroxidation induced by hepatotoxic compounds such as alcohol. To elucidate the role of iron in ethanol-induced hepatocellular damage, we developed a new experimental model in the rat. Following dietary carbonyl iron feeding for 8 weeks, animals were pair-fed a liquid ethanol diet for 4 weeks. In iron-fed animals the liver iron content was 6.4 vs. 0.5 micrograms Fe/mg protein in the controls. Blood alcohol concentrations were similar in all ethanol-fed animals. Serum alanine aminotransferase (ALT) levels were elevated to 269 +/- 49 U/l in the iron+alcohol group compared to 52 +/- 6 U/l in the other groups. There was a strong correlation between ALT levels and hepatic iron content in the ethanol-fed animals. Morphologically, the alcohol-fed rats displayed hepatic steatosis, whereas occasional inflammation and iron in Kupffer cells was seen in the iron+alcohol animals. Ultrastructurally, necrotic hepatocytes and cells phagocytosed by Kupffer cells were only encountered in the iron+alcohol group. Compared to controls, the liver content of hydroxyproline was significantly increased in the iron+alcohol group. No morphological evidence of fibrosis was noted. The present study demonstrates biochemical and morphological evidence of increased hepatocellular damage following the combination of iron and ethanol.     

Hepatotoxicity induced by iron overload and alcohol. Studies on the role of chelatable iron, cytochrome P450 2E1 and lipid peroxidation

BACKGROUND/AIMS: Clinical experience and studies with experimental animal models indicate a synergistic hepatotoxic effect of dietary iron overload and chronic alcohol ingestion. In order to elucidate the mechanism underlying this synergism, we examined the hepatic levels of ethanol-inducible cytochrome P450 2E1, glutathione and malondialdehyde, and the effect of iron chelation with desferrioxamine, in livers from rats treated with iron and/or ethanol. METHODS: Animals received diets with or without 2.5-3% carbonyl iron for 6-9 weeks, followed by an ethanol-containing diet or a liquid control diet for 5-9 weeks. Desferrioxamine was administered subcutaneously with mini-osmotic pumps. Alanine aminotransferase activity in serum and hepatic contents of glutathione and malondialdehyde were determined. The hepatic level of cytochrome P450 2E1 was determined with Western Blotting using a specific polyclonal antibody. RESULTS: The combination of iron and alcohol led to a marked increase in serum alanine aminotransferase activity as compared with all other treatment groups, and iron chelation with desferrioxamine reversed these increases. Treatment with alcohol alone led to slightly increased aminotransferases compared with controls. The level of cytochrome P450 2E1 was significantly elevated in microsomes isolated from ethanol-treated rats, but neither additional iron supplementation nor desferrioxamine influenced this level significantly. Glutathione contents were increased in the livers of animals treated with iron and/or ethanol. Malondialdehyde values were increased in iron-treated animals, whereas neither ethanol nor desferrioxamine altered malondialdehyde levels significantly. CONCLUSIONS: The toxic effects exerted by the combination of iron overload and chronic ethanol feeding on rat liver are dependent on a pool of chelatable iron. The hepatic level of cytochrome P450 2E1 is markedly induced by ethanol but not further altered by iron overload. Neither increased lipid peroxidation nor depletion of hepatic glutathione levels can explain the synergistic hepatotoxic effects of iron and ethanol in this model.     

Early alcoholic liver injury: formation of protein adducts with acetaldehyde and lipid peroxidation products, and expression of CYP2E1 and CYP3A

The formation of protein adducts with reactive aldehydes resulting from ethanol metabolism and lipid peroxidation has been suggested to play a role in the pathogenesis of alcoholic liver injury. To gain further insight on the contribution of such aldehydes in alcoholic liver disease, we have compared the appearance of acetaldehyde, malondialdehyde, and 4-hydroxynonenal adducts with the expression of cytochrome P-450IIE1, and cytochrome P-4503A enzymes in the liver of rats fed alcohol with a high-fat diet for 2 to 4 weeks according to the Tsukamoto-French procedure and in control rats (high-fat liquid diet or no treatment). Urine alcohol and serum aminotransferase levels were recorded, and the liver pathology was scored from 0 to 10 according to the presence of steatosis, inflammation, necrosis, and fibrosis. The ethanol treatment resulted in the accumulation of fat, mild necrosis and inflammation, and a mean liver pathology score of 3 (range: 1 to 5). Liver specimens from the ethanol-fed animals with early alcohol-induced liver injury were found to contain perivenular, hepatocellular acetaldehyde adducts. Malondialdehyde and 4-hydroxynonenal adducts were also present showing a more diffuse staining pattern with occasional sinusoidal reactions. In the control animals, a faint positive reaction for the hydroxynonenal adduct occurred in some of the animals fed the high fat diet, whereas no specific staining was observed in the livers from the animals receiving no treatment. Expression of both CYP2E1 and CYP3A correlated with the amount of protein adducts in the liver of alcohol-treated rats. Distinct CYP2E1-positive immunohistochemistry was seen in 3 of 7 of the ethanol-fed animals. In 5 of 7 of the ethanol-fed animals, the staining intensities for CYP3A markedly exceeded those obtained from the controls. The present findings indicate that acetaldehyde and lipid peroxidation-derived adducts are generated in the early phase of alcohol-induced liver disease. The formation of protein adducts appears to be accompanied by induction of both CYP2E1 and CYP3A.     

Some comments on the urethral syndrome

Studies were made on the mechanism by which livers of ethanol-treated rats take up an increased fraction of the total flux of unesterified fatty acid in serum. It was found that ethanol (0.7g/kg) causes a twofold rise in the serum content of liver, and that this serum is in rapid equilibrium with the general circulation. The fractional hepatic uptake from serum of group of compounds with varying uptake mechanisms and metabolic fates was studied in control and ethanol-treated animals. All the compounds tested, including unesterified fatty acid, showed an enhanced uptake when ethanol was given. For one of the compounds, carbon tetrachloride, a dose/response relationship was established between the amount administered, the amount taken up by liver, and the amount metabolized. These findings were interpreted to mean that this dose of ethanol causes the liver to receive an increased flow of blood, and as a result all compounds present and capable of being taken by liver are taken up at an increased rate. Hepatic blood flow was measured by a technique that monitors the rate of clearance of a colloidal lipid emulsion. It was found that ethanol increased hepatic blood flow by about 60%. This effect of ethanol on hepatic blood flow provides an explanation for the fatty liver and the synergistic effect between an acute dose of ethanol and carbon tetrachloride. A hypothesis to explain why a moderate dose of ethanol causes triglyceride to accumulate in liver is presented.     

Effect of glycerol on lipogenic enzyme activities and on fatty acid synthesis in the rat and chicken

The influence of glycerol on the rates of fatty acid snythesis in liver slices from rats and chickens in pieces of adipose tissue from rats was first studied. Then the effect of dietary glycerol on lipid metabolism in rats and cheickens was examined. Media containing 3 or 10 mM glycerol depressed the rate of glucose conversion to fatty acids in rat liver slices. However, media containing up to 25 mM glycerol did not influence the rate of fatty acid synthesis in chick liver slices. The inhibitory action of glycerol in rat liver slices might occur at the level of glucose (or glycogen) conversion to pyruvate because glycerol did not inhibit pyruvate or acetate conversion to fatty acids. Rats and chickens were fed glycerol containing diets for either 3 days or 3 weeks. Feeding diets containing 20.5 parts glycerol (22% of dietary energy) to rats or chickens did not influence the growth rate of the animals. However, substitution of 42.2 parts glycerol (43% of dietary energy) for glucose in the diet significantly depressed food intake and growth rate in both rats and chickens. The activities of citrate cleavage enzyme, fatty acid synthetase and malic enzyme in livers of rats fed the glycerol-containing diets were dramatically increased. However, this stimulation of enzyme activity occurred without a concomitant increase in the in vivo rate of fatty acid synthesis in the rat liver. In the chicken, unlike the rat, dietary glycerol did not stimulate but instead decreased hepatic malic enzyme and fatty acid synthetase activities. No significant differences in adipose tissue lipogenic enzyme activities or in the rates of fatty acid synthesis were observed in rats fed glycerol-containing diets. The lipogenic response to glycerol feeding depends on the species as well as the organ.     

Identification and characterisation of a superoxide dismutase and catalase from Mycobacterium ulcerans

This study investigated the role of Kupffer cells on survival and graft injury in transplanted fatty livers from rats treated acutely with ethanol. Donor rats were given ethanol (5 g/kg, by mouth) 20 hours before explantation, and liver grafts were preserved in University of Wisconsin cold storage solution for 24 to 42 hours prior to implantation. Blood samples were taken from the inferior vena cava for 3 hours after implantation. During this time, serum aspartate transaminase levels increased gradually from 122 U/L to 597 U/L in control rats, while ethanol treatment elevated values to 2,278 U/L. Gadolinium chloride (20 mg/kg, given intravenously to recipients 24 hours before explantation), a selective inactivator of Kupffer cells, minimized the increase in aspartate transaminase levels significantly. After implantation of grafts cold-stored for 42 hours, survival rates were 88% in control rats but only 33% in ethanol-treated rats. Gadolinium chloride improved survival nearly to control values. Ethanol nearly doubled white blood cell adhesion, an effect also largely blocked by gadolinium chloride. Further, alpha-(4-pyridyl 1-oxid)-N-tert-butylnitrone radical adducts detected in the bile were increased twofold by ethanol treatment. This effect was also reversed by gadolinium chloride. Taken together, these data indicate that survival is poorer and graft injury is greater in fatty livers from ethanol-treated rats. Inactivation of Kupffer cells minimized graft damage, most likely by improving hepatic microcirculation and diminishing lipid peroxidation.     

The effect of therapeutic doses of paracetamol on liver function in the rat perfused liver

The isolated liver perfusion technique was used to study the effect of therapeutic doses of paracetamol on hepatic gluconeogenesis and bromosulphthalein clearance from the perfusate and biliary excretion of the dye in the rat. Six groups of rats were studied; those in the three experimental groups were given 0.02 g kg-1 paracetamol daily for ninety days. The livers of animals in the control group and in one of the experimental groups were perfused with a medium containing pyruvate. The animals in the second experimental and control group were perfused with a medium containing bromosulphthalein (10 mg/100 mL). The livers of the third experimental and control group were subjected to histological examination. The rate of glucose formation and glucose concentrations were decreased, while, lactate levels and lactate: pyruvate ratios were increased in paracetamol-treated rats. The mean concentration of bromosulphthalein in the perfusate and biliary excretion of the dye were decreased. Macro and micro vesicular fatty change was present in the livers of paracetamol-treated rats. This study demonstrates that chronic administration of therapeutic doses of paracetamol to rats adversely affects liver function, as evidenced by impaired gluconeogenesis and bromosulphthalein clearance from the perfusate, and excretion of the dye into the bile, and provides histological evidence of hepatic damage in rats.     

Microfluorometry using fluorescein diacetate reflects the integrity of the plasma membrane in UVA-irradiated cultured skin fibroblasts

Hepatic coenzyme A (CoA) plays an important role in cellular lipid metabolism. Because mitochondria and peroxisomes represent the two major subcellular sites of lipid metabolism, the present study was designed to investigate the specific impact of hepatic CoA deficiency on peroxisomal as well as mitochondrial beta-oxidation of fatty acids. CoA deficiency (47% decrease in free CoA and 23% decrease in total CoA) was produced by maintaining weanling male Sprague-Dawley rats on a semipurified diet deficient in pantothenic acid (the precursor of CoA) for 5 weeks. Hepatic mitochondrial fatty acid oxidation of short-chain and long-chain fatty acids were not significantly different between control and CoA-deficient rats. Conversely, peroxisomal beta-oxidation was significantly diminished (38% inhibition) in livers of CoA-deficient rats compared to control animals. Peroxisomal beta-oxidation was restored to normal levels when hepatic CoA was replenished. It is postulated that since the role of hepatic mitochondrial beta-oxidation is energy production while peroxisomal beta-oxidation acts mainly as a detoxification system, the mitochondrial pathway of beta-oxidation is spared at the expense of the peroxisomal pathway when liver CoA plummets. The present study may offer an animal model to investigate mechanisms involved in peroxisomal diseases.     

In vivo induction of tyrosylprotein sulfotransferase by ethanol: role of increased enzyme synthesis

Tyrosine sulfation is a posttranslational modification involved in the synthesis, secretion, and biological activity of proteins and peptides. Our previous studies have demonstrated that the enzyme activity was induced by ethanol. In the present work, the induction was studied in detail. Initial experiments were conducted to examine the time course of tyrosylprotein sulfotransferase (TPST) induction in rats pair-fed liquid diets containing either ethanol or carbohydrate substitute (controls). Marked elevation of TPST activity (3-fold) was measured on day 10 in the liver and gastric mucosa of ethanol-fed rats. Ethanol-mediated enhancement was also noticed by Western-blot analysis with anti-TPST antibody in both the liver and gastric mucosa on days 5 and 10. We then determined the steady-state TPST protein turnover in ethanol-fed and control animals that were given 35S-methionine after 10 days of pair-feeding with liquid diet. The rates of TPST synthesis assessed by measuring initial rates of incorporation of 35S-methionine into TPST was increased in the liver and gastric mucosa of animals fed with ethanol. Monophasic exponential decay curves showed that TPST protein half-lives for liver (control: 34 hr, ethanol: 32 hr) and gastric mucosa (control: 52 hr, ethanol: 48 hr) did not differ between control and ethanol groups. Our overall results indicate that the in vivo induction of TPST by ethanol involves increased enzyme synthesis rather than decreased enzyme degradation.     

Studies on genotoxic effects of iron overload and alcohol in an animal model of hepatocarcinogenesis

BACKGROUND/AIMS: In order to examine whether iron and alcohol act synergistically during tumor initiation in vivo, we investigated the effects of dietary iron overload and a liquid ethanol-containing diet on the initiation phase of the Solt & Farber model of chemical hepatocarcinogenesis. METHODS: Following dietary supplementation with carbonyl iron for 8 weeks and ethanol pair-feeding according to Lieber deCarli for 5 weeks, animals were subjected to partial hepatectomy in order to induce regenerative cell proliferation and thereby "fix" putative DNA lesions. Levels of malondialdehyde, reduced and oxidized ubiquinone-9, alpha-tocopherol and 8-oxo-2'-deoxyguanosine were analyzed in liver tissue removed at the time of partial hepatectomy, and blood was collected for determination of alanine amino-transferase activities. Following a 2-week recovery period, promotion was achieved with 0.02% dietary 2-acetylaminofluorene and carbon tetrachloride. Two weeks after the completion of promotion, animals were sacrificed and the number of preneoplastic, glutathione S-transferase 7,7-positive lesions counted. Animals initiated with diethylnitrosamine served as a positive control group. RESULTS: Serum aminotransferase activities were significantly increased, and hepatic contents of ubiquinol-9 (reduced ubiquinone-9) were significantly decreased in animals exposed to the combination of iron and ethanol in comparison to the other groups. Livers from iron-treated animals had decreased levels of alpha-tocopherol and increased contents of malondialdehyde, whereas treatment with ethanol did not further enhance these alterations. Levels of 8-oxo-2'-deoxyguanosine were not significantly different in animals treated with iron, ethanol or iron + ethanol as compared with controls. The number of preneoplastic foci at the time of sacrifice was not increased in livers exposed to iron and/or ethanol as compared with those from control animals. As expected, the number of foci was significantly increased in positive controls which were initiated with diethylnitrosamine. CONCLUSIONS: Iron potentiated the cytotoxic effects of ethanol, resulting in increased serum aminotransferase activities and decreased hepatic contents of ubiquinol. However, the combination of iron and ethanol did not exert genotoxic effects detectable as enhanced hepatic levels of 8-oxo-2'-deoxyguanosine, or increased formation of preneoplastic, glutathione S-transferase 7,7-positive lesions in the Solt & Farber model of chemical hepatocarcinogenesis.     

Chemokine and cell adhesion molecule mRNA expression and neutrophil infiltration in lipopolysaccharide-induced hepatitis in ethanol-fed rats

Neutrophil infiltration is a feature of alcoholic hepatitis (AH), and although the mechanism by which this occurs is unclear, it may involve a chemotactic gradient. We used lipopolysaccharide (LPS) to induce, in ethanol-fed rats, liver damage similar to that seen in AH. To our knowledge, this study is the first to examine the effect of ethanol on LPS-stimulated chemokine mRNA expression in this model. Hepatic cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-2, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1beta, MIP-2, and eotaxin mRNA levels were elevated 1 to 3 hr post-LPS in both groups. Maximal expression of MIP-2 and MCP-1 mRNA was higher in ethanol-fed rats 1 hr post-LPS, whereas CINC-2 mRNA expression was elevated above controls at 12 to 24 hr. Hepatic intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 mRNA levels were elevated in both groups at 1 hr, whereas L-selectin expression in ethanol-fed rats was elevated above controls at 12 to 24 hr. Hepatic neutrophil infiltration was highest during maximal hepatocyte necrosis. These data suggest that cell adhesion molecules, in conjunction with elevated cytokines and the subsequently induced chemokines, may assist in the formation of a chemotactic gradient within the liver, causing the neutrophil infiltration seen both in this model and possibly in AH.     

Soybean protein suppresses hepatic lipogenic enzyme gene expression in Wistar fatty rats

The effects of dietary soybean protein on lipogenic enzyme gene expression in livers of genetically fatty rats (Wistar fatty) have been investigated. When Wistar fatty rats and their lean littermates (7-8-wk old) were fed a casein or soybean protein isolate diet containing hydrogenated fat (4% hydrogenated fat plus 1% corn oil) or corn oil (5%) for 3 wk, the hepatic messenger RNA concentrations and activities of lipogenic enzymes were significantly lower in rats fed soybean protein than in those fed casein, regardless of genotype or dietary fat. The conversion rates of thyroxine to triiodothyronine by liver microsomes and plasma triiodothyronine concentrations were lower in the fatty rats than in the lean rats and were significantly greater in rats fed soybean protein than in those fed casein. Conversely, plasma and liver triacylglycerol concentrations were lower in soybean protein-fed fatty and lean rats than in those fed casein. The body weight was less in the fatty rats fed soybean protein than in those fed casein after 3 wk of feeding. Moreover, dietary polyunsaturated fatty acids suppressed lipogenic enzyme gene expression in the lean rats but did not in the fatty rats. Dietary soybean protein appeared to be useful for the reduction of obesity.