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Ethanol consumption-induced oxidative stress that is a major etiological factor has been proven to play important roles in organs' injury. In the present study, we investigated the protective effect of fish protein hydrolysate prepared from the heads and viscera of sardinelle (Sardinella aurita) (SPH) against the toxicity of ethanol on the liver and kidney of adult male rats. Animals were divided into four groups of six animals each: group C served as control, group Eth received 30 % ethanol solution at the dose of 3 g/kg body weight, group SPH received only 7.27 mg of SPH/kg body weight, and group Eth-SPH received ethanol and SPH simultaneously at the doses of 30 % and 7.27 mg/kg body weight, respectively. All groups were treated by gavage way for 15 days. Ethanol treatment decreased the defense enzymatic system including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), which increased after the co-administration of SPH. Malondialdehyde (MDA) and toxicity biomarker levels such as aspartate transaminase (AST) and alanine transaminase (ALT) and alcaline phosphatase (ALP) and gamma-glutamyl transaminase (GGT) activities were enhanced after chronic ethanol treatment and reduced by co-treatment with SPH. The histological examination of the liver and kidney confirmed biochemical changes in ethanol-treated rats and demonstrated the protective role of SPH.

Background: Evidence has emerged demonstrating that ethanol (EtOH) influences cytokine expression within the central nervous system, although most studies have examined long-term exposure. Thus, the cytokine response to an acute EtOH challenge was investigated, in order to characterize profiles of cytokine changes following acute exposure., Methods: Rats pups were injected intraperitoneally (i.p.) with 2-g/kg EtOH, and IL-1 mRNA and protein were assessed 0, 60, 120, 180, and 240 minutes post injection (Experiment 1). In Experiments 2 to 5, the expression of several cytokines was examined in adult male rats during acute intoxication (3 hours after 4-g/kg EtOH), as well as withdrawal (18 hours post injection), after i.p. or intragastric (i.g.) EtOH administration., Results: Early in ontogeny, acute EtOH significantly decreased brain IL-1 mRNA and protein. Subsequently, when adult rats were examined, significant and temporally dynamic alterations in central and peripheral cytokines were observed following acute i.p. EtOH exposure (4 g/kg). Although cytokine- and region-dependent central IL-6 expression was generally increased and tumor necrosis factor alpha decreased during intoxication, IL-1 expression exhibited increases during withdrawal. In the periphery, acute i.p. EtOH elevated expression of all cytokines, with the response growing in magnitude as the time post injection increased. Following acute i.g. EtOH (4 g/kg), intoxication-related increases in IL-6 expression were again observed in the paraventricular nucleus of the hypothalamus (PVN), although to a lesser extent. Long-term, voluntary, intermittent EtOH consumption resulted in tolerance to the effects of an i.g. EtOH challenge (4 g/kg) on PVN IL-6 expression, whereas these same elevations in IL-6 expression were still seen in the amygdala in rats with a history of moderate EtOH intake. Treatment with minocycline did not significantly attenuate i.p. or i.g. EtOH-induced changes in central cytokine expression., Conclusions: Together, these studies provide a foundation for understanding fluctuations in central and peripheral cytokines following acute EtOH as potential contributors to the constellation of neural and behavioral alterations observed during EtOH intoxication and withdrawal., (Copyright © 2014 by the Research Society on Alcoholism.)

Ethanol consumption can lead to hepatic steatosis that contributes to late-stage liver diseases such as cirrhosis and hepatocellular carcinoma. In this study, we investigated the potential protective effect of a flavonoid, luteolin, on ethanol-induced fatty liver development and liver injury. Six-wk-old male C57BL/6 mice were divided into 3 groups: a control group; a group exposed to alcohol by using a chronic and binge ethanol feeding protocol (EtOH); and a group that was administered daily 50 mg/kg of luteolin in addition to ethanol exposure (EtOH + Lut). A chronic and binge ethanol feeding protocol was used, including chronic ethanol consumption (1%, 2%, and 4% for 3 d, and 5% for 9 d) and a binge (30% ethanol) on the last day. Compared with the control group, the EtOH group had a significant elevation in serum concentrations of alanine aminotransferase (ALT) (561%), triglyceride (TG) (47%), and LDL cholesterol (95%), together with lipid accumulation in the liver. Compared with the EtOH group, the EtOH + Lut group had significant reductions in serum concentrations of ALT (43%), TG (22%), LDL cholesterol (52%), and lipid accumulation in the liver. Ethanol elevated liver expression of lipogenic genes including sterol regulatory element-binding protein 1c (Srebp1c) (560%), fatty acid synthase (Fasn) (190%), acetyl-CoA carboxylase (Acc) (48%), and stearoyl-CoA desaturase 1 (Scd1) (286%). Luteolin reduced ethanol-induced expression of these genes in the liver: Srebp1c (79%), Fasn (80%), Acc (60%), and Scd1 (89%). In cultured hepatocytes, luteolin prevented alcohol-induced lipid accumulation and increase in the expression of lipogenic genes. The transcriptional activity of the master regulator of lipid synthesis, sterol regulatory element-binding protein (SREBP), was enhanced by ethanol treatment (160%) and reduced by luteolin administration (67%). In addition, ethanol-induced reduction of AMP-activated protein kinase and SREBP-1c phosphorylation was abrogated by luteolin. Collectively, our study indicates that luteolin is effective in ameliorating ethanol-induced hepatic steatosis and injury., (© 2014 American Society for Nutrition.)

Background and Aim: Alcohol-related liver disease (ALD) is mediated in part by insulin resistance. Attendant dysregulation of lipid metabolism increases accumulation of hepatic ceramides that worsen insulin resistance and compromise the structural and functional integrity of the liver. Insulin and insulin growth factor (IGF) stimulate aspartyl-asparaginyl-β-hydroxylase (AAH), which promotes cell motility needed for structural maintenance and remodeling of the liver. AAH mediates its effects by activating Notch, and in ALD, insulin/IGF signaling, AAH, and Notch are inhibited., Method: To test the hypothesis that in ALD, hepatic ceramide load contributes to impairments in insulin, AAH, and Notch signaling, control and chronic ethanol-fed adult Long-Evans rats were treated with myriocin, an inhibitor of serine palmitoyl transferase. Livers were used to assess steatohepatitis, insulin/IGF pathway activation, and expression of AAH-Notch signaling molecules., Results: Chronic ethanol-fed rats had steatohepatitis with increased ceramide levels; impairments in signaling through the insulin receptor, insulin receptor substrate, and Akt; and decreased expression of AAH, Notch, Jagged, Hairy-Enhancer of Split-1, hypoxia-inducible factor 1α, and proliferating cell nuclear antigen. Myriocin abrogated many of these adverse effects of ethanol, particularly hepatic ceramide accumulation, steatohepatitis, and impairments of insulin signaling through Akt, AAH, and Notch., Conclusions: In ALD, the histopathology and impairments in insulin/IGF responsiveness can be substantially resolved by ceramide inhibitor treatments, even in the context of continued chronic ethanol exposure., (© 2013 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.)

Oxidative stress and lipid accumulation play important roles in alcohol-induced liver injury. Previous reports showed that, in livers of nuclear factor erythroid 2-related factor 2 (Nrf2)-activated mice, genes involved in antioxidant defense are induced, whereas genes involved in lipid biosynthesis are suppressed. To investigate the role of Nrf2 in ethanol-induced hepatic alterations, Nrf2-null mice, wild-type mice, kelch-like ECH-associated protein 1-knockdown (Keap1-KD) mice with enhanced Nrf2, and Keap1-hepatocyte knockout (Keap1-HKO) mice with maximum Nrf2 activation, were treated with ethanol (5 g/kg, po). Blood and liver samples were collected 6h thereafter. Ethanol increased alanine aminotransferase and lactate dehydrogenase activities as well as thiobarbituric acid reactive substances in serum of Nrf2-null and wild-type mice, but not in Nrf2-enhanced mice. After ethanol administration, mitochondrial glutathione concentrations decreased markedly in Nrf2-null mice but not in Nrf2-enhanced mice. H(2)DCFDA staining of primary hepatocytes isolated from the four genotypes of mice indicates that oxidative stress was higher in Nrf2-null cells, and lower in Nrf2-enhanced cells than in wild-type cells. Ethanol increased serum triglycerides and hepatic free fatty acids in Nrf2-null mice, and these increases were blunted in Nrf2-enhanced mice. In addition, the basal mRNA and nuclear protein levels of sterol regulatory element-binding protein 1(Srebp-1) were decreased with graded Nrf2 activation. Ethanol further induced Srebp-1 mRNA in Nrf2-null mice but not in Nrf2-enhanced mice. In conclusion, Nrf2 activation prevented alcohol-induced oxidative stress and accumulation of free fatty acids in liver by increasing genes involved in antioxidant defense and decreasing genes involved in lipogenesis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We employed a proteomic profiling strategy to examine the effects of ethanol and betaine diet supplementation on major liver protein level changes. Male Wistar rats were fed control, ethanol or betaine supplemented diets for 4 weeks. Livers were removed and liver cytosolic proteins resolved by one-dimensional and two-dimensional separation techniques. Significant upregulation of betaine homocysteine methyltransferase-1, methionine adenosyl transferase-1, and glycine N-methyltransferase were the most visually prominent protein changes observed in livers of rats fed the betaine supplemented ethanol diet. We hypothesise that this concerted upregulation of these methionine metabolic pathway enzymes is the protective mechanism by which betaine restores a normal metabolic ratio of liver S-adenosylmethionine to S-adenosylhomocysteine. Ethanol also induced significant downregulation of carbonic anhydrase-III protein levels which was not restored by betaine supplementation. Carbonic anhydrase-III can function to resist oxidative stress, and we therefore hypothesise that carbonic anhydrase-III protein levels compromised by ethanol consumption, contribute to ethanol-induced redox stress.

The current study was designed to investigate the effect and potential mechanism of exogenous administration of S-adenosyl-l-methionine (SAM) on the enhanced hepatotoxicity induced by the Fas agonistic Jo2 antibody plus acute ethanol pretreatment in C57BL/6 mice. Acute ethanol plus Jo2 treatment produces liver toxicity under conditions in which ethanol alone or Jo2 alone do not. SAM significantly attenuated this elevated hepatotoxicity in mice as manifested by a decrease of serum aminotransferases and morphological amelioration. Levels of SAM and activity of methionine adenosyltransferase were lowered by the ethanol plus Jo2 treatment but restored by administration of SAM. The ethanol plus Jo2 treatment increased activity and content of CYP2E1, iNOS content and TNF-alpha levels; these increases were blunted by SAM. SAM also protected against the elevated oxidative and nitrosative stress found after ethanol plus Jo2, likely due to the decreases in CYP2E1, iNOS and TNF-alpha. Calcium-induced swelling of mitochondria was enhanced by the ethanol plus Jo2 treatment and this was prevented by SAM. JNK and P38 MAPK were activated by the ethanol plus Jo2 treatment; JNK activation was partially prevented by SAM. It is suggested that SAM protects against the ethanol plus Jo2 toxicity by restoring hepatic SAM levels, preventing the increase in iNOS, CYP2E1 and TNF-alpha and there by lowering the elevated oxidative/nitrosative stress and activation of the JNK signal pathway, ultimately preventing mitochondrial damage.

Ethanol is a widely used drug that is consumed in large amounts for pharmacologic effects. Elimination of ethanol alters metabolism in the liver and throughout an organism. Ethanol's effect on metabolites can influence the regulation of key pathways such as gluconeogenesis. We adopted a proton NMR-based metabolomic approach to study ethanol-induced metabolic changes in liver, blood, and brain tissue from rats exposed to either a single dose of ethanol or a chronic 4 day binge-ethanol protocol. Both acute and binge ethanol caused (i) decreased glucose, lactate, and alanine in liver and serum; (ii) increased acetate in liver and serum; and (iii) increased acetoacetate in serum. Binge-ethanol increased liver beta-hydroxybutyrate and decreased betaine. Pretreatment with the antioxidant butylated hydroxytoluene (BHT) increased betaine and reduced ethyl glucuronide (EtG) in livers of binge-ethanol animals, as compared to those not pretreated with BHT. We found no change in brain metabolites after a single dose of ethanol. Unsupervised principal component (PC) analysis of spectral data from liver and serum successfully discriminated treatment groups, based largely on the biochemical differences outlined above, confirming the results of manual analysis. To explain the observed lack of gluconeogenesis following ethanol treatment and to resolve apparently discordant results from previous studies, we propose a model in which decreased hepatic alanine removes inhibition on pyruvate kinase, thus permitting a futile cycle that diverts phosphoenolpyruvate away from gluconeogenesis. This is a new mechanism that biochemically elucidates the well-known, yet unexplained, "empty calorie" phenomenon of ethanol. Reduction of EtG by pretreatment with BHT suggests that BHT and perhaps other compounds may alter the pharmacokinetics of EtG so that EtG may not always be a sensitive marker for ethanol abuse.

Alcoholic fatty liver results from an impaired fatty acid catabolism due to blockade of PPARalpha and increased lipogenesis due to activation of sterol regulatory element-binding protein (SREBP)-1c. Because both oxidized fats (OF) and conjugated linoleic acids (CLA) have been demonstrated in rats to activate hepatic PPARalpha, we tested the hypothesis that these fats are able to prevent ethanol-induced triacylglycerol accumulation in the liver by upregulation of PPARalpha-responsive genes. Forty-eight male rats were assigned to 6 groups and fed isocaloric liquid diets containing either sunflower oil (SFO) as a control fat, OF prepared by heating of SFO, or CLA, in the presence and absence of ethanol, for 4 wk. Administration of ethanol lowered mRNA concentrations of PPARalpha and the PPARalpha-responsive genes medium chain acyl-CoA dehydrogenase, long chain acyl-CoA dehydrogenase, acyl-CoA oxidase, carnitine palmitoyl-CoA transferase I, and cytochrome P450 4A1 and increased triacylglycerol concentrations in the liver (P < 0.05). OF increased hepatic mRNA concentrations of PPARalpha-responsive genes and lowered hepatic triacylglycerol concentrations compared with SFO (P < 0.05) whereas CLA did not. Rats fed OF with ethanol had similar mRNA concentrations of PPARalpha-responsive genes and similar triacylglycerol concentrations in the liver as rats fed SFO or CLA without ethanol. In contrast, hepatic mRNA concentrations of SREBP-1c and fatty acid synthase were not altered by OF or CLA compared with SFO. This study shows that OF prevents an alcohol-induced triacylglycerol accumulation in rats possibly by upregulation of hepatic PPARalpha-responsive genes involved in oxidation of fatty acids, whereas CLA does not exert such an effect.

Alcohol use is contributing to an unprecedented decline in life expectancy. Damage to the liver after ethanol administration is a well-known phenomenon. Free radical mechanisms have been proposed to play a part in ethanol-induced liver toxicity. Ingestion of diets rich in polyunsaturated fatty acids (PUFAs) along with alcohol is known to result in enhanced liver damage. The present work is aimed at evaluating the protective role of ferulic acid, a naturally occurring plant component, on alcohol- and PUFA-induced liver toxicity. Three different doses of ferulic acid (10, 20, and 40 mg/kg of body weight) were administered to rats given alcohol, heated PUFA (DeltaPUFA), and alcohol + DeltaPUFA. Influence of ferulic acid on alcohol-and PUFA-induced liver damage was evaluated by analyzing the activities of the liver marker enzymes alkaline phosphatase, gamma-glutamyl transferase, alanine transaminase, and aspartate transaminase. The activities of these liver marker enzymes were increased in the alcohol, DeltaPUFA, and alcohol + DeltaPUFA groups but were decreased significantly on treatment with ferulic acid. The low dose (10 mg/kg of body weight) was not effective, but both 20 mg and 40 mg/kg of body weight were found to be effective. The 20 mg/kg of body weight dose was found to be more effective than 40 mg/kg of body weight (the high dose). The administration of ferulic acid to normal rats did not produce any harmful effects. Thus our results show that ferulic acid is an effective anti-hepatotoxic agent without side effects and may be a good candidate in the current search for a natural hepatoprotective agent.

Purpose: Alcoholic liver disease is a major medical complication of alcohol abuse and a common liver disease in western countries. Increasing evidence demonstrates that oxidative stress plays an important etiologic role in the development of alcoholic liver disease. Alcohol alone or in combination with high fat is known to cause oxidative injury. The present study therefore aims at evaluating the protective role of curcumin, an active principle of turmeric and a synthetic analog of curcumin (CA) on alcohol and thermally oxidised sunflower oil (DeltaPUFA) induced oxidative stress., Methods: Male albino Wistar rats were used for the experimental study. The liver marker enzymes: gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), the lipid peroxidative indices: thiobarbituric acid reactive substances (TBARS) and hydroperoxides (HP) and antioxidants such as vitamin C, vitamin E, reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) were used as biomarkers for testing the antioxidant potential of the drugs., Results: The liver marker enzymes and lipid peroxidative indices were increased significantly in alcohol, DeltaPUFA and alcohol + DeltaPUFA groups. Administration of curcumin and CA abrograted this effect. The antioxidant status which was decreased in alcohol, DeltaPUFA and alcohol + DeltaPUFA groups was effectively modulated by both curcumin and CA treatment. However, the reduction in oxidative stress was more pronounced in CA treatment groups compared to curcumin., Conclusion: In conclusion, these observations show that CA exerts its protective effect by decreasing the lipid peroxidation and improving antioxidant status, thus proving itself as an effective antioxidant.

This work studied the effects of ethanol in the absence and presence of haloperidol under two experimental conditions. In protocol 1, rats were treated daily with ethanol (4 g/kg, p.o.) for 7 days, and received only haloperidol (1 mg/kg, i.p.) from the 8th day to the 14th day. In protocol 2, animals received ethanol, and the treatment continued with ethanol and haloperidol from the 8th day to the 14th day. Results show increases in alanine transaminase (ALT; 48% and 55%) and aspartate transaminase (AST; 32% and 22%) levels after ethanol or haloperidol (14 days) treatments, as compared with controls. Apolipoprotein A-1 (APO A1) levels were increased by haloperidol, after 7- (148%) but not after 14-day treatments, as compared with controls. Levels of lipoprotein (high-density lipoprotein (HDL-C)) tended to be increased only by ethanol treatment for 14 days. ALT (80%) and AST (43%) levels were increased in the haloperidol plus ethanol group (protocol 2), as compared with controls. However, an increase in APO A1 levels was observed in the haloperidol group pretreated with ethanol (protocol 1), as compared with controls and ethanol 7-day treatments. Triglyceride (TG) levels were increased in the combination of ethanol and haloperidol in protocol 1 (234%) and 2 (106%), as compared with controls. Except for a small decrease in haloperidol groups, with or without ethanol, as related to ethanol alone, no other effect was observed in HDL-C levels. In conclusion, we showed that haloperidol might be effective in moderating lipid alterations caused by chronic alcohol intake.

To test the hypothesis that ethanol-induced hepatic apoptosis is secondary to the oxidative stress generated by cytochrome P4502E1 (CYP2E1), we assessed the effects of the carotenoid lycopene, a potent antioxidant extracted from tomatoes, on oxidative stress and apoptosis in HepG2 cells overexpressing CYP2E1 (2E1 cells). These were exposed for 5 days to 100mM ethanol and 10 microM lycopene or an equal volume of placebo (vehicle). Ethanol significantly increased apoptosis measured by flow cytometry and by TUNEL assay. This was accompanied by an ethanol-induced oxidative stress: hydrogen peroxide production was significantly increased and mitochondrial GSH was strikingly decreased. Both were restored by lycopene, with a significant decrease in apoptosis. The placebo had no protective effect. In conclusion, Lycopene opposes the ethanol-induced oxidative stress and apoptosis in 2E1 cells. The parallelism between these effects suggests a causal link. Furthermore, these beneficial effects and the innocuity of lycopene now justify an in vivo trial.

Apoptosis is critically involved in hepatic pathogenesis induced by acute alcohol exposure. This study was undertaken to test the hypothesis that zinc interferes with an important Fas ligand-mediated pathway in the liver, leading to the inhibition of ethanol-induced apoptosis. Male 129/Sv(PC)J mice were injected subcutaneously with ZnSO4 (5 mg of Zn ion/kg) in 12-hr intervals for 24 hr before intragastric administration of ethanol (5 g/kg) in 12-hr intervals for 36 hr. Ethanol-induced apoptosis in the liver was detected by a terminal deoxynucleotidyl transferase nick-end labeling assay and was further confirmed by electron microscopy. The number of apoptotic cells in the livers pretreated with zinc was significantly decreased, being only 15% of that found in the animals treated with ethanol only. Characteristic apoptotic morphological changes observed by electron microscopy were also inhibited by zinc. Importantly, zinc inhibited ethanol-induced activation of caspase-3, the primary executioner protease responsible for alcohol-induced liver apoptosis, and caspase-8 as determined by enzymatic assay. Immunohistochemical analysis revealed that zinc inhibited ethanol-induced endogenous Fas ligand activation, which is a key component in signaling pathways leading to hepatic caspase-8 and subsequent caspase-3 activation and apoptosis. These results demonstrate that zinc is a potent inhibitor of acute ethanol-induced liver apoptosis, and this effect occurs primarily through zinc interference with Fas ligand pathway and the suppression of caspase-3.

Aim: To investigate free-radical scavenger effect of n-acetylcysteine in rats intragastrically fed with ethanol., Methods: Twenty-four rats divided into three groups were fed with ethanol (6 g/kg/day, Group 1), ethanol and n-acetylcysteine (1 g/kg, Group 2), or isocaloric dextrose (control group, Group 3) for 4 weeks. Then animals were sacrificed under ether anesthesia, and intracardiac blood and liver tissues were obtained. Measurements were made in both serum and homogenized liver tissues. Malondialdehyde (MDA) level was measured by TBARS method. Glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) levels were studied by commercial kits. Kruskal-Wallis test was used for statistical analysis., Results: ALT and AST in Group 1 (154 U/L and 302 U/L, respectively) were higher than those in Group 2 (94 U/L and 155 U/L) and Group 3 (99 U/L and 168 U/L) (P=0.001 for both). Serum and tissue levels of MDA in Group 1 (1.84 nmol/mL and 96 nmol/100 mg-protein) were higher than that in Group 2 (0.91 nmol/mL and 64 nmol/100 mg protein) and Group 3 (0.94 nmol/mL and 49 nmol/100 mg-protein) (P<0.001 for both). On the other hand, serum GSH-Px level in Group 1 (8.21 U/g Hb) was lower than that in Group 2 (16 U/g Hb) and Group 3 (16 U/g-Hb) (P<0.001). Serum and liver tissue levels of SOD in Group 1 (11 U/mL and 26 U/100 mg-protein) were lower than that in Group 2 (18 U/mL and 60 U/100 mg protein) and Group 3 (20 U/mL and 60 U/100 mg-protein) (P<0.001 for both)., Conclusion: Ethanol-induced liver damage was associated with oxidative stress, and co-administration of n-acetylcysteine attenuates this damage effectively in rat model.

Retinoic acid (RA) supplementation suppresses ethanol-enhanced hepatocyte hyperproliferation in rats; however, little is known about the mechanism(s). Here, we investigated whether RA affects the protein kinase signaling pathways in the liver tissues of rats fed with a high dose of ethanol for a prolonged period of time (6 months). Results show that there were greater levels of phosphorylated Jun N-terminal kinase (JNK) and phosphorylated c-Jun protein, but not total JNK protein, in livers of ethanol-fed rats vs those of controls. Moreover, ethanol feeding to rats increased the levels of phosphorylated mitogen-activated protein kinase kinase-4 (MKK-4) and decreased the levels of mitogen-activated kinase phosphatase-1 (MKP-1) in liver tissue. However, hepatic levels of phosphorylated-p38 protein and total-p38 protein were not altered by the ethanol treatment. In contrast, all-trans-RA supplementation at two doses in ethanol-fed rats greatly attenuated the ethanol-induced hepatic phosphorylation of MKK-4, phosphorylated-JNK and c-Jun proteins. The level of MKP-1 was increased in ethanol-fed rats supplemented with all-trans-RA. Further, ethanol-induced hepatocyte hyperproliferation, measured by immunostaining for proliferating cell nuclear antigen, were markedly decreased by all-trans-RA supplementation. Interestingly, hepatic apoptosis in the liver of ethanol-fed rats after 6 months of treatment decreased significantly. This decrease of hepatic apoptosis in ethanol-fed rats was prevented by all-trans-RA supplementation in a dose-dependent manner. The results from these studies indicate that restoration of RA homeostasis is critical for the regulation of JNK-dependent signaling pathway and apoptosis in the liver of ethanol-fed rats.

Centrilobular hypoxia has been suggested to contribute to hepatic damage caused by alcohol intoxication. However, the mechanisms involved are still poorly understood. We have investigated whether alterations of Na(+) homeostasis might account for ethanol-mediated increase in hepatocyte sensitivity to hypoxia. Addition of ethanol (100 mmol/l) to isolated rat hepatocytes incubated under nitrogen atmosphere greatly stimulated cell death. An increase in intracellular Na(+) levels preceded cell killing and Na(+) levels in hepatocytes exposed to the combination of ethanol and hypoxia were almost twice those in hypoxic cells without ethanol. Na(+) increase was also observed in hepatocytes incubated with ethanol in oxygenated buffer. Ethanol addition significantly lowered hepatocyte pH. Inhibiting ethanol and acetaldehyde oxidation with, respectively, 4-methylpyrazole and cyanamide prevented this effect. 4-methylpyrazole, cyanamide as well as hepatocyte incubation in a HCO(3)(-)-free buffer or in the presence of Na(+)/H(+) exchanger blocker 5-(N,N-dimethyl)-amiloride also reduced Na(+) influx in ethanol-treated hepatocytes. 4-methylpyrazole and cyanamide similarly prevented ethanol-stimulated Na(+) accumulation and hepatocyte killing during hypoxia. Moreover, ethanol-induced Na(+) influx caused cytotoxicity in hepatocytes pre-treated with Na(+), K(+)-ATPase inhibitor ouabain. Also in this condition 4-methylpyrazole and 5-(N,N-dimethyl)-amiloride decreased cell killing. These results indicate that ethanol can promotes cytotoxicity in hypoxic hepatocytes by enhancing Na(+) accumulation.

Geranylgeranylacetone (GGA) has been introduced into the clinical field as an anti-ulcer drug. In addition to protective effects on gastric mucosal cells, GGA also has anti-apoptotic effects against ischemia and reperfusion injury in hepatocytes and intestinal cells. However, the molecular mechanisms of the cytoprotective or anti-apoptotic effect of GGA are largely unknown. To explore the molecular mechanism of GGA action, we focused on thioredoxin (TRX), an endogenous-redox-acting molecule. We have demonstrated that GGA induces the messenger RNA and protein of TRX and affects the activation of transcription factors, AP-1 and NF-kappaB, and that GGA blunted ethanol-induced cytotoxicity of cultured hepatocytes. These results provide evidence suggesting that a possible novel molecular mechanism of GGA is to protect cells via the induction of TRX and the activation of transcription factors such as NF-kappaB and AP-1., (Copyright 2000 Academic Press.)

The purpose of this investigation was to assess the effect of chlormethiazole treatment on liver damage in the experimental rat intragastric ethanol-feeding model of alcoholic liver disease. Chlormethiazole has been used in the treatment of alcoholic withdrawal and has been shown to inhibit cytochrome P4502E1. Since treatment of experimental alcoholic liver disease with CYP2E1 inhibitors had an ameliorating effect on liver injury in the rat, chlormethiazole was used to see if it had a similar effect. Rats fed ethanol for 2 months had significantly less liver injury when chlormethiazole was added to the diet, fed intragastrically. The CYP2E1 apoprotein levels, which were increased by ethanol feeding, were also increased when chlormethiazole was fed with ethanol. Chlormethiazole inhibited the increase in the ethanol-induced CYP2E1 activity in vivo, as measured by chlorzoxazone 6-hydroxylation, but did not affect the level of CYP2E1 apoprotein. Likewise, the reduction in proteasome proteolytic enzyme activity produced by ethanol feeding was blunted in chlormethiazole-fed rats. These results support the conclusion that chlormethiazole treatment partially protects the liver from injury by inhibiting CYP2E1 activity in vivo.

Ursolic acid is the active material isolated from the leaves of the Eucalyptus hybrid E. tereticornis. In the present study, it has shown a significant preventive effect in vitro against ethanol-induced toxicity in isolated rat hepatocytes. Compared with the incubation of isolated hepatocytes with ethanol only, the simultaneous presence of ursolic acid in the cell suspension preserved the viability of hepatocytes and reversed the ethanol-induced loss in the level of all the marker enzymes (AST, ALT and AP) studied. Ethanol alone resulted in a 48%-54% decrease in the viability and a 42%-54% reduction in the biochemical parameters of the hepatocytes. Ursolic acid showed a concentration dependent (1-100 microg/mL) preventive effect (12%-76%) on alcohol-induced hepatocyte toxicity by restoring the altered parameters. The results thus suggest the effective use of an in vitro test system as an alternative for in vivo assessment of hepatoprotective activity of purified material., (Copyright 2000 John Wiley & Sons, Ltd.)

Background: Alcohol consumption increases apoptosis of hepatocytes. This effect appears to be mediated by the induction of hepatic cytochrome P-4502E1(CYP2E1) and its generation of free radicals, which results in an enhanced lipid peroxidation that initiates apoptosis. Because polyenylphosphatidylcholine (PPC), a soybean extract rich in polyunsaturated phosphatidylcholines, decreases the induction of ethanol-specific CYP2E1 and opposes oxidative stress, we hypothesized that PPC supplementation may attenuate hepatocyte apoptosis caused by ethanol ingestion., Methods: Twenty-eight male Sprague Dawley rats were pair-fed Lieber-DeCarli liquid diets containing 36% of energy as alcohol or an isocaloric amount of carbohydrate for 28 days. Half of the rats were given PPC (3 g/liter), whereas the other half received the same amount of linoleate (as safflower oil) and of choline as the bitartrate. An additional dose of alcohol (3 g/kg) was given intragastrically 90 min before the livers were removed. We assessed apoptosis in formalin-fixed, paraffin-embedded liver sections by using the TUNEL (terminal transferase dUTP nick end labeling) assay. Apoptotic hepatocytes were identified by positive TUNEL staining in conjunction with condensation of nucleoplasm or margination of chromatin. In each rat, 20,000 to 60,000 hepatocytes were counted by light microscopy by using Image-Pro Plus computer software, and the incidence of apoptosis was expressed as the percentage of total hepatocytes., Results: Alcohol feeding resulted in a 4.5-fold increase in apoptosis of hepatocytes compared to pair-fed control rats; PPC supplementation decreased the alcohol-induced apoptosis to less than half. No difference in the incidence of apoptosis between the control and PPC-supplemented rats was found in the absence of alcohol. Apoptosis was distributed randomly in the liver lobules of the rats fed the control diet, whereas the alcohol-induced apoptosis was significantly increased in the perivenular area. PPC supplementation strikingly reduced this effect., Conclusions: PPC attenuates alcohol-induced apoptosis of hepatocytes; this effect may provide a mechanism for PPC's protection against liver injury, possibly in association with its antioxidative action via the down-regulation of ethanol-mediated CYP2E1 induction.

Increase in oxidative stress after chronic ethanol consumption can result in hepatic injury. Because polyphenolic compounds can offer antioxidant protection to the cardiovascular system, this study was designed to investigate whether dietary supplementation of polyphenols from grapes may ameliorate hepatic injury resulting from chronic ethanol consumption. Male Sprague-Dawley rats were administered the following diets for 2 mo: 1) Lieber-DeCarli (L-D) diet with isocaloric amount of maltose instead of ethanol (Basal), 2) the L-D diet with 50g/L ethanol (EtOH); 3) L-D diet with 50 mg/L of grape polyphenols (GP) and 4) ethanol diet with GP (EtOH + GP). Rats given EtOH or EtOH + GP diets had significantly more hepatic triacylglycerols (P < 0.0001) and lipid peroxidation products (P < 0.01) compared with those given the Basal and GP diets. In addition, ethanol ingestion also decreased significantly (P < 0.01) the proportion of 16:0 and increased 18:0 and 18:1 in hepatic phospholipids, suggesting a perturbation of the de novo fatty acid biosynthesis pathways. However, GP supplementation alone and GP added to the ethanol diet did not alter the lipid changes mediated by ethanol except for the levels of 22:6(n-3) which were significantly (P < 0.05) higher in the EtOH + GP group than in the EtOH group. Despite a lack of gross lipid changes, histologic assessment showed significantly (P < 0.05) less hepatic damage in the GP + EtOH group compared with the EtOH group. These results clearly distinguished ethanol-mediated changes in hepatic morphology from the changes in hepatic lipids and further demonstrated the ability of GP to ameliorate hepatic damage resulting from chronic ethanol consumption.

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 (6-30x) 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.

Chronic ethanol and paracetamol consumption, both individually and in combination, caused hepatic changes in rats. Treatment of testosterone (2.5 mg/kg body wt.) to the alcoholic and paracetamol administered rats showed decreased activities of serum transaminases, serum acid and alkaline phosphatases, and decreased levels of hepatic triglycerides, cholesterol and free fatty acids. Concentration of the lipid peroxidation product-malondialdehyde was significantly decreased in the liver after testosterone treatment in alcohol and paracetamol administered groups. Histopathological observations further confirm that testosterone could offer protection against alcohol and paracetamol induced damage to liver in animals.

The process of lipid peroxidation and the system of antioxidant defense in brain and liver of fetuses on the 20th day of gestation and 1 day old rats after antenatal exposure to ethanol and limontar were studied. It was shown that antenatal exposure to ethanol led to activation of the process of lipid peroxidation in brain and to inhibition of of the enzyme system of antioxidant defense in liver of fetal and newborn rats. Limontar promoted the normalization of both the process of lipid peroxidation and the system of antioxidant defense.

An acute ethanol load (50 mmol/kg, i.p.) induces an increase in the total non-heme iron and in the low-molecular-weight non-heme iron complexes (LMW-Fe) content both in liver and cerebellum. This increase in LMW-Fe is associated with a decrease in some essential trace elements (selenium, zinc, copper) playing a role in the anti-oxidant system. These changes could contribute to the enhancement in lipid peroxidation which occurs at the hepatic and cerebellar level following the ethanol administration. The administration of allopurinol prior to the ethanol load prevents the changes in non-heme iron and trace elements. This prevention may contribute to the protective effects of allopurinol on the ethanol-induced oxidative stress.

The effects of chronic administration of ethanol on the lipid composition of erythrocytes and liver of rats were investigated. Ethanol was chronically administered alone or in combination with Evening Primrose Oil (containing 10% v/v gamma-linolenic acid) in a nutritionally balanced milk diet. Chronic administration of ethanol alone significantly decreased the content of arachidonic acid in erythrocyte membranes, whereas the concomitant administration of Evening Primrose Oil reversed this effect. The triacylglycerol content was significantly increased in the microsomal fraction of the liver after chronic ethanol administration. Ethanol also significantly increased the ratio of cholesterol:cholesteryl esters in the microsomal fraction. The arachidonic acid content of the whole liver fraction was significantly reduced after chronic administration of ethanol, whereas concomitant administration of Evening Primrose Oil did not reverse this effect. The administration of Evening Primrose Oil during chronic ethanol intake may have beneficial effects, as it reverses some of the effects of ethanol on erythrocyte and hepatocyte membrane lipids which may be detrimental to health.

Acute addition of ethanol or acetaldehyde to the isolated, perfused rat liver leads to an increase in ethane and n-pentane release. These volatile hydrocarbons are known to originate from the peroxidation of polyunsaturated fatty acids. The effects are half-maximal at 0.5 mM ethanol or 20 microM acetaldehyde in the entering perfusate. Propionaldehyde and benzaldehyde are also able to elicit ethane release. Pargyline and disulfiram, inhibitors of aldehyde oxidation, inhibited the extra ethane release in all cases. The inhibitory effect of pargyline is suppressed during addition of metyrapone. The study indicates that the oxidation of acetaldehyde and not of ethanol itself is the step responsible for increased ethane formation by the perfused rat liver during ethanol infusion.

1. The galactose-elimination rate in perfused livers from starved rats was decreased in the presence of ethanol (2-28mM) to one-third of the control values. Orotate injections partly reversed the effect of ethanol, so that the galactose-elimination rate was about two-thirds of the control values. Orotate alone had no effect on the galactose-elimination rate. 2. Ethanol increased [galactose 1-phosphate] and [UDP-galactose], and decreased (UDP-glucose] and [UTP], both with and without orotate. Orotate increased [UTP], [UDP-galactose], both with and without ethanol. The increase of [galactose 1-phosphate] in the presence of ethanol was inhibited by orotate. Orotate alone had no appreciable effect on [galactose 1-phosphate]. 3. Both the effect of ethanol and that of orotate on the galactose-elimination rate can be accounted for by assuming inhibition of galactokinase by galactose 1-phosphate with Ki about 0.2mM, the inhibition being either non-competitive or uncompetitive. 4. The primary effect of ethanol seems to be inhibition of UDP-glucose epimerase (EC 5.1.3.2), followed by accumulation of UDP-galactose, trapping of UDP-glucose and increase of [galactose 1-phosphate]. Orotate decreased the effect of ethanol, probably by increasing [UDP-glucose].

The serum total lipids, triglycerides, total cholesterol and free fatty acid concentrations, as well as hepatic triglyceride and cholesterol levels, were increased in acutely ethanol-treated rats. Treatment of ethanol-given animals with o-ethylrutoside resulted in a significant reduction in all examined fractions of serum lipids and in the hepatic total cholesterol level.

The incorporation of 14C-valine into liver protein was studied in isolated rat liver parenchymal cells. Various glucose levels in the incubation media did not affect the rate of 14C-valine incorporation into proteins. The insulin stimulated incorporation of 14C-valine into proteins was also unaffected by the various glucose levels. Ethanol decreased the incorporation of 14C-valine into liver proteins, affecting stationary and export proteins to the same extent. This inhibitory action of ethanol on valine incorporation was reversed by increasing exogenous glucose concentrations. The combination of insulin and high glucose level totally prevented the ethanol inhibition.

Ethanol, when tested alone, inhibited proteolysis by about 20%; however, no effect was detected when it was combined with exogenous oxidizable fuels which inhibited proteolysis by themselves. Ethanol was effective in inhibiting proteolysis in the presence of protease inhibitors like ammonia, leupeptin or methylamine, indicating that its mechanism of action involves a non-lysosomal pathway of degradation. Ethanol oxidation is mandatory for it to have effect on proteolysis, however, its action is not related to an increased state of reduction of the NAD system. In contrast to other reductants of the NAD system, ethanol effect is accompanied by a rise in the phosphorylation state of the adenine nucleotides, suggesting that its action might be related to the cellular energy state.

1. The administration of a single oral dose of 2, 3, 4 or 5 g of ethanol/kg (43.5, 65.2, 87.0 or 108.7 mmol/kg respectively) to rats increases the rate of oxygen consumption by liver slices from animals killed 24--48 h later. 2. The increase in the rate of hepatic respiration can be blocked by incubation in a medium containing ouabain, an inhibitor of the sodium pump, or in a calcium-free medium. 3. The enhancement of oxygen uptake caused by a single dose of ethanol can be abolished by adrenalectomy or by prior administration of the alpha-adrenergic blocking agent phentolamine, and is markedly less in thyroidectomized animals. 4. It is suggested that the effect which is elicited by acute ethanol administration on respiration by liver slices is mediated by adrenaline and by throid hormones, both of which appear to exert a calorigenic effect by activation of the sodium pump. The results are discussed in relation to the changes in liver oxidative capacity induced by chronic alcohol ingestion.

The effect of the glucocorticoid-antagonist RU486 (Roussel-Uclaf, France) on the increased activity of hepatic tryptophan oxygenase (TO) after administration of corticosterone and ethanol in rats was studied. RU486 (40 mg/kg per os) inhibited completely the effect of corticosterone (5-15 mg/kg injected intraperitoneally) on TO. Ethanol (4 g/kg) given intraperitoneally is followed by peak corticosterone concentrations comparable to those seen after the administration of 5-10 mg exogenous corticosterone, and increased the TO activity 3-fold 4 h after the injection. RU486 inhibited completely the ethanol-induced increase of TO, indicating that this increase is mediated by corticosterone.

The influence of alcohol on albumin synthesis was studied in the isolated perfused rabbit liver. Carbonate-(14)C was used to label the intracellular arginine pool which serves as the precursor of both the carbon of urea and the guanido carbon of arginine in albumin. The control group synthesized albumin at a rate of 33 mg/100 g of wet liver weight during 2.5 hr of perfusion. When alcohol, 220 mg/100 ml, was added to the perfusate, albumin synthesis decreased to between 7 and 11 mg, less than one-third the control rate. The addition of 10 mM tryptophan to perfusates containing alcohol prevented most of the inhibitory effects and albumin synthesis increased to average 24 mg. Further, the addition of alcohol to the perfusate decreased the hepatic protein/DNA ratio from 70 to 54 and the RNA/DNA ratio from 2.3 to 1.8, changes equivalent to those seen after a 24 hr fast. The addition of tryptophan to the perfusate prevented these findings in both instances. Endoplasmic membrane-bound polysomes were examined for aggregation. Alcohol decreased the quantity of heavier aggregates. Reaggregation occurred when tryptophan was added but quantitative changes in albumin synthesis could not be related to the degree of reaggregation.