Dilinoleoylphosphatidylcholine decreases ethanol-induced cytochrome P4502E1.

Cytochrome P4502E1 (CYP2E1) induction by ethanol contributes to alcoholic liver disease and we found that a mixture of polyunsaturated phosphatidylcholines (PPC), which protects against alcohol-induced liver injury, also decreases CYP2E1. Since dilinoleoylphosphatidylcholine (DLPC) is the major component of PPC, we assessed here whether it is responsible for the protection of PPC by feeding rats for 8 weeks our liquid diet containing ethanol (36% of energy) or isocaloric carbohydrates, with either DLPC (1.5 g/1000 cal), PPC (3 g/1000 cal), or linoleate. CYP2E1 was assessed by Western blots and by two of its enzyme activities: the microsomal ethanol-oxidizing system (MEOS) and p-nitrophenolhydroxylase (PNP). With ethanol, CYP2E1 increased 10-fold, with corresponding rises in PNP and MEOS activities. Compared to linoleate, DLPC significantly decreased cytochrome b(5), total cytochromes P450, CYP2E1 content and its corresponding activities. DLPC decreases ethanol-induced CYP2E1 and should be considered for the prevention of alcoholic liver disease.     

Oxidation of LDL in baboons is increased by alcohol and attenuated by polyenylphosphatidylcholine.

Alcohol taken in moderation may prevent atherosclerosis, whereas heavy drinking has the opposite effect, in part by promoting oxidation of low density lipoproteins (LDL), a pathogenetic factor in atherogenesis. We assess here: 1 ) whether similar alterations can be reproduced in baboons fed 50% of energy as ethanol (the average intake of alcoholics) for 7- 8 years, and 2 ) whether such alterations are affected by supplementation with polyenylphosphatidylcholine (PPC), a mixture of polyunsaturated phosphatidylcholines, shown to prevent alcoholic fatty liver, fibrosis, and cirrhosis. Ten animals were given the ethanol-containing diet and ten were pair-fed isocaloric control diets. In half of the pairs, the diets were supplemented with 2.8 g of polyenylphosphatidylcholine/1000 kcal. Alcohol feeding increased LDL-lipoperoxides and made LDL-proteins more negatively charged, changes that were attenuated or prevented by PPC. The oxidizability of LDL was determined in vitro by the formation of conjugated dienes after oxidation with copper. Alcohol shortened the lag time (which measures LDL antioxidant capacity); this effect was normalized by PPC supplementation. By contrast, PPC produced no changes in the controls. Thus polyenylphosphatidylcholine, by markedly attenuating the ethanol-induced increase in LDL oxidation, opposes one of the effects whereby alcohol promotes atherosclerosis.     

Effects of ethanol on lipid metabolism.

Alcohol promotes accumulation of fat in the liver mainly by substitution of ethanol for fatty acids as the major hepatic fuel. The degree of lipid accumulation depends on the supply of dietary fat. Progressive alteration of the mitochondria, which occurs during chronic alcohol consumption, decreases fatty acid oxidation by interfering with citric acid cycle activity. This block is partially compensated for by increased ketone body production, which results in ketonemia. Thus, mitochondrial damage perpetuates fatty acid accumulation even in the absence of ethanol oxidation. Alcohol facilitates esterification of the accumulated fatty acids to triglycerides, phospholipids, and cholesterol esters, all of which accumulate in the liver. The accumulated lipids are disposed of in part as serum lipoprotein, resulting in moderate hyperlipemia. In some individuals with pre-existing alterations of lipid metabolism, small ethanol dose may provoke marked hyperlipemia which responds to alcohol withdrawal. Inhibition of the catabolism of cholesterol to bile salt may contribute to the hepatic accumulation and hypercholesterolemia. The capacity of lipoprotein production and hyperlipemia development increases during chronic alcohol consumption, probably as a result of the concomitant hypertrophy of the endoplasmic reticulum and Golgi apparatus. However, this compensation is relatively inefficient in ridding the liver of fat. This inefficiency may be linked to alterations of hepatic microtubules induced by ethanol or its metabolites, which interfere with the export of protein from liver to serum, promoting hepatic accumulation of proteins as well as fat. As liver injury aggravates, hyperlipemia wanes and liver steatosis is exaggerated. Derangements of serum lipids similar to those found in other types of liver disease also become apparent. The changes in serum lipids may be a sensitive indicator of the progression of liver damage in the alcoholic.     

Mitochondria and hepatotoxicity of ethanol

The current understanding of the effects of alcohol intoxication on the basic mitochondrial functions has been presented. Both, the direct toxic effect of ethanol on biological membranes and various cellular systems and the toxicity of acetaldehyde and reactive oxygen species (the products of ethanol oxidation) are discussed, with emphasis on the effect of ethanol on the basic functions of mitochondria and Ca(2+)-dependent mitochondrial permeability transition. Based on the available experimental data, it is demonstrated that acute alcohol intoxication causes a global mitochondrial dysfunction in the liver, resulting in considerable disturbance of the whole cellular metabolism. Alcohol poisoning of the liver leads to a decreased ability of cells to withstand oxidative stress, to support the synthesis of vital metabolic intermediates (e.g., methyl groups), as well as to produce urea from ammonia, due to a decreased permeability of the outer membrane and impaired exchange of substrates between the cytoplasm and the mitochondrial matrix. This review emphasizes the role of the voltage-dependent anion channels of the outer mitochondrial membrane in ethanol-mediated disturbances of basic mitochondrial functions and its consequences for the entire cell metabolism in the liver.     

Role of mitochondria in hepatotoxicity of ethanol

The current understanding of the effects of alcohol intoxication on the basic mitochondrial functions has been presented. Both, the direct toxic effect of ethanol on biological membranes and various cellular systems and the toxicity of acetaldehyde and reactive oxygen species (the products of ethanol oxidation) are discussed, with emphasis on the effect of ethanol on the basic functions of mitochondria and Ca²⁺-dependent mitochondrial permeability transition. Based on the available experimental data, it is demonstrated that acute alcohol intoxication causes a global mitochondrial dysfunction in the liver, resulting in considerable disturbance of the whole cellular metabolism. Alcohol poisoning of the liver leads to a decreased ability of cells to withstand oxidative stress, to support the synthesis of vital metabolic intermediates (e.g., methyl groups), as well as to produce urea from ammonia, due to a decreased permeability of the outer membrane and impaired exchange of substrates between the cytoplasm and the mitochondrial matrix. This review emphasizes the role of porin channels of the outer mitochondrial membrane in ethanol-mediated disturbances of basic mitochondrial functions and its consequences for the entire cell metabolism in the liver.     

Overexpression of CYP2E1 in mitochondria sensitizes HepG2 cells to the toxicity caused by depletion of glutathione

Induction of CYP2E1 by ethanol is one mechanism by which ethanol causes oxidative stress and alcohol liver disease. Although CYP2E1 is predominantly found in the endoplasmic reticulum, it is also located in rat hepatic mitochondria. In the current study, chronic alcohol consumption induced rat hepatic mitochondrial CYP2E1. To study the role of mitochondrial targeted CYP2E1 in generating oxidative stress and causing damage to mitochondria, HepG2 lines overexpressing CYP2E1 in mitochondria (mE10 and mE27 cells) were established by transfecting a plasmid containing human CYP2E1 cDNA lacking the hydrophobic endoplasmic reticulum targeting signal sequence into HepG2 cells followed by G418 selection. A 40-kDa catalytically active NH2-terminally truncated form of CYP2E1 (mtCYP2E1) was detected in the mitochondrial compartment in these cells by Western blot analysis. Cell death caused by depletion of GSH by buthionine sulfoximine (BSO) was increased in mE10 and mE27 cells as compared with cells transfected with empty vector (pCI-neo). Antioxidants were able to abolish the loss of cell viability. Increased levels of reactive oxygen species and mitochondrial 3-nitrotyrosine and 4-hydroxynonenal protein adducts and decreased mitochondrial aconitase activity and mitochondrial membrane potential were observed in mE10 and mE27 cells treated with BSO. The mitochondrial membrane stabilizer, cyclosporine A, was also able to protect these cells from BSO toxicity. These results revealed that CYP2E1 in the mitochondrial compartment could induce oxidative stress in the mitochondria, damage mitochondria membrane potential, and cause a loss of cell viability. The accumulation of CYP2E1 in hepatic mitochondria induced by ethanol consumption might play an important role in alcohol liver disease.     

S-adenosylmethionine prevents chronic alcohol-induced mitochondrial dysfunction in the rat liver

An early event that occurs in response to alcohol consumption is mitochondrial dysfunction, which is evident in changes to the mitochondrial proteome, respiration defects, and mitochondrial DNA (mtDNA) damage. S-adenosylmethionine (SAM) has emerged as a potential therapeutic for treating alcoholic liver disease through mechanisms that appear to involve decreases in oxidative stress and proinflammatory cytokine production as well as the alleviation of steatosis. Because mitochondria are a source of reactive oxygen/nitrogen species and a target for oxidative damage, we tested the hypothesis that SAM treatment during alcohol exposure preserves organelle function. Mitochondria were isolated from livers of rats fed control and ethanol diets with and without SAM for 5 wk. Alcohol feeding caused a significant decrease in state 3 respiration and the respiratory control ratio, whereas SAM administration prevented these alcohol-mediated defects and preserved hepatic SAM levels. SAM treatment prevented alcohol-associated increases in mitochondrial superoxide production, mtDNA damage, and inducible nitric oxide synthase induction, without a significant lessening of steatosis. Accompanying these indexes of oxidant damage, SAM prevented alcohol-mediated losses in cytochrome c oxidase subunits as shown using blue native PAGE proteomics and immunoblot analysis, which resulted in partial preservation of complex IV activity. SAM treatment attenuated the upregulation of the mitochondrial stress chaperone prohibitin. Although SAM supplementation did not alleviate steatosis by itself, SAM prevented several key alcohol-mediated defects to the mitochondria genome and proteome that contribute to the bioenergetic defect in the liver after alcohol consumption. These findings reveal new molecular targets through which SAM may work to alleviate one critical component of alcohol-induced liver injury: mitochondria dysfunction.     

Ethanol perfusion increases the yield of oxidative phosphorylation in isolated liver of fed rats

The question arises as to the effect of ethanol on the actual yield of oxidative phosphorylation in the whole liver because of contradictory results reported in isolated hepatic mitochondria.The adenosine triphosphate (ATP) content of liver isolated from fed rats and perfused in the presence (10 mM) and absence of ethanol was continuously evaluated using ³¹P Nuclear Magnetic Resonance (NMR). An accurate estimation of mitochondrial ATP synthesis in the whole organ was obtained by subtracting the glycolytic ATP supply from the total ATP production. Simultaneously, the respiratory activity was assessed using O₂ Clark electrodes.The data indicate that ethanol enhanced the net consumption of ATP, leading to a new steady state of the ATP content. ATP synthesis was also found higher under ethanol [1.86±0.02 μmol/min g wet weight (min g ww)] than in control [1.44±0.18 μmol/min g ww]. However, mitochondrial respiration remained unchanged [2.20±0.13 μmol/min g ww] and, consequently, the in situ mitochondrial ATP/O ratio increased from 0.33±0.035 (control) to 0.42±0.015 (ethanol).The increase of the oxidative phosphorylation yield in the whole liver may be linked to the decrease in cytochrome oxidase activity induced by ethanol [FEBS Lett. 468 (2000) 239]. The significant raise (27%) of the ATP/O ratio was not sufficient to maintain the ATP level following ethanol-increased ATP consumption.     

Protective effects of cassia seed ethanol extract against carbon tetrachloride-induced liver injury in mice

Oxidative stress has been recognized as a critical pathogenetic mechanism for the initiation and the progression of hepatic injury in a variety of liver disorders. Antioxidants, including many natural compounds or extracts, have been used to cope with liver disorders. The present study was designed to investigate the hepatoprotective effects of cassia seed ethanol extract (CSE) in carbon tetrachloride (CCl(4))-induced liver injury in mice. The animals were pre-treated with different doses of CSE (0.5, 1.0, 2.0 g/kg body weight) or distilled water for 5 days, then were injected intraperitoneally with CCl(4) (0.1% in corn oil, v/v, 20 ml/kg body weight), and sacrificed at 16 hours after CCl(4) exposure. The serum aminotransferase activities, histopathological changes, hepatic and mitochondrial antioxidant indexes, and cytochrome P450 2E1 (CYP2E1) activities were examined. Consistent with previous studies, acute CCl(4) administration caused great lesion to the liver, shown by the elevation of the serum aminotransferase activities, mitochondria membrane permeability transition (MPT), and the ballooning degeneration of hepatocytes. However, these adverse effects were all significantly inhibited by CSE pretreatment. CCl(4)-induced decrease of the CYP2E1 activity was dose-dependently inhibited by CSE pretreatment. Furthermore, CSE dramatically decreased the hepatic and mitochondrial malondialdehyde (MDA) levels, increased the hepatic and mitochondrial glutathione (GSH) levels, and restored the activities of superoxide dismutase (SOD), glutathione reductase (GR), and glutathione S-transferase (GST). These results suggested that CSE could protect mice against CCl(4)-induced liver injury via enhancement of the antioxidant capacity.     

Ethanol consumption and liver mitochondria function

The mitochondrion is the subcellular organelle affected earliest during the development of alcoholic liver disease. As a result of chronic ethanol consumption mitochondrial protein synthesis is decreased significantly due to a depression in the functioning of the mitochondrial ribosome. This causes a significant decrease in the concentrations of the thirteen mitochondria gene products, all of which are components of the oxidative phosphorylation system. Consequently, there is a depression in the rate at which ATP is synthesized in hepatic mitochondria. In addition to this loss in function, hepatic mitochondria either acutely or chronically exposed to ethanol generate increased levels of reactive oxygen species (ROS). This elevation in ROS has been demonstrated in both isolated mitochondria and hepatocytes. The increase in mitochondrial ROS production accompanying acute ethanol exposure is due to mitochondrial associated reoxidation of NADH produced during ethanol and acetaldehyde metabolism. The elevation in ROS generation observed in mitochondria from chronic ethanol consumers is likely due to decreases in mitochondrial-derived electron transport components, which in turn results in higher levels of the semiquinone forms of flavin mononucleotide and ubiquinone. Both these semiquinones readily donate electrons to molecular oxygen to form superoxide.     

Mitochondrial alterations in livers of Sod1-/- mice fed alcohol

Chronic alcohol consumption induced liver injury in Cu,Zn-superoxide dismutase-deficient mice (Sod1-/-), with extensive centrilobular necrosis and inflammation and a reduction in hepatic ATP content. Mechanisms by which ethanol decreased ATP in these mice remain unclear. We investigated alterations in mitochondria of Sod1-/- mice produced by chronic ethanol treatment. These mitochondria had an increase in State 4 oxygen consumption with succinate and especially with glutamate plus malate compared to mitochondria from pair-fed Sod1-/- mice or mitochondria from wild-type mice fed dextrose or ethanol. This uncoupling was associated with a decrease in ADP/O and respiratory control ratios, a decline in mitochondrial membrane potential, enhanced mitochondrial permeability transition, and decreased aconitase activity. Total thiols and uncoupling protein 2 levels were elevated in the pair-fed Sod1-/- mitochondria, perhaps an adaptive response to oxidant stress. However, no such increases were found with the ethanol-fed Sod1-/- mitochondria, suggesting a failure to develop these adaptations. The mitochondria from the ethanol-fed Sod1-/- mice had elevated levels of cleaved Bax, Bak, Bcl-xl, and adenine nucleotide translocator. Immunoprecipitation studies revealed increased association of Bax and Bak with the adenine nucleotide translocator. ADP-ATP exchange was very low in the ethanol-fed Sod1-/- mitochondria. These results suggest that ethanol treatment of Sod1-/- mice produces uncoupling and a decline in Deltapsi, swelling, increased association of proapoptotic proteins involved in the permeability transition, and decreased adenine nucleotide translocator activity, which may be responsible for the decline in ATP levels and development of necrosis in this model of alcohol-induced liver injury.     

Stabilization of hsp70 mRNA on prolonged cell exposure to hypertonicity

The question arises as to the effect of ethanol on the actual yield of oxidative phosphorylation in the whole liver because of contradictory results reported in isolated hepatic mitochondria.The adenosine triphosphate (ATP) content of liver isolated from fed rats and perfused in the presence (10 mM) and absence of ethanol was continuously evaluated using 31P Nuclear Magnetic Resonance (NMR). An accurate estimation of mitochondrial ATP synthesis in the whole organ was obtained by subtracting the glycolytic ATP supply from the total ATP production. Simultaneously, the respiratory activity was assessed using O(2) Clark electrodes.The data indicate that ethanol enhanced the net consumption of ATP, leading to a new steady state of the ATP content. ATP synthesis was also found higher under ethanol [1.86+/-0.02 micromol/min g wet weight (min g ww)] than in control [1.44+/-0.18 micromol/min g ww]. However, mitochondrial respiration remained unchanged [2.20+/-0.13 micromol/min g ww] and, consequently, the in situ mitochondrial ATP/O ratio increased from 0.33+/-0.035 (control) to 0.42+/-0.015 (ethanol).The increase of the oxidative phosphorylation yield in the whole liver may be linked to the decrease in cytochrome oxidase activity induced by ethanol [FEBS Lett. 468 (2000) 239]. The significant raise (27%) of the ATP/O ratio was not sufficient to maintain the ATP level following ethanol-increased ATP consumption.     

Effects of exercise and ethanol on liver mitochondrial function

Rates of ADP stimulated respiration for various substrates were determined in mitochondria isolated from the livers of female Sprague-Dawley rats following 8 weeks of treatment with daily swimming, ethanol consumption, or both. All rats were fed an American Institute of Nutrition (AIN) type liquid diet with the ethanol treated rats receiving 35% of the calories as ethanol. Chronic exposure to ethanol depressed both state 3 respiration with glutamate as a substrate and cytochrome oxidase activity. Respiratory control ratios and P:O ratios, however, were unaffected by the ethanol exposure. Exercise alone had no effect on hepatic mitochondrial function. There were also no significant alterations in oxidative function of hepatic mitochondria from rats which were endurance-trained by swimming while receiving the ethanol diet. This lack of alteration in mitochondrial function was in spite of the fact that these rats consumed an identical amount of ethanol as those which incurred mitochondrial dysfunction. These results indicate that regular exercise has the potential to attenuate the ethanol induced decline in hepatic mitochondria.     

Contribution of “substrate shuttles” in the transport of extramitochondrial reducing equivalents by hepatic mitochondria from chronic alcohol-fed mice

Effects of chronic alcohol treatment have been investigated on the rates of extramitochondrial NADH utilization by hepatic mitochondria in the presence or absence of “malate-aspartate shuttle,” oxidation of ethanol, α-glycerophosphate, and the activity of succinic dehydrogenase, along with the changes in the intrahepatic distribution of aspartate aminotransferase. The rates of blood alcohol clearance, hepatic alcohol dehydrogenase activity, and NADPH-dependent microsomal ethanol oxidation were also studied after different time intervals of alcohol withdrawal from chronically alcohol-fed animals. Hepatic mitochondria from chronically ethanol-fed mice (ethanol withheld 20 hr before sacrifice) utilized extramitochondrial NADH at rates 25–40% higher than the corresponding pair-fed controls. Addition of malateaspartate shuttle components to mitochondria from control and ethanol-fed groups resulted in 70 and 90% stimulation of NADH utilization, respectively. Mitochondria from both groups showed respiratory control upon ADP addition (state 3). Preincubation with amino-oxyacetate or hydrazine, which inhibit aspartate aminotransferase activity, prevented the stimulatory effect of malate-aspartate shuttle on NADH utilization. Mitochondria from livers of chronic ethanol-fed mice in the presence of reconstituted malate-aspartate shuttle showed 30–40% higher utilization of ethanol than the corresponding pair-fed control animals. The rate of mitochondrial α-glycerophosphate utilization by alcohol-fed animals was significantly higher than the control group. Succinic dehydrogenase activity measured as an index of mitochondrial permeability in the absence of Ca²⁺ showed 85% higher activity in alcoholtreated group than the control animals. Chronic ethanol feeding for 4 weeks resulted in an increase in the activity of hepatic aspartate aminotransferase in the cytoplasmic fraction and a corresponding decrease in the mitochondrial fraction. Alcohol withdrawal from chronic alcohol-fed animals resulted in a decrease in the blood alcohol clearance rate after 10 days. Furthermore, a lack of correlation was observed between the rates of blood alcohol clearance and the activity of hepatic alcohol dehydrogenase on one hand, and between the rates of blood alcohol clearance and the microsomal ethanol-oxidizing activity on the other.     

S-adenosylmethionine (SAMe) protects against acute alcohol induced hepatotoxicity in mice small star, filled

Although S-Adenosylmethionine (SAMe) has beneficial effects in many hepatic disorders, the effects of SAMe on acute alcohol-induced liver injury are unknown. In the present study, we investigated effects of SAMe on liver injury in mice induced by acute alcohol administration. Male C57BL/6 mice received ethanol (5 g/kg BW) by gavage every 12 hrs for a total of 3 doses. SAMe (5 mg/kg BW) was administrated i.p. once a day for three days before ethanol administration. Subsequent serum ALT level, hepatic lipid peroxidation, enzymatic activity of CYP2E1 and hepatic mitochondrial glutathione levels were measured colorimetrically. Intracellular SAMe concentration was measured by high-performance liquid chromatography (HPLC). Histopathological changes were assessed by H&E staining. Our results showed that acute ethanol administration caused prominent microvesicular steatosis with mild necrosis and an elevation of serum ALT activity. SAMe treatment significantly attenuated the liver injury. In association with the hepatocyte injury, acute alcohol administration induced significant decreases in both hepatic SAMe and mitochondrial GSH levels along with enhanced lipid peroxidation. SAMe treatment attenuated hepatic SAMe and mitochondrial GSH depletion and lipid peroxidation following acute alcohol exposure. These results demonstrate that SAMe protects against the liver injury and attenuates the mitochondrial GSH depletion caused by acute alcohol administration. SAMe may prove to be an effective therapeutic agent in many toxin-induced liver injuries including those induced by alcohol.     

The responses of mitochondrial proteome in rat liver to the consumption of moderate ethanol: the possible roles of aldo-keto reductases

A large body of evidence supports the view that mitochondria are a primary target of alcohol stress. Changes in mitochondrial proteins due to moderate ethanol intake, however, have not been broadly and accurately estimated. For this study, rats were fed low doses of ethanol and the mitochondria were isolated from heart, kidney, and liver, using ultracentrifugation with Nycodenz density gradient. The mitochondrial proteins were well resolved upon two-dimensional electrophoresis (2DE), and the alcohol-responsive 2DE spots were identified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Compared with the control group, the proteins extracted from liver mitochondria of ethanol-fed rats exhibited the significant changes on 2DE images, whereas the 2DE images obtained from the kidney and the heart mitochondria remained almost unchanged by ethanol feeding. Significantly, over 50% of the alcohol-responsive proteins in liver mitochondria were members of aldo-keto reductase family (AKR), which were usually present in cytoplasm. The organelle distributions of AKR proteins in liver mitochondria were further confirmed by Western blot analysis as well as by confocal microscopy. In addition, translocations of AKR were examined in the CHANG cell line, which was cultured with and without ethanol. The results of Western blot strongly suggested that the abundances of AKR proteins in the mitochondria were greatly reduced by the presence of ethanol in culture medium. The results of this study show that, even with moderate ethanol feeding, the mitochondrial proteome in rat liver was more sensitive to alcohol stress than that of either the kidney or the heart. The translocation of AKR proteins may be involved in the detoxification of liver cells.     

Combined effect of ascorbic acid and selenium supplementation on alcohol-induced oxidative stress in guinea pigs

Oxidative stress is a key step in the pathogenesis of ethanol associated liver injury. Ethanol administration induces an increase in lipid peroxidation either by enhancing the production of oxygen reactive species or by decreasing the level of endogenous antioxidants. In this present study, four groups of male guinea pigs (Cavia porcellus) were maintained for 45 days as follows: Control group (1 mg ascorbic acid (AA)/100 g body wt./day); Ethanol group (1 mg AA/100 g body wt./day+900 mg ethanol/100 g body wt./day); Selenium+AA group (25 mg AA+0.05 mg sodium selenite/100 g body wt./day); Ethanol+Se+AA group (25 mg AA+0.05 mg sodium selenite/100 g body wt.+900 mg ethanol/100 g body wt./day). Malondialehyde (MDA), hydroperoxides (HP) and conjugated dienes (CD) were significantly increased, while the activities of scavenging enzymes superoxide dismutase (SOD) and catalase were reduced in the alcohol administered groups. Co-administration of Se+AA along with alcohol increased the activities of scavenging enzymes and reduced the lipid peroxidation products level in hepatic tissues of guinea pigs. Activities of glutathione peroxidase (GPX) and glutathione reductase (GR) were enhanced in co-administered group. gamma-Glutamyl transpeptidase (GGT), a marker enzyme of alcohol induced toxicity, was also reduced, as was the glutathione content. This study suggests that the combined effect of Se+AA, provides protection against alcohol-induced oxidative stress as evidenced from the decreased levels of lipid peroxidation products and enhanced activities of scavenging enzymes.     

Ethanol increases mitochondrial cytochrome P450 2E1 in mouse liver and rat hepatocytes

Enhanced hepatic levels of cytochrome P450 2E1 (CYP2E1) may play a key role in the pathogenesis of some liver diseases because CYP2E1 represents a significant source of reactive oxygen species. Although a large fraction of CYP2E1 is located in the endoplasmic reticulum, CYP2E1 is also present in mitochondria. In this study, we asked whether ethanol, a known inducer of microsomal CYP2E1, could also increase CYP2E1 within mitochondria. Our findings indicated that ethanol increased microsomal and mitochondrial CYP2E1 in cultured rat hepatocytes and in the liver of lean mice. This was associated with decreased levels of glutathione, possibly reflecting increased oxidative stress. In contrast, in leptin-deficient obese mice, ethanol administration did not increase mitochondrial CYP2E1, nor it depleted mitochondrial glutathione, suggesting that leptin deficiency hampers mitochondrial targeting of CYP2E1. Thus, ethanol intoxication increases CYP2E1 not only in the endoplasmic reticulum but also in mitochondria, thus favouring oxidative stress in these compartments.     

Effects of 4-hydroxynonenal on mitochondrial 3-hydroxy-3-methylglutaryl (HMG-CoA) synthase

Chronic ethanol consumption causes increased production of reactive oxygen species in hepatic mitochondria accompanied by elevations in products of lipid peroxidation such as 4-hydroxynonenal (4-HNE). In the current study we investigated the effects of chronic ethanol consumption on a prominent protein-4-HNE adduct in liver mitochondria. Male Sprague-Dawley rats were fed a liquid diet for 31 days in which ethanol constituted 36% of total calories. Immunoblot analyses of liver mitochondria from ethanol-fed and control animals, using an antibody to a 4-HNE-protein adduct, demonstrated elevated 4-HNE binding (+50%) to a mitochondrial protein of approximately 55 kDa due to chronic ethanol consumption. Analysis of this protein using AspN digestion and tandem mass spectrometry identified it as the mitochondrial form of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. Activity of the activated form of this enzyme was unchanged in livers from ethanol-fed animals, but the protein level was elevated by 36%, which suggests a compensatory mechanism to maintain constant levels of synthase activity in the mitochondrion in the face of continuous inactivation by 4-HNE. Treatment of isolated mitochondria with 4-HNE demonstrated that the enzyme activity decreased as a function of 4-HNE concentration and with time of exposure. This study demonstrates that ethanol consumption increases the formation of a 4-HNE adduct with mitochondrial HMG-CoA synthase, which has the potential to inactivate the enzyme in situ.     

Resveratrol, a red wine polyphenol, attenuates ethanol-induced oxidative stress in rat liver

The involvement of oxidative stress in the pathogenesis of alcoholic diseases in the liver has been repeatedly confirmed. Resveratrol, a natural phytoalexin present in grape skin and red wine possesses a variety of biological activities including antioxidant. This study was conducted to evaluate whether resveratrol has a preventive effect on the main indicators of hepatic oxidative status as an expression of the cellular damage caused by free radicals, and on antioxidant defence mechanism during chronic ethanol treatment. Wistar rats were treated daily with 35% ethanol solution (3 g/kg/day i.p.) during 6 weeks and fed basal diet or basal diet containing 5 g/kg resveratrol. Control rats were treated with i.p. saline and fed basal diet. Experimentally, chronic ethanol administration leads to hepatotoxicity as monitored by the increase in the level of hepatic marker enzymes and the appearance of fatty change, necrosis, fibrosis and inflammation in liver sections. Ethanol also enhanced the formation of MDA in the liver indicating an increase in lipid peroxidation, a major end-point of oxidative damage, and caused drastic alterations in antioxidant defence systems. Particularly the activities of hepatic superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) were found reduced by ethanol treatment while glutathione reductase (GR) activity was unchanged. Dietary supplementation with resveratrol during ethanol treatment inhibited hepatic lipid peroxidation and ameliorated SOD, GPx and CAT activities in the liver. Conclusively, we can suggest that resveratrol could have a beneficial effect in inhibiting the oxidative damage induced by chronic ethanol administration, which was proved by the experiments that we conducted on rats.