A review of the role of reactive oxygen and nitrogen species in alcohol-induced mitochondrial dysfunction

Our understanding of the mechanisms involved in the development of alcohol-induced liver disease has increased substantially in recent years. Specifically, reactive oxygen and nitrogen species have been identified as key components in initiating and possibly sustaining the pathogenic pathways responsible for the progression from alcohol-induced fatty liver to alcoholic hepatitis and cirrhosis. Ethanol has been demonstrated to increase the production of reactive oxygen and nitrogen species and decrease several antioxidant mechanisms in liver. However, the relative contribution of the proposed sites of ethanol-induced reactive species production within the liver is still not clear. It has been proposed that chronic ethanol-elicited alterations in mitochondria structure and function might result in increased production of reactive species at the level of the mitochondrion in liver from ethanol consumers. This in turn might result in oxidative modification and inactivation of mitochondrial macromolecules, thereby contributing further to mitochondrial dysfunction and a loss in hepatic energy conservation. Moreover, ethanol-related increases in reactive species may shift the balance between pro- and anti-apoptotic factors such that there is activation of the mitochondrial permeability transition, which would lead to increased cell death in the liver after chronic alcohol consumption. This article will examine the critical role of these reactive species in ethanol-induced liver injury with specific emphasis on how chronic ethanol-associated alterations to mitochondria influence the production of reactive oxygen and nitrogen species and how their production may disrupt hepatic energy conservation in the chronic alcohol abuser.     

Brain mitochondrial alterations after chronic alcohol consumption

The aim of this study was to demonstrate the existence of alterations in glutathione and cholesterol homeostasis in brain mitochondria from alcoholic rats. Glutathione concentration decreased, whereas oxidized glutathione and cholesterol contents increased in these organelles, suggesting the ethanol-induced generation of reactive oxygen species, and the impairment of mitochondrial uptake of glutathione, possibly due to the increase in cholesterol deposition. The release of apoptogenic proteins was increased after stimulating mitochondria from the brain of alcoholic rats with atractyloside. As a conclusion, chronic alcohol consumption might sensitize brain mitochondria to apoptotic stimuli, and promote the subsequent release of apoptotic proteins.     

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.     

Contribution of mitochondria to oxidative stress associated with alcoholic liver disease.

The importance of oxidative stress in the development of alcoholic liver disease has long been appreciated. The mechanism by which ethanol triggers an increase in reactive oxygen species in the liver is complex, however, recent findings suggest that the mitochondrion may contribute significantly to the overall increase in oxidant levels in hepatocytes exposed to ethanol acutely or chronically. This review is focused on observations which indicate that the ability of ethanol to increase mitochondrial reactive oxygen species production is linked to its metabolism via oxidative processes and/or ethanol-related alterations to the mitochondrial electron transport chain. Furthermore, the capacity of ethanol-elicited increases in reactive oxygen species to oxidatively modify and inactivate mitochondrial proteins is highlighted as a mechanism by which ethanol might further disrupt the structure and function of mitochondria.     

Mitochondrial adaptations to obesity-related oxidant stress

It is not known why viable hepatocytes in fatty livers are vulnerable to necrosis, but associated mitochondrial alterations suggest that reactive oxygen species (ROS) production may be increased. Although the mechanisms for ROS-mediated lethality are not well understood, increased mitochondrial ROS generation often precedes cell death, and hence, might promote hepatocyte necrosis. The aim of this study is to determine if liver mitochondria from obese mice with fatty hepatocytes actually produce increased ROS. Secondary objectives are to identify potential mechanisms for ROS increases and to evaluate whether ROS increase uncoupling protein (UCP)-2, a mitochondrial protein that promotes ATP depletion and necrosis. Compared to mitochondria from normal livers, fatty liver mitochondria have a 50% reduction in cytochrome c content and produce superoxide anion at a greater rate. They also contain 25% more GSH and demonstrate 70% greater manganese superoxide dismutase activity and a 35% reduction in glutathione peroxidase activity. Mitochondrial generation of H(2)O(2) is increased by 200% and the activities of enzymes that detoxify H(2)O(2) in other cellular compartments are abnormal. Cytosolic glutathione peroxidase and catalase activities are 42 and 153% of control values, respectively. These changes in the production and detoxification of mitochondrial ROS are associated with a 300% increase in the mitochondrial content of UCP-2, although the content of beta-1 ATP synthase, a constitutive mitochondrial membrane protein, is unaffected. Supporting the possibility that mitochondrial ROS induce UCP-2 in fatty hepatocytes, a mitochondrial redox cycling agent that increases mitochondrial ROS production upregulates UCP-2 mRNAs in primary cultures of normal rat hepatocytes by 300%. Thus, ROS production is increased in fatty liver mitochondria. This may result from chronic apoptotic stress and provoke adaptations, including increases in UCP-2, that potentiate necrosis.     

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.     

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.     

Suppression of acute ethanol-induced hepatic steatosis by docosahexaenoic acid is associated with downregulation of stearoyl-CoA desaturase 1 and inflammatory cytokines

Excessive alcohol consumption can lead to hepatic steatosis. Omega-3 (n-3) polyunsaturated fatty acids (PUFA) have been shown to be effective in reducing hepatic accumulation of triglycerides (TG) by downregulation of TG biosynthesis in the liver. The aim of this study was to examine whether supplementation with the n-3 PUFA, docosahexaenoic acid (DHA), can effectively reduce acute alcohol-induced hepatic steatosis. Acute alcohol-induced hepatic steatosis was generated in 9-week-old male mice (C57BL/6J) by oral gavage of ethanol (4.7 g/kg BW) diluted in water (60%, v/v), with or without DHA (250 mg/kg BW), every 12 h for 3 administrations. Compared to the control (ethanol-alone) group, animals supplemented with DHA were protected against ethanol-induced TG accumulation in the liver. Accordingly, hepatic stearoyl-CoA desaturase-1 (SCD-1) expression, serum alanine aminotransferase (ALT) activity, and the levels of inflammatory cytokines (such as IL-6 and TNF-α) in the liver were significantly reduced, whereas the expression of heme oxygenase-1 (HO-1), an enzyme that can improve cell survival in liver tissue, was markedly increased in DHA-supplemented mice compared to the control animals. There were no differences in serum TG level and hepatic production of reactive oxygen species (ROS) between the two groups. Our findings demonstrate that DHA supplementation protects against acute ethanol-induced hepatic steatosis, which may be associated with reduced expression of SCD-1 and inflammatory cytokines.     

Oxidative stress and antioxidant defenses in ethanol-induced cell injury

Although in the past several mechanisms and factors have been proposed to be responsible for alcoholic liver disease (ALD), at present the involvement of oxygen free radicals and consequently of oxidative stress has acquired remarkable credit. In numerous experimental studies it has been shown the occurrence of alcohol-induced generation of oxygen- and ethanol-derived free radicals through different pathways and from different sources. Mitochondria appear to be both an important source of reactive oxygen species (ROS) and also a primary target of ethanol-induced damage. The consistent induction of the mitochondrial antioxidant enzyme manganese superoxide dismutase (Mn-SOD) observed in experimental animals after acute and chronic ethanol administration has all the characteristics of a "stress response" to an oxidative insult.     

Dynamic Adaptation of Liver Mitochondria to Chronic Alcohol Feeding in Mice: BIOGENESIS,REMODELING, AND FUNCTIONAL ALTERATIONS*

Liver mitochondria undergo dynamic alterations following chronic alcohol feeding to mice. Intragastric alcohol feeding to mice resulted in 1) increased state III respiration (109% compared with control) in isolated liver mitochondria, probably due to increased levels of complexes I, IV, and V being incorporated into the respiratory chain; 2) increased mitochondrial NAD⁺ and NADH levels (∼2-fold), with no change in the redox status; 3) alteration in mitochondrial morphology, with increased numbers of elongated mitochondria; and 4) enhanced mitochondrial biogenesis in the liver, which corresponded with an up-regulation of PGC-1α (peroxisome proliferator-activated receptor γ coactivator-1α). Oral alcohol feeding to mice, which is associated with less liver injury and steatosis, slightly enhanced respiration in isolated liver mitochondria (30.8% compared with control), lower than the striking increase caused by intragastric alcohol feeding. Mitochondrial respiration increased with both oral and intragastric alcohol feeding despite extensive N-acetylation of mitochondrial proteins. The alcohol-induced mitochondrial alterations are probably an adaptive response to enhance alcohol metabolism in the liver. Isolated liver mitochondria from alcohol-treated mice had a greater rate of acetaldehyde metabolism and respiration when treated with acetaldehyde than control. Aldehyde dehydrogenase-2 levels were unaltered in response to alcohol, suggesting that the greater acetaldehyde metabolism by isolated mitochondria from alcohol-treated mice was due to increased mitochondrial respiration that regenerated NAD⁺, the rate-limiting substrate in alcohol/acetaldehyde metabolism. Overall, our work suggests that mitochondrial plasticity in the liver may be an important adaptive response to the metabolic stress caused by alcohol intake and could potentially play a role in many other vital functions performed by the liver.     

Catechin suppresses an array of signalling molecules and modulates alcohol-induced endotoxin mediated liver injury in a rat model

Induction of nuclear factor kappa B (NF-κB)-mediated gene expression has been implicated in the pathogenesis of alcoholic liver disease through enhanced production of reactive oxygen species and pro-inflammatory mediators. The present study was carried out to investigate the role of catechin as a chain breaking inhibitor against experimental alcoholic liver injury. Rats were administered 35% v/v ethanol orally at a dose of 10 g/Kg/day for two weeks, followed by 14 g/Kg/day for 10 weeks. Catechin (50 mg/Kg) was co-supplemented after 4 weeks of alcohol treatment till the end of the dosing period. Following chronic alcohol exposure, rats developed endotoxemia and severe pathological changes in the liver such as pronounced fatty change, vacuolar degeneration and inflammation. These changes were accompanied by activation of NF-κB and induction of inflammatory and cytotoxic mediators leading to increased level of tumor necrosis factor-alpha, enhanced formation of malondialdehyde in the liver followed by drastic alterations in the hepatic antioxidant defense systems. Additionally, nitrite levels and lactate dehydrogenase activities were also significantly elevated on chronic alcohol consumption. Alcohol exposure also increased the number of micronucleated cells indicating that alcohol abuse may again be associated with the nuclear changes. Supplementation with catechin ameliorated the alcohol-induced liver injury by downregulating the endotoxin-mediated activation of initial signalling molecule NF-κB and further going downstream the signalling cascade including tumor necrosis factor-alpha, nitric oxide and reactive oxygen species and by enhancing the antioxidant profile. These observations correlated well with the histological findings. Moreover, a remarkable decrease in the percentage of micronucleated cells was observed with catechin supplementation indicating an apparent protection against alcohol-induced toxicity. These findings suggest that catechin may alleviate experimental alcoholic liver disease by suppressing induction of NF-κB, a key component of signalling pathway, thus forming a pharmacological basis for designing novel therapeutic agents against alcohol induced endotoxin-mediated liver injury.     

Protective effects of cilostazol on ethanol-induced damage in primary cultured hepatocytes

Alcoholic liver disease (ALD) caused by excessive alcohol consumption is associated with oxidative stress, mitochondrial dysfunction, and hepatocellular apoptosis. Cilostazol, a licensed clinical drug used to treat intermittent claudication, has been reported to act as a protective agent in a spectrum of diseases. However, little information regarding its role in ethanol-induced hepatocellular toxicity has been reported. In the current study, we investigated the protective effects and mechanisms of cilostazol on ethanol-induced hepatocytic injury. Rat primary hepatocytes were pretreated with cilostazol prior to ethanol treatment. MTT and LDH assay indicated that ethanol-induced cell death was ameliorated by cilostazol in a dose-dependent manner. Our results display that overproduction of intracellular reactive oxygen species (ROS) and 4-hydroxy-2-nonenal (4-HNE) induced by ethanol was attenuated by pretreatment with cilostazol. Furthermore, cilostazol significantly inhibited ethanol-induced generation of ROS in mitochondria. Importantly, it was shown that cilostazol could improve mitochondrial function in primary hepatocytes by restoring the levels of ATP and mitochondrial membrane potential (MMP). Additionally, cilostazol was found to reduce apoptosis induced by ethanol using a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Mechanistically, we found that cilostazol prevented mitochondrial pathway-mediated apoptotic signals by reversing the expression of Bax and Bcl2, the level of cleaved caspase-3, and attenuating cytochrome C release. These findings suggest the possibility of novel ALD therapies using cilostazol.     

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.     

Zinc prevention and treatment of alcoholic liver disease

Alcoholic liver disease (ALD) is associated with decreases in zinc (Zn) and its major binding protein, metallothionein (MT), in the liver. Studies using animal models have shown that Zn supplementation prevents alcohol-induced liver injury under both acute and chronic alcohol exposure conditions. There are hepatic and extrahepatic actions of Zn in the prevention of alcoholic liver injury. Zn supplementation attenuates ethanol-induced hepatic Zn depletion and suppresses ethanol-elevated cytochrome P450 2E1 (CYP2E1) activity, but increases the activity of alcohol dehydrogenase in the liver; an action that is likely responsible for Zn suppression of alcohol-induced oxidative stress. Zn also enhances glutathione-related antioxidant capacity in the liver. At the cellular level, Zn inhibits alcohol-induced hepatic apoptosis partially through suppression of the Fas/FasL-mediated pathway. Zn supplementation preserves intestinal integrity and prevents endotoxemia, leading to inhibition of endotoxin-induced tumor necrosis factor-alpha (TNF-alpha) production in the liver. Zn also directly inhibits the signaling pathway involved in endotoxin-induced TNF-alpha production. These hepatic and extrahepatic effects of Zn are independent of MT. However, low levels of MT in the liver sensitize the organ to alcohol-induced injury, and elevation of MT enhances the endogenous Zn reservoir and makes Zn available when oxidative stress is imposed. Zn has a high potential to be developed as an effective agent in the prevention and treatment of ALD.     

Mitochondrial dysfunction in hepatitis C virus infection

The mechanisms of liver injury in chronic hepatitis C virus (HCV) infection are poorly understood though HCV induces a state of hepatic oxidative stress that is more pronounced than that present in many other inflammatory diseases. This mini-review will focus on recent findings revealing an unexpected role of mitochondria in providing a central role in the innate immunity and in addition will illustrate the application of stably transfected human-derived cell lines, inducibly expressing the entire HCV open reading frame for in vitro studies on mitochondria. Results obtained by a comparative analysis of the respiratory chain complexes activities along with mitochondrial morpho-functional confocal microscopy imaging show a detrimental effect of HCV proteins on the cell oxidative metabolism with specific inhibition of complex I activity, decrease of mtDeltaPsi, increased production of reactive oxygen species. A possible de-regulation of calcium recycling between the endoplasmic reticulum and the mitochondrial network is discussed to provide new insights in the pathogenesis of hepatitis C.     

Oxidative mechanisms in the pathogenesis of alcoholic liver disease

Although the capacity of ethanol to induce oxidative stress in the liver is well established, the mechanisms by which oxidative damage contributes to the pathogenesis of alcoholic liver disease (ALD) is still incompletely understood. Recent reports have implicated oxidative mechanisms in the onset of alcoholic steatosis and in the formation of Mallory's bodies. Moreover, by inducing mitochondrial alterations, oxidative stress promotes hepatocyte necrosis and contributes to alcohol-induced sensitization of hepatocyte to the pro-apoptotic action of TNF-alpha. Oxidative mechanisms play also a role in the progression of liver fibrosis by triggering the release of pro-fibrotic cytokines and activating collagen gene expression in hepatic stellate cells. Finally, immune responses towards antigens originating from the reactions of lipid peroxidation products with hepatic proteins might represent one of the mechanisms that contribute to perpetuate chronic hepatic inflammation in ALD. Altogether these observations give a rationale to the possible clinical application of antioxidants in the therapy of ALD.     

Cytochrome P4502E1, oxidative stress, JNK, and autophagy in acute alcohol-induced fatty liver

Binge alcohol drinking induces hepatic steatosis. Recent studies showed that chronic ethanol-induced fatty liver was, at least in part, CYP2E1 dependent. The mechanism of acute alcohol-induced steatosis and whether CYP2E1 plays any role are still unclear. Increasing oxidative stress by alcohol can activate the JNK MAP kinase signaling pathway, suggesting that JNK might be a target for prevention of alcohol-induced steatosis. We used CYP2E1 knockout (KO) mice, a JNK inhibitor, and JNK1 or JNK2 knockout mice to test the role of CYP2E1, JNK, and the individual role of JNK1 and JNK2 in acute alcohol-induced steatosis. In wild-type (WT) mice, acute alcohol activates CYP2E1 and increases oxidative stress, which reciprocally increases activation of the JNK signaling pathway. Acute alcohol-induced fatty liver and oxidative stress were blunted in CYP2E1 KO mice and by the JNK inhibitor in WT mice. The antioxidant N-acetylcysteine decreased the acute alcohol-induced oxidative stress, the activation of JNK, and the steatosis but not the activation of CYP2E1. Acute alcohol decreased autophagy and increased expression of SREBP, effects blocked by the JNK inhibitor. Acute alcohol-induced fatty liver was the same in JNK1 and JNK2 KO mice as in WT mice; thus either JNK1 or JNK2 per se is sufficient for induction of steatosis by acute alcohol. The results show that acute alcohol elevation of CYP2E1, oxidative stress, and activation of JNK interact to lower autophagy and increase lipogenic SREBP resulting in fatty liver.     

Ethanol-induced dysfunction of hepatocytes and leukocytes in patients without liver failure

The repeated intake of a great amount of ethanol is followed by functional and organic changes in the body. The intestinal absorption of alcohol is accompanied by an increased absorption of Gram negative bacteria endotoxins in the portal blood. In the liver, endotoxins stimulate CD14 receptors on the membrane of Kupffer cells, with a secondary inflammatory liver response, consisting in the secretion of proinflammatory cytokines and acute phase proteins. Simultaneously, alcohol metabolism in the hepatocytes by alcohol dehydrogenase, microsomal enzymes and catalase pathways determines a large production of ROS (reactive oxygen species), with secondary oxidative aggression on all liver cells: hepatocytes, Kupffer cells, endothelial sinusoidal cells, hepatic stellate cells and liver s lymphocytes. The oxidative aggression, as well as the intermediary products of the alcohol metabolism, cause a structural change of the antigenic structures of the liver and of the released proteins, that induces an immune response on the both pathways (humoral and cellular). The pathophysiological mechanisms and the paraclinical characteristics of the ethanol-induced liver failure are well known, so we were interested to study the patients with chronic alcoholism, but no clinical or paraclinical sign of liver failure, in order to describe the liver's protective mechanisms. For this reason, 153 patients with chronic alcoholism were divided into four test lots, in order to determine: the activity and the serum level of ceruloplasmin, plasma level of MDA (malondialdehyde), lactic and pyruvic acids, serum level of transferrin, alpha1-antitrypsin, CRP (C reactive protein), C3 fraction of the complement, IgA, IgG, IgM, IL-1beta, IL-6 and IL-8, cytosolic level of the cytochrome c in the circulating leukocytes. An immunophenotype study (as normal markers) on the peripheral blood lymphocytes was performed, too. The results demonstrate an important oxidative aggression induced by three sources: the alcohol metabolism in the hepatocytes, activated Kupffer cells and activated neutrophils that have infiltrated the liver, due to the chemoattractant effect of IL-8. This aggression induces apoptosis and necrosis of the liver cells. The major liver protective factor is, in our opinion, IL-6, due to its important antioxidant, antiapoptotic and proregenerative demonstrated actions. This protective effect of IL-6 is accompanied by antioxidant and antiprotease actions of ceruloplasmin, alpha1-antitrypsin and transferrin. We consider that an increased serum level of IL-6 accompanied by a decreased level of IL-1beta signify that antiapoptotic, antioxidant and proregenerative liver mechanisms prevail against proapoptotic and necrotic mechanisms. On the other hand, the ethanol-induced apoptosis of leukocytes (especially of the B cells) is very important, probably due to the absence of IL-6 protective action on these cells. The apoptosis of the circulating leukocytes is proved by their significant increase of the cytochrome c cytosolic level. The ethanol-induced liver immune response is predominantly cellular, as proved by the decreased ratio T helper (CD4+)/T cytotoxic (CD8+) in the peripheral blood. It is very important to observe that these significant immunologic changes appear before clinical or paraclinical signs of hepatic failure start. All these parameters were investigated in three groups of patients: chronic alcoholics, chronic alcoholics in the first 24 hours of the withdrawal and chronic alcoholics with acute alcohol intoxication, so the aggression types and the protective mechanisms were measured and differentiated in each "ethanolic status".     

Alteration of mitochondrial supercomplexes assembly in metabolic diseases

Deregulation of nutrient, hormonal, or neuronal signaling produces metabolic alterations that result in increased mitochondrial reactive oxygen species (ROS) production. The associations of the mitochondrial respiratory chain components into supercomplexes could have pathophysiological relevance in metabolic diseases, as supramolecular arrangements, by sustaining a high electron transport rate, might prevent ROS generation. In this review, the relationship between mitochondrial dysfunction and supercomplex arrangement of the mitochondrial respiratory chain components in obesity, insulin resistance, hepatic steatosis and diabetes mellitus is summarized and discussed.     

Lipid oxidation and autophagy in yeast

Autophagy, a process involved in the degradation and the recycling of long-lived proteins and organelles to survive nitrogen starvation, is generally non-selective. However, recent data suggest that selective forms of autophagy exist, that are able to specifically target several organelles, including mitochondria. Conversely, mitochondrial alterations could trigger autophagy. Such a selective form of autophagy might be involved in the elimination of damaged mitochondria. We reported previously that, mitochondria were early targets of rapamycin-induced autophagy. Here we report that rapamycin-induced autophagy is accompanied by the early production of reactive oxygen species and by the early oxidation of mitochondrial lipid. Inhibition of these oxidative effects by resveratrol largely impaired autophagy of both cytosolic proteins and mitochondria, and delayed subsequent cell death. These results support a role of mitochondrial oxidation events in the activation of autophagy.