Supplementary Materials from Cisplatin-Induced Apoptosis Involves Membrane Fluidification via Inhibition of NHE1 in Human Colon Cancer Cells

A growing body of evidences indicate the major role of extracellular vesicles (EVs) as players of cell communication in the pathogenesis of liver diseases. EVs are membrane-enclosed vesicles released by cells into the extracellular environment. Oxidative stress is also a key component of liver disease pathogenesis, but no role for hepatocyte-derived EVs has yet been described in the development of this process. Recently, some polycyclic aromatic hydrocarbons (PAHs), widespread environmental contaminants, were demonstrated to induce EV release from hepatocytes. They are also well-known to trigger oxidative stress leading to cell death. Therefore, the aim of this work was to investigate the involvement of EVs derived from PAHs-treated hepatocytes (PAH-EVs) in possible oxidative damages of healthy recipient hepatocytes, using both WIF-B9 and primary rat hepatocytes. We first showed that the release of EVs from PAHs -treated hepatocytes depended on oxidative stress. PAH-EVs were enriched in proteins related to oxidative stress such as NADPH oxidase and ferritin. They were also demonstrated to contain more iron. PAH-EVs could then induce oxidative stress in recipient hepatocytes, thereby leading to apoptosis. Mitochondria and lysosomes of recipient hepatocytes exhibited significant structural alterations. All those damages were dependent on internalization of EVs that reached lysosomes with their cargoes. Lysosomes thus appeared as critical organelles for EVs to induce apoptosis. In addition, pro-oxidant components of PAH-EVs, e.g. NADPH oxidase and iron, were revealed to be necessary for this cell death.

Extracellular vesicles (EVs) are membrane-enclosed nanostructures released by cells into the extracellular environment. As major actors of physiological intercellular communication, they have been shown to be pathogenic mediators of several liver diseases. Extracellular vesicles also appear to be potential actors of drug-induced liver injury but nothing is known concerning environmental pollutants. We aimed to study the impact of polycyclic aromatic hydrocarbons (PAHs), major contaminants, on hepatocyte-derived EV production, with a special focus on hepatocyte death. Three PAHs were selected, based on their presence in food and their affinity for the aryl hydrocarbon receptor (AhR): benzo[a]pyrene (BP), dibenzo[a,h]anthracene (DBA), and pyrene (PYR). Treatment of primary rat and WIF-B9 hepatocytes by all 3 PAHs increased the release of EVs, mainly comprised of exosomes, in parallel with modifying exosome protein marker expression and inducing apoptosis. Moreover, PAH treatment of rodents for 3 months also led to increased EV levels in plasma. The EV release involved CYP metabolism and the activation of the transcription factor, the AhR, for BP and DBA and another transcription factor, the constitutive androstane receptor, for PYR. Furthermore, all PAHs increased cholesterol levels in EVs but only BP and DBA were able to reduce the cholesterol content of total cell membranes. All cholesterol changes very likely participated in the increase in EV release and cell death. Finally, we studied changes in cell membrane fluidity caused by BP and DBA due to cholesterol depletion. Our data showed increased cell membrane fluidity, which contributed to hepatocyte EV release and cell death.

Hepatic steatosis (i.e. lipid accumulation) and steatohepatitis have been related to diverse etiologic factors, including alcohol, obesity, environmental pollutants. However, no study has so far analyzed how these different factors might interplay regarding the progression of liver diseases. The impact of the co-exposure to the environmental carcinogen benzo[a]pyrene (B[a]P) and the lifestyle-related hepatotoxicant ethanol, was thus tested on in vitro models of steatosis (human HepaRG cell line; hybrid human/rat WIF-B9 cell line), and on an in vivo model (obese zebrafish larvae). Steatosis was induced prior to chronic treatments (14, 5 or 7 days for HepaRG, WIF-B9 or zebrafish, respectively). Toxicity and inflammation were analyzed in all models; the impact of steatosis and ethanol towards B[a]P metabolism was studied in HepaRG cells. Cytotoxicity and expression of inflammation markers upon co-exposure were increased in all steatotic models, compared to non steatotic counterparts. A change of B[a]P metabolism with a decrease in detoxification was detected in HepaRG cells under these conditions. A prior steatosis therefore enhanced the toxicity of B[a]P/ethanol co-exposure in vitro and in vivo; such a co-exposure might favor the appearance of a steatohepatitis-like state, with the development of inflammation. These deleterious effects could be partly explained by B[a]P metabolism alterations.

We previously demonstrated that co-exposing pre-steatotic hepatocytes to benzo[a]pyrene (B[a]P), a carcinogenic environmental pollutant, and ethanol, favored cell death. Here, the intracellular mechanisms underlying this toxicity were studied. Steatotic WIF-B9 hepatocytes, obtained by a 48h-supplementation with fatty acids, were then exposed to B[a]P/ethanol (10 nM/5 mM, respectively) for 5 days. Nitric oxide (NO) was demonstrated to be a pivotal player in the cell death caused by the co-exposure in steatotic hepatocytes. Indeed, by scavenging NO, CPTIO treatment of co-exposed steatotic cells prevented not only the increase in DNA damage and cell death, but also the decrease in the activity of CYP1, major cytochrome P450s of B[a]P metabolism. This would then lead to an elevation of B[a]P levels, thus possibly suggesting a long-lasting stimulation of the transcription factor AhR. Besides, as NO can react with superoxide anion to produce peroxynitrite, a highly oxidative compound, the use of FeTPPS to inhibit its formation indicated its participation in DNA damage and cell death, further highlighting the important role of NO. Finally, a possible key role for AhR was pointed out by using its antagonist, CH-223191. Indeed it prevented the elevation of ADH activity, known to participate to the ethanol production of ROS, notably superoxide anion. The transcription factor, NFκB, known to be activated by ROS, was shown to be involved in the increase in iNOS expression. Altogether, these data strongly suggested cooperative mechanistic interactions between B[a]P via AhR and ethanol via ROS production, to favor cell death in the context of prior steatosis.

The rise in prevalence of non-alcoholic fatty liver disease (NAFLD) constitutes an important public health concern worldwide. Including obesity, numerous risk factors of NAFLD such as benzo[a]pyrene (B[a]P) and ethanol have been identified as modifying the physicochemical properties of the plasma membrane in vitro thus causing membrane remodeling—changes in membrane fluidity and lipid-raft characteristics. In this study, the possible involvement of membrane remodeling in the in vivo progression of steatosis to a steatohepatitis-like state upon co-exposure to B[a]P and ethanol was tested in obese zebrafish larvae. Larvae bearing steatosis as the result of a high-fat diet were exposed to ethanol and/or B[a]P for seven days at low concentrations coherent with human exposure in order to elicit hepatotoxicity. In this condition, the toxicant co-exposure raised global membrane order with higher lipid-raft clustering in the plasma membrane of liver cells, as evaluated by staining with the fluoroprobe di-4-ANEPPDHQ. Involvement of this membrane’s remodeling was finally explored by using the lipid-raft disruptor pravastatin that counteracted the effects of toxicant co-exposure both on membrane remodeling and toxicity. Overall, it can be concluded that B[a]P/ethanol co-exposure can induce in vivo hepatotoxicity via membrane remodeling which could be considered as a good target mechanism for developing combination therapy to deal with steatohepatitis.

Fucoxanthin is a carotenoid present in brown micro- and macro algae, that induces apoptosis or autophagy in cancer cells grown in vitro. In vivo studies confirmed its interest as a natural anticancer compound, as it exerts antitumoral, antimetastatic and antiangiogenic activities in animal models. Studies focused on the pharmacology of fucoxanthin in cancer cells and tumors have revealed that it affects a wide panel of cellular, molecular and tissular processes, suggesting that its biological activity may be related in part to a nonspecific integration in cell membranes, and possible interaction with lipid rafts. Thus, preliminary data confirming this interaction of fucoxanthin with lipid rafts were obtained in mast cells and hepatoma WIF-B9 cells. We here discuss this hypothesis, in view of the critical function of lipid rafts in cancer cell survival, invasivity and communication with the tumoral microenvironment.

The easy-to-use in vivo model, zebrafish larva, is being increasingly used to screen chemical-induced hepatotoxicity, with a good predictivity for various mechanisms of liver injury. However, nothing is known about its applicability in exploring the mechanism called membrane remodeling, depicted as changes in membrane fluidity or lipid raft properties. The aim of this study was, therefore, to substantiate the zebrafish larva as a suitable in vivo model in this context. Ethanol was chosen as a prototype toxicant because it is largely described, both in hepatocyte cultures and in rodents, as capable of inducing a membrane remodeling leading to hepatocyte death and liver injury. The zebrafish larva model was demonstrated to be fully relevant as membrane remodeling was maintained even after a 1-week exposure without any adaptation as usually reported in rodents and hepatocyte cultures. It was also proven to exhibit a high sensitivity as it discriminated various levels of cytotoxicity depending on the extent of changes in membrane remodeling. In this context, its sensitivity appeared higher than that of WIF-B9 hepatic cells, which is suited for analyzing this kind of hepatotoxicity. Finally, the protection afforded by a membrane stabilizer, ursodeoxycholic acid (UDCA), or by a lipid raft disrupter, pravastatin, definitely validated zebrafish larva as a reliable model to quickly assess membrane remodeling involvement in chemical-induced hepatotoxicity. In conclusion, this model, compatible with a high throughput screening, might be adapted to seek hepatotoxicants via membrane remodeling, and also drugs targeting membrane features to propose new preventive or therapeutic strategies in chemical-induced liver diseases. Copyright © 2016 John Wiley & Sons, Ltd.

The bacterial communication through signaling molecules termed quorum sensing has gained a lot of attention due to its applicability to different fields of study. Quorum sensing regulates several bacterial activities such as biofilm formation, antibiotic resistance, bioluminescence and bacterial virulence. These coordinated activities are only realized when the bacterial population reaches a high population density. The signaling molecules used in quorum sensing occur at very low concentrations which call for very sensitive methods for their identification and characterization. Many studies have been performed on the use of inhibitors in attempt to disrupt quorum sensing and consequently alleviate its negative effects. This article gives a description of the types of auto-inducers involved in quorum sensing and the fields of study they greatly affect especially the environmental one. It also covers types of analytical sample preparation and detection methods, and different types of inhibitors used for quorum quenching.

Most tumors undergo metabolic reprogramming towards glycolysis, the so-called Warburg effect, to support growth and survival. Overexpression of IF1, the physiological inhibitor of the F0F1ATPase, has been related to this phenomenon and appears to be a relevant marker in cancer. Environmental contributions to cancer development are now widely accepted but little is known about the underlying intracellular mechanisms. Among the environmental pollutants humans are commonly exposed to, benzo[a]pyrene (B[a]P), the prototype molecule of polycyclic aromatic hydrocarbons (PAHs), is a well-known human carcinogen. Besides apoptotic signals, B[a]P can also induce survival signals in liver cells, both likely involved in cancer promotion. Our previous works showed that B[a]P elicited a Warburg-like effect, thus favoring cell survival. The present study aimed at further elucidating the molecular mechanisms involved in the B[a]P-induced metabolic reprogramming, by testing the possible involvement of IF1. We presently demonstrate, both in vitro and in vivo , that PAHs, especially B[a]P, strongly increase IF1 expression. Such an increase, which might rely on β2-adrenergic receptor activation, notably participates to the B[a]P-induced glycolytic shift and cell survival in liver cells. By identifying IF1 as a target of PAHs, this study provides new insights about how environmental factors may contribute to related carcinogenesis.

La stéatose est la pathologie hépatique la plus répandue dans les pays occidentaux, affectant environ 80 % de la population obèse. Elle se caractérise par l’enrichissement des hépatocytes en gouttelettes lipidiques contenant principalement des triglycérides. La stéatose, réversible, est considérée comme bénigne d’un point de vue clinique. Cependant, elle représente une étape de sensibilisation des hépatocytes aux agressions ultérieures. En effet, 20 % des personnes atteintes de stéatose développent une maladie du foie plus sévère, comme la stéatohépatite, se traduisant par une mort cellulaire et une inflammation. En outre, la stéatohépatite favorise la progression vers des pathologies encore plus graves telles que la cirrhose. Pourtant, les causes et mécanismes impliqués dans la transition de la stéatose vers la stéatohépatite sont encore mal connus. Notre étude vise à tester in vitro l’impact de faibles doses de contaminants environnementaux sur la progression pathologique de la stéatose hépatique. Un modèle in vitro de stéatose a été obtenu par supplémentation (48 h) d’hépatocytes de la lignée WIF-B9 en acide oléique et en acide palmitique. La toxicité de deux contaminants, le benzo[a]pyrène (BaP) et le mono (2-éthylhexyl) phtalate (MEHP), en combinaison binaire ou non avec l’éthanol, a été étudiée après établissement de la stéatose. Nous avons démontré que la stéatose potentialise la mort cellulaire induite par le BaP ou le MEHP et que la co-exposition avec l’éthanol augmente cette toxicité. En outre, des expériences préliminaires suggèrent que les toxiques testés puissent induire une inflammation, notamment en augmentant l’expression du TNFα. L’évolution de la stéatose sous l’effet des toxiques a aussi été étudiée afin de vérifier la présence d’acides gras libres, potentiellement responsables d’une lipotoxicité. En condition de stéatose, le BaP accroît la teneur en triglycérides et la co-exposition MEHP/éthanol réduit la concentration en cholestérol. Afin d’étudier une potentielle prolifération compensatoire, un test d’incorporation à la thymidine tritiée a été réalisé. L’exposition au BaP diminue la prolifération cellulaire en cas de stéatose préalable. Une telle diminution est également observée sous l’effet du MEHP et de sa co-exposition avec l’éthanol mais semble indépendante de la présence d’une stéatose préalable. Le remodelage membranaire et le stress oxydant étant connus pour être induits par l’éthanol et le BaP, l’implication de ces mécanismes a été testée ; nos résultats indiquent que ceux-ci interviennent dans la mort cellulaire induite par les contaminants dans le contexte de stéatose. La présence d’une stéatose augmente les effets toxiques du BaP et du MEHP et ces effets sont d’autant plus marqués en présence d’éthanol. Ces toxiques pourraient donc favoriser la transition vers la stéatohépatite.

Several epidemiologic studies have shown an interactive effect of heavy smoking and heavy alcohol drinking on the development of hepatocellular carcinoma. It has also been recently described that chronic hepatocyte death can trigger excessive compensatory proliferation resulting later in the formation of tumors in mouse liver. As we previously demonstrated that both benzo[a]pyrene (B[a]P), an environmental agent found in cigarette smoke, and ethanol possess similar targets, especially oxidative stress, to trigger death of liver cells, we decided to study here the cellular and molecular mechanisms of the effects of B[a]P/ethanol coexposure on cell death. After an 18-h incubation with 100nM B[a]P, primary rat hepatocytes were supplemented with 50mM ethanol for 5 or 8h. B[a]P/ethanol coexposure led to a greater apoptotic cell death that could be linked to an increase in lipid peroxidation. Plasma membrane remodeling, as depicted by membrane fluidity elevation and physicochemical alterations in lipid rafts, appeared to play a key role, because both toxicants acted with specific complementary effects. Membrane remodeling was shown to induce an accumulation of lysosomes leading to an important increase in low-molecular-weight iron cellular content. Finally, ethanol metabolism, but not that of B[a]P, by providing reactive oxygen species, induced the ultimate toxic process. Indeed, in lysosomes, ethanol promoted the Fenton reaction, lipid peroxidation, and membrane permeabilization, thereby triggering cell death. To conclude, B[a]P exposure, by depleting hepatocyte membrane cholesterol content, would constitute a favorable ground for a later toxic insult such as ethanol intoxication. Membrane stabilization of both plasma membrane and lysosomes might be a potential target for further investigation considering cytoprotective strategies.

Plasma membrane is an early target of polycyclic aromatic hydrocarbons (PAH). We previously showed that the PAH prototype, benzo[a]pyrene (B[a]P), triggers apoptosis via DNA damage-induced p53 activation (genotoxic pathway) and via remodeling of the membrane cholesterol-rich microdomains called lipid rafts, leading to changes in pH homeostasis (non-genotoxic pathway). As omega-3 (n-3) fatty acids can affect membrane composition and function or hamper in vivo PAH genotoxicity, we hypothesized that addition of physiologically relevant levels of polyunsaturated n-3 fatty acids (PUFAs) might interfere with B[a]P-induced toxicity. The effects of two major PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), were tested on B[a]P cytotoxicity in the liver epithelial cell line F258. Both PUFAs reduced B[a]P-induced apoptosis. Surprisingly, pre-treatment with DHA increased the formation of reactive B[a]P metabolites, resulting in higher levels of B[a]P-DNA adducts. EPA had no apparent effect on B[a]P metabolism or related DNA damage. EPA and DHA prevented B[a]P-induced apoptotic alkalinization by affecting Na+/H+ exchanger 1 activity. Thus, the inhibitory effects of omega-3 fatty acids on B[a]P-induced apoptosis involve a non-genotoxic pathway associated with plasma membrane remodeling. Our results suggest that dietary omega-3 fatty acids may have marked effects on the biological consequences of PAH exposure.

Les espèces réactives de l'oxygène (ERO) et de l'azote (ERN) produites par les cellules ont été longtemps vues comme des produits toxiques du métabolisme pouvant altérer les constituants lipidiques, protéiques ou le DNA de la cellule. Toutefois, en permettant la modulation de l'expression de gènes, les ERO/ERN sont avant tout des acteurs importants de la signalisation cellulaire et de la régulation des métabolismes. La production des ERO dans les cellules mammifères est essentiellement d'origine enzymatique : la NAD(P)H oxydase membranaire et le complexe enzymatique mitochondrial de la chaîne respiratoire en sont les principales sources. Les NO synthases, dont trois types sont actuellement décrits, sont quant à elles la source du radical monoxyde d'azote °NO. Deux niveaux principaux assurent la régulation des taux intracellulaires d'ERO/ERN : les systèmes anti-oxydants constitutifs de la cellule et l'induction de gènes dont le produit possède une activité anti-oxydante ou la répression de gènes dont le produit exprime une activité pro-oxydante lors d'un veritable stress oxydant cellulaire. Plusieurs facteurs de transcription fondamentaux pour la cellule sont les cibles des ERO/ERN parmi lesquels le Nuclear Factor-KB (NF-KB) et l' Activator Protein-1 (AP-1) doivent être cités et sont décrits dans cette revue.The history of studies regarding reactive oxygen and nitrogen species (ROS/RNS) is approximatively of 50 years. ROS were shown initially for their deleterious effects on marcormolecules such as DNA and proteins, leading to deterioration of cellular functions as an oxidative stress. On the other hand, recent studies have demonstrated that ROS/RNS act as oxidative signalling in cells, resulting in various gene expressions. This brief review focuses on the main cellular origins of ERO/ERN, such as mitochondrial respiratory chain, NAD(P)H oxidase and NO synthases, and describe the modulation by the reactive species of two major signal transduction pathways, NF-KB and AP-1 pathways.

Previously, we demonstrated that eicosapentaenoic acid enhanced ethanol-induced oxidative stress and cell death in primary rat hepatocytes via an increase in membrane fluidity and lipid raft clustering. In this context, another n-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA), was tested with a special emphasis on physical and chemical alteration of lipid rafts. Pretreatment of hepatocytes with DHA reduced significantly ethanol-induced oxidative stress and cell death. DHA protection could be related to an alteration of lipid rafts. Indeed, rafts exhibited a marked increase in membrane fluidity and packing defects leading to the exclusion of a raft protein marker, flotillin. Furthermore, DHA strongly inhibited disulfide bridge formation, even in control cells, thus suggesting a disruption of protein-protein interactions inside lipid rafts. This particular spatial organization of lipid rafts due to DHA subsequently prevented the ethanol-induced lipid raft clustering. Such a prevention was then responsible for the inhibition of phospholipase C-γ translocation into rafts, and consequently of both lysosome accumulation and elevation in cellular low-molecular-weight iron content, a prooxidant factor. In total, the present study suggests that DHA supplementation could represent a new preventive approach for patients with alcoholic liver disease based upon modulation of the membrane structures.

Dietary n-3 polyunsaturated fatty acids (n-3 PUFAs) have been reported to modulate lipid raft-dependent signaling, but not yet lipid raft-dependent oxidative stress. Previously, we have shown that ethanol-induced membrane remodeling, i.e., an increase in membrane fluidity and alterations in physical and biochemical properties of lipid rafts, participated in the development of oxidative stress. Thus, we decided to study n-3 PUFA effects in this context, by pretreating hepatocytes with eicosapentaenoic acid (EPA), a long-chain n-3 PUFA, before addition of ethanol. EPA was found to increase ethanol-induced oxidative stress through membrane remodeling. Addition of EPA resulted in a marked increase in lipid raft aggregation compared to ethanol alone. In addition, membrane fluidity of lipid rafts was markedly enhanced. Interestingly, EPA was found to preferentially incorporate into nonraft membrane regions, leading to raft cholesterol increase. Lipid raft aggregation by EPA enhanced phospholipase Cγ translocation into these microdomains. Finally, phospholipase Cγ was shown to participate in the potentiation of oxidative stress by promoting lysosome accumulation, a major source of low-molecular-weight iron. To conclude, the ability of EPA to modify lipid raft physical and chemical properties plays a key role in the enhancement, by this dietary n-3 PUFA, of ethanol-induced oxidative stress.

Alcohol: Clinical and Experimental ResearchVolume 34, Issue s3 p. 89A-170A Abstracts – Posters First published: 23 July 2010 https://doi.org/10.1111/j.1530-0277.2010.01292_4.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Volume34, Issues3Special Issue: Abstracts from the International Society for Biomedical Research on Alcoholism World Congress, September 13-16, 2010 Paris, FranceAugust 2010Pages 89A-170A RelatedInformation

Quantitative data from experiments of gene expression are often normalized through levels of housekeeping genes transcription by assuming that expression of these genes is highly uniform. This practice is being questioned as it becomes increasingly clear that the level of housekeeping genes expression may vary considerably in certain biological samples. To date, the validation of reference genes in aging has received little attention and suitable reference genes have not yet been defined. Our aim was to evaluate the expression stability of frequently used reference genes in human peripheral blood mononuclear cells with respect to aging. Using quantitative RT-PCR, we carried out an extensive evaluation of five housekeeping genes, i.e. 18s rRNA, ACTB, GAPDH, HPRT1 and GUSB, for stability of expression in samples from donors in the age range 35-74 years.The consistency in the expression stability was quantified on the basis of the coefficient of variation and two algorithms termed geNorm and NormFinder. Our results indicated GUSB be the most suitable transcript and 18s the least for accurate normalization in PBMCs. We also demonstrated that aging is a confounding factor with respect to stability of 18s. HPRT1 and ACTB expression, which were particularly prone to variability in aged donors. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

The role of the hepatocyte plasma membrane structure in the development of oxidative stress during alcoholic liver diseases is not yet fully understood. Previously, we have established the pivotal role of membrane fluidity in ethanol-induced oxidative stress, but no study has so far tested the involvement of lipid rafts. In this study, methyl-β-cyclodextrin or cholesterol oxidase, which were found to disrupt lipid rafts in hepatocytes, inhibited both reactive oxygen species production and lipid peroxidation, and this suggested a role for these microstructures in oxidative stress. By immunostaining of lipid raft components, a raft clustering was detected in ethanol-treated hepatocytes. In addition, we found that rafts were modified by formation of malondialdehyde adducts and disulfide bridges. Interestingly, pretreatment of cells by 4-methyl-pyrazole (to inhibit ethanol metabolism) and various antioxidants prevented the ethanol-induced raft aggregation. In addition, treatment of hepatocytes by a stabilizing agent (ursodeoxycholic acid) or a fluidizing compound [2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate] led to inhibition or enhancement of raft clustering, respectively, which pointed to a relationship between membrane fluidity and lipid rafts during ethanol-induced oxidative stress. We finally investigated the involvement of phospholipase C in raft-induced oxidative stress upon ethanol exposure. Phospholipase C was shown to be translocated into rafts and to participate in oxidative stress by controlling hepatocyte iron content. Conclusion: Membrane structure, depicted as membrane fluidity and lipid rafts, plays a key role in ethanol-induced oxidative stress of the liver, and its modulation may be of therapeutic relevance. (HEPATOLOGY 2007.)