Ethanol Induces Oxidative Stress in Primary Rat Hepatocytes Through the Early Involvement of Lipid Raft Clustering

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1 Ethanol Induces Oxidative Stress in Primary Rat Hepatocytes Through the Early Involvement of Lipid Raft Clustering Philippe Nourissat, 1 * Marion Travert, 1 * Martine Chevanne, 1 Xavier Tekpli, 2,3 Amélie Rebillard, 2,3 Gwenaelle Le Moigne-Müller, 2,3 Mary Rissel, 2,3 Josiane Cillard, 1 Marie-Thérèse Dimanche-Boitrel, 2,3 Dominique Lagadic-Gossmann, 2,3 and Odile Sergent 1 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- -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 2008;47:59-70.) Abbreviations: 4-MP, 4-methyl pyrazole; A 2 C, 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate; CHOL, cholesterol; CHOX, cholesterol oxidase; CTX, cholera toxin; DTT, dithiothreitol; EPR, electron paramagnetic resonance; GM1, monosialotetrahexosylganglioside; GPI, glycosylphosphatidylinositol; HPLC-UV, high-performance liquid chromatography/ultraviolet; LPO, lipid peroxidation; MAL-6, tetramethyl-4-maleimidopiperidine-1-oxyl; MCD, methyl- -cyclodextrin; MDA, malondialdehyde; NCDC, 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate; PI-PLC, phosphatidyl inositol specific phospholipase C; PLC, phospholipase C; ROS, reactive oxygen species; SH, sulfhydryl; TH, thiourea; TRITC, tetramethyl rhodamine isothiocyanate; UDCA, ursodeoxycholate sodium salt; Vit E, vitamin E; W/S, ratio of the height of the low-field weakly immobilized line to the height of the low-field strongly immobilized line. From 1 Unité Propre de Recherche de l Enseignement Supérieur Equipe d Accueil (UPRES EA) 3891, Unité de Formation et de Recherche des Sciences Pharmaceutiques et Biologiques, Université de Rennes 1, Rennes, France; 2 Institut National de la Santé et de la Recherche Médicale 620, Rennes, France; and 3 Université de Rennes 1, Institut Fédératif de Recherche 140, Rennes, France. Received April 19, 2007; accepted July 31, Supported by the Institut de Recherches Scientifiques sur les Boissons (contract number 2005/27) and the Région Bretagne (which provided a grant to P.N.). *These authors contributed equally to this work. Address reprint requests to: Odile Sergent, Unité de Formation et de Recherche des Sciences Pharmaceutiques et Biologiques, 2 Avenue du Professeur Léon Bernard, Rennes Cedex, France. osergent@univ-rennes1.fr; fax: Copyright 2007 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience ( DOI /hep Potential conflict of interest: Nothing to report. Supplementary material for this article can be found on the HEPATOLOGY Web site ( 59

2 60 NOURISSAT, TRAVERT, ET AL. HEPATOLOGY, January 2008 Liver injury in alcoholic liver diseases is the result of multiple overlapping mechanisms including alcohol metabolism, inflammation, altered intracellular signaling, and oxidative stress. 1 The occurrence of oxidative stress in these diseases and its relationship with ethanol liver damage have been extensively documented, 2-5 but the mechanisms responsible for oxidative stress development are still a matter of debate. Oxidative stress occurs when the balance between oxidation, as the result of reactive oxygen species (ROS), and antioxidation is tilted in favor of the former. In hepatocytes, ethanol-induced ROS generation is well described as involving mitochondria, endoplasmic reticulum with the cytochrome P450, cytosolic free iron, and cytosolic enzymes such as xanthine oxidase or aldehyde oxidases. 6,7 In comparison, less is known about the possible role of the plasma membrane in this oxidative injury, although this barrier is recognized as the first target of ethanol. Indeed, acute ethanol intoxication increases membrane fluidity, 8,9 which can then induce oxidative stress. 9 Ethanol is also well described as interfering with many signal transduction systems in the plasma membrane, such as adenylate cyclase, phospholipase C, or calcium pathways, 10 and impairing receptor-mediated endocytosis. 11 In addition, more recently, the plasma membrane has been described not as a random formation of lipids based on the fluid-mosaic model but rather as laterally organized in lipid-specific domains. 12 More specifically, the presence of some nonionic detergent insoluble, sphingolipid, and cholesterol-rich microdomains, called lipid rafts, with a highly ordered spatial structure has raised the possibility that lipid lateral organization of the plasma membrane can participate in cell signaling. Thus, several examples exist in the literature showing that lipid rafts play a pivotal role via the recruitment or release of signaling proteins in response to extracellular stimuli. 13,14 In this context, one might suppose these microdomains to participate in the secondary development of an oxidative stress triggered by ethanol. Besides, a new hypothesis about the toxicity of this molecule has been very recently proposed consisting of a possible disrupting effect of ethanol on lipid-protein interaction in lipid rafts from macrophages. 15 The aim of this study was to test whether lipid rafts were involved in ethanol-induced oxidative stress. For this purpose, lipid rafts of primary rat hepatocytes were disrupted before the study of ethanol-induced oxidative stress, and different lipid raft markers were examined after ethanol treatment. Our results showed, for the first time, that lipid rafts may play a pivotal role in ethanol-induced oxidative stress according to changes in their biochemical and physical properties. Materials and Methods Chemicals See Supplementary Materials and Methods. Cell Isolation and Culture Adult rat hepatocytes were isolated from 2-month-old Sprague-Dawley animals by perfusion of the liver as previously described, except that a liberase solution (17 g/ ml) was used for dissociation. 16 In most cases, cell viability was above 90%. See Supplementary Materials and Methods for detailed protocols and treatments. Lipid Raft Fractionation by Flotation Method Lipid rafts are defined as being resistant toward solubilization by nonionic detergents at a low temperature. On the basis of this property, lipid rafts float and concentrate in a low-density sucrose gradient upon centrifugation. Thus, adapted from a previously described method, 17 lipid raft fractions were prepared by discontinuous sucrose gradient ultracentrifugation. One-milliliter fractions were collected from the top of the gradient and were then analyzed for protein and lipid content. For additional information, see Supplementary Materials and Methods. Determination of Lipid Raft Markers Protein Assays. Flotillin-1 and transferrin receptor (CD71) expression was analyzed by western blotting with antiflotillin-1 (1:2000) or anti-cd71 (1:500) monoclonal antibodies, respectively. For additional information, see Supplementary Materials and Methods. Cholesterol Dosage. The cholesterol content of each gradient fraction was evaluated spectrophotometrically by a cholesterol oxidase (CHOX)/peroxidase assay with the Infinity Cholesterol kit (Thermo Electron Corp., Eragny sur Oise, France) after lipid extraction by a chloroform/ methanol mixture (2:1 vol/vol) according to the method of Folch. Determination of Structural Perturbation of Lipid Rafts Spin-Labeling Techniques. Using paramagnetic reporter groups incorporated into lipid rafts, electron paramagnetic resonance (EPR) spectroscopy can evaluate changes in both membrane fluidity and protein interactions. Membrane fluidity was measured, as previously described, 9 with the spin label 12-doxyl stearic acid. The fluidity of the labeled membranes was quantified by the calculation of the order parameter S, which is inversely related to membrane fluidity. Protein structural changes were examined with tetramethyl-4-maleimidopiperidine-

3 HEPATOLOGY, Vol. 47, No. 1, 2008 NOURISSAT, TRAVERT, ET AL oxyl (MAL-6), a thiol-specific protein spin label. 18 The relevant EPR parameter used for the evaluation of protein structural changes is the ratio of the height of the low-field weakly immobilized line to the height of the low-field strongly immobilized line (W/S). A decrease in the W/S ratio reveals conformational compactions or increased interactions between membrane proteins. For additional information, see Supplementary Materials and Methods. Fluorescent Staining of Lipid Rafts. Lipid rafts were stained by the binding of the green-fluorescent Alexa Fluor 488 conjugate of cholera toxin subunit B to the pentasaccharide chain of lipid raft ganglioside monosialotetrahexosylganglioside (GM1) with the Vybrant lipid raft labeling kit (Molecular Probes, Invitrogen, Cergy- Pontoise, France). In the case of dual staining, hepatocytes were incubated simultaneously with the indicated primary monoclonal mouse antibody (flotillin, 1:50, or phospholipase C, 10 g/ml). For additional information, see Supplementary Materials and Methods. Evaluation of Oxidative Stress For additional information, see Supplementary Materials and Methods. Determination of ROS Production. Intracellular levels of ROS were measured with the nonfluorescent probe dihydrofluorescein diacetate as previously described. 9 Evaluation of Lipid Peroxidation. Lipid peroxidation of rat hepatocytes was estimated with the fluorescent probe C11-BODIPY 581/591 as previously described. 19 Malondialdehyde (MDA) is a secondary end product of degradation of oxidized polyunsaturated fatty acids; this highly reactive molecule can then bind to primary amino groups of various biomolecules such as proteins and phospholipids in membranes, leading to the formation of conjugated Schiff bases. Lipid raft bound MDA was quantified by high-performance liquid chromatography/ ultraviolet (HPLC-UV) 20 after hydrolysis of raft pools at 60 C and ph 13. Measurement of Low-Molecular-Weight Iron. Intracellular low-molecular-weight iron was measured by EPR, as previously described. 21 Statistical Analysis See Supplementary Materials and Methods. Results Ethanol-Induced Oxidative Stress Was Dependent on Lipid Rafts Lipid Raft Disrupters Inhibited Ethanol-Induced Oxidative Stress. In order to evaluate the involvement of lipid rafts in ethanol-induced oxidative stress, we first decided to test two known disrupters of these structures: methyl- -cyclodextrin (MCD), a water-soluble cyclic heptasaccharide that binds cholesterol and can therefore extract cholesterol from membranes, and CHOX, which produces membrane cholestenone, which is thought to break the bonding of cholesterol with sphingolipids. As shown in Fig. 1A,B, pretreatment with MCD protected hepatocytes from ethanol-induced oxidative stress; indeed, an inhibition of both ROS formation (Fig. 1A) and lipid peroxidation (Fig. 1B) was observed at 1 and 5 hours of incubation with ethanol, respectively. The specificity of the cholesterol-depleting effect of this agent was then checked by replenishment of the plasma membrane cholesterol content. Thus, in cultures pretreated with MCD, the consecutive cholesterol (50 g/ml) supplementation of media was found to reestablish the membrane cholesterol concentration without modifying the membrane fluidity of bulk membranes (data not shown). Regarding oxidative stress, we found that, in MCD preexposed hepatocytes, cholesterol addition restored the ROS production (Fig. 1A) and lipid peroxidation (Fig. 1B) due to ethanol. Similar results were observed with CHOX (Fig. 1A,B). Together, these data strongly point to a role for lipid rafts in ethanol-induced oxidative stress. Because we have previously shown that, in rat hepatocytes, ethanol induced very rapid ROS production, as soon as 15 minutes, 9 the effect of lipid raft disrupting agents was also studied at this early time point. Interestingly, lipid raft disrupting agents displayed, at that time, no effect on ROS formation, in contrast to 4-methyl-pyrazole, an inhibitor of ethanol metabolism (Fig. 1C). With the aim of seeking the mechanism by which lipid rafts can participate in the initiation of oxidative stress, we next measured the level of low-molecular-weight iron in intact cells, using the EPR technique. Low-molecular-weight iron consists of iron species that are not contained in high-molecular-weight molecules, such as ferritin or mitochondrial ferroproteins, but are able to trigger oxidative stress by catalyzing the formation of a highly free radical, the hydroxyl radical, via a Fenton or Haber-Weiss reaction. As expected from previous studies, 9,21,22 ethanol increased the content of low-molecular-weight iron by nearly 40% (Fig. 2). Our data further show that lipid raft disrupters prevented this elevation. Note that, at 15 minutes of incubation with ethanol, no elevation of low-molecular-weight iron was detected (data not shown). Because ethanol-induced ROS formation and lipid peroxidation were previously demonstrated to be due to low-molecular-weight iron elevation, 9,23 the aforementioned results indicated that lipid raft disrupters protected hepatocytes from oxidative stress by preventing the increase in low-molecular-weight iron. Disruption of Lipid Rafts by MCD and CHOX. See Supplementary Data.

4 62 NOURISSAT, TRAVERT, ET AL. HEPATOLOGY, January 2008 Ethanol Induced Alterations in Biochemical and Physical Properties of Lipid Rafts Effect of Ethanol on Lipid Raft Markers. In order to test whether ethanol modified the composition of lipid Fig. 2. Lipid rafts were involved in the elevation of low-molecularweight iron due to ethanol. Low-molecular-weight iron was determined by electron paramagnetic resonance analysis after chelation by 3 mm deferiprone. Rat hepatocytes were preincubated with 7.5 mm methyl- cyclodextrin (MCD) or 0.1 U/mL cholesterol oxidase (CHOX) for 1 hour before the addition of ethanol for a further 1-hour incubation time. Values are given as mean standard deviation of three independent experiments. Ethanol-treated versus untreated cultures: *P rafts, a part of this study addressed the characterization of lipid rafts prepared from hepatocytes after incubation or not with ethanol (50 mm, 1 hour) by comparing positive and negative markers of these membrane microdomains. The characterization of both soluble and detergent-resistant fractions by immunoblotting with antibodies against flotillin-1 and CD71 (positive and negative raft markers, respectively; Fig. 3A,B) revealed that ethanol did not modify the distribution of CD71; however, a slight shift toward a higher buoyancy fraction could be observed for flotillin-1, which might suggest a higher size of lipid rafts. In addition, no change in flotillin-1 expression was observed between total lysates from control and ethanoltreated hepatocytes (data not shown). We further showed that ethanol remained ineffective for the cholesterol con- 4 Fig. 1. Lipid raft disrupters affected ethanol-induced oxidative stress as evaluated by (A) reactive oxygen species (ROS) production (1-hour treatment) and (B) lipid peroxidation (5-hour treatment) without any effect on (C) early ROS production due to ethanol metabolism (15- minute treatment). ROS production was followed by the fluorescence of fluorescein, and lipid peroxidation was measured with the relative fluorescence of the probe C 11 -BODIPY 581/591. Rat hepatocytes were incubated or not with 50 mm ethanol for 15 minutes, for 60 minutes, or for 5 hours. Some cultures were pretreated for 1 hour with lipid raft disrupters [7.5 mm methyl- -cyclodextrin (MCD) or 0.1 U/mL cholesterol oxidase (CHOX)] or for 0.5 hours with 1 mm 4-methyl pyrazole (4-MP). Another set of cultures was supplemented with 50 g/ml cholesterol (CHOL) for 0.5 hours after pretreatment with MCD. Then, ethanol was added for 1 hour (ROS production) or 5 hours (lipid peroxidation). Values are given as mean standard deviation of three independent experiments. Ethanol-treated versus untreated cultures: *P 0.05 and ***P NS indicates not significant.

5 HEPATOLOGY, Vol. 47, No. 1, 2008 NOURISSAT, TRAVERT, ET AL. 63 Fig. 3. Effect of ethanol or lipid raft disrupters on lipid raft [(A) flotillin-1 expression and (D) alkaline phosphatase activity] or non lipid raft markers [(B) transferrin receptor (CD71)] and (C) on total amounts of cholesterol. Rat hepatocytes were incubated or not with 50 mm ethanol, 7.5 mm methyl- -cyclodextrin (MCD), or 0.1 U/mL cholesterol oxidase (CHOX) for 1 hour. Then, cells were lysed in 1% Triton X-100 and fractionated on a sucrose density gradient by ultracentrifugation. An equal protein amount of each fraction was then submitted to western blotting for analysis of flotillin-1 or transferrin receptor (CD71). One representative of three independent experiments is shown. Total amounts of cholesterol and alkaline phosphatase activities were determined in fractions 1 (pool I), 2-6 (pool II; detergent-resistant raft fractions), and 7-11 (pool III; soluble fractions). A typical distribution of cholesterol in individual fractions from control hepatocytes is given in the inset. Values are given as mean standard deviation of three independent experiments. Ethanol-treated versus untreated cultures: *P 0.05 and **P tent of raft fractions (Fig. 3C) but induced a significant decrease of alkaline phosphatase activity in lipid rafts (Fig. 3D). This decrease was not observed in nonraft fractions. Ethanol Induced Clustering of Lipid Rafts. As clustering of lipid rafts and hence rearrangement of cell surfaces in large signaling platforms have been described as an important step in many signaling pathways, 17,24,25 we next stained the raft-associated ganglioside GM1 with fluorescent-labeled cholera toxin B subunit. In hepatocytes treated for 1 hour with ethanol, fluorescent-labeled cholera toxin accumulated according to a punctuated pattern in the plasma membrane, thus suggesting a redistribution of GM1 into clusters, whereas, in control hepatocytes, GM1 distributed throughout the membrane as indicated by the diffused green fluorescence (Fig. 4A). In addition, the size of the patches increased at 5 hours, whereas their number was reduced (Fig. 4A). To further demonstrate the ability of ethanol to induce raft aggregation, we also stained another positive raft marker, flotillin-1 (Fig. 4B). After 1 hour of ethanol exposure, flotillin was also redistributed as a punctuated pattern and colocalized with ganglioside GM1. The pretreatment by MCD prevented the formation of these punctuations, and this indicated that they might correspond to lipid raft aggregation (Fig. 5A). Such an aggregation was also completely blocked by the pretreatment of hepatocytes with 4-methylpyrazole, an ethanol metabolism inhibitor (Fig. 5A), and this suggested the involvement of ethanol metabolism. To further understand how ethanol can aggregate lipid rafts, we tested the effects of several antioxidants: thiourea, a ROS scavenger; vitamin E, a free-radical chainbreaking antioxidant capable of inhibiting lipid peroxidation; and dithiothreitol, a reducing agent that can prevent the formation of disulfide bonds. All these compounds blocked ethanol-induced raft aggregation at both 1 (Fig. 5B) and 5 hours (data not shown), and this indicated that ethanol-induced oxidative stress was

6 64 NOURISSAT, TRAVERT, ET AL. HEPATOLOGY, January 2008 involved. The involvement of ROS was further confirmed by the study of flotillin expression in fractions of sucrose gradients obtained after preincubation of hepatocytes with thiourea (Fig. 5C). Indeed, this pretreatment before ethanol exposure prevented the shift of flotillin toward higher buoyancy fractions. Ethanol Caused Oxidative Changes in Lipid Rafts. As oxidative stress seems to be involved in raft aggregation, oxidative alterations of some raft components were measured. First, oxidized protein structural changes in lipid rafts were studied with MAL-6, a thiol-specific protein spin label, which binds covalently to free sulfhydryl (SH) groups. In lipid rafts isolated from ethanol-treated hepatocytes, the W/S ratio decreased, revealing an increase in interactions between membrane proteins (Fig. 6A). This W/S decrease could be due to protein oxidation leading to the formation of disulfide bridges between sites closer to the surface of the protein; in this case, thiol groups were not accessible anymore for MAL-6, and this lowered the W value. This hypothesis was confirmed by the experiments performed in the presence of dithiothreitol, a reducing agent well known to reduce disulfide bridges (Fig. 6A). Indeed, dithiothreitol treatment led to an increase of the W/S ratio. Second, the formation of MDA adducts to raft components was detected by HPLC-UV following hydrolysis of raft membranes to release MDA from its binding with lipids and proteins. MDA is a secondary end product of the degradation of oxidized polyunsaturated fatty acids that can diffuse from its production site to react with free amino groups in proteins and lipids. Ethanol treatment of rat hepatocytes led to an elevation of bound MDA content in lipid rafts that increased with time (Fig. 6B). Note that thiourea and vitamin E pretreatment inhibited the MDA adduct formation in lipid rafts (data not shown). Simultaneously, an increase in free MDA in ethanol (1 hour)-treated hepato- Fig. 4. Ethanol induced lipid raft aggregation visualized by fluorescence microscopy analysis of lipid raft markers [(A) ganglioside monosialotetrahexosylganglioside (GM1) and (B) flotillin-1]. (A) Membrane lipid rafts were visualized by fluorescence microscopy with cholera toxin conjugated with Alexa Fluor 488, which binds the raft-associated glycosphingolipid GM1. Rat hepatocytes were incubated or not with ethanol for 1 or 5 hours. (B) Membrane lipid rafts were costained by Alexa Fluor cholera toxin (CTX; green fluorescence at left) and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated anti flotillin-1 (red fluorescence in the middle). The merge of both images exhibits yellow areas (at right), which represent colocalization of GM1 and flotillin-1. Rat hepatocytes were incubated or not with ethanol for 1 hour. One representative of three independent experiments is shown. Fig. 5. Effects of a lipid raft disrupter, an ethanol metabolism inhibitor, and various antioxidants on ethanol-induced lipid raft aggregation. Lipid raft aggregation was visualized (A,B) by fluorescence microscopy with cholera toxin conjugated with Alexa Fluor 488, which binds the raft-associated monosialotetrahexosylganglioside, or (C) by analysis of flotillin-1 expression in each fraction of a sucrose gradient (a shift toward a higher buoyancy fraction reflecting a higher size of lipid rafts and hence aggregation). Rat hepatocytes were incubated or not with ethanol for 1 hour. (A) Some cultures were pretreated for 1 hour with 7.5 mm methyl- -cyclodextrin (MCD), a lipid raft disrupter, or for 0.5 hours with 1 mm 4-methyl-pyrazole (4-MP), an ethanol metabolism inhibitor. (B,C) In another set of cultures, cells were preincubated with either 100 mm thiourea (TH), a reactive oxygen species scavenger, for 1 hour, 250 M vitamin E (Vit E), a free radical chain-breaking antioxidant, for 12 hours, or 500 mm dithiothreitol (DTT), a reducing agent able to reduce disulfide bridges, for 1 hour. One representative of three independent experiments is illustrated.

7 HEPATOLOGY, Vol. 47, No. 1, 2008 NOURISSAT, TRAVERT, ET AL. 65 the bulk membrane fluidizing effect of ethanol following its metabolism is responsible for an enhancement of oxidative stress in rat hepatocytes. 9 We first tested whether such an increase might also occur in lipid rafts from ethanol-treated hepatocytes. With EPR analysis, ethanol (1 hour) was found to increase fluidity in these microdomains as well (Fig. 7A). Note that raft fluidity was still lower than that in bulk membranes in both untreated and ethanol-treated cells, in agreement with the highly packed structure of lipid rafts. In addition, thiourea and vitamin E were found to inhibit the ethanol-induced raft fluidization (data not shown). Finally, to gain further insight into the role of membrane fluidity in ethanol toxicity, raft aggregation was analyzed when hepatocytes were pretreated with either ursodeoxycholate sodium salt (UDCA; 100 M), a membrane stabilizing agent, or with 2-(2- methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A 2 C; 5 M), a membrane fluidizer. Our results clearly showed that UDCA protected against ethanol-induced raft aggregation, whereas A 2 C enhanced it (Fig. 7B). Note also that A 2 C alone induced a slight increase in raft aggregation (Fig. 7B). Thus, the fluidizing effect of ethanol appeared to be involved in the raft aggregation process. Fig. 6. Ethanol induced oxidative changes in lipid rafts as evaluated by the formation of (A) disulfide bridges and (B) malondialdehyde (MDA) adducts. Rat hepatocytes were incubated or not with ethanol for 1 hour (W/S ratio and MDA) or 5 hours (MDA only). Some cultures were pretreated with 500 mm dithiothreitol (DTT), a reducing agent capable of reducing disulfide bridges. Values are the mean standard deviation of three independent experiments. Ethanol-treated versus untreated cultures: *P 0.05, **P 0.01, and ***P cytes was detected by HPLC-UV of the cell lysate ultrafiltrate without hydrolysis (control, 22 6 ng/mg protein, versus ethanol, ng/mg protein); this suggested that MDA was not necessarily produced in lipid rafts. Altogether, these data show that lipid raft components can rapidly undergo oxidative damage, as revealed by the formation of both disulfide bridges and MDA adducts, which may then lead to raft aggregation. An Increase in Membrane Fluidity Was Involved in Lipid Raft Clustering. We have previously shown that Relocation of Phosphatidyl Inositol Specific Phospholipase C (PI-PLC) to Lipid Rafts Participated in the Ethanol-Induced Oxidative Stress Alkaline phosphatase is a glycosylphosphatidylinositol (GPI)-anchored protein tethered to the extracellular face of a plasma membrane and PI-PLC is an enzyme that can cleave the GPI anchor of such proteins, leading to their release in the extracellular medium. On the basis of the fact that alkaline phosphatase activity was markedly reduced only in lipid rafts isolated from ethanol-treated hepatocytes (Fig. 3D), we finally decided to test a role for PI-PLC in the ethanol effects. Translocation of phospholipase C (PLC) to rafts was first studied by an analysis of the PLC 1 expression in raft fractions (Fig. 8A) and by the staining of hepatocytes with tetramethyl rhodamine isothiocyanate anti-plc 1 (Fig. 8B). Changes were observed only following a 60-minute ethanol exposure. Indeed, in a gradient from ethanol-treated hepatocytes, nonraft fraction 9 contained less PLC 1, whereas expression increased in raft fractions 4, 5, and 6. As indicated after merging of the fluorescence stainings, GM1 and PLC 1 colocalized and coaggregated as shown by the appearance of yellow patches. Next, we examined the role of PI-PLC by using 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC), a specific inhibitor. In lipid rafts, NCDC pretreatment, prior to ethanol, prevented the de-

8 66 NOURISSAT, TRAVERT, ET AL. HEPATOLOGY, January 2008 In this article, we show, for the first time in hepatocytes, that lipid rafts, when modified by oxidative reactions, are then involved in the development of oxidative stress due to ethanol, as characterized by both ROS production and lipid peroxidation. Such an involvement of lipid rafts has previously been described in other cells, such as murine lymphocytes 26 and keratinocytes, 27 but only for ROS production and with other inducers such as arsenite and ultraviolet radiation, respectively. In comparison with those articles, we now demonstrate that oxidative changes within lipid rafts constitute a prerequisite for the oxidative stress to develop. Previously, it was also suggested that lipid rafts might be a target for ethanol in RAW macrophages because similar effects were obtained with ethanol and with lipid raft disrupters on lipopolysaccharide-induced production of tumor necrosis factor or receptor clustering. 28,29 Similar observations were made in Chinese hamster ovary cells. 30 However, in all these works, the nature of the changes that affected lipid rafts remained imprecise, and furthermore, no clear evidence was presented showing that rafts were disrupted. Our data strongly indicated no disruption of lipid rafts in Fig. 7. (A) Ethanol increased membrane fluidity in both bulk membranes and lipid rafts, a phenomenon involved in the aggregation of lipid rafts, as shown by (B) the use of either a membrane stabilizing agent [ursodeoxycholate sodium salt (UDCA)] or a membrane fluidizer [2-(2- methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A 2 C)]. Membrane lipid rafts were visualized by fluorescence microscopy with cholera toxin conjugated with Alexa Fluor 488, which binds the raft-associated glycosphingolipid monosialotetrahexosylganglioside. One representative of three independent experiments is shown. The membrane fluidity was monitored by electron paramagnetic resonance analysis of 12-doxyl stearic acid embedded in membranes. This analysis resulted in the calculation of the order parameter S, which is inversely related to membrane fluidity. Rat hepatocyte cultures were treated for 1 hour with ethanol. Values are the mean standard deviation of three independent experiments. Ethanol-treated versus untreated cultures: ***P crease of alkaline phosphatase activity (Fig. 9A). In addition, NCDC inhibited the related oxidative stress as shown by the decrease of ROS production (Fig. 9B), lipid peroxidation (Fig. 9C) and low-molecular-weight iron (Fig. 9D). All these results strongly emphasized that lipid rafts contributed to ethanol-induced oxidative stress by promoting, via the activation of PI-PLC, the elevation of low-molecular-weight iron. Discussion Fig. 8. Ethanol induced phospholipase C 1 translocation into lipid rafts after 60 minutes of incubation. Rat hepatocytes were incubated or not with 50 mm ethanol for 15 or 60 minutes. Then, cells were lysed in 1% Triton X-100 and fractionated on a sucrose density gradient by ultracentrifugation. (A) An equal protein amount of each fraction was then submitted to western blotting for analysis of flotillin-1. (B) Hepatocytes were costained by Alexa Fluor cholera toxin (CTX; green fluorescence at left) and tetramethyl rhodamine isothiocyanate (TRITC) conjugated antigen to phospholipase C 1 (anti-plc 1; red fluorescence in middle). The merge of both images exhibits yellow areas (at right) that represent colocalization of monosialotetrahexosylganglioside and PLC 1. One representative of three independent experiments is shown.

9 HEPATOLOGY, Vol. 47, No. 1, 2008 NOURISSAT, TRAVERT, ET AL. 67 Fig. 9. Effect of 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC), a phospholipase C 1 inhibitor, on (A) alkaline phosphatase activity in lipid rafts, (B) reactive oxygen species (ROS) production, (C) lipid peroxidation, and (D) low-molecular-weight iron. ROS production was followed by the fluorescence of fluorescein, and lipid peroxidation was measured with the relative fluorescence of the probe C 11 -BODIPY 581/591. Rat hepatocytes were incubated or not with 50 mm ethanol for 1 hour. Some cultures were pretreated with 50 M NCDC for 30 minutes. Values are the mean standard deviation of three independent experiments. Ethanol-treated versus untreated cultures: *P 0.05 and **P ethanol-treated hepatocytes as demonstrated by the absence of a decrease in cholesterol content and flotillin-1 expression in raft fractions. In addition, when compared with control cultures, flotillin content in the sucrose fractions of the lowest density (2 and 3) was increased in ethanol-treated hepatocytes. This different buoyant density with ethanol may reflect an elevation of raft size and hence a raft aggregation. This was confirmed by clustering of the raft-positive marker ganglioside GM1, as evidenced by fluorescence microscopy. In our model, we showed that raft aggregation was dependent on ROS production by ethanol metabolism because both 4-methylpyrazole, an inhibitor of ethanol metabolism, and thiourea, an ROS scavenger, inhibited the formation of aggregates. Regarding thiourea, note that the flotillin shift among raft fractions was also inhibited, these results being consistent with those by Lu and coworkers 31 using similar molecular tools in activated T lymphocytes. To our knowledge, only two mechanisms, both occurring in rafts and dependent on ROS, have been described to merge these rafts into larger membrane microdomains: (1) the activation of acid sphingomyelinase by ROS and hence generation within rafts of ceramide, 32 which has the tendency to self-associate, 25 and (2) crosslinks between oxidized proteins. 33,34 As pretreatment of hepatocytes by desipramine, an acid sphingomyelinase inhibitor, did not affect ethanol-induced oxidative stress (data not shown), we then focused on the second mechanism. Protein crosslinkage can be mediated by oxidative modifications at SH groups and/or free amino (NH2) groups. By EPR, an increase in disulfide bridges (S-S bond) was observed in rafts isolated from ethanol-treated hepatocytes, with dithiothreitol preventing both this increase and raft aggregation. In this context, one might then propose that ethanol-induced ROS production would trigger formation of disulfide bridges from two intermolecular SH groups from several rafts leading to protein crosslinks and raft clustering (Fig. 7). Our data are consistent with reports on the dithiothreitol inhibition of raft aggregation due to arsenite exposure of T lymphocytes, 35 but we further show the occurrence of oxidative changes within lipid rafts. Indeed, we detected not only disulfide bridges within rafts but also formation of adducts with MDA, a well-known end product of lipid peroxidation. Interestingly, vitamin E, a lipid peroxidation inhibitor, prevented the formation of raft aggregates. In addition, the increase in aggregation observed at 5 hours compared to 1 hour was associated with an elevation of the MDA content in lipid rafts. It is well known that unsaturated aldehydes produced during lipid peroxidation, such as MDA or 4-hydroxynonenal, can react with cysteine, lysine, and arginine residues in proteins to form carbonyl groups, which may then form a Schiff base with a lysine of another protein. Such a protein can be included in another raft, leading to protein crosslinkage and raft clustering (Fig. 7). The involvement of carbonyl compounds, produced by oxidative stress, in raft aggrega-

10 68 NOURISSAT, TRAVERT, ET AL. HEPATOLOGY, January 2008 Fig. 10. Proposed mechanism for the role of lipid rafts in the amplification of oxidative stress due to ethanol. In hepatocytes, ethanol metabolism rapidly leads to reactive oxygen species (ROS) production and lipid peroxidation (LPO), which in turn may trigger a biophysical alteration of the plasma membrane. More precisely, this mild and early oxidative stress due to ethanol metabolism would lead to oxidative damage and increased membrane fluidity of lipid rafts, which then would promote raft clustering. Finally, raft aggregation by activating phospholipase C signaling pathways may facilitate an elevation of low-molecularweight (LMW) iron. Lipid raft disrupters [methyl- -cyclodextrin (MCD) or cholesterol oxidase (CHOX)], by preventing raft aggregation, would inhibit ethanol-induced oxidative stress. 4-MP indicates 4-methyl pyrazole; A 2 C, 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate; DTT, dithiothreitol; NCDC, 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate; TH, thiourea; and UDCA, ursodeoxycholate sodium salt. tion has been strongly suggested by Nakashima et al. 36 and Akhand et al., 33 as treatment of cells by these compounds was capable of activating membrane raft-associated cell surface receptors. Our results further support such an hypothesis and show, for the first time, that these compounds, when produced inside hepatocytes undergoing an oxidative stress, led to aggregation of lipid rafts. The predicted low membrane fluidity of lipid rafts due to their specific lipid composition and the results of our previous study, demonstrating the involvement of the elevation of membrane fluidity in ethanol-induced oxidative stress and cell death 9 by the use of a membrane stabilizing agent (UDCA) or a membrane fluidizer compound (A 2 C), encouraged us to test the effect of ethanol on lipid raft fluidity. Using an EPR technique, we were able to evaluate membrane fluidity directly in rafts and in bulk membranes. Although we confirmed lower fluidity for lipid rafts compared to bulk membranes, ethanol was found to increase membrane fluidity in these microdomains. Therefore, we decided to test the effect of UDCA or A 2 C on raft aggregation, as detected by GM1 staining. UDCA prevented ethanol-induced lipid raft aggregation, whereas A 2 C enhanced it, and this suggested that the ethanol-induced fluidization was also involved in raft aggregation. In a previous article, 17 we also found that an increase in membrane fluidity might be required for lipid raft clustering in human colon cancer cells treated by cisplatin. The involvement of membrane fluidization for lipid raft clustering might explain how membrane fluidity can enhance ethanol-induced oxidative stress. Previously, we postulated that the involvement of membrane fluidity in ethanol-induced oxidative stress proceeded through a two-step mechanism 9 : (1) ethanol metabolism, by triggering early ROS production, promotes a rapid increase in membrane fluidity, and (2) such an increase is then responsible for the enhancement of ROS production and lipid peroxidation. Considering this chronology of events, we tested lipid raft disrupters following very short incubation times with ethanol. The beneficial effects of raft disrupters toward ethanol-induced ROS production were obtained only when ROS production was evaluated after 1 hour of incubation. At 15 minutes, no protection was provided by these compounds, unlike the inhibitor of ethanol metabolism, 4-methyl-pyrazole. In addition, no elevation of low-molecular-weight iron was detected. This reinforced our assumption that the early ROS formation was mainly due to ethanol metabolism, whereas the late phase relied upon membrane events such as membrane fluidization and lipid raft clustering, these last events being dependent on ethanol metabolism for their occurrence (Fig. 10). Such early ROS production has also been detected by others and has been attributed to mitochondrial dysfunction due to an increased cellular reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide ratio upon ethanol metabolism. 37,38 It has been extensively discussed that the aggregation of lipid rafts leads to the activation of cell signaling, with rafts helping the formation of large signal platforms with the accumulation of tyrosine phosphorylated proteins and cytosolic proteins. 14,24 Among these proteins, PI- PLC is conspicuous for the following reasons: PI-PLC is well known to be activated during ethanol intoxication, 10 its activity increased with an elevation of membrane fluidity and the aggregation of its substrates, 39 and PLC has been reported to be located in rafts after activation by ROS such as H 2 O PI-PLC can cleave the GPI anchor of proteins, such as alkaline phosphatase, leading to the release of the protein in the extracellular medium. Here we found that alkaline phosphatase activity decreased only in lipid rafts isolated from ethanol-treated hepatocytes, thus suggesting an increase of PI-PLC activity. In

11 HEPATOLOGY, Vol. 47, No. 1, 2008 NOURISSAT, TRAVERT, ET AL. 69 addition, translocation of PLC 1 into rafts was observed in ethanol-treated hepatocytes following a 1-hour treatment. As no translocation was detected after 15 minutes, this reinforced the idea that a first step with weak production of ROS was necessary to induce oxidative changes consequently altering the physical and biochemical properties of rafts such as membrane fluidization and raft clustering. Only then could PLC be translocated and activated (Fig. 10). Inhibition of PLC by NCDC prevented the ethanol-induced oxidative stress evidenced by ROS production, lipid peroxidation, and the elevation of low-molecular-weight iron. It is noteworthy that thiourea, vitamin E, and dithiothreitol, which prevented both raft clustering and fluidization, also inhibited the elevation of low-molecular-weight iron (data not shown). It remains to be determined how PLC would facilitate this elevation. Much evidence has been accumulated that activation of PLC can be coupled to endocytic uptake mechanisms. First, it has been reported that cleavage of GPI-anchored proteins can activate endocytosis related to caveolin, a lipid raft marker, 41,42 and PLC has been reported to have this GPI cleavage activity. 43 Second, PLC is also well known to cleave phosphatidylinositol 4,5-biphosphate (PIP2) in second messengers (inositol 1,4,5- triphoshate and diacylglycerol), which lead to an increase in cytosolic calcium concentration. Such an elevation of calcium can promote endocytosis. In this context, activation of PLC by ethanol might facilitate endocytosis, thus leading to iron uptake. Regarding endocytosis, it is important to stress that among lipid rafts, caveolae have been implicated in endocytosis processes. 44 Nonetheless, we were unable to detect any caveolin expression under our experimental conditions, whatever the detection means used (western blotting or immunolocalization; data not shown), in contrast to previous studies. 45 Actually, other studies have shown that the caveolin expression would be much lower in hepatocytes than in nonparenchymal liver cells. 46,47 Nevertheless, numerous studies have evidenced that another pathway of endocytosis, which involves neither clathrin nor caveolin, could be identified as dependent on lipid rafts. It is clear that further experimentation is needed, but one might speculate that low-molecular-weight iron can enter hepatocytes in this way. 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