Acides gras polyinsaturés oméga 3 et toxicité hépatique de l'éthanol : rôle du remodelage membranaire

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A role for lipid rafts in the protection afforded by docosahexaenoic acid against ethanol toxicity in primary rat hepatocytes.

International audience: 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

Rôles des hydrocarbures aromatiques sur la régulation de l interleukine 8 et de la protéine Niemann-Pick type C1 dans les macrophages humains en culture primaire et leurs effets sur l accumulation de lipides dans différents modèles cellulaires

resumeRENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Effet des acides gras polyinsaturés à longue chaîne n-3 sur le remodelage membranaire induit par les toxiques chimiques : retentissement sur la mort cellulaire

National audienceHomeostasis of plasma membrane is crucial for the regulation of many cell processes such as inflammation, cell proliferation, migration or death. This especially dynamic cell structure can undergo alteration when exposed to a wide range of chemical toxicants, thus triggering a membrane stress signal that can change the activation of intracellular signalling pathways, involved in such phenomena. Yet, long chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) are described as capable of changing physicochemical properties of membranes. Thus, LC n-3 PUFA can possibly counteract or potentiate the effects of chemical toxicants with a membrane action. In this paper, we will mainly be interested in hepatotoxicants, notably benzo(a)pyrene, an environmental pollutant of the polycyclic aromatic hydrocarbon family, also found in cigarette smoke, and ethanol, the hepatoxicant prototype, whose human exposure is linked to lifestyle.L’homéostasie de la membrane plasmique est essentielle à la régulation de plusieurs processus cellulaires comme l’inflammation, la prolifération, la migration ou la mort cellulaire. Cette structure très dynamique peut subir des altérations lors de son exposition à de nombreux toxiques chimiques, déclenchant un signal de stress membranaire, qui peut modifier l’activation de voies de signalisation intracellulaire impliquées dans ces différents phénomènes. Or, les acides gras polyinsaturés à longue chaîne n-3 (AGPI LC n-3) sont décrits comme capables de modifier les propriétés physicochimiques des membranes. Les AGPI LC n-3 sont donc susceptibles de limiter ou de potentialiser les effets des toxiques chimiques à action membranaire. Dans cet article, nous nous intéresserons principalement aux hépatotoxiques, notamment le benzo(a)pyrène, un contaminant de l’environnement de la famille des hydrocarbures aromatiques polycycliques, trouvé également dans la fumée de cigarette, et l’éthanol, le prototype des toxiques du foie, dont l’exposition humaine est liée au mode de vie

NPC1 repression contributes to lipid accumulation in human macrophages exposed to environmental aryl hydrocarbons.

International audienceAIMS: Aryl hydrocarbons (AHs), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo(a)pyrene (BP), are environmental contaminants promoting the development of atherosclerosis-related cardiovascular diseases. In order to identify molecular mechanisms involved in these effects, we have analysed AH-mediated regulation of the lipid trafficking Niemann-Pick type C1 protein (NPC1) and its contribution to AH-induced macrophage lipid accumulation. METHODS AND RESULTS: Exposure of primary human macrophages to TCDD and BP decreased NPC1 mRNA expression in a time-dependent manner. NPC1 protein expression and NPC1-related acid sphingomyelinase activity were reduced in parallel. NPC1 was also similarly down-regulated in mice exposed to BP. Moreover, TCDD and BP were demonstrated to trigger lipid accumulation in human macrophages, as assessed by Oil Red O and Nile Red staining and cholesterol determination. Such lipid loading occurred at least partly in endosomal/lysosomal compartments as demonstrated by immunolabelling of lipid vesicles by the lysosome-associated membrane protein 1. These cellular phenotypic effects were found to be similar to those triggered by knock-down of NPC1 expression using siRNAs and were counteracted by NPC1 overexpression, thus supporting the contribution of NPC1 to AH-mediated lipid accumulation in macrophages. Finally, both NPC1 down-expression and lipid accumulation in response to TCDD were found to be abolished through knock-down of the AH receptor (AHR), a ligand-activated transcription factor mediating many effects of AHs. CONCLUSION: Our data have shown that contaminants such as TCDD and BP repress NPC1 expression in macrophages in an AHR-dependent manner, which likely contributes to macrophage lipid accumulation caused by these environmental chemicals. Thus, NPC1 appears to be a new molecular target regulated by environmental AHs and putatively involved in their deleterious cardiovascular effects

Inhibition of human mesenchymal stem cell-derived adipogenesis by the environmental contaminant benzo(a)pyrene.

International audiencePolycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BP) are environmental contaminants exerting various toxic effects. PAHs have notably been found to inhibit adipogenesis in rodent species. To determine whether a similar process concerns human cells, we have analyzed the effects of BP towards differentiation of human cultured mesenchymal stem cells (MSC) into adipocytes, triggered by a pro-adipogenic culture medium. BP was found to markedly prevent formation of lipid vesicles, cellular lipid accumulation and up-regulation of adipogenic markers such as fatty acid binding protein-4 and glyceraldehyde-3-phosphate dehydrogenase, which represent major hallmarks of human MSC-derived adipocytes. The aryl hydrocarbon receptor (AhR), known to mediate most of the toxic effects of PAHs, was demonstrated to be present and functional in human MSC. 2,3,7,8-tetrachlorodibenzo-p-dioxin, an AhR agonist like BP, was found to inhibit lipid accumulation in human MSC cultured with adipogenic medium, in contrast to the PAH benzo(e)pyrene, known to not, or only poorly, interact with AhR. Moreover, BP inhibitory effect toward lipid accumulation in MSC exposed to adipogenic medium was fully counteracted by co-treatment with the AhR antagonist alpha-naphtoflavone. Taken together, these data indicate that environmental PAHs like BP can likely inhibit human adipogenesis in an AhR-dependent manner

P12-03 Hepatotoxicity induced by a mixture of PAHs: role of extracellular vesicles

International audienc

A New In Vivo Zebrafish Bioassay Evaluating Liver Steatosis Identifies DDE as a Steatogenic Endocrine Disruptor, Partly through SCD1 Regulation

International audienceNon-alcoholic fatty liver disease (NAFLD), which starts with liver steatosis, is a growing worldwide epidemic responsible for chronic liver diseases. Among its risk factors, exposure to environmental contaminants, such as endocrine disrupting compounds (EDC), has been recently emphasized. Given this important public health concern, regulation agencies need novel simple and fast biological tests to evaluate chemical risks. In this context, we developed a new in vivo bioassay called StAZ (Steatogenic Assay on Zebrafish) using an alternative model to animal experimentation, the zebrafish larva, to screen EDCs for their steatogenic properties. Taking advantage of the transparency of zebrafish larvae, we established a method based on fluorescent staining with Nile red to estimate liver lipid content. Following testing of known steatogenic molecules, 10 EDCs suspected to induce metabolic disorders were screened and DDE, the main metabolite of the insecticide DDT, was identified as a potent inducer of steatosis. To confirm this and optimize the assay, we used it in a transgenic zebrafish line expressing a blue fluorescent liver protein reporter. To obtain insight into DDE’s effect, the expression of several genes related to steatosis was analyzed; an up-regulation of scd1 expression, probably relying on PXR activation, was found, partly responsible for both membrane remodeling and steatosis

Membrane Remodeling as a Key Player of the Hepatotoxicity Induced by Co-Exposure to Benzo[a]pyrene and Ethanol of Obese Zebrafish Larvae

International audienceThe 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