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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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Various drugs and chemicals can cause a glutathione (GSH) depletion in the liver. Moreover, nitric oxide (NO) can be generated in response to physiological and pathological situations such as inflammation. The aim of this study was to estimate oxidative stress when primary rat hepatocytes were exposed to GSH depletion after NO production. For this purpose, cells were preincubated with lipopolysaccharide (LPS) and gamma-interferon (IFN) for 18 h in order to induce NO production by NO synthase and then L-buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, was added for 5 h. In hepatocyte cultures preincubated with LPS and IFN before BSO addition, an increase in lipid peroxidation was noted. In those cells, an elevation of iron-bound NO and a decrease in free NO led us to suggest the involvement of low-molecular-weight iron (LMW iron) in the enhancement of oxidative stress. Indeed, addition of deferiprone, a chelator of LMW iron, reduced iron-bound NO levels and the extent of oxidative stress. Moreover, an important elevation of LMW iron levels was also observed. As both, N-acetylcysteine, a GSH precursor, and N(G)-monomethyl-L-arginine, a NO synthase inhibitor, totally inhibited the elevation of LMW iron and oxidative stress, a cooperative role could be attributed to NO production and GSH depletion.