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.)

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