30, 31, 33, 55, 57 In support of our hypothesis, we found MitoQ inhibited the formation 4-HNE protein adduct formation

and 3-NT levels, indicators of the antioxidant action of MitoQ (Figs. 1, 2). The pattern of 4-HNE and 3-NT staining demonstrate a strong gradient extending from the pericentral region deep into the periportal region of the liver and is consistent with similar studies of ethanol-induced liver injury.58 The enhancement of the oxidative/nitrosative mTOR inhibitor stress gradient is linked to several factors including exacerbation of the hypoxic gradient developed in the liver acini and LPS-induced cytokine production in chronic ethanol consumption. MitoQ treatment in LPS-induced inflammation has been shown to involve decreases in proinflammatory cytokines such as interleukin (IL)-1β, IL-6, and IL-8 and an increase in the antiinflammatory cytokine IL-10 levels.59 However, in the present study we found that MitoQ did not significantly change the expression of iNOS, suggesting that its mode of action is downstream of cytokine signaling PDGFR inhibitor (Fig. 2B). Because it has been shown that mitochondria and specifically MitoQ can modify the cellular response to hypoxia, we next examined this pathway.30,

33, 40 Induction of tissue hypoxia and HIF1α in the liver is a hallmark of alcohol-induced liver disease.29, 60 Furthermore, iNOS-derived NO has been shown to inhibit prolyl hydroxylase enzyme activity by competing for the iron(II) in the catalytic site of the enzymes during normoxia and changes mitochondrial function with increased ROS formation.7, 50, 61 Our data are consistent with this literature because we found chronic ethanol-induced HIF1α expression/stabilization Akt inhibitor (Fig. 3A), increased ROS, and increased iNOS (Fig. 2B). MitoQ treatment inhibited ethanol-induced HIF1α expression in the liver (Fig. 3A), whereas iNOS expression remained

unaltered (Fig. 2B). Recently, it has been shown that HIF1α in hepatocytes is a major determinant in the pathogenesis of alcoholic steatosis.29 Taken together, we propose that MitoQ inhibits the mitochondria-dependent induction of HIF1α through suppression of increased mitochondrial ROS in response to NO exposure or damage to the mitochondrion by peroxynitrite. To form peroxynitrite, superoxide must also be formed in response to ethanol consumption and could come from a number of sources, including the mitochondrion or NADPH oxidase.10, 14 Because it has been shown that MitoQ can directly scavenge peroxynitrite, this is a likely mechanism through which activation of HIF1α is prevented.36 Ethanol feeding leads to inhibition of mitochondrial protein synthesis, which is largely responsible for the changes in the activities of the mitochondrial respiratory chain complexes.