EtOH KO mice had 2-fold lower hepatic NADPH levels than corresponding WT mice (Fig. 3B; Supporting Figure 4). Thus, the oxidative stress generated in KO livers by ethanol ingestion likely depletes hepatic NADPH, an important donor of reducing equivalents in antioxidant defense pathways. Mitochondrial dysfunction resulting from hepatic oxidative stress can mediate alcohol-induced liver injury. To determine whether the increased oxidative stress in EtOH KO mice was associated with mitochondrial dysfunction, we assayed the activities of key enzymes in isolated mitochondria from freshly harvested liver tissue (Fig. 4). KO mice had no change in complex I, II, and IV activities, but Fulvestrant molecular weight had lower activity

of citrate synthase, the first enzyme of the tricarboxylic acid (TCA) cycle, than the corresponding WT groups. Additionally, activity of aconitase, which

catalyzes the second step in the TCA cycle, was lower in PF KO mice and in both EtOH groups. Citrate synthase check details activity is known to be susceptible to oxidative damage from peroxyl radicals. 25 We conclude from these results that EtOH KO mice have mitochondrial dysfunction associated with increased oxidative stress. β-Catenin has been implicated in the oxidative stress response via binding to forkhead box (FOXO) transcription factors and regulating expression of antioxidant genes. 23 Manganese superoxide dismutase (SOD2) is a mitochondrial enzyme that is critical for protection against oxidative stress. We hypothesized that β-catenin participates

in protection against alcohol-mediated oxidative stress by regulating the expression of SOD2. Western blotting analysis showed significantly lower SOD2 protein levels in KO mice in both treatment groups (Fig. 5A,B). Real-time polymerase SPTLC1 chain reaction (PCR) analysis showed that expression of Sod2 was lower in KO mice, suggesting transcriptional regulation by β-catenin (Fig. 5C). Expression of Sod2 is up-regulated by the forkhead transcription factor, FoxO3a. Therefore, we performed immunoprecipitation studies in WT livers and found protein-protein interaction between FoxO3 and β-catenin (Fig. 5D). Expression of two other targets of FoxO3, Cdkn1b and GADD45, were lower in KO mice (Supporting Fig. 5). We conclude from these results that β-catenin transcriptionally regulates the critical mitochondrial oxidative stress response protein, SOD2, via binding to its upstream regulator, FoxO3. We then asked whether treatment with the antioxidant, N-acetylcysteine (NAC), could prevent mortality associated with ethanol ingestion in KO mice. KO mice were given NAC via twice-daily intraperitoneal injection (500 mg/kg). We found that NAC could not prevent either death (data not shown) or liver steatosis (Supporting Fig. 6) in EtOH KO mice. We then asked whether KO mice would exhibit differences in ethanol metabolism in vivo.