4B) and by the hepatic triglycerides (Fig. 4C). Alanine aminotransferase (ALT) activity increased by 2-fold only in ethanol-binged WT mice (Fig. 4D). In contrast, chronic ethanol feeding caused greater inflammation, necrosis, JQ1 research buy and ductular reaction in Ass+/− than in WT mice (Fig. 4E,F). The steatosis grade (Fig. 4F),

oil red O staining, and morphometry analysis (Supporting Fig. 4A-4B) demonstrated more neutral fat in chronic ethanol-fed Ass+/− than in WT mice, suggesting more liver injury by partial Ass ablation in the chronic ethanol feeding model. In order to investigate the effect of Ass deficiency on NOS2 and NO· generation, immunohistochemistry (IHC) was performed. There was more intense staining for NOS2 LY294002 ic50 (5-fold) and 3-NT residues (10-fold)—the footprint for nitrosative stress—in WT given an ethanol binge compared with Ass+/− mice, which was quantified by morphometry analysis (Fig. 5A-C). Chronic ethanol feeding

elevated NOS2 (2-fold, not statistically significant) and 3-NT protein adducts (3-fold) both in WT and in Ass+/− mice (Fig. 5D-F). Western blot analysis showed a 4- and a 2-fold increase in NOS2 in binged WT and Ass+/− mice, respectively (Supporting Fig. 5A, left), whereas there was only a 2-fold increase in NOS2 expression in both genotypes after chronic ethanol feeding. NOS1 and NOS3 expression remained similar with binge or chronic ethanol feeding in both WT and Ass+/− mice (Supporting Fig. 5A). However, serum nitrites plus nitrates, considered surrogate markers of NOS3 activity, remained similar in the binge model (Supporting Fig. 5B, left), but were lower in chronic ethanol-fed Ass+/− than in WT mice (Supporting Fig. 5B, right). ROS—key players in ethanol toxicity—are generated among others by microsomal CYP2E1, which is induced by ethanol itself. 15, 16 Because alcohol intake stabilizes CYP2E1 against 17-DMAG (Alvespimycin) HCl degradation contributing to liver injury, we examined CYP2E1 expression. Western blot analysis showed similar CYP2E1 induction by ethanol binge (Supporting Fig. 6A, left) and by chronic ethanol feeding (Supporting Fig. 6A, right) in WT and in Ass+/− mice. Lastly, IHC for 4-HNE—a lipid peroxidation end-product—was

similarly increased by the ethanol binge in both groups of mice (not statistically significant) (Supporting Fig. 6B); however, the increase was much higher in chronically ethanol-fed Ass+/− than in WT mice (Supporting Fig. 6C). Glutathione (GSH) is a key endogenous antioxidant participating in detoxification reactions. 17 WT and Ass+/− mice showed similar basal GSH, whereas binge drinking reduced GSH level by 50% in both WT and Ass+/− mice (Supporting Fig. 7). Total and mitochondrial GSH were higher in Ass+/− than in WT mice in the control group chronically fed a high-fat diet (Fig. 6A). This may have served as a protective mechanism in the ethanol binge model in addition to decreased NO· generation due to impairment of the L-citrulline/NO· cycle.