Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and ethanol were assayed using Infinity kits (Thermo Electron, Melbourne, Australia) and an ethanol assay kit (Biovision, Mountain View, CA), respectively as previously described [20]. Liver tumor necrosis factor α (TNFα) was assayed using a TNFα ELISA kit (Biosource, Camarillo, CA). Liver triglyceride (TG) was determined using an Infinity kit (Thermo Electron, Melbourne, Australia). Hepatic levels of GSH and TBARs were determined as previously described [20,25]. CYP2E1 activity was measured by the rate of oxidation of p-nitrophenol to p-nitrocatechol by isolated hepatic microsomes [26]. Immunoblots for β-actin, CYP2E1, peroxisome proliferator-activated receptor α (PPARα), acyl CoA oxidase (AOX), stearoyl CoA desaturase-1 (SCD-1), Bcl-2, Bcl XL, pJNK, JNK and antioxidant enzymes were carried out as previously described [20,25]. The antibody against acyl-CoA oxidase was a gift from Professor Paul Van Veldhoven, (K.U. Leuven, Belgium). Polyclonal antibody against SCD was from Cell Signaling Technology, Inc (Danvers MA). Antibodies against PPARα, Bcl 2, Bcl-XL, pJNK, JNK, catalase, GPx-4, thioredoxin, SOD-1 and SOD-2 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).Blots were quantified using the Image J (version 1.37v) software program from NIH. Ratios were expressed as protein/actin or pJNK/JNK.
