Chronic Alcohol Feeding Stimulates the Formation of Mutagenic Etheno-Adducts in a Rodent model of NASH Via Induction of Cytochrome P450 2E1

Abstract

Background and Aims: Chronic alcohol consumption may cause liver fibrosis and hepatocellular carcinoma (HCC) in non-alcoholic fatty liver disease (NAFLD). Synergistic effects of insulin resistance and alcohol triggering fibrogenesis and HCC are implicated. Formation of mutagenic etheno adducts binding to adenosin (EdA) generated via induced CYP2E1 has been suggested as a key mechanisms of hepatocarcinogenesis. Methods: Obese Zucker rats and lean littermates (4 groups) received Lieber-De Carli liquid diets containing either ethanol or carbohydrates for 3 months. Fibrosis and steatosis were quantified by Sirius Red and Oil Red O, respectively. EdA immunohistology was performed using a monoclonal antibody, and fibrosis-related gene and protein expression and hydroxyproline quantified. CYP2E1 and 4A2 were assayed at transcription and protein level. Results: Obese rats had more steatosis than lean animals but showed no aggravating impact of chronic alcohol intake. Histology and liver hydroxyproline showed similar extent of fibrosis in all groups. Procollagen alpha1(I), alpha smooth muscle actin, and TGFbeta-1 gene expression were not different. CYP2E1 protein was induced by 35% in both groups of obese rats, while corresponding mRNA levels were upregulated 3.5-fold in alcoholic lean rats. CYP4A2 was lower in obese rats, but upregulated 2-fold in those fed alcohol. In alcoholic obese and lean rats hepatocyte nuclei stained positive for EdA in 36% and 23%, respectively, but only 14% in obese rats without alcohol. EdA immunostaining was absent in lean non-alcoholic rats. EdA labelling correlated positively with CYP2E1 and 4-HNE immunostaining. Conclusions: Chronic alcohol feeding in obese, insulin-resistant rats exerts no effect on liver fibrosis formation, but potentiates the formation of mutagenic EdA possibly due to CYP2E1 induction. This observation may explain the increased risk of developing HCC in patients with NAFLD who chronically drink alcohol.