CHRONIC ENTERAL ETHANOL TREATMENT CAUSES HYPOXIA IN RAT-LIVER TISSUE IN-VIVO

It is known that ethanol increases oxygen consumption in in vitro live r models, which could lead to hypoxia. Although it was shown recently that one large dose of ethanol caused hypoxia in rat liver in vivo, wh ether ethanol produces hypoxia in a clinically relevant chronic model remains unclear. In the present study, therefore, the effect of chroni c ethanol on hypoxia was investigated in vivo using the 2-nitroimidazo le hypoxia marker, pimonidazole. Male Wistar rats (300-325 g) were exp osed to enteral ethanol continuously for 4 weeks. In this model, rats develop steatosis, inflammation, and necrosis characteristic of early stages of clinical alcoholic liver disease in humans. One hour before they were killed, rats were injected with pimonidazole (120 mg/kg intr avenously), and livers were surgically isolated, removed, and fixed. P rotein-bound pimonidazole adducts were identified on formalin-fixed, p araffin-embedded tissue with immunohistochemistry. Ethanol administrat ion for 4 weeks significantly increased serum aspartate transaminase l evels and hepatic pathology scores for steatosis, inflammation, and ne crosis, as expected. Ethanol treatment significantly increased both th e extent and number of cells that stained positive for pimonidazole co mpared with control animals given an enteral diet without ethanol. Qua ntitative image-analysis of pimonidazole binding showed that 4 weeks o f ethanol administration nearly doubled the pimonidazole positive area in tissue. Ethanol also increased pimonidazole binding significantly at 7 days, long before inflammation and necrosis could be detected. Th ese results indicate that chronic ethanol causes hypoxia at the cellul ar level in rat liver in vivo and lend support to the hypothesis that hypoxia is involved in mechanisms of early alcoholic liver injury.