Rapid changes in cytochrome P4502E1 (CYP2E1) activity and other P450 isozymes following ethanol withdrawal in rats

Abstract

This study describes the effects of chronic ethanol (ETOH) treatment and withdrawal on the rat hepatic mixed-function mono-oxygenase system. Male Sprague-Dawley rats (150–200 g, 10 per group) were administered ETOH as part of the Lieber-deCarli liquid diet for 3 weeks. Ethanol was removed, and the animals were euthanized at 0, 24, 48, 72 and 168 hr post-withdrawal. Microsomes were prepared, and ethanol-inducible cytochrome P4502E1 (CYP2E1) activity was measured using the enzyme markers N-nitrosodimethylamine demethylase (NDMAd), p-nitrophenol hydroxylase (PNPH) and aniline hydroxylase (AH). Activities were found to be induced significantly after chronic ETOH feeding using all three assays (NDMAd, 5-fold; PNPH, 3.5-fold; AH, 9-fold). Upon ETOH withdrawal, all three activities dropped markedly, with NDMAd and PNPH at control values at 24 hr and all subsequent time points. AH activity remained 3-fold higher than controls at 24, 48 and 72 hr. Western blot analyses showed that immunoreactive CYP2E1 returned to control at 24 hr, consonant with NDMAd and PNPH activities. The prolonged induction of AH activity following ETOH withdrawal indicates that it is not a specific marker of CYP2E1-catalyzed reactions. Collectively, these data are suggestive of a rapid mechanism of CYP2E1 degradation in the rat liver. Of the other parameters investigated in this study, total cytochrome P450 content was increased 2.5-fold after ETOH feeding, with levels dropping markedly 24 hr post-withdrawal. NADPH-dependent cytochrome c reductase activity was unchanged throughout the course of the study. CYP1A1, CYP2B1 and CYP3A activities were assessed by the substrate probes ethoxyresorufin O-dealkylase (EROD), pentoxyresorufin O-dealkylase (PROD) and erythromycin N-demethylase (ERNd). EROD and PROD were induced significantly by ETOH administration (2-fold) at 0 hr, with EROD remaining elevated over controls 24 hr post-withdrawal. Quantitative western blot analysis of CYP1A1 and CYP2B1 revealed a pattern of immunostaining generally consistent with but less variable than levels predicted by the respective substrate markers. Both proteins were induced significantly by chronic ethanol administration (CYP1A1, 1.9-fold; CYP2B1, 4-fold). Induction of these P450 isoforms persisted for several days following withdrawal. In contrast, immunoreactive CYP1A2 was found to decrease significantly (by 30–40%) during ethanol withdrawal (24, 48, 72, 168 hr). ERNd activity was induced significantly by chronic ETOH feeding (2.5-fold) and remained so for 24 hr into the withdrawal period (2-fold). Immunoreactive CYP3A1 was also induced significantly following ETOH administration (0 hr) and 24 hr following withdrawal. Collectively, the data presented suggest that chronic ethanol feeding induces in vivo at least four distinct P450 isoforms: CYP2E1, CYP1A1, CYP2B1 and CYP3A1. Ethanol withdrawal resulted in a 30–40% loss of CYP1A2. CYP1A1, CYP2B1 and CYP3A1 were elevated during ethanol withdrawal, unlike CYP2E1, a protein that undergoes rapid degradation following the removal of ethanol. Such findings are consistent with several distinct mechanisms of P450 isoform induction/ degradation by ethanol.