BILE-ACIDS PRODUCE A GENERALIZED REDUCTION OF THE CATALYTIC ACTIVITY OF CYTOCHROMES P450 AND OTHER HEPATIC-MICROSOMAL ENZYMES IN-VITRO - RELEVANCE TO DRUG-METABOLISM IN EXPERIMENTAL CHOLESTASIS

In bile duct-ligated male rats, there is a reduction of total hepatic microsomal cytochrome P450 (P450) levels and of NADPH-cytochrome P450 reductase (P450-reductase) activity, but the changes in activity of in dividual microsomal enzymes are nonuniform. We have proposed that the initial effect of cholestasis on microsomal proteins is a non-specific reduction caused by bile acid-mediated destruction, whereas the dispr oportionate lowering of male-specific P450 enzymes results from second ary down-regulation of some cytochrome P450 (CYP) genes. We report her ein the results of experiments to test the first part of this hypothes is, at least as indicated by enzyme inhibition. Hepatic microsomal fra ctions from normal male rats were incubated at 37 degrees C with incre asing concentrations of a range of bile acids selected for their varyi ng physicochemical properties. The endpoints were catalytic activity o f three individual CYP proteins, CYP 2A1 (measured as testosterone 7 a lpha-hydroxylase activity), 2C11 (testosterone 2 alpha-hydroxylase and 16 alpha-hydroxylase) and 3A2 (testosterone 6 beta-hydroxylase), and the non-CYP enzymes, steroid 17 beta-dehydrogenase and P450-reductase. With 0.25 mmol/L cholic acid, a concentration exceeded in serum follo wing bile duct ligation, there was a significant reduction in the acti vity of all enzymes at 4 h. Cholic acid-mediated inhibition was dose-d ependent and there was no difference in inhibitory activity towards th e male sex-dependent CYP 2C11 and 3A2 and the non-sex-dependent CYP 2A 1 and other microsomal enzymes. Taurocholic acid was twice as potent a n inhibitor as unconjugated cholic acid, the respective apparent I-50 values being similar to 0.6 mmol/L compared with similar to 1.2 mmol/L . The dihydroxy bile acids, chenodeoxycholic acid and deoxycholic acid , were also more potent inhibitors than cholic acid, exhibiting I-50 v alues in the range of 0.3-0.5 mmol/L, but the monohydroxy bile acid, l ithocholic acid, was the most potent inhibitor (I-50 similar to 0.2 mm ol/L). Thus, the inhibitory potential of bile acids towards microsomal enzymes was inversely related to their extent of hydroxylation, while taurine conjugation enhanced the inhibitory potential of cholic acid. These data confirm the potential of bile acids to inhibit the activit y of microsomal enzymes in livers of bile duct-ligated rats and indica te that such changes can occur with concentrations of bile acids that are physiologically relevant. Further, the results are consistent with the proposal that the disproportionately greater reduction of the mal e sex-dependent CYP, 2C11 and 3A2, is not explained by a destructive m echanism.