Flavones as in vivo inducers and in vitro inhibitors of cytochrome p-450-dependent arylhydrocarbon hydroxylase and affinity labeling of microsomal epoxide hydrase by effector-site-directed ligands
Part I. Induction of cytochrome P-450 dependent 7-ethoxycoumarin deethylase activity in rat liver microsomes by intraperitoneal administration of flavones (viz. (alpha)-napthoflavone ((alpha)NF), (beta)-napthoflavone ((beta)NF), flavone and 4'-bromoflavone) and of 3-methylcholanthrene (3MC) was studied. Kinetic plots showed that whereas (beta)NF and 3MC exposure produced microsomes with enzymatic activity corresponding to one (or mainly one) form of the enzyme, exposure to flavone, (alpha)NF or 4'-bromoflavone showed enzymatic activity with contributions due to at least two distinct forms of the enzyme. Two distinct forms were also detected in control microsomes. These enzyme forms were also distinguishable by a careful analysis of the inhibition constants obtained by using flavones ((alpha)NF, (beta)NF, (gamma)NF, 4'-bromoflavone and flavone) and selected arylpyrroles and arylimidazoles, as in vitro inhibitors of liver microsomal 7-ethoxycoumarin deethylase activity 1-Arylpyrroles were determined to be significantly less potent in vitro inhibitors of the microsomal monooxygenase system than the corresponding imidazoles, and the inhibition patterns obtained suggest that the former preferentially bind to the free enzyme, while the latter bind about equally well to the free enzyme and the enzyme-7-ethoxycoumarin complex The observed inhibition by (alpha)NF and by (beta)NF seemed comparable in all types of microsomal preparations examined, while the induction patterns by these compounds exhibited significant differences. This suggests that the differential effects of these compounds towards arylhydrocarbon-induced tumors is more likely to be the result of differential induction rather than differential inhibition Differences observed in inhibition mechanisms by various inhibitors indicates that K(,i) values would be more meaningful than I(,50) values in comparing the potency of various inhibitors of AHH activity Part II. Photoaffinity labeling of microsomal epoxide hydrase by 2-azido-9-fluorenone or by 4-azido-9-fluorenone (both being analogs of the in vitro stimulator, 9-fluorenone) lead to differential stimulation of the styrene oxide hydration, catalyzed by the labeled biological system. This indicates differential labeling of the microsomal enzyme system by these two azido analogs of fluorenone. Labeling by 2-azido-9-fluorenone decreased the K(,M) and increased the V(,max) value Addition of fluorenone to the 2-azido-9-fluorenone labeled microsomes resulted in further in vitro activation of styrene oxide hydration, suggesting that labeling is not complete in terms of all possible effector binding sites Affinity labeling by 1-bromobenzyl phenyl ketone also leads to a stimulation of epoxide hydrase catalyzed styrene oxide hydration, suggesting that nucleophilic centers might be present at the effector ligand binding site