Stimulation of tolbutamide hydroxylation by acetone and acetonitrile in human liver microsomes and in a cytochrome P-450 2C9-reconstituted system.
To examine the response of individual cytochrome P-450 species catalysing the epoxidation of aldrin (Wolff T and Guengerich FP, Biochem Pharmacol 36: 2581-2588, 1987), monooxygenase systems reconstituted from these species were assayed in the presence of 5% (v/v) = 0.87 M ethanol. The activity of cytochromes P-450PB-B and P-450PB-D, two enzymes inducible by phenobarbital was increased seven-fold. The activity of two other P-450 enzymes purified from these animals was either inhibited by 50%, as observed for cytochrome P-450PB-C or remained unchanged, as noted with cytochrome P-450PCN-E. Two P-450 enzymes purified from untreated rats, cytochromes P-450UT-F and P-450UT-H, showed an inhibition by 50 and 20%, respectively, while the activity of cytochrome P-450UT-A was slightly increased by 50%. Indirect evidence that solvents enhance aldrin epoxidation by interacting with the hemoprotein was obtained by the finding that ethanol stimulated the activity of cytochrome P-450PB-B already, before addition of the lipid component, L-alpha-1,2-dilauroyl-sn-glycero-3-phosphocholine. The Km of cytochrome P-450PB-B for NADPH cytochrome P-450 reductase was not altered by ethanol indicating that the interaction between the two enzymes was not affected by the solvent. Other results indicate that the stimulatory solvent binds to a site, apart from the type I or type II binding site. The potency of various hydrophylic solvents to modify aldrin epoxidase activity was assayed in microsomes of rats pretreated with phenobarbital and of untreated male rats. Ethanol, n-propranol, n-butanol, acetone and tetrahydrofuran enhanced enzyme activity of phenobarbital pretreated rats to a maximal extent of two-fold and, at similar concentrations, inhibited the enzyme activity of untreated rats by 50%. The potency of these solvents correlated with their lipophilicity. Methanol and dimethylsulfoxide only slightly modified the activity of induced and noninduced animals. In the presence of 0.5 M n-propranol as the modifying agent, microsomal epoxidase activity of rats pretreated with pregnenolone-16 alpha-carbonitrile, dexamethasone, 3-methylcholanthrene and of control rats was inhibited by 60-80%, whereas the activity of animals pretreated with phenobarbital, DDT, or the polychlorinated biphenyl mixture, Clophen A 50, was stimulated between two- and three-fold. The results reveal that organic solvents frequently used to dissolve monooxygenase substrates may considerably modify the activity of cytochrome P-450 dependent reactions, in particular when purified enzymes are assayed.