Monkey hepatic microsomal alcohol oxygenase: purification and characterization of a cytochrome P450 enzyme catalyzing the stereoselective oxidation of 7alpha- and 7beta-hydroxy-delta8-tetrahydrocannabinol to 7-oxo-delta8-tetrahydrocannabinol.

The formation of 7-oxo-delta8-tetrahydrocannabinol (7-Oxo-delta8-THC) from 7alpha- or 7beta-hydroxy-delta8-THC (7alpha- or 7beta-OH-delta8-THC) was found in hepatic microsomes of monkeys. The activity in 7beta-OH-delta8-THC was stereoselectively 2.5- to 4.6-fold higher than that from 7alpha-OH-delta8-THC. The oxidative activities of 7alpha- and 7beta-OH-delta8-THC to 7-Oxo-delta8-THC were inhibited to 35% and 10%, respectively, of the control value by the antibody against P450GPF-B (CYP3A), a major enzyme responsible for the formation of 7-Oxo-delta8-THC in guinea pigs. In the Lineweaver-Burk double-reciprocal plot analysis, testosterone 6beta-hydroxylase activity was competitively inhibited by 7beta-OH-delta8-THC. Two cytochrome P450 enzymes, called P450JM-D and P450JM-E, were purified from hepatic microsomes of Japanese monkeys. P450JM-E, assumed to be CYP3A8, immunologically reacted with the antibody against P450GPF-B and showed high forming activity of 7-Oxo-delta8-THC from 7-OH-delta8-THC. On the other hand, 7-Oxo-delta8-THC forming activity of P450JM-D, assumed to be CYP2C, was less than 10% of that of P450JM-E (CYP3A8). Oxygen-18 (18O) derived from atmospheric oxygen was incorporated into about 40% of the corresponding ketone formed from 7alpha-OH-delta8-THC or 8beta-OH-delta9-THC by P450JM-E (CYP3A8), although the incorporation of the stable isotope into the oxidized metabolite from 7beta-OH-delta8-THC or 8alpha-OH-delta9-THC was negligible. These results indicate that P450JM-E (CYP3A8) is a major enzyme of the oxidation of 7-OH-delta8-THC in monkey hepatic microsomes. The oxidation mechanism may proceed as follows: the alpha- and beta-epimers of 7-OH-delta8-THC or 8-OH-delta9-THC may be converted to ketone through dehydration of an enzyme-bound gem-diol by P450JM-E (CYP3A8), although this stereoselective dehydration differentiates between two epimers.