Monkey Hepatic Microsomal Alcohol Oxygenase: Purification and Characterization of a Cytochrome P450 Enzyme Catalyzing the Stereoselective Oxidation of 7.ALPHA.- and 7.BETA.-Hydroxy-.DELTA.8-tetrahydrocannabinol to 7-Oxo-.DELTA.8-tetrahydrocannabinol.

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Abstract
The formation of 7-oxo-Δ8-tetrahydrocannabinol (7-Oxo-Δ8-THC) from 7α- or 7β-hydroxy-Δ8-THC (7α- or 7β-OH-Δ8-THC) was found in hepatic microsomes of monkeys. The activity in 7β-OH-Δ8-THC was stereoselectively 2.5- to 4.6-fold higher than that from 7α-OH-Δ8-THC. The oxidative activities of 7α- and 7β-OH-Δ8-THC to 7-Oxo-Δ8-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-Δ8-THC in guinea pigs. In the Lineweaver-Burk double-reciprocal plot analysis, testosterone 6β-hydroxylase activity was competitively inhibited by 7β-OH-Δ8-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-Δ8-THC from 7-OH-Δ8-THC. On the other hand, 7-Oxo-Δ8-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 7α-OH-Δ8-THC or 8β-OH-Δ9-THC by P450JM-E (CYP3A8), although the incorporation of the stable isotope into the oxidized metabolite from 7β-OH-Δ8-THC or 8α-OH-Δ9-THC was negligible. These results indicate that P450JM-E (CYP3A8) is a major enzyme of the oxidation of 7-OH-Δ8-THC in monkey hepatic microsomes. The oxidation mechanism may proceed as follows: the α- and β-epimers of 7-OH-Δ8-THC or 8-OH-Δ9-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.