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.

Abstract

Tetrahydrocannabinol (THC) is the major psychoactive constituent of marijuana. It is well known that D 8 -THC is oxidized to numerous metabolites in the liver of various mammals including monkeys and humans.1,2) Most metabolites have been suggested to contribute to the psychoactivity of the parent compound. 7-Oxo-D 8 -THC shows almost equivalent pharmacological activity to D 8 -THC in mice, whereas 7a-and 7b-hydroxy-D 8 -THC (7a-and 7b-OH-D 8 -THC) were without significant activity.3) Several recent studies have clarified that cytochrome P450 (P450) plays a major role in the oxidation of THC. [4][5][6][7][8] However, the metabolic reaction is complicated and the isoforms responsible for specific metabolic reactions have not been completely elucidated. We have previously found that a guinea pig hepatic microsomal enzyme, called microsomal alcohol oxygenase (MALCO), is able to oxidize 7-OH-D 8 -THC to 7-Oxo-D 8-THC (Fig. 1), 9) and have recently clarified that P450 isoforms belonging to the 3A subfamily are the major enzymes of 7-OH-D 8 -THC MALCO in guinea pigs (P450GPF-B), 10) mice (CYP3A11), 11) rats (CYP3A1 and CYP3A2), 12) and humans (CYP3A4). 13)CYP3A is a highly expressed enzyme in rodents, monkeys, and humans and it has been demonstrated to play a prominent role in the metabolism of many clinically important drugs. 14) CYP3A shows selectivity for the chemical substituents rather than selectivity for substrates. 15,16) For example, the allylic positions, in spite of structurally unrelated compounds, are the major site of oxidation catalyzed by CYP3A.17-20) Species-related differences exist in the catalytic properties of P450 enzymes belonging to the same family or subfamily, but it is difficult to elucidate the properties of the enzyme responsible for a specific reaction across species. 12,21) To our best knowledge, no detailed study has been reported with respect to the specific isoform(s) involved in the formation of 7-oxo-D 8 -THC from 7a-and 7b-OH-D 8 -THC in monkeys.In the present study, we purified a P450 enzyme catalyzing the stereoselective oxidation of 7a-and 7b-OH-D 8 -THC to 7-Oxo-D 8 -THC from hepatic microsomes of Japanese monkeys and considered the oxidation mechanisms of 7-OH-D 8 -THC and 8-OH-D 9 -THC using oxygen-18 gas. The formation of 7-oxo-D D 8 -tetrahydrocannabinol ( -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. MATERIALS AND METHODS Materials

Effective NADH-Dependent Oxidation of 7.BETA.-Hydroxy-.DELTA.8-tetrahydrocannabinol to the Corresponding Ketone by Japanese Monkey Hepatic Microsomes

“…7) In the reconstituted system, we used a microsomal lipid system including NADPH-P450 reductase, cytochrome b 5 and sodium cholate as described previously. When The data are expressed as the meanϮS.E.…”

“…5,6) We have purified CYP3A8 as a major enzyme of MALCO from hepatic microsomes of monkey. 7) The activity was stereoselective and the rate of conversion of 7b-hydroxy-D 8 -THC to 7-oxo-D 8 -THC was higher than that from 7a-hydroxy-D 8 -THC. 7,8) In general, the P450-dependent monooxygenase system has an absolute requirement for NADPH.…”

“…7) The activity was stereoselective and the rate of conversion of 7b-hydroxy-D 8 -THC to 7-oxo-D 8 -THC was higher than that from 7a-hydroxy-D 8 -THC. 7,8) In general, the P450-dependent monooxygenase system has an absolute requirement for NADPH. A few studies have indicated that NADH can also accelerate the oxidative metabolism of p-nitrophenetole 9) and p-nitroanisole, 10,11) and the bioactivation of 2-acetylaminofluorene 12) and nitrosoamines.…”

“…7,8) In general, the P450-dependent monooxygenase system has an absolute requirement for NADPH. A few studies have indicated that NADH can also accelerate the oxidative metabolism of p-nitrophenetole 9) and p-nitroanisole, 10,11) and the bioactivation of 2-acetylaminofluorene 12) and nitrosoamines.…”

“…7) NADPH-cytochrome c (P450) reductase and cytochrome b 5 were purified from hepatic microsomes of ddY male mice (Hokuriku Experimental Animals Lab., Kanazawa, Japan) by the methods of Yasukochi and Masters, 19) and Funae and Imaoka,20) respectively. One unit of the reductase was defined as the amount of reductase catalyzing the reduction of 1 mmol of cytochrome c per minute.…”