Introduction

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Tetrahydrocannabinol (THC), a psychoactive constituent of marijuana, is known to be extensively metabolized in various animal species (Harvey and Paton, 1984). Allylic oxidation appears to be the most important route for the biotransformation of ⁸-THC. 11Hydroxy-⁸-THC has been observed as a major metabolite in mammals (Foltz et al., 1970). This metabolite has been reported to have a higher pharmacological activity than ⁸-THC itself (Watanabe et al., 1980). We have previously demonstrated that the pharmacological activity of 7-oxo-⁸-THC formed from 7-hydroxy-⁸-THC was comparable to that of ⁸-THC, although other allylic alcohols, 7- and 7-hydroxy-⁸-THC were without significant activity (Narimatsu et al., 1984).

It has been generally known that secondary alcohols are oxidized to the corresponding ketones by alcohol dehydrogenase in cytosol (Kageura and Toki, 1974, 1975). However, we have previously found that a guinea pig hepatic microsomal enzyme, named microsomal alcohol oxygenase (MALCO), is able to oxidize 7-hydroxy-⁸-THC to 7-oxo-⁸-THC (Narimatsu et al., 1988). Recently we purified a P450, named P450GPF-B, which is a major enzyme responsible for the formation of 7-oxo-⁸-THC in guinea pig hepatic microsomes and is estimated to be a member of the 3A subfamily (Matsunaga et al., 1997). Bornheim et al. have reported that the antibody against P450 3A inhibits the formation of 8-oxo-⁹-THC from ⁹-THC in hepatic microsomes of mice (Bornheim et al., 1991). However, they have not directly demonstrated the contribution of the mouse 3A enzyme to the formation of 8-oxo-⁹-THC from 8-hydroxy-⁹-THC, since 8-oxo-⁹-THC are thought to be formed from 8-hydroxylated metabolites by further enzymatic oxidation.

In the present study, we purified and elucidated a P450 that plays a major role in the formation of 7-oxo-⁸-THC from 7- and 7-hydroxy-⁸-THC in mouse liver.

	Materials and Methods

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Materials. Sepharose 4B and 2',5'-ADP-Sepharose 4B were obtained from Pharmacia Fine Chemicals (Uppsala, Sweden); hydroxylapatite for an open column was obtained from Bio-Rad (Richmond, CA): preparative DEAE-5PW and hydroxylapatite columns for HPLC were obtained from Tosoh (Tokyo, Japan). Emulgen 911 was kindly provided by Kao-Atlas Co. (Tokyo, Japan). 7- and 7-Hydroxy-⁸-THC (Mechoulam et al., 1972), 7-oxo-⁸-THC (Narimatsu et al., 1984), and 5'-nor-⁸-THC-4'-oic acid (Ohlsson et al., 1979) were prepared by the methods previously reported. Microsomal lipids were extracted from hepatic microsomes obtained from adult male mice with chloroform:methanol (2:1), and the solvent was evaporated to dryness in vacuo. Other chemicals and solvents used were of the highest quality commercially available.

Animal Treatment and Preparation of Microsomes. Male mice of the ddY strain (8 weeks old; Hokuriku Experimental Animals Lab., Kanazawa, Japan) were used in all experiments. Phenobarbital sodium (100 mg/kg in saline) and 3-methylcholanthrene (40 mg/kg in salad oil) were administered intraperitoneally every 24 hr for 2 days. Dexamethasone (500 mg/kg in salad oil) was injected intraperitoneally at a single dose. Acetone was given in 5%(v/v) solution in drinking water for 10 days until the mice were killed. After fasting for 12 hr, the animals were killed by decapitation 48 hr after the first injection of phenobarbital, 3-methylcholanthrene, and dexamethasone. Hepatic microsomal pellets were prepared by the method reported previously (Matsunaga et al., 1996).

Purification of P450 from Hepatic Microsomes of Dexamethasone-Treated Male Mice. Microsomes (P450: 1.93 nmol/mg protein; total, 3094 nmol) were suspended in buffer A (0.1 M potassium phosphate buffer [pH 7.25] containing 20% glycerol, 1 mM EDTA, and 0.5 mM dithiothreitol). Then 20% sodium cholate solution (pH 7.4) was added to a final concentration of 0.7%. This mixture was stirred for 30 min at 0°C to solubilize microsomes, and the resulting suspension was centrifuged at 105,000g for 60 min. The supernatant fraction of the cholate-solubilized hepatic microsomes was put on an -aminooctyl-Sepharose 4B column (4×30 cm) equilibrated with buffer A containing 0.5% sodium cholate. The column was washed with equilibration buffer, and P450 was eluted with buffer A, containing 0.4% sodium cholate and 0.1% Emulgen 911. The P450 fractions were pooled, concentrated with an ultrafiltration membrane (UK-50; Toyo Roshi, Tokyo, Japan), and dialyzed against 20 mM Tris-acetate buffer (pH 7.5) containing 20% glycerol. The dialyzed solution was subjected to HPLC with a preparative DEAE-5PW anion-exchange column (2.15 × 15 cm; Tosoh, Tokyo, Japan) previously equilibrated with buffer B (20 mM Tris-acetate buffer [pH 7.5] containing 20% glycerol and 0.4% Emulgen 911). The column chromatography was performed with a linear gradient of sodium acetate from 0 to 0.2 M in buffer B. The fractions were combined, concentrated and dialyzed against buffer C (10 mM potassium phosphate buffer [pH 7.4] containing 20% glycerol and 0.2% sodium cholate). The dialyzed sample was subjected to HPLC with a hydroxylapatite column (0.75 × 7 cm; Tosoh) previously equilibrated with buffer C. P450 was eluted with a linear gradient of potassium phosphate buffer (pH 7.4) from 10 to 350 mM containing 20% glycerol, 0.2% sodium cholate and 0.4% Emulgen 911. The electrophoretically homogeneous fractions were combined and concentrated.

Purification of Other Enzymes. NADPH-cytochrome c (P450) reductase and cytochrome b₅ were purified from hepatic microsomes of ddY mice by the methods of Yasukochi and Masters (Yasukochi and Masters, 1976), and Funae and Imaoka (Funae and Imaoka, 1985), respectively. One unit of the reductase was defined as the amount of reductase catalyzing the reduction of 1 mmol of cytochrome c per min. The detergent was removed by using a small hydroxylapatite column.

Measurement of Oxidative Activity. The formation of 7-oxo-⁸-THC was measured essentially as previously described (Matsunaga et al., 1997), except for the conditions in the reconstitution studies. 7-Hydroxy-⁸-THC (12 µg) was incubated with purified P450 (50 pmol), 0.5 units of NADPH-cytochrome c (P450) reductase, 50 pmol of cytochrome b₅, 50 µg of microsomal lipids, 100 µg of sodium cholate, 1 mM NADPH, and 100 mM potassium phosphate buffer (pH 7.4) to make a final volume of 0.5 ml. The mixture was incubated at 37°C for 20 min after preincubation at 37°C for 2 min. Metabolites were extracted and analyzed by electron capture detector-gas chromatography as described previously (Matsunaga et al., 1997).

Other Methods. Polyclonal antibody against the purified P450 was raised in rabbits as described previously (Narimatsu et al., 1990). Western blotting (Towbin et al., 1979) and immunoinhibition of microsomal enzyme activities (Matsunaga et al., 1997) were performed as described previously. Protein concentration was estimated by the method of Lowry et al. (Lowry et al., 1951), using bovine serum albumin as a standard. P450 and cytochrome b₅ contents were determined by the methods of Omura and Sato (Omura and Sato, 1964), and Omura and Takesue (Omura and Takesue, 1970), respectively. -Aminooctyl-Sepharose 4B was prepared as described previously (Nishikawa and Bailon, 1975). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out according to the method of Laemmli (Laemmli, 1970). The statistical significance of differences was determined by means of the Bonferroni test.

	Results and Discussion

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Table 1 shows the effect of treatment with various P450 inducers, dexamethasone, phenobarbital, 3-methylcholanthrene, and acetone on P450 content, and 7- and 7-hydroxy-⁸-THC MALCO activities in mouse liver. P450 content was significantly increased by these inducers except for 3-methylcholanthrene. 7- and 7-Hydroxy-⁸-THC MALCO activities were also significantly increased by the treatment with dexamethasone or phenobarbital. Especially, dexamethasone induced the MALCO activities more than 10-fold of control. Meehan et al. reported that phenobarbital and dexamethasone were able to induce mRNA species from the CYP2B, CYP2C, and CYP3A gene subfamilies in vivo in mice (Meehan et al., 1988). Corcos also showed that dexamethasone is generally a stronger inducer of CYP3A mRNAs than phenobarbital in mouse, although induction levels of phenobarbital and dexamethasone on CYP2B mRNAs were comparable (Corcos, 1992). On the other hand, 7- and 7-hydroxy-⁸-THC MALCO activities were suppressed to 67% and 25%, respectively, of control by treatment with acetone, although P450 content was increased 1.6-fold, compared with the untreated group. Besides alcohol dehydrogenase, the oxidation of ethanol and other aliphatic alcohols to the corresponding aldehydes is also catalyzed by CYP2E1 (Morgan et al., 1982). The lack of induction with acetone, a typical inducer of CYP2E1 (Freeman et al., 1992), indicates that acetone-inducible forms of P450 may not participate in the oxidative catalytic activity of 7-hydroxy-⁸-THC in the mouse hepatic microsomes. The oxidative activities of 7- and 7-hydroxy-⁸-THC to 7-oxo-⁸-THC in dexamethasone-treated mouse liver were significantly inhibited by SKF 525-A and metyrapone, while barbital and pyrazole, inhibitors of aldehyde reductase and alcohol dehydrogenase, did not show any inhibition for the oxidation of the alcohol (data not shown). These results indicate that the formation of 7-oxo-⁸-THC increased by dexamethasone pretreatment depends on induction of P450. Recently, we purified a P450 belonging to 3A subfamily as a major enzyme responsible for the oxidation of 7-hydroxy-⁸-THC to 7-oxo-⁸-THC in hepatic microsomes of guinea pigs (Matsunaga et al., 1997). The antibody against P450GPF-B inhibited 7- and 7-hydroxy-⁸-THC MALCO activities in dexamethasone-treated mouse liver up to approximately 20% of the control value.

On the basis of the above results, we carried out the purification of MALCO from hepatic microsomes of dexamethasone-treated mice by chromatography on columns consisting of -aminooctyl-Sepharose 4B, DEAE-5PW, and hydroxylapatite, using the immunological crossreaction with antibody against P450GPF-B as an indicator. The purified P450 showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the apparent molecular mass of 51,000. The purified P450 had a specific content of 13.8 nmol/mg protein. The characterized NH₂-terminal amino acid sequence up to the first 20 residues of P450MDX-B was identical to that of CYP3A11 estimated from cDNA (Yanagimoto et al., 1992). P450MDX-B showed high oxidative activities for 7- and 7-hydroxy-⁸-THC in the reconstituted system (table 2). This purified enzyme also showed comparable activity to CYP3A1 (Halvorson et al., 1990) and CYP3A4 (Yamazaki et al., 1996) for testosterone 6-hydroxylation, which is thought to be one of specific reactions for the CYP3A enzyme in rodents and primates. 7- and 7-Hydroxy-⁸-THC MALCO activities in dexamethasone-treated mice liver were markedly inhibited by antibody against P450MDX-B (fig. 1). This antibody also inhibited the MALCO activities in untreated mice liver to about 10% of control value when the antiserum was added at a protein ratio of 6 mg/mg microsomes. These results indicate that P450MDX-B is a major enzyme responsible for the oxidative transformation of 7- and 7-hydroxy-⁸-THC to 7-oxo-⁸-THC in mouse liver.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Effects of antibody against P450MDX-B on 7- and 7-hydroxy-8-THC MALCO activities in dexamethasone-treated mouse liver. Hepatic microsomes were preincubated with various amounts of the sera for 30 min at 37°C and then incubated with 7- (A) or 7-hydroxy-8-THC (B) in the presence of an NADPH-generating system. 7- and 7-Hydroxy-8-THC MALCO activities without serum (100% as the control) were 4.05 and 8.38 nmol/min/mg protein, respectively. Open and closed circles indicate the addition of preimmune serum and antiserum against P450MDX-B, respectively.