Effective NADH-Dependent Oxidation of 7b -Hydroxy-D 8 - tetrahydrocannabinol to the Corresponding Ketone by Japanese Monkey Hepatic Microsomes

646 Biol. Pharm. Bull. 28(4) 646 651 (2005) Vol. 28, No. 4 Effective NADH-Dependent Oxidation of 7b -Hydroxy-D 8 - tetrahydrocannabinol to the Corresponding Ketone by Japanese Monkey Hepatic Microsomes Tamihide MATSUNAGA,*,a Shinsuke HIGUCHI, b Kazuhito WATANABE, b Takashi KAGEYAMA, c Shigeru OHMORI, a and Ikuo YAMAMOTO d a Division of Pharmacy, Shinshu University Hospital; 3 1 1 Asahi, Matsumoto 390 8621, Japan: b Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University; Ho-3 Kanagawa-machi, Kanazawa 920 1181, Japan: c Department of Biochemistry, Primate Research Institute, Kyoto University; Kanrin, Inuyama 484 8506, Japan: and d Department of Hygienic Chemistry, School of Pharmaceutical Sciences, Kyushu University of Health and Welfare; 1714 1 Yoshino-chou, Nobeoka 822 8508, Japan. Received August 9, 2004; accepted December 27, 2004 The NADH-dependent activity by hepatic microsomes of Japanese monkeys for 7-oxo-D 8 -tetrahydrocannabinol (7-oxo-D 8 -THC) formation from 7b -hydroxy-d 8 -THC exhibited about 70% of the NADPH-dependent activity (100%) at the substrate concentration of 72.7 mm, although NADPH was an obligatory cofactor for maximal activity. Both NADH- and NADPH-dependent activities were significantly inhibited by the typical P450 inhibitors, such as SKF525-A and metyrapone. Both activities were almost completely inhibited by the NADPH- P450 reductase inhibitor diphenyliodonium chloride. The ratio of NADH- and NADPH-dependent activities varied significantly according to the substrate concentration. Interestingly, the NADH-dependent activity was higher than that of NADPH at low substrate concentrations of 13 50 mm. The ratio was also affected by the cofactor concentration. In the reconstituted system of CYP3A8 purified from hepatic microsomes of Japanese monkeys as a major enzyme responsible for the NADPH-dependent oxidation, NADH as well as NADPH could sustain the oxidation of 7b-hydroxy-D 8 -THC to the corresponding ketone. The NADH-dependent oxidation of 7b-hydroxy-D 8 -THC by monkey livers is mainly catalyzed by CYP3A8 as well as the NADPH-dependent oxidation. These results indicate that NADH as a cofactor may be also useful for the oxidation of 7b-hydroxy-D 8 -THC, and that the cofactor requirement for the reaction is varied by the concentrations of substrate and/or cofactor. Key words 7-hydroxy-D 8 -tetrahydrocannabinol; CYP3A8; monkey; NADH; P450; microsomal alcohol oxygenase Tetrahydrocannabinol (THC) is a major psychoactive constituent of marijuana, Cannabis sativa L. It is well known that THC is oxidized to a number of metabolites in the liver of mammals. 1) Recent studies have clarified that P450 plays a major role in the oxidation of THC. 2 4) We have previously reported that a hepatic microsomal enzyme, named microsomal alcohol oxygenase (MALCO), is able to oxidize 7aand 7b-hydroxy-D 8 -THC to 7-oxo-D 8 -THC in the presence of NADPH and molecular oxygen. 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. 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. 13,14) During our studies on the microsomal oxidative metabolism of D 8 -THC, we observed that 7b-hydroxy-D 8 -THC was effectively biotransformed to 7-oxo-D 8 - THC by using NADH as a cofactor, without NADPH (Fig. 1). The present study was undertaken to define the basic characteristics of the microsomal NADH-mediated oxidation of 7b-hydroxy-D 8 -THC to 7-oxo-D 8 -THC in the liver of monkeys and compare it to the NADPH-dependent systems. MATERIALS AND METHODS Materials NADH, NAD and NADP were purchased from Boehringer-Mannheim GmbH (Darmstadt, Germany); NADPH was from Oriental Yeast Co., Ltd. (Tokyo, Japan); metyrapone and 5a-cholestane were from Sigma Chemical Co. (St Louis, MO, U.S.A.); and diphenyliodonium chloride (DPIC), potassium cyanide and cholic acid were obtained from Wako Pure Chemicals (Osaka, Japan). SKF525-A and Emulgen 911 were kindly provided by Smith, Kline and French Laboratories (Philadelphia, PA) and Kao-Atlas Co. (Tokyo, Japan), respectively. 7b-Hydroxy-D 8 -THC, 15) 7-oxo- D 8 -THC 16) and 5 -nor-d 8 -THC-4 -oic acid 17) were prepared by the methods previously reported. The purity of the cannabinoids was verified to be more than 98% by gas chromatography. Microsomal lipids were extracted from hepatic microsomes 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. Animals and Preparation of Microsomes Liver samples from Japanese monkeys were provided by the Primate Research Institute, Kyoto University (Inuyama, Japan). Liver tissue homogenized using a Teflon-homogenizer was centrifuged to obtain the microsomes as described previously. 18) The microsomal pellets obtained were suspended in 100 mm potassium phosphate buffer (ph 7.4) containing 20% glycerol and 5 mm EDTA. Enzyme Assays In the study of the microsomal fraction, To whom correspondence should be addressed. e-mail: t-matsu@hsp.md.shinshu-u.ac.jp 2005 Pharmaceutical Society of Japan

April 2005 647 Fig. 1. Oxidation Metabolism of D 8 -THC at the C-7 Position by Hepatic Microsomes the substrate was incubated with microsomes, 1 mm NAD, NADP, NADH and/or NADPH and 100 mm potassium phosphate buffer (ph 7.4) to make a final volume of 0.5 ml. In the reconstitution studies of the complete system, each substrate was incubated with CYP3A8 (30 pmol), 0.33 units of NADPH-cytochrome c (P450) reductase, 30 pmol of cytochrome b 5, 50 mg of microsomal lipids, 100 mg of sodium cholate, 1 mm NADPH or NADH, and 100 mm potassium phosphate buffer (ph 7.4) to make a final volume of 0.5 ml. The reaction was allowed to proceed for 20 min at 37 C. The metabolites were determined using previously reported methods. Other Methods CYP3A8 was purified from hepatic microsomes of Japanese female monkeys as previously reported. 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. The detergent was removed using a hydroxylapatite column (f5mm 10 mm ). Protein concentration was estimated by the method of Lowry et al., 21) using bovine serum albumin as a standard. P450 and cytochrome b 5 contents were determined by the methods of Omura and Sato, 22) and Omura and Takesue, 23) respectively. RESULTS Cofactor Requirements and Optimum ph in Oxidation of 7b-Hydroxy-D 8 -THC to 7-Oxo-D 8 -THC The cofactor requirement for 7-oxo-D 8 -THC formation from 7b-hydroxy- D 8 -THC with hepatic microsomes of monkeys was examined (Table 1). NADPH was an obligatory cofactor to show the maximal activity, while NAD and NADP, cofactors of dehydrogenase, were virtually inactive. The NADH-dependent activity exhibited about 70% of the NADPH-dependent Table 1. Cofactor Requirement for 7-Oxo-D 8 -THC Formation from 7b- Hydroxy-D 8 -THC with Liver Microsomes of Male Japanese Monkeys Monkey Year-old Activity (nmol/min/mg protein) NAD NADP NADH NADPH 71Mf 6 0.04 0.07 0.19 0.18 73Mf 2 0.07 0.04 0.28 0.31 74Mf 2 0.02 0.04 0.29 0.41 The data are expressed as the mean of two experiments. 7b-Hydroxy-D 8 -THC (72.7 m M) was incubated with the microsomes for 20 min at 37 C. Each cofactor was added to the incubation mixture at 1 mm. Fig. 2. Effect of ph on 7b-Hydroxy-D 8 -THC Oxidative Activity of Japanese Monkey Hepatic Microsomes 7b-Hydroxy-D 8 -THC (72.7 m M) was incubated with the microsomes for 20 min at 37 C in the presence of various cofactors. NAD (open triangles), NADP (closed triangles), NADH (open circles) or NADPH (closed circles) was added to the incubation mixture at 1 mm. Potassium phosphate buffer, Tris HCl buffer, and glycine sodium hydroxy buffer were used for ph 5.5 7.5, 7.5 9.0, and 9.0 10.5, respectively. The data are expressed as the mean of two experiments. activity at the substrate concentration of 72.7 m M. Figure 2 shows the effect of ph on 7-oxo-D 8 -THC formation. The optimum ph in the NADH system was 7.5 and was the same as that in the NADPH system.

648 Vol. 28, No. 4 Table 2. Effects of Inhibitors on NADH- or NADPH-Dependent Oxidative Activity of 7b-Hydroxy-D 8 -THC in Monkey Hepatic Microsomes Inhibitors Concentration NADH Activity (nmol/min/mg protein) NADPH Experiment 1 Control 0.29 0.03 (100) 0.41 0.07 (100) SKF525-A 1.0 mm 0.13 0.02 (45) 0.15 0.01 (38) Metyrapone 1.0 mm 0.13 0.01 (45) 0.20 0.01 (50) Potassium cyanide 0.1 mm 0.31 0.03 (105) 0.39 0.09 (97) 1.0 mm 0.31 0.03 (106) 0.32 0.08 (79) Experiment 2 Control 0.21 0.02 (100) 0.29 0.03 (100) DPIC 1.0 mm 0.02 0.00 (10) 0.02 0.00 (7) The data are expressed as the mean S.E. of three experiments. Numbers in parentheses represent the relative activities. 7b-Hydroxy-D 8 -THC (72.7 m M) was incubated with the microsomes for 20 min at 37 C in the presence of various inhibitors. Each cofactor was added to the incubation mixture at 1 mm. Fig. 3. Effect of Substrate Concentration on NADH- or NADPH-Dependent 7b-Hydroxy-D 8 -THC Oxidative Activity of Japanese Monkey Hepatic Microsomes 7b-Hydroxy-D 8 -THC (13.3 to 150 m M) was incubated with the microsomes for 20 min at 37 C in the presence of 1 mm NADH (open circles) or NADPH (closed circles). The data are expressed as the mean S.E. of three experiments. Effects of Inhibitors on 7-Oxo-D 8 -THC Formation Table 2 summarizes the effects of various inhibitors on 7-oxo-D 8 -THC formation in the presence of NADH or NADPH. SKF525-A and metyrapone inhibitors of P450 inhibited the formation of 7-oxo-D 8 -THC from 7b-hydroxy- D 8 -THC in the NADH system by 45%. DPIC, which is an inhibitor of NADPH-P450 reductase, 24) almost completely inhibited the activity. These inhibitors also inhibited to an extent similar to that in the NADPH system. On the other hand, potassium cyanide, an inhibitor of a terminal oxidase in fatty acid desaturase, had no significant effect on the formation of 7-oxo-D 8 -THC. Effects of Substrate and Cofactor Concentration on the Formation of 7-Oxo-D 8 -THC Figure 3 shows the effect of substrate concentration (13.3 150 m M) on 7-oxo-D 8 -THC forming activity in the presence of NADH or NADPH, at a final concentration of 1 mm. The activity in the NADH system rose with an increasing concentration of the substrate and reached a maximum at about 55 m M. In the NADPH system, the activity increased slowly compared with the NADH system until the substrate concentration reached 40 m M, however, marked increases in activity were observed Fig. 4. Effect of Substrate Concentration on NADH Plus NADPH-Dependent 7b-Hydroxy-D 8 -THC Oxidative Activity of Japanese Monkey Hepatic Microsomes 7b-Hydroxy-D 8 -THC (13.3 to 150 m M) was incubated with the microsomes for 20 min at 37 C in the presence of 1 mm NADH plus NADPH. The data are expressed as the mean of two experiments. when the substrate concentration increased to more than 40 m M. As the result, the NADH-dependent activity was higher than that of NADPH at substrate concentrations of 13 50 m M (Fig. 3). When both NADH and NADPH are used as cofactors at lower concentrations of the substrate (below 40 m M), 7-oxo-D 8 -THC forming activity was almost the same as the activity when using NADH alone, whereas at a higher concentration (above 55 m M) it was the same as NADPH alone (Fig. 4). Figure 5 shows the effect of cofactor concentration (0.01 2 mm ) on the oxidative activity. When NADPH was used as the cofactor, maximum activity at the substrate concentrations of 40 and 80 m M was achieved at the cofactor concentrations of 0.2 and 1 mm, respectively, and then declined. In contrast, when NADPH was replaced with NADH for any substrate concentration, the activity was increased according to the cofactor concentration up to 2 mm. 7-Oxo-D 8 -THC Formation by CYP3A8 in a Reconstituted Enzyme System CYP3A8 was purified from hepatic microsomes of Japanese monkeys as a major enzyme responsible for the NADPH-dependent oxidation of 7b-hydroxy-D 8 - THC. 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

April 2005 649 Fig. 5. Effect of Cofactor Concentration on NADH- or NADPH-Dependent 7b-Hydroxy-D 8 -THC Oxidative Activity of Japanese Monkey Hepatic Microsomes The microsomes were incubated with 40 m M (A), 80 m M (B), and 150 m M (C) of 7b-hydroxy-D 8 -THC for 20 min at 37 C in the presence of NADH (open circles) or NADPH (closed circles). The data are expressed as the mean S.E. of three experiments. Table 3. Component Requirements and Effect of Substrate Concentration on Oxidative Activity of 7b-Hydroxy-D 8 -THC in Reconstituted System of CYP3A8 NADPH was used as the cofactor, the oxidative activity of 7b-hydroxy-D 8 -THC to 7-oxo-D 8 -THC by CYP3A8 at the substrate concentrations of 40 and 72.7 m M was 4.56 and 8.03 nmol/min/nmol P450, respectively (Table 3). NADH alone could also sustain the oxidation of 7b-hydroxy-D 8 - THC in the reconstituted system as indicated the microsomal fraction, although the activity was lower than the NADPHdependent activity. However, the activity could not be detected in the absence of CYP3A8 or NADPH-cytochrome c (P450) reductase. Cytochrome b 5 was an absolute requirement, especially in the NADH-dependent reaction, because the activity in the absence of cytochrome b 5 was decreased to about 10% of the activity in its presence. DISCUSSION NADH Activity (nmol/min/nmol P450) NADPH Ratio (NADH/NADPH) Complete 2.13 4.56 0.47 (Substrate 40 m M) Complete 2.54 8.03 0.32 (Substrate 72.7 m M) P450 0.01 0.01 NADPH-P450 reductase 0.01 0.01 b 5 0.28 2.79 0.10 The data are expressed as the mean of two experiments. The complete system contained purified CYP3A8 (30 pmol), NADPH-cytochrome c (P450) reductase (0.33 units), cytochrome b 5 (30 pmol), microsomal lipids (50 mg), sodium cholate (100 mg), NADPH or NADH (1 mm) and potassium phosphate buffer (ph 7.4, 100 mm) to make a final volume of 0.5 ml. The reaction was allowed to proceed for 20 min at 37 C. The metabolites were determined by previously reported methods. Shigematsu et al. 9,11,25) described the NADH-dependent O-dealkylation of p-nitrophenetole by rabbit hepatic microsomes and concluded that the reaction is probably P450-independent because it was not inhibited by carbon monoxide. On the other hand, Kamataki et al. 10,26) reported that the antibody against P450 inhibited the NADH-dependent O-demethylation of p-nitroanisole; thus it would appear that the reaction is in fact mediated by a species of P450 even though it is atypical in not being inhibited by carbon monoxide. Kuwahara and Mannering 27) concluded that NADHand NADPH-dependent O-deethylation of p-nitrophenetole involve different P450 enzymes and separate electron transfer systems. Murray and Butler 28) also reported that NADH, as well as NADPH, is able to support the efficient biotransformation of parathion to paraoxon and 4-nitrophenol in vitro and that P450 is involved in parathion oxidation mediated by both cofactors. In a previous study, we have found that 7b-hydroxy-D 8 - THC is NADPH- and O 2 -dependently oxidized to 7-oxo-D 8 - THC by hepatic microsomes 5) and clarified that CYP3A is a major enzyme responsible for the NADPH-dependent oxidation in various animals. 6,7,29 31) The P450-dependent monooxygenase system preferentially requires NADPH as a source of reducing equivalents; the NADPH-P450 reductase transfers two electrons from this nucleotide to the P450. In the presence of NADPH, however, NADH can function as the source of the second electron, transferred to the P450 through cytochrome b 5 reductase, following incorporation of the oxygen to the enzyme-substrate complex. 32) Furthermore, some P450 monooxygenase reactions are supported by NADH at only 10 20%. 18,33,34) This has usually been interpreted to mean that NADH can supply an electron to a given P450-substrate complex at a rate only 10 20% of that supported by NADPH. Actually, when NADPH is the cofactor, this process is less efficient. A report demonstrated an approximate 4000-fold greater Michaelis constant for the reduction of cytochrome c by NADH than for the NADPHmediated reaction. 35) However, the study did not evaluate whether inclusion of P450 and its substrates may enhance the rate of reduction supported by NADH. On the other hand, extensive NADH-dependent oxidation of 7b-hydroxy-D 8 - THC was observed in hepatic microsomes of monkeys (Table 1). Indeed, the oxidative activity of 7b-hydroxy-D 8 - THC in the presence of NADH was about 70% of that of NADPH. Moreover, in the low-substrate concentration system or high-cofactor concentration system, the NADH was more effective, under the study conditions, than NADPH in supporting the oxidation of 7b-hydroxy-D 8 -THC to 7-oxo-

650 Vol. 28, No. 4 Fig. 6. NADH- or NADPH-Dependent Electron Transport in 7b-Hydroxy-D 8 -THC Oxidation The NADH- and NADPH-dependent electron transport systems are indicated by solid and dotted lines, respectively. D 8 -THC (Figs. 3, 5). The typical P450 inhibitors SKF 525-A and metyrapone decreased the rates of NADH-dependent 7b-hydroxy-D 8 -THC oxidation. These inhibitors also inhibited to an extent similar to that with NADPH. The NADPH-P450 reductase inhibitor DPIC almost completely inhibited 7b-hydroxy-D 8 -THC metabolism mediated by both NADH and NADPH. These results indicate the involvement of P450 in the NADH-dependent oxidation. It is not still unknown why the NADPH-dependent activity, especially at low substrate concentration, is dramatically decreased depending on the cofactor concentration (Fig. 5A). It is known that the NADPH-dependent aldo-keto reductase was present in the microsomal fraction. 36) There is a possibility that the resulting 7-oxo-D 8 -THC is reduced to 7-hydroxy- D 8 -THC by aldo-keto reductase. However, we could not detect the reduced metabolites such as 7a- or 7b-hydroxy-D 8 - THC when 7-oxo-D 8 -THC was incubated with hepatic microsomes in the presence of NADPH (data not shown). THC and many of the metabolites are highly lipophilic compounds. The n-octanol/water partition ratio of THC at neutral ph is in the order of 6000. It is well known that the primary and secondary hydroxylations occur sequentially. 37) The 7-oxo-D 8 -THC that resulted might be further metabolized by CYP enzymes preferentially that used NADPH as an electron donor. Further experiments may be required to clarify the above mechanisms. Certain hepatic microsomal P450 reactions may be enhanced by NADH, which appears to involve electron transfer via cytochrome b 5. 27,38) There was no synergism in the reaction in the presence of either NADH or NADPH. When both NADH and NADPH are used as cofactors, 7-oxo-D 8 -THC forming activity was almost the same as the activity when using NADH alone at lower concentrations of the substrate, whereas it was the same as NADPH alone at higher concentrations (Fig. 4). These results suggest that NADH is utilized as a cofactor in preference to NADPH in the low 7bhydroxy-D 8 -THC concentration system (Fig. 6). In the reconstituted system, the oxidation activity of 7bhydroxy-D 8 -THC in the NADH-dependent system at a low substrate concentration (40 m M) was about 50 % of that in NADPH, although it was not sufficiently reflected in the results of the microsomal fractions (Table 3). CYP3A8 is a major enzyme responsible for testosterone 6b-hydroxylation in monkey liver microsomes. 39) In the reconstituted system, however, the rate of testosterone 6b-hydroxylation supported by NADH was only 10 20% of that by NADPH (data not shown). This result was consistent with the data from monkey liver microsomes (data not shown). It is possible that the NADH-dependent oxidation of 7b-hydroxy-D 8 -THC is also catalyzed by enzyme(s) other than CYP3A8. Yamazaki et al. 34) have reported, however, that NADH-dependent testosterone 6b-hydroxylation and nifedipine oxidation were about one-eighth and one-third, respectively, of the levels obtained in the NADPH-supported system in human liver microsomes. The cofactor requirements of CYP3A8 may be changed by the substrates. These observations support the possibility that NADPH may be replaced by NADH as a cofactor for the microsomal oxidation of 7b-hydroxy-D 8 -THC, especially at a low substrate concentration or high cofactor concentration (Fig. 6). It has been suggested that NADH can transfer electrons directly to the NADPH-P450 reductase and P450, because sufficient activity was detected in the absence of NADH-cytochrome b 5 reductase. The NADH-dependent reaction may be a significant pathway of 7b-hydroxy-D 8 - THC metabolism since NAD(H) is the predominant pyridine nucleotide in liver. 40) Further extensive studies including kinetic experiments are required to clarify the NADH-dependent oxidation mechanisms of 7b-hydroxy-D 8 -THC in monkey liver microsomes. Acknowledgements This work was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, and by the Spe-

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