Introduction

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Cytochrome P450 (P450¹) 2D isoforms have been identified in several mammalian species and are involved in the monooxygenation of various chemicals including antidepressants (e.g. desipramine), -blockers (e.g. propranolol), antiarrhythmics (e.g. sparteine), and others (e.g. dextromethorphan and methadone) in the liver (Eichelbaum and Gross, 1990; Gonzalez, 1996). In humans, only one isoform, CYP2D6, is expressed in various tissues including the liver, kidney, brain, placenta, breast, and lung (Niznik et al., 1990; Romkes-Sparks et al., 1994; Carcillo et al., 1996; Hakkola et al., 1996; Guidice et al., 1997; Huang et al., 1997). On the other hand, in rats, six isoforms (CYP2D1, CYP2D2, CYP2D3, CYP2D4, CYP2D5, and CYP2D18) have been identified by genomic analysis (Kawashima and Strobel, 1995; Gonzalez, 1996; Nelson et al., 1996). Among these six, CYP2D5 and CYP2D18 have over 95% similarity in amino acid sequence to CYP2D1 and CYP2D4, respectively (Gonzalez et al., 1987; Matsunaga et al., 1989; Kawashima and Strobel, 1995). Similar to human CYP2D6, the six rat CYP2D isoforms are expressed in various tissues such as liver, kidney, and brain (Komori, 1993; Hellmold et al., 1995; Kawashima and Strobel, 1995; Masubuchi et al., 1996; Zhang et al., 1996; Hiroi et al., 1998). Interestingly, the mRNA of each isoform showed a specific tissue distribution in our previous study (Hiroi et al., 1998). CYP2D2 and CYP2D3 mRNAs are mainly expressed in the liver, kidney, and small intestinal mucosa, which are constantly exposed to xenobiotics such as drugs, food components, and environmental contamination. In contrast, CYP2D1/5 mRNA is expressed systemically in various tissues. CYP2D4/18 mRNA is expressed in brain, adrenal gland, ovary, testis, and gonecystis, in addition to liver, kidney, and small intestinal mucosa. CYP2D4 has also been identified in rat breast (Hellmold et al., 1995). The specific tissue distributions of the rat CYP2D isoforms suggest that each isoform has specific catalytic properties and plays specific roles in various tissues. However, very little information on the similarities and/or differences in catalytic properties among these six isoforms is available at present. It is difficult to purify each CYP2D isoform from rat tissues, because of their highly homologous amino acid sequences (Gonzalez et al., 1987; Matsunaga et al., 1989; Matsunaga et al., 1990; Kawashima and Strobel, 1995). To solve this problem, we isolated full-length cDNAs of rat CYP2D1, CYP2D2, CYP2D3, and CYP2D4 and human CYP2D6. Each enzymatic protein was expressed in yeast cells, and each recombinant enzyme was used as an isolated enzyme (Imaoka et al., 1996; Wan et al., 1997).

In the present study, we focused our effort on the catalytic similarities and differences between rat CYP2D isoforms and investigated the catalytic activities of five recombinant isoforms (human CYP2D6 and rat CYP2D1, CYP2D2, CYP2D3, and CYP2D4) toward bufuralol, debrisoquine, and propranolol, typical substrates for CYP2D. We found specific activity for rat CYP2D2 and CYP2D4 and the activities to be useful as a probe for these isoforms.

	Materials and Methods

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Chemicals. Debrisoquine, 4-hydroxydebrisoquine, bufuralol and 1'-hydroxybufuralol were purchased from Daiichi Pure Chemicals Co., Ltd. (Tokyo, Japan). 4-Hydroxypropranolol, 5-hydroxypropranolol, 7-hydroxypropranolol, and N-desisopropylpropranolol were kindly provided by Dr. Narimatsu (Okayama University, Okayama, Japan). NADPH was obtained from the Oriental Yeast Co., Ltd. (Tokyo, Japan). Propranolol, other reagents and organic solvents were obtained from Wako Pure Chemical Industries (Osaka, Japan).

Expression of Human and Rat CYP2D Isoforms in Yeast Cells. Rat CYP2D1 and CYP2D3 cDNAs were isolated from a rat liver cDNA library. Rat CYP2D2 cDNA was amplified from the rat liver cDNA library by PCR. Rat CYP2D4 cDNA was amplified from rat brain total RNA by reverse transcription-PCR. Human CYP2D6 cDNA was amplified from a human liver cDNA library by PCR. CYP2D1, CYP2D2, CYP2D3, CYP2D4, and CYP2D6 enzymes were expressed in Saccharomyces cerevisiae, as reported previously (Imaoka et al., 1996; Wan et al., 1997), and the microsomal fraction was prepared from yeast cells and used as recombinant enzyme. The total P450 contents of recombinant CYP2D1, CYP2D2, CYP2D3, CYP2D4, and CYP2D6 were 4.88, 0.25, 1.08, 4.91, and 2.16 nmol/ml, respectively.

Rat and Human Hepatic Microsomes and Purified P450 Isoforms. Male Sprague-Dawley rats weighing 200 to 250 g were obtained from Nippon Clea (Tokyo, Japan). Rat hepatic microsomes were prepared as reported previously (Funae and Imaoka, 1985). Human hepatic microsomes were obtained from the International Institute for the Advancement of Medicine (Exton, PA). Eight isoforms (CYP1A1, CYP1A2, CYP2A2, CYP2B1, CYP2C11, CYP2D1, CYP2E1, and CYP3A2) of rat P450s were purified from rat hepatic microsomes as described previously (Funae and Imaoka, 1985; Funae and Imaoka, 1987; Funae et al., 1988; Ohishi et al., 1993). Antibodies against CYP2D isoforms were raised in a female Japanese white rabbit using rat CYP2D1 purified from rat hepatic microsomes as the immunogen (Ohishi et al., 1993). Anti-CYP2D antibodies recognized all rat and human CYP2D isoforms and inhibited the catalytic activity of CYP2D (Wan et al., 1997) but not that of other CYP2D isoforms.

Assays of Catalytic Activities. Bufuralol (10 nmol) or debrisoquine (50 nmol) was incubated with recombinant CYP2D isoform (10 pmol) or hepatic microsomes (0.2 mg) and NADPH (0.2 µmol) in a final volume of 0.5 ml of 0.1 M potassium phosphate buffer, pH 7.4, at 37°C for 10 or 15 min. The reaction was started by adding NADPH and stopped with 20 µl of 60% perchloric acid. After centrifugation, the supernatant was injected into a high-performance liquid chromatography (HPLC) system with a TSKgel ODS-120T column (4.6 × 250 mm; TOSOH Corp., Tokyo, Japan). For the bufuralol oxidation assay, the column was isocratically eluted with 1 mM perchloric acid/30% acetonitrile at a flow rate of 1.0 ml/min at 50°C. The detection of metabolites was carried out using a fluorometric detector (FS-8011; TOSOH Corp.) with the excitation (Ex) and emission (Em) wavelength set at 252 and 302 nm, respectively. 1'-Hydroxybufuralol was identified by comparing its retention time with that of the authentic compound. For the debrisoquine oxidation assay, the column was isocratically eluted with 2 mM tetrafluoroacetic acid and acetonitrile (88:22) at a flow rate of 1.0 ml/min at 45°C. The metabolites were monitored at 219 nm (Ex) and 286 nm (Em). Propranolol (50 nmol) was incubated with recombinant CYP2D isoform (10 pmol) and NADPH (0.2 µmol) in a final volume of 0.5 ml of 0.1 M potassium phosphate buffer, pH 7.4, at 37°C for 10 min. The reaction was started by adding NADPH and stopped with 1 ml of 1 N NaOH. The metabolites were extracted with 3 ml of ethyl acetate. The organic phase was evaporated under a vacuum, and the residue was dissolved in the mobile phase for HPLC. Thereafter, 100 µl of the solution was injected into an HPLC system with a TSKgel ODS-120A column (4.6 × 250 mm; TOSOH Corp.). The column was isocratically eluted with methanol, acetic acid, acetonitrile, and H₂O (10:0.9:20:70, v/v) at a flow rate of 1.0 ml/min at 45°C. The metabolites were monitored at 310 nm (Ex) and 380 nm (Em).

Qualitative Analysis by Liquid Chromatography/Mass Spectrometry (LC/MS). 1'-Oxobufuralol (M1-bufuralol), 1'2'-ethenylbufuralol (M2-bufuralol), and 3-hydroxydebrisoquine (M1-debrisoquine) were identified by LC/MS. Mass spectral data on 1'2'-ethenylbufuralol and 1'-oxobufuralol were obtained with an LCQ ion-trap mass spectrometer (Thermo Finnigan, San Jose, CA) operating in the positive ion spray mode, set with a spray needle voltage of 4.5 kV, capillary temperature of 225°C, sheath gas flow of 80 U, and auxiliary gas flow of 10 U. A TSKgel ODS-80Ts column (4.6 × 250 mm; TOSOH Corp.) was used for separation. The column temperature was maintained at 40°C. The mobile phase consisting of 0.1% trifluoroacetic acid and acetonitrile (90:10, v/v) was delivered at a flow rate of 1 ml/min. Mass spectral data for 3-hydroxydebrisoquine were obtained with the same mass spectrometer also operating in the positive ion spray mode, set with a spray needle voltage of 4.5 kV, capillary temperature of 275°C, sheath gas flow of 85 U, and auxiliary gas flow of 15 U.

NMR Analysis of 3-Hydroxydebrisoquine. Debrisoquine (20 µmol) was incubated with recombinant CYP2D1 (5 nmol) and NADPH (13 µmol) in a final volume of 10 ml of 0.1 M potassium phosphate buffer, pH 7.4, at 37°C for 20 min. The reaction was started by adding NADPH and stopped with 200 µl of 60% perchloric acid. After centrifugation, the supernatant was evaporated to dryness and dissolved in 0.5 ml of water. The solution was applied to a preparative HPLC column (21.5 × 300 mm, TOSOH Corp.), and the column temperature was maintained at 40°C. The mobile phase containing 0.1% trifluoroacetic acid and acetonitrile (90:10, v/v) was delivered at a flow rate of 6 ml/min. The eluate containing 3-hydroxydebrisoquine was collected by monitoring at 215 nm and evaporated to dryness. The residue was dissolved in [²H]acetonitrile (CH₃CN) and subjected to ¹H-NMR measurements with a NMR spectrometer (UNITY INOVA 500; Varian Inc., Palo Alto, CA).

Others. Enzyme kinetic parameters (K_m and V_max) were analyzed according to a nonlinear least-square regression based on the Michaelis-Menten equation using the computer software Microcal Origin (version 5.0J, Origin LabCorp, Northampton, MA).

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

Bufuralol Metabolism. Bufuralol is a typical substrate for CYP2D isoforms. The level of 1'-hydroxybufuralol, a major metabolite of bufuralol, is often measured as an index of CYP2D activity and/or levels, and the amount of 1'-hydroxybufuralol formed from bufuralol is known to be small in CYP2D6-deficient metabolizers (Gonzalez, 1996). The HPLC profile of bufuralol metabolism by rat hepatic microsomes is shown in Fig. 1a. Bufuralol was metabolized to three metabolites, namely 1'-hydroxybufuralol, M1-bufuralol, and M2-bufuralol. The structures of M1-bufuralol and M2-bufuralol were deduced by LC/MS/MS/MS (Fig. 2). In the case of M1-bufuralol, the LC/MS analysis indicated an apparent [M + H⁺] at m/z 276. The LC/MS/MS analysis indicated fragments at m/z 220 and at m/z 202. The LC/MS/MS/MS analysis indicated a fragment at m/z 160. Judging from these results and the spectra of bufuralol (data not shown), M1-bufuralol was identified as 1'-oxobufuralol. 1'-Oxobufuralol has been reported as a bufuralol metabolite in humans, eluted just after 1'-hydroxybufuralol on HPLC (Yamazaki et al., 1994). In the case of M2-bufuralol, the LC/MS analysis indicated an apparent [M + H⁺] at m/z 260. The LC/MS/MS analysis indicated a fragment at m/z 242. The LC/MS/MS/MS analysis indicated a fragment at m/z 186. From these results, M2-bufuralol was identified as 1'2'-ethenylbufuralol. Recently 1'2'-ethenylbufuralol has been demonstrated to be a metabolite formed by CYP2D6 (Hanna et al., 2001). However, this is the first report that 1'2'-ethenylbufuralol was formed from bufuralol by rat CYP2D isoforms.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   HPLC profiles of the metabolism of bufuralol and debrisoquine by rat hepatic microsomes. Bufuralol (10 nmol) (a) or debrisoquine (50 nmol) (b) was incubated with rat hepatic microsomes (0.2 mg) and NADPH (0.2 µmol) in a final volume of 0.5 ml of 0.1 M potassium phosphate buffer, pH 7.4, at 37°C for 10 min or 15 min. Metabolites and substrates were isolated by HPLC.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Identification of M1-bufuralol and M2-bufuralol. LC/MS, LC/MS/MS, and LC/MS/MS/MS analyses of M1-bufuralol (a) and M2-bufuralol (b) were carried out as described under Materials and Methods.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Inhibition of bufuralol metabolism by anti-CYP2D antibodies in rat and human hepatic microsomes. Rat (, ) and human (, ) hepatic microsomes were preincubated with various amounts of anti-CYP2D antibodies (, ) or control antibodies (, ) at room temperature for 15 min. After preincubation, bufuralol and 0.1 M potassium phosphate buffer, pH 7.4 were added to the mixture on ice. The reaction was initiated by adding NADPH (0.2 µmol). Control activity was measured without any antibodies and is shown as 100% on the y-axis.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Catalytic activities of rat and human P450 isoforms. Bufuralol was incubated with recombinant CYP2D isoforms (10 pmol) and NADPH (0.2 µmol) in a final volume of 0.5 ml of 0.1 M potassium phosphate buffer, pH 7.4, at 37°C for 10 min. In reconstitution systems using purified P450 isoforms, bufuralol was incubated with purified P450 isoforms (10 pmol), NADPH-P450 reductase (0.4 units), dilauroylphosphatidylcholine (5 µg), sodium cholate (0.1 mg), and NADPH (0.2 µmol) in a final volume of 0.5 ml of 0.1 M potassium phosphate buffer, pH 7.4, at 37°C for 10 min. Isoforms 1 to 5 are recombinant CYP2D isoforms. Isoforms 6 to 13 are P450 isoforms purified from rat tissues. 1, recombinant CYP2D1; 2, recombinant CYP2D2; 3, recombinant CYP2D3; 4, recombinant CYP2D4; 5, recombinant CYP2D6; 6, purified CYP1A1; 7, purified CYP1A2; 8, purified CYP2A2; 9, purified CYP2B1; 10, purified CYP2C11; 11, purified CYP2D1; 12, purified CYP2E1; 13, purified CYP3A2

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Metabolic pathways of bufuralol.

Debrisoquine Metabolism. Debrisoquine is a classical substrate for CYP2D isoforms and a probe for CYP2D activity as well as bufuralol. The level of 4-hydroxydebrisoquine formed from debrisoquine is used in the judgment of CYP2D6 polymorphism in vivo (Woolhouse et al., 1979). The HPLC profile of debrisoquine metabolism by rat hepatic microsomes is shown in Fig. 1b. Human hepatic microsomes also showed the same profile (data not shown). Debrisoquine was metabolized to 4-hydroxydebrisoquine and M1-debrisoquine. 4-Hydroxydebrisoquine was eluted at a retention time of about 8.5 min and was a major metabolite. M1-Debrisoquine eluted at a retention time of 11 min. The structure of M1-debrisoquine was identified by LC/MS and NMR analysis. The MS spectrum of M1-debrisoquine gave a protonated molecular ion at m/z 192, and collision-induced decomposition led to a fragment at 174 (Fig. 6). The ion at m/z 174, a loss of 18 Da, suggested cleavage of a hydroxy group from the protonated molecular ion [M + H⁺]. Because the mass number of debrisoquine was 175, the ion at m/z 192 was suggested to be a protonated ion of hydroxylated debrisoquine at the 1-, 3-, or 4-position. To identify the hydroxylated position on debrisoquine, a ¹H-NMR spectrum of M1-debrisoquine was measured. The peaks of 1-CH2 (sharp AB quartet), 3-CH-OH (broad singlet) and 4-CH2 (broad AB quartet) were observed at 4.4, 5.6, and 3.1 ppm, respectively (data not shown). Based on these data, M1-debrisoquine was assigned as 3-hydroxydebrisoquine. 3-Hydroxydebrisoquine has been reported to be one of the debrisoquine metabolites produced by CYP2D6 (Eiermann et al., 1998).

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Identification of M1-debrisoquine. LC/MS (top) and LC/MS/MS (bottom) analyses of M1-debrisoquine found in reaction mixtures containing debrisoquine and CYP2D1. LC/MS/MS analysis was carried out by collision-induced decomposition of m/z 192 base peaks as described under Materials and Methods.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Inhibition studies of debrisoquine hydroxylation using rat hepatic microsomes. a, rat hepatic microsomes were preincubated with various amounts of anti-CYP2D antibodies (, ) or control antibodies (, ) at room temperature for 15 min. After preincubation, debrisoquine and 0.1 M potassium phosphate buffer, pH 7.4, were added to the mixture on ice. The reaction was initiated by adding NADPH (0.2 µmol). (, ) and (, ) indicate 4-hydroxylation activity and 3-hydroxylation activity, respectively. Control activity was measured without any antibodies and is shown as 100% on the y-axis. b, debrisoquine was incubated with rat hepatic microsomes (0.2 mg) and NADPH (0.2 µmol) in a final volume of 0.5 ml of 0.1 M potassium phosphate buffer, pH 7.4, at 37°C for 10 min in the presence of quinine. () and () indicate 4-hydroxylation activity and 3-hydroxylation activity, respectively.

View larger version (22K): [in this window] [in a new window]   Fig. 8.   Metabolism of debrisoquine by CYP2D isoforms. Debrisoquine was incubated with recombinant CYP2D isoforms and NADPH (0.2 µmol) in a final volume of 0.5 ml of 0.1 M potassium phosphate buffer, pH 7.4, at 37°C for 15 min. Debrisoquine and its metabolites were detected using HPLC-fluorometry.

View larger version (15K): [in this window] [in a new window]   Fig. 9.   Metabolic pathways of debrisoquine. 2D6*: Eierman et al. (1998) demonstrated this pathway in their study.

Propranolol Metabolism by Rat Hepatic Microsomes. We further investigated the metabolic properties of CYP2D using propranolol (Table 3). Propranolol is used clinically as a -blocker and is one of the substrates of CYP2D (Masubuchi et al., 1993; Masubuchi et al., 1994). Propranolol is known to be metabolized to four main metabolites (4-, 5-, 7-hydroxylpropranolol and N-desisopropylpropranolol) by rat hepatic microsomes (Masubuchi et al., 1993). In humans, propranolol was metabolized to three main metabolites (4-, 5-hydroxylproranolol and N-desisopropylpropranolol) (Masubuchi et al., 1994). In humans and rats, the N-desisopropylation has been reported to be catalyzed by CYP1A and/or CYP2C isoforms, not CYP2D isoforms (Fujita et al., 1993; Yoshimoto et al., 1995). CYP2D6 had high 4- and 5-hydroxylation activities but very low 7-hydroxylation activity. In rats, CYP2D2 and CYP2D4 had higher levels of 4-hydroxylation activity than CYP2D1 and CYP2D3. The 5-hydroxylation of propranolol was catalyzed by several CYP2D isoforms, but the levels of activity catalyzed by rat CYP2D isoforms were very low. The 7-hydroxylation was catalyzed only by CYP2D2 in rats. The 7-hydroxylation activity of rat hepatic microsomes was inhibited by anti-CYP2D antibodies to less than 10% of the control (data not shown). This suggests that the 7-hydroxylation activity was specific to CYP2D2.