Progesterone Oxidation by Cytochrome P450 2D Isoforms in the Brain

0013-7227/01/$03.00/0 Printed in U.S.A.

Endocrinology 142(9):3901–3908 Copyright © 2001 by The Endocrine Society

Progesterone Oxidation by Cytochrome P450 2D Isoforms in the Brain TOYOKO HIROI, WATARU KISHIMOTO, TOSHIO CHOW, SUSUMU IMAOKA, TAKASHI IGARASHI, AND YOSHIHIKO FUNAE Department of Chemical Biology, Osaka City University Medical School (T.H., W.K., T.C., S.I., Y.F.), Osaka 545-8585, Japan; and Department of Drug Metabolism and Pharmacokinetics, Kawanishi Pharma Research Institute, Nippon Boehringer Ingelheim Co. (W.K., T.I.), Hyogo 666-0193, Japan The existence of cytochrome P450 2D isoforms in the brain has been demonstrated, although their physiological functions remain to be elucidated. In this study we demonstrated that recombinant rat cytochrome P450 2D1 and 2D4 and human cytochrome P450 2D6 possess progesterone 6␤- and 16␣hydroxylation activities; 2␤- and 21-hydroxylation activities; and 2␤-, 6␤-, 16␣- and 21-hydroxylation activities, respectively. Cytochrome P450 2D4 had the lowest Km value and the highest maximum velocity value toward these activities. Progesterone 2␤- and 21-hydroxylation activities were also detected in rat brain microsomes, and these activities were completely inhibited by anticytochrome P450 2D antibodies. The pres-

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HE CYTOCHROME P450 (CYP) superfamily plays an important role in the oxidation of exogenous and endogenous compounds (1). The CYP11, CYP17, CYP19, and CYP21 families are expressed in steroidogenic tissues such as the adrenal gland, testis, ovary, and placenta. These isoforms are involved in the biosynthetic pathways of endogenous steroid compounds, and their expression levels are, in turn, regulated by some steroid compounds (2– 6). Recently, several steroidogenic enzymes, such as CYP11 and 3␤-hydroxysteroid dehydrogenase (3␤HSD), have been shown to be expressed in the brain (7–11). Furthermore, it has been demonstrated that the levels of some steroid compounds, such as pregnenolone and dehydroepiandrosterone, are higher in the brain than in the peripheral blood (12, 13). After gonadectomy or adrenalectomy, the levels of steroid hormones in peripheral blood decrease, but the levels of certain steroid hormones in the brain remain unchanged (14, 15). These findings strongly suggest that the central nervous system is, in fact, a steroidogenic tissue, and that several steroid compounds are biosynthesized in the central nervous system, independently of their biosynthesis in peripheral steroidogenic tissues. Steroid compounds that are biosynthesized in the central nervous system and have neural effects are referred to as neurosteroids. CYP2D isoforms have been identified in several mammalian species. Only one isoform (CYP2D6) in humans and six isoforms (CYP2D1, CYP2D2, CYP2D3, CYP2D4, CYP2D5, and CYP2D18) in rats have been identified (16 –19). Among Abbreviations: Alfaxalone, 3␣-Hydroxy-5␣-pregnan-11,20-dione; CYP, cytochrome P450; epiallopregnanolone, 3␣-hydroxy-5␣-pregnan-20one; 3␤HSD, 3␤-hydroxysteroid dehydrogenase; LC/MS, liquid chromatography/mass spectrometry; rCYP, recombinant CYP.

ence of endogenous 2␤- and 21-hydroxyprogesterones in rat brain tissues was also demonstrated. The mRNAs of cytochrome P450 2D4, CYP11A, and 3␤-hydroxysteroid dehydrogenase were detected in the rat brain, suggesting that progesterone was generated from cholesterol by CYP11A and 3␤-hydroxysteroid dehydrogenase and then underwent hydroxylation to hydroxyprogesterones by cytochrome P450 2D4 in rat brain. Collectively, our findings support the idea that cytochrome P450 2D may be involved in the regulation (metabolism and/or synthesis) of endogenous neuroactive steroids, such as progesterone and its derivatives, in brain tissues. (Endocrinology 142: 3901–3908, 2001)

these six isoforms, CYP2D5 and CYP2D18 have high similarity in their amino acid sequences to CYP2D1 and CYP2D4, respectively (17, 19, 20). Human and rat CYP2D isoforms are known to contribute to the metabolism of a large number of clinically relevant drugs, including antidepressants, antipsychotics, analgesics, neuroleptics, and serotonin receptor blockers in the liver. In addition, human and rat CYP2D isoforms have been reported to be expressed in several tissues, such as the brain and placenta (21–28). We demonstrated previously that rat CYP2D4/18 mRNA was expressed in brain and gonadal tissues such as ovary and testis (29). Some studies have suggested that steroid hormones regulate the expression of CYP2D4 and CYP2D6 (30 –32). However, the physiological functions of CYP2D isoforms in these steroidogenic tissues have not been clarified. Because these isoforms exist in several steroidogenic tissues and are regulated by steroid hormones, similar to other steroidogenic enzymes, it is possible that CYP2D isoforms may be involved in the synthesis and/or metabolism of steroid compounds in steroidogenic tissues. In this study we focused our efforts on CYP2D isoforms in the brain and investigated the potential roles of human and rat CYP2D isoforms in the brain in oxidation of neural steroid compounds, namely neurosteroids. Materials and Methods Chemicals and animals 2␣-, 2␤- 6␣-, and 6␤-Hydroxyprogesterones were purchased from Steraloids, Inc. (Newport, RI). Progesterone; 11␣-, 11␤-, 16␣-, 17␣-, 18-, 19-, 20␣-, 20␤-, and 21-hydroxyprogesteones; pregnenolone; pregnanolone; 3␣-hydroxy-5␣-pregnan-11,20-dione (alfaxalone); 3␣hydroxy-5␣-pregnan-20-one (epiallopregnanolone); and 17␤-estradiol were purchased from Sigma (St. Louis, MO). Testosterone was pur-

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Endocrinology, September 2001, 142(9):3901–3908

chased from Research Biochemicals International (Natick, MA). Bufuralol and 1⬘-hydroxybufuralol were purchased from Daiichi Pharmaceutical Co. Ltd. (Tokyo, Japan). NADPH was obtained from the Oriental Yeast Co., Ltd. (Tokyo, Japan). Other reagents and organic solvents were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Male Sprague-Dawley rats (7 wk old) were obtained from Nippon Crea (Kyoto, Japan). After decapitation, brains were removed from these rats and stored at ⫺80 C until used. Animal treatments were performed under the standard methods of humane animal care. The protocol for this study was approved by the committee on the animal care and use of Osaka City University Medical School.

Expression of recombinant CYP2D isoforms and the preparation of tissue microsomes Rat CYP2D1 and CYP2D3 cDNAs were isolated from a rat liver cDNA library. Rat CYP2D2 cDNA was amplified from a rat liver cDNA library by PCR. Rat CYP2D4 cDNA was amplified from rat brain total RNA by RT-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 (33, 34), and the microsomal fraction was prepared from yeast cells and used as recombinant enzymes. The total CYP contents of recombinant CYP2D1, CYP2D2, CYP2D3, CYP2D4, and CYP2D6 were 4.88, 0.25, 1.08, 4.91, and 2.16 nmol/ml, respectively. These recombinant CYP2D isoforms had bufuralol 1⬘-hydroxylation activity, a typical activity for CYP2D isoforms (16, 33). Rat brain microsomes were prepared as described previously, with some minor modification (35). Briefly, rat brains were homogenized with a Potter Teflon homogenizer in 4 vol 0.05 m Tris-hydrochloride buffer, pH 7.5, containing 1 mm EDTA, 1 mm dithiothreitol, 0.15 m KCl, and 0.1 mm phenylmethylsulfonylfluoride. The homogenates were centrifuged, and the microsomal pellets were washed with the homogenizing buffer. The microsomal pellet was then suspended in 0.1 m sodium phosphate buffer, pH 7.4, containing 1 mm EDTA, 1 mm dithiothreitol, and 20% (vol/vol) glycerol to yield a concentration of 30 –50 mg protein/ml and was stored at ⫺80 C.

Progesterone hydroxylation activity Progesterone (0.5–50 nmol) was incubated at 37 C for 30 or 3 min with rat brain microsomes (0.5 mg) or recombinant CYP2D isoforms (2–5 pmol) and NADPH (0.25 mmol) in a final volume of 0.5 ml 0.1 m potassium phosphate buffer, pH 7.4. The reaction was initiated by adding NADPH and was terminated by the addition of 1 ml ethyl acetate. After extraction with ethyl acetate, the organic phase was collected and evaporated under vacuum on a rotary evaporator. The resulting residue was dissolved in 50% methanol and injected onto a HPLC apparatus with an ODS-80Ts column (2.0 ⫻ 150 mm; Tosoh Corp., Tokyo, Japan). The column temperature was maintained at 40 C. The mobile phase was eluted at a flow rate of 0.3 ml/min as follows. The initial eluent profile was 50% methanol, and then the methanol concentration was linearly increased to 65% over 20 min. Progesterone and its metabolites were monitored at 240 nm using a spectrometer. Thirteen derivatives of hydroxyprogesterones (2␣-, 2␤-, 6␣-, 6␤-, 11␣-, 11␤-, 16␣-, 17␣-, 18-, 19-, 20␣-, 20␤-, and 21-hydroxyprogesteones) and progesterone were detected at different retention times under these HPLC conditions. We verified each progesterone metabolite from its retention time compared with those of authentic chemicals.

Identification of progesterone metabolites Progesterone and progesterone metabolites formed by CYP2D were identified by HPLC (HPLC/UV) and liquid chromatography/mass spectrometry (LC/MS) using an LCQ Instrument (Finnigan Co., San Jose, CA). Quantification of progesterone hydroxylation activity was calculated based on the peak area of authentic standards. Simultaneously, the column outlet was connected to a fused silica capillary, which transferred the entire eluent into the ion source, to determine the mass number of progesterone metabolites. Atmospheric pressure chemical ionization was performed in the positive ion mode with nitrogen as the nebulizer (60 arb) at 450 C. The response was optimal with a spray voltage setting of 4.55 kV, which produced a spray current of 5.4 ␮A for this specific mobile phase. The heated capillary voltage was set at 5.4 V,

Hiroi et al. • Oxidation of Progesterone by CYP2D4 in the Brain

and the temperature at 180 C. To determine a mass number of progesterone metabolites, the instrument was operated at unit resolution in the total ion-monitoring mode, monitoring the transition of the protonated molecular ion m/z from 150.0 to 400.0. For high sensitivity detection, the column eluent was monitored at the selected ion monitoring mode with the protonated molecular ion m/z 331.2. The LCQ instrument was interfaced to a computer workstation running Finnigan Mat LCQ Navigator software. Rat brain microsomal progesterone hydroxylation activity was quantified from the peak area of linear calibration plots obtained by LC/MS.

Hydroxyprogesterone in rat brains Endogenous progesterone and hydroxyprogesterones were extracted from rat brains. Rat brains were homogenized in 0.1 m potassium phosphate buffer, pH 7.4. A 2-fold volume of methanol was added, the mixture was centrifuged at 10,000 ⫻ g for 40 min, and the supernatant was collected and evaporated to half the original volume under nitrogen gas at 40 C. Next, ethyl acetate was added and mixed. After centrifugation at 3,000 rpm for 10 min, the organic phase was transferred into a clean tube. The residues evaporated by nitrogen gas at 40 C were recovered by methanol. An aliquot of the residues was analyzed by LC/MS to identify and quantify the endogenous progesterone and hydroxyprogesterones in rat brains.

RNA isolation, synthesis of cDNA, and PCRs Total RNA was extracted from frozen whole brain homogenates using the total RNA isolation reagent (Nippon Gene, Toyama, Japan). The resulting RNA was dissolved in diethylpyrocarbonate-treated water. Total RNA concentration and purity were determined spectrophotometrically at 260 nm (Beckman DU-650, Beckman Coulter, Inc., Fullerton, CA). After deoxyribonuclease treatment, total RNA was reverse transcribed using an RNA PCR kit (Takara Shuzo Co., Ltd., Kyoto, Japan) according to the manufacturer’s instructions. For conversion of total RNA to cDNA, a 20-␮l reaction mixture was prepared containing 5 U reverse transcriptase avian myoblastosis virus (AMV, Takara Shuzo Co., Ltd., Kyoto, Japan), 1 ⫻ RT-PCR buffer (10 mm Tris-HCl, pH 8.3, and 50 mm KCl), 1.5 mm MgCl2, 1 mm deoxy-NTPs, 2.5 ␮m random 9-mer primer, 20 U ribonuclease inhibitor, and 2 ␮g total RNA. The reaction was carried out at 55 C for 60 min. RT was terminated by heating the reaction mixture to 99 C for 5 min, followed by rapid chilling on ice. RT reaction mixtures, including cDNA products, were stored at ⫺20 C until used. A single cDNA produced from total RNA was amplified by PCR with primers for CYP2D4, CYP11A, and 3␤HSD. Specific oligonucleotide primer pairs against these isoforms were synthesized. Sense and antisense primer sequences against CYP2D4 were 5⬘-GACCAGTCGGGCTTTGGACCAC-3⬘ and 5⬘-CGAAGGCCTTCTTTCCAGAG-3⬘ (nucleotide positions 3094 –3115 and 4179 – 4198 in the rat CYP2D4 gene sequence), respectively (18). Sense and antisense primer sequences against CYP11A were 5⬘-CAACATCACAGAGATGCTGGCAGG -3⬘ and 5⬘-CTCAGGCATCAG GATGAGGTTGAA-3⬘ (nucleotide positions 965–988 and 1500 –1523 in the rat CYP11A gene sequence), respectively (36). Sense and antisense primer sequences against 3␤HSD were 5⬘-TGCCTGGTGACAGGAGCAGGA-3⬘ and 5⬘-AGCACTGCCTTCTCGGCCAT-3⬘ (nucleotide positions 187–207 and 652– 671 in the rat 3␤HSD gene sequence), respectively (37). The expected product sizes of CYP2D4, CYP11A, and 3␤HSD are 420, 559, and 485 bp, respectively. For PCR amplification of cDNA, the PCR was allowed to proceed for 35 cycles in 50 ␮l reaction mixtures containing 1 ⫻ polymerase reaction buffer (10 mm Tris-HCl, pH 8.3, and 50 mm KCl), 1.5 mm MgCl2, 0.2 mm deoxy-NTPs, 1 U Gold Taq DNA polymerase (PE Applied Biosystems, Foster City, CA), 0.1 ␮g cDNA, and a 200-nm concentration of the specific primers. Aliquots (10 ␮l) of amplified cDNA products were separated by electrophoresis using 2.0% agarose gels. Gels were stained with ethidium bromide and visualized under UV light. Images of the gels were recorded with a Color Image Freezer (Atto Corp., Tokyo, Japan).

Bufuralol hydroxylation activity We measured bufuralol 1⬘-hydroxylation as an index of the catalytic activities of rat and human CYP2D isoforms by the methods described


Endocrinology, September 2001, 142(9):3901–3908 3903

previously (33). To study immunoinhibition, we preincubated rat brain microsomes with various amounts of anti-CYP2D 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. Anti-CYP2D antibodies were raised in a female Japanese White rabbit using rat CYP2D1 purified from rat hepatic microsomes as the immunogen (38). Anti-CYP2D antibodies recognized all rat and human CYP2D isoforms (33) and inhibited CYP2D catalytic activity.

Result Inhibitory effects of neurosteroids on CYP2D catalytic activities

To search for candidate substrates for CYP2D, we examined the effects of various steroid compounds on CYP2D catalytic activities. Eight neurosteroids, namely, progesterone, testosterone, pregnanolone, pregnenolone, 17␤-estradiol, 17␣-hydroxyprogesterone, epiallopregnanolone, and alfaxalone, were evaluated for their inhibitory effects on bufuralol 1⬘-hydroxylation activity, which is considered a typical activity for rat or human CYP2D isoforms. Recombinant CYP2D6 (rCYP2D6) expressed in yeast cells was used as a CYP2D isoform enzyme. Assessment of competitive inhibition was performed by the comparison of Dixon plots. Among eight neurosteroids, progesterone, testosterone, pregnanolone, pregnenolone, 17␤-estradiol, and 17␣hydroxyprogesterone competitively inhibited rCYP2D6 activity, whereas epiallopregnanolone and alfaxalone noncompetitively inhibited the activity (Table 1). These results suggest that competitive inhibitors such as progesterone, testosterone, pregnanolone, pregnenolone, 17␤-estradiol, and 17␣-hydroxyprogesterone might be substrates for CYP2D isoforms. In support of this view, testosterone has been reported to be hydroxylated by CYP2D6 (39). More importantly, the Ki value of progesterone for bufuralol 1⬘TABLE 1. Inhibition manners and Ki values of various compounds toward CYP2D6 activity Compounds

Inhibition manner

Ki value (␮M)

Progesterone Testosterone Pregnanolone Pregnenolone 17␤-Estradiol 17␣-Hydroxyprogesterone Epiallopregnanolone Alfaxalone

Competitive Competitive Competitive Competitive Competitive Competitive Noncompetitive Noncompetitive

33.2 62.6 134.1 163.1 306.7 391.3

The Ki values were determined by using bufuralol as a specific CYP2D substrate. The experiments were performed in duplicate. The assignation of the inhibition manner was performed by means of a comparison of Dixon plots obtained in the presence of different concentrations of inhibitors.

hydroxylation activity was 33.2 ␮m and, in fact, was the lowest of all neurosteroids tested, indicating that progesterone has the highest affinity for rCYP2D6. Progesterone oxidation by rCYP2D isoforms

We next investigated whether rat and human rCYP2D isoforms are capable of oxidizing progesterone. As shown in Table 2, progesterone was hydroxylated to four metabolites, 2␤-, 6␤-, 16␣-, and 21-hydroxyprogesterones, by rCYP2D6. Typical HPLC profiles of progesterone and its metabolites are shown in Fig. 1. These progesterone metabolites were identified by LC/MS and confirmed by comparison to authentic compounds. Progesterone 2␤-, 6␤-, 16␣-, and 21hydroxylation activities by CYP2D6 were 0.03 ⫾ 0.01, 0.29 ⫾ 0.16, 0.08 ⫾ 0.04, and 0.18 ⫾ 0.09 nmol product/min䡠nmol P450, respectively. The progesterone 6␤-hydroxylation activity was the highest among these hydroxylation activities of rCYP2D6. In rats, rCYP2D1 and rCYP2D4 had progesterone 6␤- and 16␣-hydroxylation activities and 2␤- and 21hydroxylation activities, respectively (Table 2 and Fig. 1). rCYP2D1, rCYP2D4, and rCYP2D6 also had hydroxylation activities toward testosterone (data not shown). In contrast, rCYP2D2 and rCYP2D3 had no demonstrable hydroxylation activity toward either progesterone or testosterone (data not shown). Progesterone 2␤- and 21-hydroxylation activities catalyzed by rCYP2D4 were 1.59 ⫾ 0.49 and 1.06 ⫾ 0.17 nmol product/min䡠nmol P450, respectively, which were significantly higher than the activities catalyzed by rCYP2D1 and rCYP2D6 (see Table 2). The kinetic parameters of rCYP2D1-, rCYP2D4-, and rCYP2D6-mediated progesterone hydroxylation activities were calculated (Table 3). The Km values of rCYP2D6 for 6␤-, 16␣-, and 21-hydroxylation activities were 28.7 ⫾ 7.0, 31.0 ⫾ 18.8, and 22.1 ⫾ 4.3 ␮m, respectively. The Km value of rCYP2D6 for 2␤-hydroxylation activity could not be calculated, as the activity level was too low. The Km values of rCYP2D1 for 6␤- and 16␣-hydroxylation activities were 50.1 ⫾ 9.3 and 25.5 ⫾ 10.7 ␮m, respectively. The Km values of rCYP2D4 for 2␤- and 21-hydroxylation activities were 17.7 ⫾ 6.6 and 16.6 ⫾ 2.9 ␮m, respectively. It is important to note that the Km values of rCYP2D4 were lower than those of rCYP2D1 and rCYP2D6. The maximum velocity values of rCYP2D4 for 2␤- and 21-hydroxylation activities were 2.52 ⫾ 0.47 and 1.62 ⫾ 0.12 nmol/min䡠nmol P450, respectively, which were higher than those of rCYP2D1 and rCYP2D6. The rCYP3A9 enzyme has been previously reported to display progesterone 6␤-, 16␣-, and 21-hydroxylation activities (40). However, the Km values of rCYP3A9 for progesterone 6␤and 21-hydroxylation activities were 800 and 130 ␮m, re-

TABLE 2. Progesterone hydroxylation activities of CYP2D isoforms Metabolites

Human: 2D6

2␤-OH 6␤-OH 16␣-OH 21-OH

0.03 ⫾ 0.01 0.29 ⫾ 0.16 0.08 ⫾ 0.04 0.18 ⫾ 0.09

Rat 2D1

2D2

2D3

2D4

ND 0.16 ⫾ 0.08 0.04 ⫾ 0.02 ND

ND ND ND ND

ND ND ND ND

1.59 ⫾ 0.49 ND ND 1.06 ⫾ 0.17

Substrate, progesterone (25 ␮M). Activities are expressed as nanomoles per min/nmol P450. Values are expressed as the mean ⫾ experiments performed in triplicate. ND, Not detected.

SD

of

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FIG. 1. HPLC profiles of progesterone oxidation by CYP2D4. Progesterone was incubated at 37 C for 3 min with recombinant CYP2D4 (5 pmol) and NADPH (0.25 mmol) in a final volume of 0.5 ml 0.1 M potassium phosphate buffer, pH 7.4. Other conditions and procedures for the assay are described in Materials and Methods.

TABLE 3. Kinetic parameters of CYP2D isoforms toward progesterone hydroxylation activities CYP2D6

2␤-OH 6␤-OH 16␣-OH 21-OH

CYP2D1

Km

Vmax

NC 28.7 ⫾ 7.0 31.0 ⫾ 18.8 22.1 ⫾ 4.3

NC 0.54 ⫾ 0.25 0.15 ⫾ 0.04 0.31 ⫾ 0.12

Km

CYP2D4 Vmax

50.1 ⫾ 9.3 25.5 ⫾ 10.7

ND ND

0.51 ⫾ 0.30 0.07 ⫾ 0.03

Km

Vmax

17.7 ⫾ 6.6

2.52 ⫾ 0.47

16.6 ⫾ 2.9

ND ND

1.62 ⫾ 0.12

The experiments were performed in triplicate. Km values and Vmax values are expressed as micromoler concentrations and nanomoles per min/nmol P450, respectively. Each value is expressed as the mean ⫾ SD. NC, Not calculated; ND, not detected.

FIG. 2. mRNA expression of CYP2D4, CYP11A, and 3␤HSD in rat brains. The expression of CYP2D4, CYP11A, and 3␤HSD in rat brains was examined by RT-PCR. PCR was carried out for 35 cycles. Amplified cDNA products were separated by electrophoresis using 2.0% agarose gels. Lane 1, DNA molecular markers (1353, 1078, 872, and 603 bp); lane 2, CYP2D4; lane 3, CYP11A; lane 4, 3␤HSD.

spectively, and these Km values were markedly higher than those of CYP2D isoforms. The levels of steroid hormones in blood circulation are very low (14); however, their levels in the steroidogenic tissues are thought to be relatively high. In fact, the Km value of steroidogenic CYP isoforms such as CYP7 for steroid hormones was reported to be about 13 ␮m (41). The Km values of CYP2D isoforms for these progesterone hydroxylations are probably sufficient for in vivo reaction. Steroidogenic enzymes and CYP2D4 in rat brains

Progesterone is formed from cholesterol via pregnenolone by CYP11A and 3␤HSD in steroidogenic tissues. CYP11A

FIG. 3. Inhibition of bufuralol 1⬘-hydroxylation activities on rat brain microsomes by anti-CYP2D antibodies. Rat brain microsomes were preincubated with various amounts of anti-CYP2D antibodies (F) or control antibodies (E) 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 (0.5 mmol).

catalyzes pregnenolone formation from cholesterol, and 3␤HSD catalyzes progesterone formation from pregnenolone. Using RT-PCR, we confirmed that the mRNAs of CYP2D4, CYP11A, and 3␤HSD enzymes were expressed in the rat brain (Fig. 2). For both CYP2D4 and 3␤HSD, a single DNA fragment was amplified. These DNA fragment sizes were consistent with the expected product sizes of CYP2D4 and 3␤HSD, respectively. In the case of CYP11A, two DNA


fragments were amplified. The size of the upper one of these two fragments was consistent with the expected product size of CYP11A. These results indicate that the mRNAs of CYP2D4, CYP11A, and 3␤HSD are expressed in rat brains. CYP2D4 activity in rat brains

We and others have shown that CYP2D4 is expressed in rat brains (26, 27, 29); however, very little information is available on the existence of CYP2D proteins. To verify the existence of CYP2D proteins in rat brains, we determined bufuralol 1⬘-hydroxylation activity using rat brain microsomes and then performed inhibition studies of bufuralol 1⬘-hydroxylation activity using anti-CYP2D antibodies. Bufuralol 1⬘-hydroxylation activity on rat brain microsomes was 1.78 ⫾ 0.21 pmol product/min䡠mg protein. This activity was inhibited almost completely by the addition of antiCYP2D antibodies (Fig. 3). These results confirmed that CYP2D proteins with enzymatic activities are indeed expressed in rat brains.

FIG. 4. Progesterone hydroxylation activities on rat brain microsomes. Progesterone (50 nmol) was incubated at 37 C for 30 min with rat brain microsomes (0.5 mg) and NADPH (0.25 mmol) in a final volume of 0.5 ml 0.1 M potassium phosphate buffer, pH 7.4. The reaction was initiated by adding NADPH and terminated with 1 ml ethyl acetate. Hydroxyprogesterones were detected and identified by LC/MS.

FIG. 5. Inhibition studies of progesterone hydroxylation activities on rat brain microsomes by anti-CYP2D antibodies. Rat brain microsomes were preincubated with various amounts of anti-CYP2D antibodies (F) or control antibodies (E) at room temperature for 15 min. After preincubation, progesterone and 0.1 M potassium phosphate buffer, pH 7.4, were added to the mixture on ice. The reaction was initiated by adding NADPH (0.5 mmol).


Progesterone hydroxylation by CYP2D isoforms in brain tissues

We further measured the progesterone hydroxylation activities using rat brain microsomes. 2␤- and 21-Hydroxyprogesterones were detected in the presence of NADPH in the brain microsomes (Fig. 4); however, other hydroxymetabolites were not detected. These progesterone 2␤- and 21hydroxylation activities were 0.064 and 0.058 pmol product/ min䡠mg protein, respectively. To clarify the contribution of CYP2D isoforms to these activities, we performed inhibition studies using anti-CYP2D antibodies. Anti-CYP2D antibodies completely inhibited both activities in rat brain microsomes (Fig. 5). Taken together with the data shown in Table 2 demonstrating the ability of CYP2D4 to generate both 2␤and 21-hydroxyprogesterones, these results support the view that progesterone 2␤- and 21-hydroxylation activities in the rat brain are probably mediated by CYP2D4. Hydroxyprogesterone in rat brains

We also examined whether 21- and 2␤-hydroxyprogesterones in addition to progesterone exist endogenously in rat brains. The levels of progesterone are usually measured by immunochemical methods such as ELISA using antiprogesterone antibodies; however, antiprogesterone antibodies could not selectively differentiate progesterone from hydroxyprogesterones (data not shown). Recently, Liere et al. (42) measured trace amounts of neurosteroids in brain tissue by gas chromatography-mass spectrometry. However, it should be noted that the presence of hydroxyprogesterones was not determined in that study. As shown in Table 4, we identified and quantitated progesterone and hydroxyprogesterone in the brain tissues using HPLC/UV and LC/MS. Progesterone and its derivatives were extracted from male rat brain tissues. From the chromatogram obtained by selected ion monitoring mode at protonated molecular ion m/z 331.2, two hydroxyprogesterones were found in rat brain. These hydroxyprogesterones were identified as 21- and 2␤hydroxylprogesterones by comparing them with authentic compounds. The recoveries of these hydroxyprogesterones and progesterone were about 33% and 56%, respectively, and the concentrations of 21- and 2␤-hydroxyprogesterones in rat brains were 2.99 and 1.20 ng/g brain tissue, respectively

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(Table 4). The progesterone concentration in rat brains was 0.32 ng/g brain tissues. Thus, the levels of hydroxyprogesterones were higher than those of progesterone in rat brain tissues. Discussion

CYP2D4 is one of the rat CYP2D isoforms and is known to be expressed in rat brain (26, 27, 29). CYP2D6 is a human isoform belonging to the CYP2D subfamily and is also expressed in human brain tissues (23, 24). However, the function of rat CYP2D4 or, for that matter, human CYP2D6 in the brain has not been clarified until now. In this study we demonstrated that the rat isoforms CYP2D1 and CYP2D4 as well as the human isoform CYP2D6 possess progesterone hydroxylation activities. Our observation of the presence of 2␤- and 21-hydroxylation activities in the rat microsomes that can be effectively inhibited by anti-CYP2D antibodies indicates that these activities are imparted by CYP2D isoforms. This is further strengthened by our finding that the TABLE 4. Progesterone and hydroxyprogesterone in the rat brain Concentration (ng/g brain tissue)

Progesterone 21-OH progesterone 2␤-OH progesterone

0.32 2.99 1.20

Endogenous progesterone and hydroxyprogesterones were extracted from rat brains. LC/MS was used to identify and quantify the endogenous progesterone and hydroxyprogesterones in rat brains.


recombinant CYP2D4 indeed was capable of displaying significant 2␤- and 21-hydroxylation activities in our assay system. Taken together with the presence of endogenous 2␤hydroxyprogesterone and 21-hydroxyprogesterone in the rat brain, these results make a compelling argument that the CYP2D4 in the rat brain is probably involved in the conversion of endogenous progesterone to 2␤-hydroxyprogesterone and 21-hydroxyprogesterone. The presence of CYP2D5 and CYP2D18 in the rat brain has been demonstrated (19, 43), and these isoforms might also contribute to the hydroxylation of progesterone in the rat brain. Several steroid compounds are generated from cholesterol via progesterone, which is a precursor of several steroid compounds. Progesterone itself is one of the female steroid hormones secreted from the placenta and corpus luteum. Moreover, progesterone exists in the brain and is considered to have various functions in the nervous system as a neurosteroid (14, 44 – 46). Progesterone not only influences growth and differentiation, but also increases the expression of myelin-specific proteins in oligodendrocyte (44) and enhances ␥-aminobutyric acid-induced chloride current (45). Although, the presence of progesterone in rat brains (⬃2.2 ng/g brain tissue) has been described previously (14), until now the presence of endogenous hydroxyprogesterones has not been documented. In the previous studies the levels of progesterone measured using antiprogesterone antibodies probably included these hydroxyprogesterones, because antiprogesterone antibodies are generally unable to differen-

FIG. 6. Synthetic pathways of progesterone and its hydroxy derivatives.


tiate progesterone from hydroxyprogesterones. In fact, the total levels of progesterone and hydroxyprogesterones detected in this study were comparable to the levels of progesterone reported previously (14). Recently, several steroid hormones have been shown to display nongenomic effects via receptors and/or channels on the plasma membranes in addition to genomic effects via intracellular receptors (47, 48). The nongenomic effects are caused by parent steroid compounds and/or their metabolites generated locally in target tissues and have a more rapid onset. In the case of progesterone, the parent compound and its metabolites, such as 5␣-pregnane-3,20-dione have several nongenomic effects on neural tissues (47). In this study we not only demonstrated the ability of CYP2D4 isoform to generate 2␤-hydroxyprogesterone and 21-hydroxyprogesterone from progesterone, but we also documented the presence of these derivatives in the rat brain endogenously. If 2␤-hydroxyprogesterone and 21-hydroxyprogesterone are novel active forms of progesterone, similar to 5␣-pregnane-3,20-dione and 3␣-hydroxy5␣-pregnan-20-one, then CYP2D4 may be one of the steroidogenic enzymes. The expression levels of steroidogenic isoforms such as CYP7, CYP11, CYP17, CYP19, and CYP21, are regulated by steroid hormones (2–7). Interestingly, the mRNA levels of CYP2D isoforms in the rat brain have been shown to be modulated by progesterone and testosterone (32). In humans, CYP2D metabolic activity is increased during pregnancy, although CYP3A activity remains unchanged (30, 31). Thus, it is quite possible that the expression of CYP2D4 in rat brain tissues and CYP2D6 in human brain tissues may also be regulated by steroid hormones. On the other hand, if 2␤-hydroxyprogesterone and 21-hydroxyprogesterone are inactive forms of progesterone, then CYP2D4 may contribute to the inactivation of progesterone, thereby affecting the progesterone-mediated effects. In either event, it is likely that CYP2D4 may function in the brain as an important enzyme involved in the regulation of neuroactive steroids. Similarly, considering the ability of CYP2D6 to convert progesterone to its hydroxylated derivatives, CYP2D6 in human brain is also likely to be involved in the regulation of neuroactive steroids. Obviously, further work is needed to validate this prediction. Progesterone is generated from cholesterol via pregnenolone by CYP11A and 3␤HSD. CYP11A catalyzes pregnenolone formation from cholesterol, and 3␤HSD catalyzes progesterone formation from pregnenolone. Evidence from the present study suggests that progesterone can be further hydroxylated to hydroxyprogesterone in the brain by CYP2D isoforms (Fig. 6). In conclusion, this study demonstrates that CYP2D4 promotes the conversion of endogenous progesterone to its hydroxy derivatives in the rat brain, thus providing a novel function for CYP2D4 in the nervous system. Acknowledgments We thank Mr. Kazuhiro Yoshimura and Ms. Atsuko Tominaga for their excellent technical assistance. Received February 1, 2001. Accepted May 8, 2001. Address all correspondence and requests for reprints to: Toyoko Hiroi, Ph.D., Department of Chemical Biology, Osaka City University


Medical School, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan. E-mail: [email protected]. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science, and Culture of Japan.

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