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Progesterone Oxidation by Cytochrome P450 2D Isoforms in the Brain

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

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: toyoko-loy{at}med.osaka-cu.ac.jp

	Abstract

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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 6ß- and 16- hydroxylation activities; 2ß- and 21-hydroxylation activities; and 2ß-, 6ß-, 16- and 21-hydroxylation activities, respectively. Cytochrome P450 2D4 had the lowest K_m value and the highest maximum velocity value toward these activities. Progesterone 2ß- and 21-hydroxylation activities were also detected in rat brain microsomes, and these activities were completely inhibited by anticytochrome P450 2D antibodies. The presence of endogenous 2ß- and 21-hydroxyprogesterones in rat brain tissues was also demonstrated. The mRNAs of cytochrome P450 2D4, CYP11A, and 3ß-hydroxysteroid dehydrogenase were detected in the rat brain, suggesting that progesterone was generated from cholesterol by CYP11A and 3ß-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.

	Introduction

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THE 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, 3, 4, 5, 6). Recently, several steroidogenic enzymes, such as CYP11 and 3ß-hydroxysteroid dehydrogenase (3ßHSD), have been shown to be expressed in the brain (7, 8, 9, 10, 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, 17, 18, 19). Among 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, 22, 23, 24, 25, 26, 27, 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, 31, 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

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Chemicals and animals
2-, 2ß- 6-, and 6ß-Hydroxyprogesterones were purchased from Steraloids, Inc. (Newport, RI). Progesterone; 11-, 11ß-, 16-, 17-, 18-, 19-, 20-, 20ß-, and 21-hydroxyprogesteones; pregnenolone; pregnanolone; 3-hydroxy-5-pregnan-11,20-dione (alfaxalone); 3- hydroxy-5-pregnan-20-one (epiallopregnanolone); and 17ß-estradiol were purchased from Sigma (St. Louis, MO). Testosterone was purchased 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 x 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-, 2ß-, 6-, 6ß-, 11-, 11ß-, 16-, 17-, 18-, 19-, 20-, 20ß-, 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 µA for this specific mobile phase. The heated capillary voltage was set at 5.4 V, 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 x 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-µl reaction mixture was prepared containing 5 U reverse transcriptase avian myoblastosis virus (AMV, Takara Shuzo Co., Ltd., Kyoto, Japan), 1 x RT-PCR buffer (10 mM Tris-HCl, pH 8.3, and 50 mM KCl), 1.5 mM MgCl₂, 1 mM deoxy-NTPs, 2.5 µM random 9-mer primer, 20 U ribonuclease inhibitor, and 2 µg 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 3ßHSD. 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 3ßHSD were 5'-TGCCTGGTGACAGGAGCAGGA-3' and 5'-AGCACTGCCTTCTCGGCCAT-3' (nucleotide positions 187–207 and 652–671 in the rat 3ßHSD gene sequence), respectively (37). The expected product sizes of CYP2D4, CYP11A, and 3ßHSD are 420, 559, and 485 bp, respectively. For PCR amplification of cDNA, the PCR was allowed to proceed for 35 cycles in 50 µl reaction mixtures containing 1 x 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 µg cDNA, and a 200-nM concentration of the specific primers. Aliquots (10 µl) 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 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

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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, 17ß-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, 17ß-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, 17ß-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 K_i value of progesterone for bufuralol 1'-hydroxylation activity was 33.2 µM and, in fact, was the lowest of all neurosteroids tested, indicating that progesterone has the highest affinity for rCYP2D6.

View larger version (22K): [in this window] [in a new window]   Figure 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.

View larger version (72K): [in this window] [in a new window]   Figure 2. mRNA expression of CYP2D4, CYP11A, and 3ßHSD in rat brains. The expression of CYP2D4, CYP11A, and 3ßHSD 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, 3ßHSD.

View larger version (17K): [in this window] [in a new window]   Figure 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 (•) 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 (0.5 mmol).

View larger version (15K): [in this window] [in a new window]   Figure 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.

View larger version (17K): [in this window] [in a new window]   Figure 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 (•) or control antibodies () 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).

	Discussion

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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, 45, 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 differentiate 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 2ß-hydroxyprogesterone and 21-hydroxyprogesterone from progesterone, but we also documented the presence of these derivatives in the rat brain endogenously. If 2ß-hydroxyprogesterone and 21-hydroxyprogesterone are novel active forms of progesterone, similar to 5-pregnane-3,20-dione and 3-hydroxy-5-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, 3, 4, 5, 6, 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 2ß-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 3ßHSD. CYP11A catalyzes pregnenolone formation from cholesterol, and 3ßHSD 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).

View larger version (16K): [in this window] [in a new window]   Figure 6. Synthetic pathways of progesterone and its hydroxy derivatives.

	Acknowledgments

 
We thank Mr. Kazuhiro Yoshimura and Ms. Atsuko Tominaga for their excellent technical assistance.

	Footnotes

 
This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science, and Culture of Japan.

Abbreviations: Alfaxalone, 3-Hydroxy-5-pregnan-11,20-dione; CYP, cytochrome P450; epiallopregnanolone, 3-hydroxy-5-pregnan-20-one; 3ßHSD, 3ß-hydroxysteroid dehydrogenase; LC/MS, liquid chromatography/mass spectrometry; rCYP, recombinant CYP.

Received February 1, 2001.

Accepted for publication May 8, 2001.

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