Possible role of cytochrome P450 in inactivation of testosterone in immortalized hippocampal neurons

Brain Research 762 Ž1997. 47–55

Research report

Possible role of cytochrome P450 in inactivation of testosterone in immortalized hippocampal neurons Christina Thuerl a

a, )

, Uwe Otten b , Rolf Knoth a , Ralf Peter Meyer a , Benedikt Volk

a

Department of Neuropathology, UniÕersity of Freiburg, Neurocenter, Breisacher Strasse 64, 79106 Freiburg, Germany b Department of Physiology, UniÕersity of Basle, Vesalgasse 1, 4051 Basel, Switzerland Accepted 11 February 1997

Abstract The hippocampus as part of the limbic system is sensitive to gonadal hormones. The time-dependent expression of steroid receptors and the testosterone converting enzyme aromatase ŽCYP19. is well studied. In contrast, little is known about other cytochrome P450 enzymes in hippocampus which inactivate the gonadal hormones. For investigation of the total cytochrome P450 content and the expression of testosterone degrading CYP2B10 we used embryonic ŽE18. in comparison to postnatal ŽP21. immortalized hippocampal neurons. These embryonic neurons were demonstrated to react to hormones according a ‘critical period’ of sexual differentiation: testosterone treatment Ž1 mM to 5 mM in the culture medium. resulted in a decrease of b-tubulin, as showed by immunocytochemistry and Western blotting. Measurements with reduced CO-difference spectrum elucidated that the P450 concentration in the embryonic neurons Ž10.2 pmolrmg protein; S.D. "1.9. was twice as high as in the postnatal ones Ž5.2 pmolrmg protein; S.D. "1.0.. Correspondingly, a high value of the mitochondrial subfraction of approx. 141 pmol P450rmg protein was found in the embryonic neurons relative to the mitochondrial value of 37.7 pmol P450rmg protein in the postnatal neurons. Our results suggest a differential expression of cytochrome P450 during development. CYP2B10 was proved by electron microscopy and hormone degrading activity. q 1997 Elsevier Science B.V. Keywords: Tubulin; Androstenedione; Differentiation; Mouse; Cell line; Cytoskeleton

1. Introduction Circulating testosterone profoundly and permanently influences the development of the sexually undifferentiated brain during late fetal and early postnatal life w37x. Most, but not all regions of the brain, which are under the influence of sex hormones during this ‘critical period’ of sexual differentiation, are involved in the control of reproductive functions. As part of the limbic system the hippocampus is involved in cognition and emotion and, therefore, is also sensitive to hormones of the thyroid, adrenals and gonads Žfor review w35,36x.. Steroid receptors and the P450 enzyme aromatase, which converts testosterone to estradiol, are transiently elevated during the ‘critical period’ of sexual differentiation in the hippocampus w30,39x. Abbreviations: BSA, bovine serum albumin; DAB, diaminobenzidine; E18, embryonic day 18; GAP-43, growth-associated protein-43; PBS, phosphate-buffered saline; P21, postnatal day 21; SD, standard deviation ) Corresponding author. Fax: q49 Ž761. 270-5050. 0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 2 5 9 - X

Furthermore, other P450 enzymes of the gene subfamily CYP2B are expressed in the murine hippocampus w46x, but little is known about their possible function in neural testosterone degradation. Hormone anabolism and catabolism seem to be simultaneously existent in hippocampus but their probable divergent role in neural differentiation has not been subject of research so far. The developing organism is confronted with many environmental factors. A lot of them are substrates for members of the CYP2B subfamily. The understanding of specific neural P450 isoforms involved both in metabolism of differentiating hormones and of xenobiotics is crucial. Research of competitive or inductive action of xenobiotics on hormone metabolism during a sensitive ‘critical period’ could shed light on disorders of the neural differentiation. In this study, we have investigated whether immortalized embryonic hippocampal neurons derived from the perinatal ‘critical period’ of sexual differentiation ŽE18. w3,31x showed a response to testosterone. b-Tubulin expression has been investigated by immunohistochemistry

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and Western blotting analysis. In comparison, we have analyzed immortalized postnatal hippocampal neurons derived from P21 mice. Afterwards, total cytochrome P450 was quantified in these hippocampal cell lines. CYP2B10 is studied as a member of the CYP2B subfamily metabolizing xenobiotics and endogenous substrates such as testosterone in liver w17,38x. It is the murine counterpart of rat CYP2B1 due to the high similarity Ž93% at the amino acid level., the same inducibility by phenobarbital and functional similarities in testosterone metabolism w38x. Both are mainly involved in the O-dealkylation of 7-pentoxyre-

sorufin. Immunoelectron microscopy has been carried out to detect testosterone metabolizing cytochrome CYP2B10 in the immortalized murine hippocampal neurons. The goal of an enzymatic test was to observe testosterone degrading activity of neural CYP2B10. The unique feature of this study is the possibility of simultaneously investigating the morphological effects of testosterone and the enzymatic system metabolizing this hormone in cultured hippocampal neurons. They represent an enzymatic status quo in E18 and P21 respectively, and provide the large quantity of neurons necessary for the

Fig. 1. DAB staining of b-tubulin in immortalized hippocampal neurons. In contrast to earlier stages of development, the postnatal ŽP21. hippocampal neurons Ža. show a weaker immunopositivity than the embryonic hippocampal neurons derived from the perinatal period ŽE18. Žc.. No difference in immunoreactivity can be observed between untreated Ža. and testosterone-treated Ž1 mM for five days. postnatal neurons Žb.. In testosterone-treated embryonic neurons Žd. the intensity of the b-tubulin immunoreaction appears weaker than in untreated embryonic neurons Žc.. Bar s 90 mm.

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Fig. 2. Western blot analysis showing a concentration dependent decrease in b-tubulin protein of testosterone treated embryonic hippocampal neurons. Comparison of b-tubulin bands obtained after electrophoresis and immunoblotting of 40 mg of homogenate from embryonic immortalized hippocampal neurons ŽE18. Ža.. The immunoreactivity of b-tubulin was lower in the postnatal neurons than in the embryonic neurons and did not change after testosterone treatment Žb.. The level of GAP-43 was not altered by testosterone neither in the embryonic nor in the postnatal neurons Žc.. No T, untreated neurons; 2T, neurons treated with 2 mM testosterone for three days; 5T, neurons treated with 5 mM testosterone for three days.

assessment of cytochrome P450 and the detection of androgen metabolism, whilst exhibiting typical electrophysical and cytoskeletal features of pyramidal neurons w31x.

2. Materials and methods 2.1. Cell culture of immortalized hippocampal neurons Immortalized embryonic and postnatal hippocampal neurons were used. The hippocampal cells were immortalized by fusion to murine neuroblastoma cells. The resulting clonal cell lines resemble hippocampal neurons rather than the neuroblastoma parent, for example, in their electrophysiological behavior and release of neurotransmitters w31x. The secondary cell culture of immortalized hippocampal neurons Žcell line HN9.1 of embryonic day 18 and HN25.1 of postnatal day 21 of mouse. were grown as a monolayer in culture flasks containing Dulbecco’s modified Eagle’s Medium ŽDMEM, Life Technologies. supplemented with 5% bovine calf serum ŽLife Technologies.,

and 1% antibiotic-antimycotic solution ŽLife Technologies.. The cultures were fed every third day with fresh medium and were maintained at 378C in a humidified atmosphere of 5% CO 2 . For treatment with androgens testosterone ŽSigma. was dissolved in ethanol ŽHPLC grade; Merck.. The cells were cultured in DMEM supplemented with 1% bovine calf serum. Investigation with HPLC confirmed that the supplemented medium contained the added steroid and that the solution is stable. The influence of ethanol on cultured hippocampal neurons has been reported to occur at much higher concentrations w15x. Preliminary experiments showed that testosterone treatment elicited no changes in cell number or cell viability in embryonic and adult hippocampal neurons at a concentration range of 1 mM to 5 mM during seven days of cultivation. 2.2. Immunocytochemical analysis of b-tubulin Cells for immuno-staining with monoclonal anti-btubulin Žmouse, clone no. TUB 2.1, Sigma. were plated on Thermanox coverslips Ždiameter 13 mm, Nunc. in 24well-plates and cultured with 1 mM testosterone for five days. The hippocampal neurons were fixed with ethanol Žy208C. for 10 min. After washing three times with phosphate-buffered saline ŽPBS. the non-specific binding sites were blocked with horse serum for 30 minutes at room temperature. The primary antibody was applied at a dilution of 1 : 1000 in 1% bovine serum albumin ŽBSA. in PBS and incubated overnight at 48C. The subsequent procedure was carried out according to the protocol of the

Table 1 P450 content in microsomal and mitochondrial subfractions

Fig. 3. Comparison of the P450 content in homogenates of embryonic and postnatal immortalized hippocampal neurons. Embryonic neurons ŽE18. derived from the hormone-sensitive ‘critical period’ of the perinatal age express cytochrome P450 twice as high as the postnatal neurons ŽP21.. Results are expressed as pmolrmg protein.

Cell line

Subfraction

P450"S.D. Žpmolrmg protein.

Embryonic

Microsomes Mitochondria Microsomes Mitochondria

15.9" 1.5 141"10.1 4.5" 0.9 37.7" 6.4

Postnatal

Ratio mitochondriar microsomes 8.7 8.4

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C. Thuerl et al.r Brain Research 762 (1997) 47–55

C. Thuerl et al.r Brain Research 762 (1997) 47–55

Vectastain ABC Kit ŽVector Laboratories.. Negative control incubations were carried out omitting the primary antibody. The biotinylated secondary antibody was stained by 0.05% DAB in 0.1 M Tris-HCl ŽpH 7.2. containing 0.01% H 2 O 2 . The cultures were mounted in glycerol gelatine. Examination was carried out with a Leica DMRX photomicroscope. 2.3. Electrophoresis and Western blot analyses for the detection of b-tubulin and GAP-43 in hippocampal neurons Western blot analyses were performed on cell homogenates of immortalized hippocampal neurons after treatment with 2 mM and 5 mM testosterone for three days and of untreated neurons. Samples were prepared in Laemmli sample buffer w28x, 40 mg of protein were loaded onto each lane and separated by 10% SDS-PAGE. Western blots were performed using a mouse monoclonal antibody against b-tubulin Žmouse, clone no. TUB 2.1, Sigma. and a monoclonal antibody against GAP-43 ŽSigma.. Transfer to Immobilon P membrane ŽMillipore. was achieved by semidry blotting w45x. Proteins were visualized with Ponceau S stain and incubated overnight with the primary antibody at a dilution of 1 : 2000 at q48C. The antibody reaction was visualized by incubation of the membrane for 1 h in alkaline phosphate-conjugated secondary antibody ŽSigma., followed by blue tetrazolium and bromo-4chloro-3-indolyl phosphate ŽSigma. for 10 min w7x. 2.4. Measurement of the cytochrome P450 content in homogenates and cellular subfractions The cells were washed, harvested, sedimented at 1000 = g at room temperature and homogenized by sonification ŽBranson sonifier B15 ‘cell disrupter’.. The cellular subfractions were obtained by differential centrifugation w12x of at least 80 mg protein and resuspended in homogenization buffer Ž10 mM Tris-HCl pH 7.4, 320 mM sucrose Dq, 1 mM EDTA, 0.5 mM dithiothreitol.. Cellular subfractions were checked by electron microscopy. Protein concentration was determined according to the method of Lowry w33x with BSA as a standard. The samples were suspended in solubilization buffer Ž0.1 M potassium phosphate buffer pH 7.4, containing 1 mM EDTA, 20% Žvrv. glycerol, 0.5% Žwrv. sodium cholate, and 0.4% Žwrv. Triton N-101.. Cytochrome P450 measurements were carried out on a Lambda 2 spectralphotometer ŽPerkin Elmer., using a second derivative

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spectrophotometric method which allows accurate determination of cytochrome P450 content in suspensions w13x. 2.5. Immunoelectron microscopy for the subcellular localization of CYP2B10 Cells were fixed with 0.2% Žvrv. glutaraldehyde ŽMerck., 4% Žwrv. paraformaldehyde ŽMerck., 0.2% Žvrv. picric acid in sodium phosphate buffer ŽpH 7.4. for 15 min. The staining of CYP2B10 with a 2B1,2-specific polyclonal antibody Ždilution 1 : 1000. was carried out according to the protocol used for the staining of b-tubulin. The CYP2B10 has been identified by amino acid sequencing of the protein purified from mouse brain by anti CYP2B1,2 immuno-controlled separation strategy Žunpublished results, R. Knoth.. Thereafter, cells were washed in PBS and postfixed in 1% OsO4 . Dehydration was performed in a gradient series of ethanol. The cells were detached from the dishes by briefly melting the upper plastic material with propylene oxide according to Kuhn w27x. The cells were sedimented by short centrifugation, washed in propylene oxide and then immersed in a mixture of Eponrpropylene oxide and finally equilibrated in Eppendorf tubes into pure Epon resin. Polymerization was achieved at 708C over night. Cones with the pellet at the tip were sectioned for electron microscopy in a Philips CM 100 electron microscope. 2.6. Enzymatic test as proof for testosterone-metabolizing cytochrome P450 An enzymatic test was applied to show the catalytic activity of neural CYP2B10 in testosterone degradation. 250 nmol testosterone ŽSigma., 10 mmol glucose-6-phosphate ŽSigma. and 1 Unit glucose-6-phosphate-dehydrogenase ŽSigma. as an NADPH-regenerating system were added to 60 pmol cytochrome P450 in testing buffer Ž3 mM MgCl 2 Žwrv., 50 mM KH 2 PO4 Žwrv., pH 7.4. according to a method of Jayyosi w20x. After pre-incubation for 2 min at 378C, the reaction was started by adding 1 mmol NADPH to an end volume of 1 ml. Control incubations were carried out by omitting either cytochrome P450 or substrate. Inhibition of testosterone metabolism was performed with 10 nmol finasteride which was generously provided by Merck, Sharp and Dohme. After 14 h the reaction was stopped with dichloromethane. The metabolites were extracted with 6 ml of dichloromethane. The sample was vigorously vortexed for 1 min before it was centrifuged. The organic

Fig. 4. Immunoelectron microscopy reveals the subcellular distribution of CYP2B10 in postnatal ŽA. and embryonic immortalized hippocampal neurons ŽB.. Immunorelated DABrosmium black labelling of the smooth endoplasmatic reticulum ŽsER., rough endoplasmatic reticulum ŽrER., the envelope of a mitochondrium ŽM. and of the plasma membrane ŽPM.. Note that the adjoining neurons show different grades of immunoreactivity. N, nucleus; G, Golgi apparatus. Bar s 1 mm.

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fraction was decanted off and evaporated to dryness with N 2 . The residue was redissolved in 300 ml of methanolrwaterracetonitrile Ž39 : 60 : 1. and sonicated for 2 min. Each sample was filtered and a volume of 100 ml was injected for HPLC analysis on a Waters model 6000 A solvent delivery system. Metabolites were separated by reversed-phase HPLC using a concave gradient from 90% solvent A Žmethanol-water-acetonitrile, 39 : 60 : 1. to 85% solvent B Žmethanol-water-acetonitrile, 80 : 18 : 2. operated over 22 min at 1.5 mlrmin. Absorbency was monitored at 254 nm with a full scale of 0.01 and metabolites were identified by comparing their peaks with those of authentic standards w42x. 2.7. Statistical analysis Assays of cytochrome P450 measurements in cell homogenates were carried out on at least four different cell preparations. Data were expressed as mean value " S.D.

3. Results 3.1. ResponsiÕeness of embryonic hippocampal neurons to testosterone

the postnatal hippocampal neurons ŽTable 1.. The P450 concentration of the mitochondria was approximately eight to nine times higher than that of the microsomes. This relationship was found in the embryonic hippocampal cell line as well as in the postnatal one ŽTable 1.. The immense prominence of the mitochondrial value in the embryonic neurons relative to that of the postnatal neurons was striking. 3.3. Ultrastructural localization of CYP2B10 in hippocampal neurons By using a CYP2B1,2-specific polyclonal antibody the subcellular localization of the immunorelated CYP2B10 isoform which degrades steroid hormones w38x was determined. Fig. 4 demonstrates the ultrastructural localization of immunoreactive sites by means of the electron dense DABrosmium label. CYP2B10 was localized predominantly on the membranes of the endomembrane system. Parts of the plasma membrane, the mitochondrial envelope, the rough and the smooth endoplasmatic reticulum showed immunopositivity of cytochrome P450. It is noticeable that the cells showed different grades of immunopositivity. 3.4. Neural cytochrome P450 metabolizes testosterone

The embryonic neurons ŽE18. were sensitive according to the perinatal ‘critical period’ of sexual differentiation they were derived from. They, but not the postnatal neurons, reacted to testosterone with a decrease of b-tubulin ŽFig. 1.. The decrease of b-tubulin immunoreactivity could be confirmed by Western blot analysis. This effect depended on the concentration of testosterone ŽFig. 2a.. Testosterone treatment did not elicit a change in the btubulin level in the postnatal hippocampal neurons ŽFig. 1a,b and Fig. 2b.. The immunoreactivity of GAP-43 as a reference protein expressed in vitro w14x and in vivo w9x in hippocampal neurons was neither altered in the embryonic nor in the postnatal neurons after testosterone treatment ŽFig. 2c..

An enzymatic assay was performed to show that androstenedione formation is catalyzed by the murine CYP2B10. NADPH-dependent androstenedione formation was observed ŽFig. 5.. NADPH is essential for P450 catalyzed reactions. Omission of NADPH diminished the oxidation of testosterone to androstenedione to 20% ŽFig. 5.. The formation of androstenedione without external NADPH is explained by endogenous NADPH in the homogenate.

3.2. Embryonic hippocampal neurons reÕeal a high cytochrome P450 content relatiÕe to postnatal neurons The cytochrome P450 content of the homogenate derived from the embryonic cell line measured by reduced CO-difference spectrum line is twice as high as in the postnatal cell line ŽFig. 3.. This is the first evidence for an elevation of total cytochrome P450 content in embryonic neurons compared to neurons derived from a later stage of development. Correspondingly, the P450 content in the cellular subfractions of the embryonic neurons was remarkably higher than in the subfractions of the postnatal neurons. The evaluation of the P450 content in the cellular subfractions elucidated that cytochrome P450 is predominantly localized mitochondrial in both the embryonic and

Fig. 5. Effect of NADPH and finasteride on testosterone conversion. Finasteride is used as a competitive substrate for cytochrome P450. 60 pmol P450 of immortalized postnatal hippocampal neurons were incubated as described in Section 2.

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Finasteride and testosterone were used as competitive substrates in an enzymatic test to determine the neural catalytic activity of CYP2B10 in testosterone catabolism. Both testosterone and finasteride are metabolized by the CYP2B subfamily w19x which catalyzes the oxidation of testosterone to androstenedione in rat w42,43x. Finasteride diminished the conversion of testosterone to androstenedione to 20% ŽFig. 5.. This effect also depended upon the NADPH content of the test solution.

4. Discussion For the investigation of the hormone sensitivity of hippocampal neurons and the hormone degrading system of cytochrome P450, we used two cell lines derived from different stages of development. This model of a secondary neuronal culture is unique in its ability to provide the enormous amount of neurons needed for the assessment of the cytochrome P450 system, since the concentration of cytochrome P450 in the central nervous system is about a hundred times lower than in the liver w49x. Until now, definitive proof of CYP2B10, which degrades gonadal hormones, in hippocampal neurons has not been forthcoming primarily because of difficulties in obtaining sufficiently large quantities of the presumed products. Our results indicate that immortalized hippocampal neurons express CYP2B10 and show that this P450 isoform is capable of degrading testosterone to androstenedione. Androstenedione formation is the first step in the inactivation of testosterone, leading to excretion of 17-keto-steroids in the urine. This oxidation at C 17 is catalyzed by CYP2B1 in rat liver and lung w42,43x. However, so far little is known about neural androstenedione formation. The embryonic neurons, but not the postnatal neurons, responded according to the hormone-sensitive perinatal age they were derived from. Androgen treatment of hippocampal neurons resulted in a specific decrease of the immunoreactivity of b-tubulin as shown by the immunohistochemical reaction ŽFig. 1. and the Western blot analysis ŽFig. 2a. in contrast to GAP-43 whose immunoreactivity did not change ŽFig. 2c.. The main form of cerebral b-tubulin, the isoform b-IItubulin w4x, is predominantly expressed in neurons w16x and decreases markedly during differentiation w8,16,32x. This correlates with our results, where the postnatal neurons express less b-tubulin than the embryonic neurons ŽFig. 1 and Fig. 2. according to the later stage of development they are derived from. This observation suggests an induction of the developmental program for b-tubulin expression by testosterone. Our results give the first evidence for a hormonal regulation of this cytoskeletal protein in early stages of development. Conversely, an induction of b-tubulin by testosterone in a sexual dimorphic spinal nucleus in adult animals w34x and in axotomized hamster facialis motoneurons during regen-

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eration w22x was observed. Jones et al. showed that during regeneration of lesioned facialis-motoneurons, the administration of testosterone resulted in a more rapid induction of b-ŽII.-tubulin mRNA changes in axotomized neurons relative to injury alone w22x. The mechanisms leading to the opposite pattern of b-tubulin expression by testosterone treatment in embryonic hippocampal neurons and lesioned facialis motoneurons are unclear. However, it is known that various tubulin genes are expressed in an opposite pattern in central versus peripheral neurons w21x. The down regulation of b-tubulin could occur at the post-transcriptional level. Since the various mechanisms that regulate the b-tubulin mRNA levels during development and regeneration are not yet known, further studies on the important molecular details of hormonal gene regulation of b-tubulin are needed. The molecular mechanisms by which testosterone elicited a response in the embryonic, but not in the postnatal immortalized hippocampal neurons are also unknown. The effect of testosterone on b-tubulin in the embryonic neurons could be mediated directly by other active metabolites or via neurotrophic factors. An aromatization of testosterone to estradiol in the embryonic neurons might be considered, since the enzyme aromatase w30x as well as estrogen-receptors w39x are transiently elevated during the early ‘critical period’ in the hippocampus. The possibility that androgen treatment effects are mediated indirectly by induction of neurotrophic factor receptors must also be considered. Our study revealed a high prominence of cytochrome P450 in the androgen-sensitive embryonic neurons relative to the postnatal neurons ŽFig. 3.. But further research is necessary to compare the expression of CYP2B10 and other isoforms during development. That the neuronal P450 hemoprotein is mainly localized in the mitochondrial fraction, both in embryonic and postnatal hippocampal neurons, is in agreement with the situation reported in total rodent brain w48x and human brain w12x. It is likely that mitochondrial P450 isoforms which metabolize steroids and xenobiotics w6,20,47x contribute to the high amount of mitochondrial P450 and not steroidogenic mitochondrial isoforms, such as P450 scc, because they have never been detected in neurons. They have mainly been reported in oligodendrocytes and to a lesser extent in astrocytes type 1 Žfor review w1x.. Electron microscopy revealed the localization of CYP2B10, that metabolizes testosterone in mouse liver w38x, in hippocampal neurons, ŽFig. 4.. In accordance with both microsomal and mitochondrial localization of CYP2B1,2 described in rat brain w6,25x, we observed a predominant localization of the murine isoform CYP2B10 in the endomembrane system and in the mitochondrial envelope ŽFig. 4.. Until now, a CYP2B10 immunopositivity of the plasma membrane has not been described for cultured neurons ŽFig. 4.. However, the localization of cytochrome CYP2B

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in the plasma membrane has been reported in cultured rat hepatocytes. Robin et al. recently showed that CYP2B followed a microtubule-dependent vesicular route from the endoplasmatic reticulum to the plasma membrane w40x. The localization of CYP2B10 in the plasma membrane might prevent lipophilic substrates, like hormones or xenobiotics, from entering the cell by inactivating the substrates to more hydrophilic molecules. Enzymatic investigations ŽFig. 5. proved that the degradation of testosterone to androstenedione is catalyzed by CYP2B10. Finasteride which is metabolized by the CYP2B subfamily w19x diminished the oxidation of testosterone to androstenedione by competition. It indicates the catalyzis of the androstenedione formation by CYP2B10 which is localized in the endomembrane system, the external mitochondrial membrane and the plasma membrane. As cytochrome P450 interacts with nitric oxide w26x, which affects neurotransmission in the hippocampus w5,41x, differential expression of cytochrome P450 enzymes might regulate this transmitter as well. Other P450 isoforms control endogenous biochemical pathways of neurosteroids w44x, which modulate neurotransmission and morphology in the hippocampus w2,10,23x. The role of ‘peroxisome proliferator-activated receptors’ expressed in the hippocampus w24x and regulating P450 genes w11x must also be clarified in the future. Further experiments by mRNA analyses are necessary to clarify the possible differential expression of CYP2B10 during development. The degradation and inactivation of testosterone might regulate the hormonal effect on hippocampal neurons as was suggested for glucocorticoids in the brain w29x. The elevated metabolism of testosterone in rat liver during the early ‘critical period’ w18x might have its counterpart in brain. The inhibition of the testosterone conversion to androstenedione could clarify the importance of CYP2B10 for neural differentiation.

Acknowledgements We wish to thank Renate Wirtz for her technical assistance and MSD for generously providing finasteride. This work was supported by the Deutsche Forschungsgemeinschaft ŽSFB 505, Project A5; B.V...

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