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Keywords:

  • CYP7A1;
  • CYP39A1;
  • 17β-hydroxysteroid dehydrogenase;
  • neurosteroids

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Source of tissue
  5. Steroids and chemicals
  6. Buffers
  7. Tissue preparation
  8. Measurement of enzymes activities
  9. Gas chromatography mass spectrometry (GC–MS) analysis
  10. Messenger RNA preparation and analysis
  11. Data analysis
  12. Results
  13. Human brain tissue DHEA metabolism with reduced pyridine nucleotides as coenzymes
  14. Subcellular distribution of 7α-hydroxylase and 17β-HSD activity
  15. GC–MS analysis
  16. Rodent brain tissue DHEA metabolism with NADPH as coenzyme
  17. Kinetic properties of cerebral 7α-hydroxylase and 17β-HSD activity, effects of different pH-values and effects of the cytochrome P450 inhibitor ketoconazole
  18. 7α-Hydroxylase and 17β-HSD activity in CX and SC preparations
  19. RT–PCR analyses of CYP7A1, CYP7B1 and CYP39A1 mRNA expression
  20. Discussion
  21. Acknowledgements
  22. References

Dehydroepiandrosterone and its sulphate are important factors for vitality, development and functions of the CNS. They were found to be subjects to a series of enzyme-mediated conversions within the rodent CNS. In the present study, we were able to demonstrate for the first time that membrane-associated dehydroepiandrosterone 7α-hydroxylase activity occurs within the human brain. The cytochrome P450 enzyme demonstrated a sharp pH optimum between 7.5 and 8.0 and a mean KM value of 5.4 µm, corresponding with the presence of the oxysterol 7α-hydroxylase CYP7B1. Real-time RT–PCR analysis verified high levels of CYP7B1 mRNA expression in the human CNS. The additionally observed conversion of dehydroepiandrosterone via cytosolic 17β-hydroxysteroid dehydrogenase activity could be ascribed to the activity of an enzyme with a broad pH optimum and an undetectably high KM value. Subsequent experiments with cerebral neocortex and subcortical white matter specimens revealed that 7α-hydroxylase activity is significantly higher in the cerebral neocortex than in the subcortical white matter (p < 0.0005), whereas in the subcortical white matter, 17β-hydroxysteroid dehydrogenase activity is significantly higher than in the cerebral neocortex (p < 0.0005). No sex differences were observed. In conclusion, the high levels of CYP7B1 mRNA in brain tissue as well as in a variety of other tissues in combination with the ubiquitous presence of 7α-hydroxylase activity in the human temporal lobe led us to assume a neuroprotective function of the enzyme such as regulation of the immune response or counteracting the deleterious effects of neurotoxic glucocorticoids, rather than a distinct brain specific function such as neurostimulation or neuromodulation.

Abbreviations used
7α-hydroxy-DHEA (7α)

androst-5-ene-3β,7α-diol-17-one

7β-hydroxy-DHEA

androst-5-ene-3β,7β-diol-17-one

17β-HSD

17β-hydroxysteroid dehydrogenase

CX

cerebral neocortex

Cy

cytosolic fraction

DHEA (D)

dehydroepiandrosterone

DHEAS

dehydroepiandrosterone sulfate

Δ5-androstenediol (Δ5)

androst-5-ene-3β,17β-diol

Δ4-androstenedione (Δ4)

androst-4-ene-3,17-dione

GC–MS

gas chromatography mass spectrometry

Me

membrane fraction

Nu

nuclear fraction

PBGD

porphobilinogen deaminase

pmol/(hmgprotein)

picomole per hour and per mg protein

Rf value

retention factor value

RT–PCR

reverse transcription polymerase chain reaction

SC

subcortical white matter

testosterone (T)

androst-4-ene-17β-ol-3-one

TLC

thin layer chromatography

TMSi

trimethylsilyl

WH

whole homogenate

Humans as well as some other primates are unique among mammals as their adrenals secrete large amounts of dehydroepiandrosterone (DHEA) and its corresponding sulfate (DHEAS; Cutler et al. 1978). Circulating levels of DHEA(S) decline with age. The relationship between lower DHEA(S) levels and the incidence of heart disease, cancer, diabetes, obesity, chronic fatigue syndrome, AIDS and Alzheimer's disease is a topic of ongoing scientific discussion (Baulieu 1996; Khorram 1996; Watson et al. 1996; Hinson and Raven 1999; Williams 2000). Both compounds were reported to accumulate in the brain and are important factors for vitality, development and functions of the CNS (Majewska 1995; Baulieu and Robel 1996; Compagnone and Mellon 1998; Herbert 1998; Garcia-Estrada et al. 1999; Li et al. 2001). They were found to be subject to a series of enzyme-mediated conversions within the rodent CNS (Baulieu and Robel 1996).

Steroid hormones are known to act via binding to specific transactivating receptor proteins. To date, no such DHEA(S) receptor has been found and it was consequently concluded that these compounds do not act like other steroid hormones, which directly regulate gene expression (Mohan and Cleary 1992). In fact, it has been suggested that the intracrine metabolism of DHEA(S) is an important factor in mediating their effects (Labrie et al. 1998). Non-genomic effects of DHEA(S) are obviously also responsible for their actions in the CNS (Wolf and Kirschbaum 1999).

With reduced pyridine nucleotides as coenzymes, DHEA is known to be primarily metabolized via 7α-hydroxylase (EC 1.14.13) and 17β-hydroxysteroid dehydrogenase (17β-HSD; EC 1.1.1.62) activity within the rodent brain (Baulieu and Robel 1996). Both reactions require reduced pyridine nucleotides as coenzymes and lead to the formation of androst-5-ene-3β,7α-diol-17-one (7α-hydroxy- DHEA) and androst-5-ene-3β,17β-diol (Δ5-androstenediol), respectively. The biological significance of the 7α-hydroxylation and the 17-ketosteroid reduction of DHEA in brain tissue remains to be elucidated. So far, it could be demonstrated that DHEA as well as Δ5-androstenediol and, in particular, their 7-hydroxylated metabolites possess immunomodulatory and antiglucocorticoid properties (Morfin and Courchay 1994; Padgett and Loria 1994; Hampl et al. 1997; Loria 1997; Lafaye et al. 1999). Increased DHEA and 7α-hydroxy-DHEA serum levels were found in patients with Alzheimer's disease (Attal-Khemis et al. 1998).

To date, the existence of three human 7α-hydroxylase isozymes has been verified (Fig. 1). CYP7A1 catalyses the first and rate-limiting step of the neutral pathway of bile acid synthesis, i.e. cholesterol 7α-hydroxylation (Chiang 1998; Russell 2000; Björkhem and Eggertsen 2001). The suggested alternative acidic pathway of bile acid biosynthesis is initiated by sterol 27-hydroxylase activity. Subsequently, the resulting 27-hydroxycholesterol is 7α-hydroxylated by the catalytic activity of an hepatic oxysterol 7α-hydroxylase. CYP7B1 was first described as a rodent brain tissue DHEA 7α-hydroxylase and was originally isolated from the hippocampus (Rose et al. 1997). Thereafter, it was identified as the hepatic oxysterol 7α-hydroxylase (Schwarz et al. 1997). Another oxysterol 7α-hydroxylase with a preference for 24S-hydroxycholesterol has previously been identified in the liver and was designated CYP39A1 (Li-Hawkins et al. 2000a). Moreover, several androgen 7α-hydroxylases were identified in rodents (Nagata et al. 1987; Waxman et al. 1990; Kurose et al. 1998, 1999). To the authors' best knowledge, the coding sequences of homologous proteins in humans have not been identified to date.

image

Figure 1. DHEA metabolism in brain tissue via 17β-HSD and 7α-hydroxylase activity: the principle reactions catalysed by the known human 7α-hydroxylase isozymes CYP7A1, CYP7B1 and CYP39A1.

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The 17-ketosteroid reduction of DHEA leads to the formation of its 17β-hydroxy-metabolite Δ5-androstenediol (Fig. 1). The interconversion of 17β-hydroxysteroids and 17-ketosteroids is catalysed by a number of human 17β-HSD isozymes differing in terms of tissue distribution, catalytic preferences, substrate specificity and subcellular localization (Peltoketo et al. 1999; Adamski and Jakob 2001). In previous studies we were able to demonstrate the presence of 17β-HSD type 1, 3, 4 and 5 mRNA expression in the human temporal lobe (Stoffel-Wagner et al. 1999; Steckelbroeck et al. 2001). Another report provided strong evidence for the catalytic activity of at least one reductive and one oxidative isozyme in the human temporal lobe using androgens and oestrogens as the substrates (Steckelbroeck et al. 1999).

Systematic studies on the DHEA metabolism in the human CNS are still lacking and 7α-hydroxylase activity in the human brain has not been investigated to date. In the present study, we examined the in vitro metabolism of DHEA in human temporal lobe biopsies with reduced pyridine nucleotides as coenzymes. We determined and characterized the 7α-hydroxylation and 17-ketosteroid reduction of the molecule using an end-product isolation assay. Moreover, we investigated possible sex-, age- and tissue-specific differences in the cerebral neocortex (CX) and the subcortical white matter (SC). Another important aim of the study was to examine the mRNA expression of the three known human 7α-hydroxylase isozymes in brain tissue as well as in a variety of other tissues and cell lines by block-cycler reverse transcription polymerase chain reaction (RT–PCR) analysis. Moreover, the levels of mRNA expression of the three isozymes in these samples were quantified by real-time RT–PCR analysis.

Source of tissue

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Source of tissue
  5. Steroids and chemicals
  6. Buffers
  7. Tissue preparation
  8. Measurement of enzymes activities
  9. Gas chromatography mass spectrometry (GC–MS) analysis
  10. Messenger RNA preparation and analysis
  11. Data analysis
  12. Results
  13. Human brain tissue DHEA metabolism with reduced pyridine nucleotides as coenzymes
  14. Subcellular distribution of 7α-hydroxylase and 17β-HSD activity
  15. GC–MS analysis
  16. Rodent brain tissue DHEA metabolism with NADPH as coenzyme
  17. Kinetic properties of cerebral 7α-hydroxylase and 17β-HSD activity, effects of different pH-values and effects of the cytochrome P450 inhibitor ketoconazole
  18. 7α-Hydroxylase and 17β-HSD activity in CX and SC preparations
  19. RT–PCR analyses of CYP7A1, CYP7B1 and CYP39A1 mRNA expression
  20. Discussion
  21. Acknowledgements
  22. References

Brain tissue was obtained from patients suffering from temporal lobe epilepsy undergoing therapeutic partial temporal lobe resections. Temporal lobe tissue was immediately chilled after surgical excision and adherent blood vessels were removed. The tissue was dissected into CX and SC. The samples were then frozen in liquid nitrogen and stored at − 80°C until further processing. No tissue situated around the presumed epileptic focus was taken for the study. Immunohistochemistry, using an antibody against glial fibrillary acidic protein (astrocyte-marker), revealed fibrillary gliosis in SC and some reactive astrocytes in CX as well as in the subpial zone (not shown). This is a common finding in samples from epileptic patients. Extensive neuronal cell loss was not evident in any specimen. Tissue, which was histologically suspicious for tumour growth or inflammation, was excluded from the study.

The surgical removal of the other macroscopically normal tissues utilized in the study was also clinically indicated in all cases. Prostate tissue was obtained from a 58-year-old patient with bladder cancer undergoing cystectomy and prostatectomy, and adrenal tissue from a 45-year-old patient with kidney cancer undergoing nephrectomy. Heart muscle tissue of a 68-year-old woman and liver tissue of a 59-year-old woman were obtained from biopsies carried out to exclude diseases. The human JEG3 choriocarcinoma and U-87 astrocytoma cell lines were purchased from ATCC (Manassas, VA, USA). A venous blood sample was obtained from a 40-year-old healthy male volunteer.

Animals (C-57BL/6 mice and Wistar rats) were deeply anaesthetized using ether and were killed by decapitation. The brains were removed rapidly and immediately prepared on ice. The tissue samples were frozen in liquid nitrogen and stored at − 80°C until further processing.

The study was approved by the local ethics committee. Informed consent was received from all tissue donors or their guardians.

Steroids and chemicals

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Source of tissue
  5. Steroids and chemicals
  6. Buffers
  7. Tissue preparation
  8. Measurement of enzymes activities
  9. Gas chromatography mass spectrometry (GC–MS) analysis
  10. Messenger RNA preparation and analysis
  11. Data analysis
  12. Results
  13. Human brain tissue DHEA metabolism with reduced pyridine nucleotides as coenzymes
  14. Subcellular distribution of 7α-hydroxylase and 17β-HSD activity
  15. GC–MS analysis
  16. Rodent brain tissue DHEA metabolism with NADPH as coenzyme
  17. Kinetic properties of cerebral 7α-hydroxylase and 17β-HSD activity, effects of different pH-values and effects of the cytochrome P450 inhibitor ketoconazole
  18. 7α-Hydroxylase and 17β-HSD activity in CX and SC preparations
  19. RT–PCR analyses of CYP7A1, CYP7B1 and CYP39A1 mRNA expression
  20. Discussion
  21. Acknowledgements
  22. References

[ 4−14C]DHEA (53.8 mCi/mmol) was obtained from Perkin-Elmer Life Sciences (Zaventem, Belgium) and purified by TLC prior to use. Unlabelled androst-5-ene-3β,7α-diol-17-one (7α-hydroxy-DHEA) was purchased from Steraloids, Inc. (Newport, RI, USA). Ketoconazole was kindly provided by Janssen-Cilag GmbH (Neuss, Germany). All other unlabelled steroids, EDTA, folin and Ciocalteu's phenol reagent, TRIZMATM (a,a,a-tris-(hydroxymethyl)-methylamin), TRIZMATM–HCl, citric acid, and sodium potassium tartrate were purchased from Sigma Chemical Company (Deisenhofen, Germany). Pyridine nucleotides, PCR buffer, the expand long template PCR system (containing a mix of Taq DNA polymerase and a proofreading polymerase), ribonuclease-free deoxyribonuclease I (DNase I), deoxynucleotides and the 50-basepairs (bp) DNA size markers were obtained from Roche Molecular Biochemicals (Mannheim, Germany). Trizol LS reagent and Superscript II pre-amplification system were purchased from Invitrogen GmbH (Karlsruhe, Germany). All specific oligonucleotide primers were synthesized on a 392/4 DNA-synthesizer (Applied Biosystems, Weiterstadt, Germany). The liquid scintillation cocktail, Ultima GoldTM, was obtained from Packard-Instrument, B.V., Chemical Operations (Groningen, the Netherlands). All other chemicals and solvents were purchased from Merck AG (Darmstadt, Germany). Solvents were distilled prior to use.

Tissue preparation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Source of tissue
  5. Steroids and chemicals
  6. Buffers
  7. Tissue preparation
  8. Measurement of enzymes activities
  9. Gas chromatography mass spectrometry (GC–MS) analysis
  10. Messenger RNA preparation and analysis
  11. Data analysis
  12. Results
  13. Human brain tissue DHEA metabolism with reduced pyridine nucleotides as coenzymes
  14. Subcellular distribution of 7α-hydroxylase and 17β-HSD activity
  15. GC–MS analysis
  16. Rodent brain tissue DHEA metabolism with NADPH as coenzyme
  17. Kinetic properties of cerebral 7α-hydroxylase and 17β-HSD activity, effects of different pH-values and effects of the cytochrome P450 inhibitor ketoconazole
  18. 7α-Hydroxylase and 17β-HSD activity in CX and SC preparations
  19. RT–PCR analyses of CYP7A1, CYP7B1 and CYP39A1 mRNA expression
  20. Discussion
  21. Acknowledgements
  22. References

All steps of tissue preparation were carried out at 4°C. To ensure optimal protein concentrations in the experiments with cell-free tissue preparations, 200 mg of thawed brain tissue samples were homogenized in 1 mL ice-cold homogenization buffer using a motor-driven TeflonTM–glass homogenizer (Potter S, B. Braun, Melsungen, Germany) at 1000 rpm and 3 × 15 strokes, followed by ultrasonication for 3 × 15 s at 50 W (Labsonic 2000, B. Braun). To provide a cell-free supernatant, the final homogenates were centrifuged at 3500 g for 15 min removing cell debris and the nuclear fraction. Aliquots were removed for protein determination (Lowry et al. 1952).

To ensure optimal protein concentrations in the experiments concerning the distribution of the enzyme activity between the subcellular fractions, 400 mg brain tissue were homogenized in 1 mL homogenization buffer. An aliquot of the final homogenate was stored at − 80°C. This was used as the ‘whole homogenate fraction’. The remainder was centrifuged at 800 g for 15 min. The resulting pellet was washed twice with homogenization buffer, then resuspended in homogenization buffer and finally stored at − 80°C. It was designated the ‘nuclear fraction’. The 800 g supernatant was centrifuged at 100 000 g for 60 min to obtain the soluble supernatant as the ‘cytosolic fraction’, which was stored at −80°C. The resulting pellet was washed twice with homogenization buffer, then resuspended in homogenization buffer and finally stored at −80°C. It was designated the ‘membrane fraction’. An aliquot of every fraction was removed for protein determination.

Measurement of enzymes activities

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Source of tissue
  5. Steroids and chemicals
  6. Buffers
  7. Tissue preparation
  8. Measurement of enzymes activities
  9. Gas chromatography mass spectrometry (GC–MS) analysis
  10. Messenger RNA preparation and analysis
  11. Data analysis
  12. Results
  13. Human brain tissue DHEA metabolism with reduced pyridine nucleotides as coenzymes
  14. Subcellular distribution of 7α-hydroxylase and 17β-HSD activity
  15. GC–MS analysis
  16. Rodent brain tissue DHEA metabolism with NADPH as coenzyme
  17. Kinetic properties of cerebral 7α-hydroxylase and 17β-HSD activity, effects of different pH-values and effects of the cytochrome P450 inhibitor ketoconazole
  18. 7α-Hydroxylase and 17β-HSD activity in CX and SC preparations
  19. RT–PCR analyses of CYP7A1, CYP7B1 and CYP39A1 mRNA expression
  20. Discussion
  21. Acknowledgements
  22. References

Stock solutions with [4−14C]DHEA as the substrate were prepared in methanol and diluted with required amounts of the assay buffer at the indicated pH. The amount of methanol in the final incubation volume did not exceed 1.5%. In a final volume of 200 µL the reaction mixture contained either 50 µL of the corresponding tissue preparation or 50 µL homogenization buffer for control incubations, 100 µL of the assay buffer containing the substrate and another 50 µL homogenization buffer containing the required coenzyme. Reaction tubes were capped, vortexed and incubated for 1.5 h with constant shaking at 37°C. Assays were carried out in duplicate. Reactions were stopped by chilling and 1 mL ice-cold chloroform : methanol (2 : 1, v/v) was added. Steroids were extracted by continuous vortexing for 10 min at room temperature. Next, the mixtures were centrifuged at 1500 g for 5 min and the aqueous phases were discarded.

For the detection and quantification of the metabolites, 350-µL aliquots of the organic phases were evaporated to dryness under a stream of nitrogen. Dried organic phases were dissolved in 50 µL chloroform : ethanol (35 : 15; v/v) containing 25 µg each of the respective non-radioactive reference steroid: androst-4-ene-3,17-dione (Δ4-androstenedione), DHEA, androst-4-ene-17β-ol-3-one (testosterone, T), Δ5-androstenediol and 7α-hydroxy-DHEA as reference steroids. Pure authentic non-radioactive androst-5-ene-3β,7β-diol-17-one (7β-hydroxy-DHEA) was not available during the study.

Chromatography was carried out at constant room temperature. For one-dimensional separation of the metabolites, the dissolved aliquots were spotted on a 20 × 20 cm LK6D Silica TLC plate (Whatman International Ltd, Kent, UK) and chromatograms were developed once in dichloromethane : acetone (92.5 : 7.5, v/v, solvent system 1), followed by a second developmental step once in benzene : EtOH (9 : 1, v/v, solvent system 2). For two-dimensional separation of the metabolites, the dissolved aliquots were spotted on a 20 × 20 cm TLC glass plate pre-coated with a 0.25-mm layer of silica gel 60 F254 (Merck AG). In order to achieve complete two-dimensional separation of the metabolites, ethyl acetate : n-hexane : acetic acid (16 : 8 : 1 v/v/vol) was used as solvent system 3. In this mobile phase, 7α-hydroxy-DHEA and the possibly formed 7β-hydroxy-DHEA are known to differ in terms of their migration on TLC plates (Martin et al. 2001). For two-dimensional separation, the plates were first developed twice in the Y-direction using the solvent system 2 and then twice in the X-direction using the solvent system 3. The silica sheets were dried and the reference steroids were stained by spraying with a mixture of acetic acid (100 mL), sulfuric acid (2 mL) and anisaldehyde (1 mL) followed by heating at 130°C.

One-dimensional (or linear) radiodistributions in the Y-direction were scanned with an automatic TLC-linear analyser (LA) LB 285 equipped with a one-dimensional position multiisotopes head detector of high resolution, LB 2821-HR (EG & G Berthold, Wildbad, Germany). A special computer program (CHROMA 1D) written by E.G. & G. Berthold was used for detection and quantitative evaluation of radiosignals emitted from the plates. The relative amount of each corresponding radioactive steroid was calculated in per cent, considering the total radioactivity recovered from a single TLC lane as 100%. Blank values were subtracted from tissue metabolism rates. Enzyme activities were expressed as picomole per hour and per mg protein [pmol/(h/mgprotein)]. 7α-Hydroxylase activity was assessed by quantifying the formation of 7α-hydroxy-DHEA, whereas 17β-HSD activity was measured by quantifying the formation of Δ5-androstenediol.

Bi-dimensional distributions had to be scanned in the X-direction and in the Y-direction, i.e. approximately 100 sections of a 20 × 20 cm TLC plate were measured in the Y-direction. Accordingly, bi-dimensional radiodistribution was reconstructed from these sections by the computer program CHROMA 2D (E.G. & G. Berthold).

Gas chromatography mass spectrometry (GC–MS) analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Source of tissue
  5. Steroids and chemicals
  6. Buffers
  7. Tissue preparation
  8. Measurement of enzymes activities
  9. Gas chromatography mass spectrometry (GC–MS) analysis
  10. Messenger RNA preparation and analysis
  11. Data analysis
  12. Results
  13. Human brain tissue DHEA metabolism with reduced pyridine nucleotides as coenzymes
  14. Subcellular distribution of 7α-hydroxylase and 17β-HSD activity
  15. GC–MS analysis
  16. Rodent brain tissue DHEA metabolism with NADPH as coenzyme
  17. Kinetic properties of cerebral 7α-hydroxylase and 17β-HSD activity, effects of different pH-values and effects of the cytochrome P450 inhibitor ketoconazole
  18. 7α-Hydroxylase and 17β-HSD activity in CX and SC preparations
  19. RT–PCR analyses of CYP7A1, CYP7B1 and CYP39A1 mRNA expression
  20. Discussion
  21. Acknowledgements
  22. References

Cytosolic as well as microsomal membrane fractions of temporal lobe tissue homogenates were incubated (5 mL final incubation volume each) for 2 h with 20 µm DHEA (2 µm[4−14C]DHEA and 18 µm non-labeled DHEA) and 3 mm NADPH at pH 7.5 as described above. Negative control incubations were conducted without the addition of DHEA. Incubations were terminated by adding 25 mL ice-cold chloroform : methanol (2 : 1, v/v) and the steroids were extracted as described above. The organic phases were evaporated to dryness; the residues were dissolved in 250 µL chloroform : ethanol (35 : 15; v/v) and spotted on a 20 × 20 cm TLC glass plate pre-coated with a 0.25-mm layer of silica gel 60 F254 (Merck AG). The cytosolic reaction products were separated using dichloromethane : acetone (9 : 1, v/v) as the mobile phase. The microsomal reaction products were separated using benzene : EtOH (9 : 1, v/v). Linear radiodistribution in the Y-direction was scanned with the automatic TLC-linear analyser as described above. The positions of signals of radioactive metabolites on the plates were marked (corresponding to the position of authentic Δ5-androstenediol in the incubations with cytosolic tissue preparations and corresponding to the position of authentic 7α-hydroxy-DHEA in the incubations with microsomal tissue preparations, respectively). These zones of the silica gel were transferred on clean filter papers and the metabolites were eluted with 2 mL ethanol, 2 mL methanol and 2 mL chloroform. The elutes were evaporated to dryness under a stream of nitrogen.

GC–MS analysis of the cerebral DHEA metabolites was performed after derivatisation of the hydroxy groups of the extracted steroids to trimethylsilyl (TMSi) ethers by adding 1.5 mL TMSi-reagent (pyridine-hexamethyldisilazan-trimethylchlorosilane 9 : 3 : 1, v/v/vol) and incubation for 1 h at 65°C. The solvents were evaporated under a stream of nitrogen. The residues were dissolved in 50 µL n-decane and transferred into microvials for GC–MS analysis. Extracted compounds were separated on a cross-linked methyl silicone DB-XLB 122–1232 capillary column (30 m × 0.25 mm i.d. × 0.25 µm film thickness; J & W, Folsom, CA, USA) in a Hewlett Packard gas chromatograph 6890 after splitless injection by an HP 7683 injector at 280°C. Hydrogen was used as carrier gas with an inlet pressure of 13.4 psi, resulting in a total gas-flow of 1.0 mL/min. The oven temperature was kept for 3 min at 150°C, then raised at a rate of 30°C/min to a final temperature of 290°C and kept at this temperature for 10.33 min. The injector of the GC and detector temperature of an HP5972 mass selective detector were set to 280°C. Multiplier voltage was set to 2700 eV. The emission current was 220 µA. Electron impact ionization was employed at 70 eV ionization energy. Total ion chromatogram was established by scanning between m/z 50 and 500. The identities of the reaction products were qualified by comparison of their retention times and spectra with those of authentic 7α-hydroxy-DHEA, 7β-hydroxy-DHEA (as a minor contaminant in the utilized pure authentic 7α-hydroxy-DHEA sample) and Δ5-androstenediol.

Messenger RNA preparation and analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Source of tissue
  5. Steroids and chemicals
  6. Buffers
  7. Tissue preparation
  8. Measurement of enzymes activities
  9. Gas chromatography mass spectrometry (GC–MS) analysis
  10. Messenger RNA preparation and analysis
  11. Data analysis
  12. Results
  13. Human brain tissue DHEA metabolism with reduced pyridine nucleotides as coenzymes
  14. Subcellular distribution of 7α-hydroxylase and 17β-HSD activity
  15. GC–MS analysis
  16. Rodent brain tissue DHEA metabolism with NADPH as coenzyme
  17. Kinetic properties of cerebral 7α-hydroxylase and 17β-HSD activity, effects of different pH-values and effects of the cytochrome P450 inhibitor ketoconazole
  18. 7α-Hydroxylase and 17β-HSD activity in CX and SC preparations
  19. RT–PCR analyses of CYP7A1, CYP7B1 and CYP39A1 mRNA expression
  20. Discussion
  21. Acknowledgements
  22. References

Total RNAs were extracted from 50 to 75 mg tissue or from about 2 × 106 cells using the Trizol reagent. Total blood RNA was extracted from venous blood using the PAXgeneTM Blood RNA Validation Kit (PreAnalytiX via Qiagen, Hilden, Germany) according to the manufacturer's protocol. Subsequent treatment with RNase-free DNase I was conducted twice to eliminate contaminating DNA traces. RNAs were taken up in RNase-free H2O and quantified spectrophotometrically at 260 nm. RT was performed using 2 µg total RNA at 42°C for 60 min with 100 U Superscript II reverse transcriptase. PCR was conducted using 100–200 ng of the resulting complementary DNA in a final volume of 50 µL PCR buffer, which contained 20–40 pmol of the respective oligonucleotide primers.

To amplify the mRNA species we used specific oligonucleotide primers crossing intron-exon boundaries as follows: CYP7A1 forward primer (F) 5′-CTGAGGCTTTCCAGTGCCT-3′, and reverse primer (R) 5′-AGGTAGTCTTTGTCTTCCCGT-3′, yielding a PCR product of 208 bp; CYP7B1 (F) 5′-GTCCTACATGGTGACCCTGA-3′, and (R) 5′-CATTTGCTGGTTCCAGTTCC-3′ (152 bp); CYP39A1 (F) 5′-GTCTTCTGGACCCATTACCC-3′, and (R) 5′-AGCTCAGGTCTAGGTGCTGC-3′ (176 bp); porphobilinogen deaminase (PBGD) (F) 5′-ACCAAGGAGCTTGAACATGC-3′, and (R) 5′-GAAAGACAACAGCATCATGAG-3′ (145 bp).

PCR was initiated by a 5-min denaturation step at 94°C. The PCR program consisted of 35 cycles of a 30-s denaturation step at 94°C, followed by a 30-s annealing step at 58°C and a 105-s extension step at 72°C. A 10-min final extension step at 72°C completed the PCR. The products were resolved on 2% agarose gel containing ethidium bromide and then visualized under ultraviolet light using the GEL Doc 1000 System (Bio-Rad Laboratories, Inc. Heidelberg, Germany).

Real-time PCR was carried out with the ABI 7700 Instrument (Applied Biosystems, Weiterstadt, Germany) using the non-specific DNA binding dye SYBR green I for the detection of PCR products. As external standard, we used purified PCR products (QiaQuick PCR Purificaton Kit, Qiagen) of reverse-transcribed total RNA from human liver. The amount of the standard was quantified photometrically and serial dilutions of standard were used for measuring the standard curve. Paying particular attention to the avoidance of the detection of spurious amplicons such as primer dimer products, the following adaptations of the block-cycler protocols for real-time have been implemented: (i) real-time PCR was set up using 12.5 µL PCR master mix (QuantiTect SYBR Green PCR Kit, Qiagen), supplemented with 10 pmol of the above mentioned primers and 4 µL of either external standards or 80 ng cDNA to give a final volume of 25 µL; (ii) after initial denaturation for 15 min at 95°C the setting of the thermal profile of the 50 amplification cycles were 15 s at 94°C, 30 s at 55°C and 30 s at 72°C. Acquisition of the fluorescent signal was adjusted to the end of each 72°C step. As an additional control of specificity, PCR products were size-fractioned on 2% agarose gels and visualized under ultraviolet light. Initial differences in the amount of RNA subjected to RT were corrected by calculating the ratios of mRNA expression of the investigated gene to mRNA expression of the low copy housekeeping gene PBGD. Results represent mean values of two independent experiments and are given as arbitrary units with the expression in liver set as 100%.

Human brain tissue DHEA metabolism with reduced pyridine nucleotides as coenzymes

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Source of tissue
  5. Steroids and chemicals
  6. Buffers
  7. Tissue preparation
  8. Measurement of enzymes activities
  9. Gas chromatography mass spectrometry (GC–MS) analysis
  10. Messenger RNA preparation and analysis
  11. Data analysis
  12. Results
  13. Human brain tissue DHEA metabolism with reduced pyridine nucleotides as coenzymes
  14. Subcellular distribution of 7α-hydroxylase and 17β-HSD activity
  15. GC–MS analysis
  16. Rodent brain tissue DHEA metabolism with NADPH as coenzyme
  17. Kinetic properties of cerebral 7α-hydroxylase and 17β-HSD activity, effects of different pH-values and effects of the cytochrome P450 inhibitor ketoconazole
  18. 7α-Hydroxylase and 17β-HSD activity in CX and SC preparations
  19. RT–PCR analyses of CYP7A1, CYP7B1 and CYP39A1 mRNA expression
  20. Discussion
  21. Acknowledgements
  22. References

To identify the reaction products, we first carried out two-dimensional TLC analyses with cell-free temporal lobe tissue preparations (equal shares of CX and SC) from a 36-year-old woman and a 24-year-old man. The conversion of 3 µm DHEA was investigated at pH 7.5, using 3 mm NADPH as the coenzyme. Figure 2 shows two-dimensional TLC autoradiograms of radioactively labelled DHEA and its metabolites. Co-chromatographied and stained non-radioactive reference steroids are plotted by rims. The radio signals obtained corresponded exactly to authentic DHEA, 7α-hydroxy-DHEA (7α-hydroxylase activity) and Δ5-androstenediol (17β-HSD activity). The solvent system used for chromatography in the X-direction is known to separate 7α-hydroxy-DHEA and 7β-hydroxy-DHEA (Martin et al. 2001). Unfortunately, authentic 7β-hydroxy-DHEA was not available as reference substance during the present study. However, two-dimensional TLC analysis failed in detecting the formation of a third metabolite. Identical results were obtained with tissue preparations of both patients (data shown for the woman only).

image

Figure 2. Digital autoradiograms of two-dimensional TLC analysis of [4−14C]DHEA metabolism in the human temporal lobe. The displayed autoradiograms show the radio signals derived from incubations of [4−14C]DHEA with a cell-free brain tissue preparation of a 36-year-old woman (a) and from a tissue-less control incubation (b). TLC was carried out as described in the Material and methods section, following incubations with 3 µm DHEA and 3 mm NADPH at pH 7.5. The plotted rims are assigned to (Δ4) Δ4-androstenedione (D) DHEA (T) testosterone (Δ5) Δ5-androstenediol and (7α) 7α-hydroxy-DHEA. They were obtained by staining non-radioactive authentic reference steroids added to the incubation extracts. The origins of TLC are marked with (OR). Acquisition parameters: gain: 3; voltage: 1380 V; step-width: 2 mm; slit-width: 2 mm; step number: 100; counting time: 200 min.

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Further investigations revealed that NADPH is the preferred coenzyme for the reactions. The use of NADH resulted in no significant metabolite formation (data not shown).

Subcellular distribution of 7α-hydroxylase and 17β-HSD activity

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Source of tissue
  5. Steroids and chemicals
  6. Buffers
  7. Tissue preparation
  8. Measurement of enzymes activities
  9. Gas chromatography mass spectrometry (GC–MS) analysis
  10. Messenger RNA preparation and analysis
  11. Data analysis
  12. Results
  13. Human brain tissue DHEA metabolism with reduced pyridine nucleotides as coenzymes
  14. Subcellular distribution of 7α-hydroxylase and 17β-HSD activity
  15. GC–MS analysis
  16. Rodent brain tissue DHEA metabolism with NADPH as coenzyme
  17. Kinetic properties of cerebral 7α-hydroxylase and 17β-HSD activity, effects of different pH-values and effects of the cytochrome P450 inhibitor ketoconazole
  18. 7α-Hydroxylase and 17β-HSD activity in CX and SC preparations
  19. RT–PCR analyses of CYP7A1, CYP7B1 and CYP39A1 mRNA expression
  20. Discussion
  21. Acknowledgements
  22. References

To determine the distribution of the enzyme activities between the subcellular fractions, whole homogenates, nuclear fractions, membrane fractions and cytosolic fractions of temporal lobe tissue (equal shares of CX and SC) from a 33-year-old woman and a 35-year-old man were prepared. 7α-hydroxylase and 17β-HSD activity was investigated using 2 µm DHEA and 3 mm NADPH at pH 7.5. As shown in Fig. 3(a), 7α-hydroxylase activity was found to be associated with the cellular membranes, whereas 17β-HSD activity was enriched in the cytosolic fraction. Analogous results were obtained with tissue preparations from both the woman and the man (data shown for the woman only).

image

Figure 3. Characteristics of cerebral 7α-hydroxylase and 17-ketosteroid reductase. (a) The subcellular distribution of 7α-hydroxylase (▪) and 17-ketosteroid reductase (□) was investigated using whole homogenate (WH), a crude nuclear fraction (Nu), a membrane fraction (Me) as well as a cytosolic fraction (Cy) from brain tissue of a 33-year-old woman. Enzyme activity was determined using 2 µm DHEA with 3 mm NADPH at pH 7.5; (b) the kinetic properties of 7α-hydroxylase and of 17-ketosteroid reductase in the human temporal lobe were determined using cell-free brain tissue preparations from a 38-year-old woman (○) and from a 38-year-old man (▪) with 3 mm NADPH at pH 7.5. Incubations were carried out at increasing DHEA concentrations; (c) the effect of the pH-value on the 7α-hydroxylation and the 17-ketosteroid reduction of DHEA in the human temporal lobe was investigated using cell-free brain tissue preparations from a 38-year-old woman (○) and from a 38-year-old man (▪) with 2 µm DHEA and 3 mm NADPH. Incubations were performed over a pH range from 4.5 to 9.5; (d) the effect of cytochrome P450 inhibition on 7α-hydroxylation (•) and on 17-ketosteroid reduction (○) of DHEA was determined using a cell-free brain tissue preparation from a 28-year-old man with 2 µm DHEA and 3 mm NADPH at pH 7.5. Incubations were carried out at increasing ketoconazole concentrations. The results represent mean values of assays performed in duplicate as described in the experimental section.

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GC–MS analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Source of tissue
  5. Steroids and chemicals
  6. Buffers
  7. Tissue preparation
  8. Measurement of enzymes activities
  9. Gas chromatography mass spectrometry (GC–MS) analysis
  10. Messenger RNA preparation and analysis
  11. Data analysis
  12. Results
  13. Human brain tissue DHEA metabolism with reduced pyridine nucleotides as coenzymes
  14. Subcellular distribution of 7α-hydroxylase and 17β-HSD activity
  15. GC–MS analysis
  16. Rodent brain tissue DHEA metabolism with NADPH as coenzyme
  17. Kinetic properties of cerebral 7α-hydroxylase and 17β-HSD activity, effects of different pH-values and effects of the cytochrome P450 inhibitor ketoconazole
  18. 7α-Hydroxylase and 17β-HSD activity in CX and SC preparations
  19. RT–PCR analyses of CYP7A1, CYP7B1 and CYP39A1 mRNA expression
  20. Discussion
  21. Acknowledgements
  22. References

To clearly identify the reaction products, cytosolic as well as microsomal membrane fractions of CX tissue from a 38-year-old woman and a 28-year-old man were incubated with 20 µm DHEA and 3 mm NADPH at pH 7.5. Metabolites were separated, detected and eluted as described in the material and methods section. GC–MS analysis of the trimethylsilyl ethers of metabolites from the incubations with cytosolic tissue preparations revealed the occurrence of a compound with a retention time and a characteristic ion fragment pattern (Fig. 4) identical to that of the trimethylsilyl ether of authentic Δ5-androstenediol (retention time of 10.06 min). GC–MS analysis of microsomal metabolites proved the formation of a metabolite with a retention time and an ion fragment pattern (Fig. 5) identical to authentic 7α-hydroxy-DHEA (retention time of 10.08 min). Identical results were obtained with tissue preparations of both patients (data shown for the man only). GC–MS analysis revealed that the authentic 7α-hydroxy-DHEA sample used in the experiments was contaminated with small amounts of 7β-hydroxy-DHEA, a molecule with a retention time of 10.71 min in GC and a characteristic ion fragment pattern identical to 7α-hydroxy-DHEA (data not shown). The in vitro formation of significant amounts of cerebral 7β-hydroxy-DHEA can be excluded, as a compound with the same retention time and the same characteristic ion fragment pattern as 7β-hydroxy-DHEA was not detected in the organic brain tissue extracts. For negative controls, respective incubations were conducted without the addition of DHEA. Here, no significant amounts of metabolites with retention times and characteristic ion fragment patterns identical to Δ5-androstenediol or 7α-hydroxy-DHEA were detectable.

image

Figure 4. (a) GC retention time of trimethylsiyl ethers of organic brain tissue metabolites with the same Rf value in TLC as authentic Δ5-androstenediol after in vitro incubation of 20 µm DHEA and 3 mm NADPH with a cytosolic preparation of temporal lobe tissue from a 28-year-old man at pH 7.5; (b) GC–MS analysis of the trimethylsilyl ether of the cerebral DHEA metabolite with the same Rf value in TLC and the same retention time in subsequent GC as authentic Δ5-androstenediol; (c) GC–MS analysis of the trimethylsilyl ether of authentic Δ5-androstenediol.

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image

Figure 5. (a) GC retention time of trimethylsiyl ethers of organic brain tissue metabolites with the same Rf value in TLC as authentic 7α-hydroxy-DHEA after in vitro incubation of 20 µm DHEA and 3 mm NADPH with a microsomal preparation of temporal lobe tissue from a 28-year-old man at pH 7.5; (b) GC–MS analysis of the trimethylsilyl ether of the cerebral DHEA metabolite with the same Rf value in TLC and the same retention time in subsequent GC as authentic 7α-hydroxy-DHEA; (c) GC–MS analysis of the trimethylsilyl ether of authentic 7α-hydroxy-DHEA.

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Rodent brain tissue DHEA metabolism with NADPH as coenzyme

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Source of tissue
  5. Steroids and chemicals
  6. Buffers
  7. Tissue preparation
  8. Measurement of enzymes activities
  9. Gas chromatography mass spectrometry (GC–MS) analysis
  10. Messenger RNA preparation and analysis
  11. Data analysis
  12. Results
  13. Human brain tissue DHEA metabolism with reduced pyridine nucleotides as coenzymes
  14. Subcellular distribution of 7α-hydroxylase and 17β-HSD activity
  15. GC–MS analysis
  16. Rodent brain tissue DHEA metabolism with NADPH as coenzyme
  17. Kinetic properties of cerebral 7α-hydroxylase and 17β-HSD activity, effects of different pH-values and effects of the cytochrome P450 inhibitor ketoconazole
  18. 7α-Hydroxylase and 17β-HSD activity in CX and SC preparations
  19. RT–PCR analyses of CYP7A1, CYP7B1 and CYP39A1 mRNA expression
  20. Discussion
  21. Acknowledgements
  22. References

To compare the results from human brain tissue DHEA metabolism with the processes in rodent brain tissue, we investigated the 7α-hydroxylation and 17-ketosteroid reduction of DHEA in cerebrum samples of rodents. For this purpose, cell-free tissue preparations of one animal of each gender of neonate and adult rats (6 days old; 286 days old) and of neonate and adult mice (9 days old; 313 days old) were incubated with 2 µm DHEA and 3 mm NADPH at pH 7.5.

7α-Hydroxylase as well as 17β-HSD activity was detected in all of the rodent brain tissue preparations (Fig. 6). In the experiment with mice brain tissue, the formation of a third and, thus far unidentified, metabolite was observed. The amounts of 7α-hydroxy-DHEA and Δ5-androstenediol formed during the incubations are specified in Table 1.

image

Figure 6. Digital autoradiogram of one-dimensional TLC analysis of rodent brain tissue [4−14C]DHEA metabolism. The displayed autoradiograms show radio signals derived from incubations with cell-free cerebrum tissue preparation of one male (m) and one female (f) neonate (neo) mouse (9 days old), one male (313 days old) and one female adult (adt) mouse (364 days old), one male and one female neonate rat (6 days old) and one male and one female adult rat (286 days old) as well as from tissue-less control incubations (C). TLC was carried out as described in the Material and methods section, following incubations with 3 µm DHEA and 3 mm NADPH at pH 7.5. The displayed radio signals correspond to authentic non-radioactive (D) DHEA (Δ5) Δ5-androstenediol and (7α) 7α-hydroxy-DHEA. Unidentified radio signals are marked with a question mark. Acquisition parameters: gain: 3; voltage: 1380 V; step-width: 2 mm; slit-width: 2 mm; step number: 100; counting time: 200 min.

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Table 1.  Metabolism of 2 μm DHEA via 7α-hydroxylase (7α) and 17β-HSD (Δ5) activity in cell-free tissue preparations of neonate and adult cerebrum samples from C-57 black mice as well as from Wistar rats
 NeonatalAdultNeonatalAdult
  1. 7α, 7α--hydroxylase activity in pmol/(h/mgprotein); Δ5, 17β-HSD activity in pmol/(h/mgprotein). Incubations were carried out at pH 7.5 using 3 mm NADPH as the coenzyme.

 Male mouseFemale mouse
Age (days)    9313    9313
257.8151.2153.0151.9
Δ 5  11.2  10.1  11.5    7.6
 Male ratFemale rat
Age (days)    6286    6286
 11.9  64.1  14.4  48.2
Δ 5  10.8  22.0  10.8  22.3

Kinetic properties of cerebral 7α-hydroxylase and 17β-HSD activity, effects of different pH-values and effects of the cytochrome P450 inhibitor ketoconazole

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Source of tissue
  5. Steroids and chemicals
  6. Buffers
  7. Tissue preparation
  8. Measurement of enzymes activities
  9. Gas chromatography mass spectrometry (GC–MS) analysis
  10. Messenger RNA preparation and analysis
  11. Data analysis
  12. Results
  13. Human brain tissue DHEA metabolism with reduced pyridine nucleotides as coenzymes
  14. Subcellular distribution of 7α-hydroxylase and 17β-HSD activity
  15. GC–MS analysis
  16. Rodent brain tissue DHEA metabolism with NADPH as coenzyme
  17. Kinetic properties of cerebral 7α-hydroxylase and 17β-HSD activity, effects of different pH-values and effects of the cytochrome P450 inhibitor ketoconazole
  18. 7α-Hydroxylase and 17β-HSD activity in CX and SC preparations
  19. RT–PCR analyses of CYP7A1, CYP7B1 and CYP39A1 mRNA expression
  20. Discussion
  21. Acknowledgements
  22. References

To determine the kinetic properties of cerebral 7α-hydroxylase and 17β-HSD activity we investigated the metabolism of DHEA in cell-free CX tissue preparations from two women (aged 33 and 38 years) and two men (aged 35 and 38 years) with rising DHEA concentrations and 3 mm NADPH at pH 7.5. As shown in Fig. 3(b), kinetic analyses revealed KM-values of 6.2, 5.1, 6.1 and 4.2 µm for 7α-hydroxylase activity, respectively. The Vmax values were 146.0, 72.9, 106.5 and 79.4 pmol/(hmgprotein), respectively. In all four experiments, the formation of Δ5-androstenediol increased approximately linearly within the range of substrate concentrations. Therefore, no attempt was made to estimate KM- and Vmax-values of 17β-HSD activity.

For further characterization of cerebral 7α-hydroxylase and 17β-HSD activity, cell-free CX tissue preparations from a 38-year-old woman and a 38-year-old man were incubated with 2 µm DHEA and 3 mm NADPH final concentrations at different pH-values. As shown in Fig. 3(c), 7α-hydroxylase activity displayed a sharp pH optimum between 7.5 and 8.0, whereas 17β-HSD activity exhibited a broad pH optimum between 6.5 and 9.0.

The effect of ketoconazole on the cerebral 7α-hydroxylation and 17-ketosteroid reduction of DHEA was tested using cell-free CX tissue preparations from a 35-year-old woman and a 28-year-old man. Incubations were carried out with 2 µm DHEA and 3 mm NADPH final concentrations at pH 7.5 using inhibitor concentrations ranging from 7.8 to 1000 nm. Computer-assisted non-linear curve-fitting analysis revealed ketoconazole as a strong inhibitor of 7α-hydroxylase activity (Fig. 3d; data shown for the man only) with IC50 values (inhibitor concentrations resulting in 50% inhibition) of 23 and 31 nm, respectively. No inhibition of 17β-HSD activity was observed.

7α-Hydroxylase and 17β-HSD activity in CX and SC preparations

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Source of tissue
  5. Steroids and chemicals
  6. Buffers
  7. Tissue preparation
  8. Measurement of enzymes activities
  9. Gas chromatography mass spectrometry (GC–MS) analysis
  10. Messenger RNA preparation and analysis
  11. Data analysis
  12. Results
  13. Human brain tissue DHEA metabolism with reduced pyridine nucleotides as coenzymes
  14. Subcellular distribution of 7α-hydroxylase and 17β-HSD activity
  15. GC–MS analysis
  16. Rodent brain tissue DHEA metabolism with NADPH as coenzyme
  17. Kinetic properties of cerebral 7α-hydroxylase and 17β-HSD activity, effects of different pH-values and effects of the cytochrome P450 inhibitor ketoconazole
  18. 7α-Hydroxylase and 17β-HSD activity in CX and SC preparations
  19. RT–PCR analyses of CYP7A1, CYP7B1 and CYP39A1 mRNA expression
  20. Discussion
  21. Acknowledgements
  22. References

To determine sex- and tissue-specific differences, we investigated 7α-hydroxylase and 17β-HSD activity in cell-free tissue preparations of CX as well as SC specimens from eight female (aged 1–60 years) and nine male patients (aged 5–58 years). Incubations were carried out using 2 µm DHEA with 3 mm NADPH at pH 7.5. Due to the limited number of tissue samples from children, no attempt was made to investigate differences between children and adults by statistical analysis. Data analysis yielded no sex differences in both CX and SC. Therefore, we combined all data from female and male patients to investigate the differences between CX and SC. As shown in Fig. 7, 7α-hydroxylase activity was found to be significantly higher (p < 0.0005) in CX [14.9 ± 2.9 pmol/(hmgprotein); (mean±SD)] than in SC [9.3 ± 2.9 pmol/(hmgprotein)], whereas 17β-HSD activity was significantly higher (p < 0.0005) in SC [33.0 ± 12.6 pmol/(hmgprotein)] than in CX [11.7 ± 3.3 pmol/(hmgprotein)].

image

Figure 7. DHEA metabolism in cell-free cerebral neocortex (solid symbols) and subcortical white matter (open symbols) preparations was investigated in relation to tissue-, sex- and age-specific differences using 2 µm DHEA with 3 mm NADPH at pH 7.5. 7α-hydroxylase (a) and 17β-HSD activity (b) was studied in temporal lobe preparations of eight female (1–60 years old; circles) and nine male patients (5–58 years old; squares). The results represent mean values of assays performed in duplicate as described in the experimental section.

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RT–PCR analyses of CYP7A1, CYP7B1 and CYP39A1 mRNA expression

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Source of tissue
  5. Steroids and chemicals
  6. Buffers
  7. Tissue preparation
  8. Measurement of enzymes activities
  9. Gas chromatography mass spectrometry (GC–MS) analysis
  10. Messenger RNA preparation and analysis
  11. Data analysis
  12. Results
  13. Human brain tissue DHEA metabolism with reduced pyridine nucleotides as coenzymes
  14. Subcellular distribution of 7α-hydroxylase and 17β-HSD activity
  15. GC–MS analysis
  16. Rodent brain tissue DHEA metabolism with NADPH as coenzyme
  17. Kinetic properties of cerebral 7α-hydroxylase and 17β-HSD activity, effects of different pH-values and effects of the cytochrome P450 inhibitor ketoconazole
  18. 7α-Hydroxylase and 17β-HSD activity in CX and SC preparations
  19. RT–PCR analyses of CYP7A1, CYP7B1 and CYP39A1 mRNA expression
  20. Discussion
  21. Acknowledgements
  22. References

In order to investigate the mRNA expression of CYP7A1, CYP7B1 as well as CYP39A1 in the human temporal lobe, we first conducted block-cycler RT–PCR analyses (35 cycles) using pooled cDNA samples of cerebral neocortex as well as of subcortical white matter from either eight female (aged 11–60 years) or eight male patients (aged 5–58 years). Moreover, mRNA expression was investigated in human leucocytes, the U-87 astrocytoma cell line (U-87), liver, prostate, adrenal cortex (AdX), the JEG3 choriocarcinoma cell line (JEG3) and heart muscle. As shown in Fig. 8(a), CYP7A1 mRNA expression was detected in liver and, to an apparently lesser extent, in prostate. Significant CYP7B1 mRNA expression was found in all samples under investigation, except for leucocytes and the U-87 cell line (Fig. 8b). Significant CYP39A1 mRNA expression was detected in liver and prostate and to an obviously lesser extent, in heart muscle and brain tissue (Fig. 8c). All results were confirmed by sequence analysis (data not shown).

image

Figure 8. RT–PCR based identification of the expression of CYP7A1, CYP7B1 and CYP39A1 in human tissues. Prior to RT, total RNA samples isolated from cerebral neocortex (CX) and subcortical white matter (SC) of three women (f) or three men (m) were pooled. Total RNA samples isolated from leucocytes (LC), the U-87 astrocytoma cell line (U-87), liver tissue, prostate tissue (PR), adrenal cortex tissue (AdCx), the human choriocarcinoma cell line JEG3 (JEG3) and heart muscle were also investigated. Oligonucleotide primers used in the RT–PCR experiments were specific for CYP7A1 (a), CYP7B1 (b), CYP39A1 (c) and for PBGD (d), respectively. Lane 2 is an H2O no-template negative control and a 50-bp ladder as DNA size marker is given on the left.

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To quantify the expression of the mRNA species in CX and SC of eight female (aged 11–60 years) and eight male patients (aged 5–58 years) in comparison to the above-mentioned tissues and cell lines we conducted real-time RT–PCR analyses as described in the Materials and methods section. As shown in Table 2, CYP7A1 is not only expressed in human liver and prostate, but, to a lesser extent, also in brain tissue (more than two orders of magnitude less than in liver). Data analysis yielded no sex- or tissue-specific differences. Significant CYP7B1 mRNA expression was confirmed in brain tissue, liver, prostate, adrenal cortex, the JEG3 cell line and heart muscle, but not in leucocytes and the U-87 cell line. The amount of mRNA in brain tissue is obviously in the same order of magnitude as in liver. Data analysis yielded no sex difference. Differences between the mRNA expression in CX and SC were not significant. Furthermore, a significant mRNA expression of CYP 39A1 in liver, prostate and heart muscle was confirmed by real-time RT–PCR. In contrast, the mRNA expression of CYP39A1 in brain tissue is obviously two orders of magnitude lower than in liver. Data analysis yielded no sex- or tissue-specific differences. CYP7A1, CYP7B1 and CYP39A1 mRNA expression could be verified in all the brain tissue samples investigated.

Table 2.  Expression levels of CYP7A1, CYP7B1 and CYP39A1 mRNAs in cerebral neocortex (CX) and subcortical white matter (SC) of eight female (11–60 years old) and eight male patients (5–58 years old) in comparison to human leucocytes (LC), the U-87 astrocytoma cell line (U-87), liver, prostate (PR), adrenal cortex (AdCx), the JEG3 choriocarcinoma cell line (JEG3) and heart muscle
 CYP7A1CYP7B1CYP39A1
  1. Data are given as arbitrary units with the expression in liver set as 100%. The mRNA expression in brain tissues is expressed as means ±SEM.

CX (female)0.7±0.156.9±6.81.3±0.2
SC (female)0.4±0.152.4±11.20.9±0.2
CX (male)0.8±0.243.0±4.81.2±0.2
SC (male)1.0±0.239.3±4.81.3±0.2
LC0.30.80.0
U-870.00.00.1
Liver100.0100.0100.0
PR98.6733.1207.0
AdCx0.09.30.2
JEG30.09.10.2
Heart4.4249.4232.2

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Source of tissue
  5. Steroids and chemicals
  6. Buffers
  7. Tissue preparation
  8. Measurement of enzymes activities
  9. Gas chromatography mass spectrometry (GC–MS) analysis
  10. Messenger RNA preparation and analysis
  11. Data analysis
  12. Results
  13. Human brain tissue DHEA metabolism with reduced pyridine nucleotides as coenzymes
  14. Subcellular distribution of 7α-hydroxylase and 17β-HSD activity
  15. GC–MS analysis
  16. Rodent brain tissue DHEA metabolism with NADPH as coenzyme
  17. Kinetic properties of cerebral 7α-hydroxylase and 17β-HSD activity, effects of different pH-values and effects of the cytochrome P450 inhibitor ketoconazole
  18. 7α-Hydroxylase and 17β-HSD activity in CX and SC preparations
  19. RT–PCR analyses of CYP7A1, CYP7B1 and CYP39A1 mRNA expression
  20. Discussion
  21. Acknowledgements
  22. References

The intracrine metabolism of DHEA is suggested to play a major role in mediating its peripheral effects (Labrie et al. 1998, 2000). In the rodent brain, the formation of DHEA metabolites with reduced pyridine nucleotides as coenzymes is primarily catalysed via 7α-hydroxylase and 17β-HSD activity (Baulieu and Robel 1996). The present study is the first to demonstrate and characterize the in vitro metabolism of DHEA via 7α-hydroxylase and 17β-HSD activity in the human CNS.

The verified occurrence of DHEA 7α-hydroxylase and DHEA 17-ketosteroid reductase activity in the human temporal lobe (Figs 2, 4 and 5) is in accordance with results obtained from rodent brain tissue experiments (Akwa et al. 1992; Doostzadeh et al. 1997; Rose et al. 1997; Fig. 6). Furthermore, the observation of 17-ketosteroid reductase activity with DHEA as the substrate corresponds to a previous study demonstrating the presence of 17-ketosteroid reductase activity in the human temporal lobe withΔ4-androstenedione as well as oestrone as the substrate (Steckelbroeck et al. 1999). Moreover, cerebral 17-ketosteroid reductase activity is in agreement with the observation of significant mRNA expression of the reductive 17β-HSD isozymes type 3 and 5 in the human temporal lobe (Stoffel-Wagner et al. 1999; Steckelbroeck et al. 2001). With these results in mind, DHEA has to be considered as an additional substrate for the reductive 17β-HSD pathway in the human CNS.

Within the rodent brain, DHEA is also known to be converted into 7β-hydroxy-DHEA (Akwa et al. 1992; Doostzadeh et al. 1997). According to this, we observed the formation of low amounts of a third polar metabolite when investigating mouse brain tissue homogenates (Fig. 6). Although this metabolite remained unidentified, it is most likely identical with 7β-hydroxy-DHEA. In contrast to the observations in mouse brain, only the formation of 7α-hydroxy-DHEA and Δ5-androstenediol from DHEA could be demonstrated in the human temporal lobe. Even when conducting two-dimensional TLC analysis with two different solvent systems, only two metabolites were detected (Fig. 2), although the solvent system used for chromatography in the X-direction is known to separate 7α-hydroxy-DHEA and 7β-hydroxy-DHEA (Martin et al. 2001). The in vitro formation of 7α-hydroxy-DHEA and Δ5-androstenediol as well as the lack of in vitro formation of 7β-hydroxy-DHEA could be verified by GC–MS analysis (Figs 4 and 5).

Both 7α-hydroxylase and 17-ketosteroid reductase require reduced pyridine nucleotides as coenzymes. We found that NADPH is the preferred coenzyme of the two human cerebral enzymes. The use of NADH resulted in no significant metabolite formation, reflecting the general principal that NADPH is consumed in biosynthetic reactions, whereas NADH is primarily used for the generation of ATP.

To further characterize the cerebral metabolism of DHEA via 7α-hydroxylase and 17β-HSD activity, the subcellular distributions, the pH optima, and the KM-values of the enzymes were investigated. The membrane-associated CYP7B1 is known to be responsible for the 7α-hydroxylation of DHEA in the rodent brain (Rose et al. 1997, 2001). The kinetic characterization of the recombinant rat protein revealed a KM-value of 13.6 µm (Rose et al. 1997). Microsomal rat brain 7α-hydroxylase activity with DHEA as the substrate showed a KM-value of 13.8 µm and a pH optimum of 7.8 (Akwa et al. 1992). According to these results, the human cerebral DHEA 7α-hydroxylase is obviously also a membrane-associated protein with a sharp pH optimum between 7.5 and 8.0 (Figs 3a and c). In comparison to the rodent brain tissue protein, it demonstrated a somewhat lower mean KM-value of 5.4 µm (Fig. 3b). The cytochrome P450 inhibitor ketoconazole was shown to be a potent inhibitor of microsomal mouse brain 7α-hydroxylase activity (Doostzadeh et al. 1997). According to these findings, the present study revealed that ketoconazole is also a strong inhibitor of human brain tissue 7α-hydroxylase activity (Fig. 3d), verifying the catalytic activity of a cytochrome P450-containing enzyme. On the whole, the biochemical properties of human cerebral DHEA 7α-hydroxylase activity suggest the presence of CYP7B1 in the human temporal lobe as it was demonstrated for the rodent brain.

Block-cyler RT–PCR analysis revealed strong CYP7B1 mRNA expression in the human temporal lobe (Fig. 8). Real-time RT–PCR analysis verified a high cerebral CYP7B1 mRNA expression and demonstrated a slightly but not significantly higher mRNA expression of the isozyme in the CX than in the SC with no sex differences (Table 2). These results coincide with the data on 7α-hydroxylase activity, which were demonstrated to be significantly higher in CX than in SC with no sex differences (Fig. 7a). Due to the cellular composition of CX and SC it might be suggested that the enzyme is a neuronal rather than a glial protein. This is corroborated by the results of in situ hybridization analysis and investigations of reporter-gene activity in gene-targeted mice, which addressed CYP7B1 expression to the neuronal cell layer of the dentate gyrus (Rose et al. 2001). The summarized findings provide clear evidence that CYP7B1 expression is responsible for DHEA 7α-hydroxylation in the human temporal lobe.

The undetectably high KM-value of 17β-HSD and its broad pH optimum between 6.5 and 9.0 (Figs 3b and c) verify the data of a previous study with Δ4-androstenedione as the substrate (Steckelbroeck et al. 1999). The unusual kinetic parameters of cerebral 17-ketosteroid reductase activity might suggest that cerebral Δ5-androstenediol formation is not based on an enzyme-catalysed process. However, the catalytic activity of an enzyme could be proved previously (Steckelbroeck et al. 1999), as kinetic analysis of the NADPH-dependent 17-ketosteroid reductase activity revealed a typical Michaelis–Menten kinetic. The presence of 17-ketosteroid reductase activity in the cytosolic fraction (Fig. 3a) coincides with significant mRNA expression of the cytosolic 17β-HSD isozyme type 5 in the human temporal lobe (Steckelbroeck et al. 2001). 17β-HSD type 5 is a cytosolic protein, which uses NADPH and NADP as cofactors and catalyses the interconversion of androgens, oestrogens as well as DHEA and Δ5-androstenediol (Lin et al. 1997; Dufort et al. 1999; Peltoketo et al. 1999). To the authors' knowledge, the pH optimum and the KM-values of human 17β-HSD type 5 activity have not been investigated to date. The formation of Δ5-androstenediol from DHEA via 17-ketosteroid reductase activity was significantly higher in SC than in CX, showing no sex differences (Fig. 7b). Previously, this was also shown for the 17-ketosteroid reduction of Δ4-androstenedione (Steckelbroeck et al. 1999). Moreover, the mRNA expression of 17β-HSD type 5 was also found to be significantly higher SC than in CX with no sex differences (Steckelbroeck et al. 2001). These data suggest that the cerebral 17-ketosteroid reductase is a glial rather than neuronal protein. The lack of inhibition of the formation of Δ5-androstenediol from DHEA by ketoconazole (Fig. 3d) reinforces the fact that human brain tissue 17-ketosteroid reductase activity depends on the expression of a protein not containing cytochrome P450, just as the aldo-keto reductase 17β-HSD type 5. When summarizing all concordances, it appears that the same enzyme is responsible for the cerebral reduction of DHEA and of Δ4-androstenedione and that cerebral expression of 17β-HSD type 5 might be responsible for the observed catalytic activity.

Three human 7α-hydroxylase isozymes are known to date (Chiang 1998; Russell 2000; Björkhem and Eggertsen 2001). CYP7A1 catalyses the first step of the neutral pathway of bile acids biosynthesis, i.e. cholesterol7α-hydroxylation. Recently, it could be demonstrated that, apart from its function as cholesterol 7α-hydroxylase, CYP7A1 is also able to function as an oxysterol7α-hydroxylase (Norlin et al. 2000). For instance, it catalyses the conversion of 27-hydroxycholesterol with about half the activity compared to the activity toward cholesterol. CYP7A1, but not CYP7B1, is induced by dietary cholesterol in mice liver and its expression is reduced to undetectable levels in response to bile acids, whereas CYP7B1 expression is only modestly regulated (Schwarz et al. 1997). Homozygous CYP7A1 knockout mice appeared normal at birth, but died within 18 days of life due to malabsorption of dietary lipids and fat-soluble vitamins (Ishibashi et al. 1996). Maintaining nursing mothers on specially supplemented chow prevented deaths of the pups. In the older animals, bile acid deficiency is then overcome by an alternate pathway involving an inducible oxysterol 7α-hydroxylase (Schwarz et al. 1996).

The oxysterol 7α-hydroxylase CYP7B1 is apparently responsible for the hepatic 7α-hydroxylation of 27-hydroxycholesterol, which represents the precursor molecule of the alternate acidic pathway of bile acids biosynthesis (Schwarz et al. 1997). CYP7B1 demonstrated broad oxysterol substrate specificity and these compounds accumulate in the plasma and tissues of CYP7B1 knockout mice (Li-Hawkins et al. 2000a,b). Interestingly, the loss of oxysterol 7α-hydroxylase activity is obviously compensated by other pathways in CYP7B1 knockout mice (Li-Hawkins et al. 2000b). The significance of CYP7B1 expression in humans was emphasized by the fatal effects of a homozygous nonsense mutation of the gene, which results in severe neonatal liver disease and is apparently incompatible with post-natal survival (Setchell et al. 1998). The lack of oxysterol 7α-hydroxylase activity due to this mutation led to an accumulation of hepatotoxic unsaturated monohydroxy bile acids, such as 27-hydroxycholesterol, in serum and urine which is apparently consistent with the concept of CYP7B1 as an important factor of bile acid biosynthesis. On the other hand, CYP7B1 is known to be responsible for the 7α-hydroxylation of DHEA within the rodent CNS (Rose et al. 1997, 2001) and the cloned human CYP7B1 isozyme converts DHEA to a one hundred times greater extent than 27-hydroxycholesterol (Wu et al. 1999).

CYP39A1 represents another oxysterol 7α-hydroxylase with a strong preference for 24S-hydroxycholesterol (Li-Hawkins et al. 2000a). Conversion into 24S-hydroxycholesterol is an important brain-specific mechanism for the elimination of cholesterol across the blood–brain barrier (Lütjohann et al. 1996; Björkhem et al. 1997, 1998; Björkhem and Eggertsen 2001). Consequently, it can be assumed that hepatic CYP39A1 expression is responsible for the subsequent 7α-hydroxylation of the cerebral metabolite in the liver.

CYP7B1 mRNA expression was primarily shown in a variety of human tissues when conducting multiple northern blot analyses (including a non-specified whole brain sample), whereas CYP7A1 mRNA expression was detected exclusively in the human liver (Wu et al. 1999). The present block-cycler RT–PCR experiments yielded similar results with the detection of CYP7A1 mRNA expression in liver and prostate only (Fig. 8a). In contrast, significant CYP7B1 expression was demonstrated in a variety of human tissues and cell lines (Fig. 8b). Subsequent real-time RT–PCR analyses verified these results and demonstrated high CYP7B1 mRNA expression in the human prostate, heart muscle, temporal lobe, and liver (Table 2).

Previous multiple northern blot analysis showed that CYP39A1 is exclusively expressed in murine liver tissue (Li-Hawkins et al. 2000a). The present block-cycler RT–PCR analysis yielded mRNA expression of the human isozyme in human liver, prostate and, as shown here for the first time, to an apparently lesser also extent in the human CNS (Fig. 8c). These results could be verified via real-time RT–PCR analysis that demonstrated a high CYP39A1 mRNA expression in the human liver, prostate and heart muscle but only low expression of the isozyme in the human temporal lobe (Table 2). Translation of the CYP39A1 mRNA transcript into active protein in the human CNS was not investigated during the present study. However, it cannot entirely be ruled out that the expression of CYP39A1 in the human brain contributes to the cerebral 7α-hydroxylation of DHEA as, to the authors' knowledge, the catalytic activity of the enzyme toward this substrate has not been studied.

The presence of DHEA 7α-hydroxylase activity has been established in many murine tissues and its production rate is highest in the brain (Morfin and Starka 2001). Humans show considerably higher plasma and brain tissue DHEA levels than rodents (de Peretti and Forest 1976; Fehér et al. 1977; Cutler et al. 1978; Corpechot et al. 1981; Lanthier and Patwardhan 1986) and the compound is known to accumulate within the brain (Baulieu and Robel 1996). Given this and assuming a catabolic function of brain tissue DHEA 7α-hydroxylase, one should expect a higher 7α-hydroxylase activity in human brain tissue than in rodent brain tissue. Instead, we found that the 7α-hydroxylation of DHEA is lower in the human cerebrum than in the rodent cerebrum (Table 1, Fig. 7). Consequently, one might presume an essential metabolic role of DHEA 7α-hydroxylase activity in the CNS. DHEA as well as Δ5-androstenediol and, in particular, their 7-hydroxylated metabolites possess immunomodulatory and antiglucocorticoid properties (Morfin and Courchay 1994; Padgett and Loria 1994; Hampl et al. 1997; Loria 1997; Lafaye et al. 1999). Therefore, it might be assumed that endocrine regulation of the immune response or counteracting the deleterious effects of neurotoxic glucocorticoids is a possible function of cerebral 7α-hydroxylase and 17-ketosteroid reductase activity. Interestingly, widespread mRNA expression of CYP7B1 in fetal mice tissues showed an overall decline towards birth (Bean et al. 2001). An increasing restriction to the hippocampus was observed in the fetal mice brain and the highest expression levels were seen when the fetus was most vulnerable to steroid excess. These results are consistent with a neuroprotective function of CYP7B1. Furthermore, and to the authors' best knowledge, neurostimulatory or neuromodulatory effects of 7α-hydroxy DHEA or Δ5-androstenediol have not been observed to date.

In conclusion, we confirmed relatively high and ubiquitous CYP7B1 mRNA expression in the human temporal lobe as well as well as in a variety of other tissues. Furthermore, ubiquitous metabolism of DHEA via oxysterol 7α-hydroxylase and 17β-HSD activity without sex differences was demonstrated in the mature human temporal lobe of all patients. These data, in the context of the above-mentioned previous studies, led us to assume a neuroprotective function of the cerebral enzymes, rather than a distinct brain-specific function such as neurostimulation or neuromodulation. The participation of subcortical white matter 17β-HSD activity and cerebral neocortex 7α-hydroxylase activity in cerebral DHEA metabolism indicates the co-operation of neurons and glia in neurosteroid metabolism.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Source of tissue
  5. Steroids and chemicals
  6. Buffers
  7. Tissue preparation
  8. Measurement of enzymes activities
  9. Gas chromatography mass spectrometry (GC–MS) analysis
  10. Messenger RNA preparation and analysis
  11. Data analysis
  12. Results
  13. Human brain tissue DHEA metabolism with reduced pyridine nucleotides as coenzymes
  14. Subcellular distribution of 7α-hydroxylase and 17β-HSD activity
  15. GC–MS analysis
  16. Rodent brain tissue DHEA metabolism with NADPH as coenzyme
  17. Kinetic properties of cerebral 7α-hydroxylase and 17β-HSD activity, effects of different pH-values and effects of the cytochrome P450 inhibitor ketoconazole
  18. 7α-Hydroxylase and 17β-HSD activity in CX and SC preparations
  19. RT–PCR analyses of CYP7A1, CYP7B1 and CYP39A1 mRNA expression
  20. Discussion
  21. Acknowledgements
  22. References
  • Adamski J. and Jakob F. J. (2001) A guide to 17β-hydroxysteroid dehydrogenases. Mol. Cell. Endocrinol. 171, 14.
  • Akwa Y., Morfin R. F., Robel P. and Baulieu E. E. (1992) Neurosteroid metabolism: 7α-hydroxylation of dehydroepiandrosterone and pregnenolone by rat brain microsomes. Biochem. J. 288, 959964.
  • Attal-Khemis S., Dalmeyda V., Michot J. L., Roudier M. and Morfin R. (1998) Increased total 7α-hydroxy-dehydroepiandrosterone in serum of patients with Alzheimer's disease. J. Gerontol. Biol. 53, B125B132.
  • Baulieu E. E. (1996) Dehydroepiandrosterone (DHEA): a fountain of youth? J. Clin. Endocrinol. Metab. 81, 31473151.
  • Baulieu E. E. and Robel P. (1996) Dehydroepiandrosterone and dehydroepiandrosterone sulphate as neuroactive neurosteroids. J. Endocrinol. 150, S221S239.
  • Bean R., Seckl J., Lathe R. and Martin C. (2001) Ontogeny of the neurosteroid enzyme Cyp7b in the mouse. Mol. Cell. Endocrinol. 174, 137144.
  • Björkhem I. and Eggertsen G. (2001) Genes involved in initial steps of bile acid synthesis. Curr. Opin. Lipidol. 12, 97103.
  • Björkhem I., Lütjohann D., Breuer O., Sakinis A. and Wennmalm A. (1997) Importance of a novel oxidative mechanism for elimination of brain cholesterol. Turnover of cholesterol and 24(S)-hydroxycholesterol in rat brain as measured with 18O2 techniques in vivo and in vitro. J. Biol. Chem. 272, 3017830184.
  • Björkhem I., Lütjohann D., Diczfalusy U., Stahle L., Ahlborg G. and Wahren J. (1998) Cholesterol homeostasis in human brain: turnover of 24S-hydroxycholesterol and evidence for a cerebral origin of most of this oxysterol in the circulation. J. Lipid Res. 39, 15941600.
  • Chiang J. Y. L. (1998) Regulation of bile acid synthesis. Front. Biosci. 3, D176D193.
  • Compagnone N. A. and Mellon S. H. (1998) Dehydroepiandrosterone: a potential signalling molecule for neocortical organization during development. Proc. Natl Acad. Sci. USA 95, 46784683.
  • Corpechot C., Robel P., Axelson M., Sjövall J. and Baulieu E. E. (1981) Characterization and measurement of dehydroepiandrosterone sulphate in rat brain. Proc. Natl Acad. Sci. USA 78, 47044707.
  • Cutler G. B. Jr, Glenn M., Bush M., Hodgen G. D., Graham C. E. and Loriaux D. L. (1978) Adrenarche: a survey of rodents, domestic animals, and primates. Endocrinology 103, 21122118.
  • Doostzadeh J., Cotillon A. C. and Morfin R. (1997) Dehydroepiandrosterone 7α- and 7β-hydroxylation in mouse brain microsomes. Effects of cytochrome P450 inhibitors and structure-specific inhibition by steroid hormones. J. Neuroendocrinol. 9, 923928.
  • Dufort I., Rheault P., Huang X. F., Soucy P. and Luu-The V. (1999) Characteristics of a highly labile human type 5 17β-hydroxysteroid dehydrogenase. Endocrinology 140, 568574.
  • Fehér T., Bodrogi L., Fehér K. G., Poteczin E. and Kölcsey I. S. (1977) Free and solvolysable dehydroepiandrosterone and androsterone in blood of mammals under physiological conditions and following administration of dehydroepiandrosterone. Acta Endocrinol. 85, 126133.
  • Garcia-Estrada J., Luquin S., Fernandez A. M. and Garcia-Segura L. M. (1999) Dehydroepiandrosterone, pregnenolone and sex steroids down-regulate reactive astroglia in the male rat brain after a penetrating brain injury. Int. J. Dev. Neurosci. 17, 145151.
  • Hampl R., Morfin R. and Starka L. (1997) Minireview: 7-hydroxylated derivatives of dehydroepiandrosterone: what are they good for? Endocr. Regul. 31, 211218.
  • Herbert J. (1998) Neurosteroids, brain damage, and mental illness. Exp. Gerontol. 33, 713727.
  • Hinson J. P. and Raven P. W. (1999) DHEA deficiency syndrome: a new term for old age? J. Endocrinol. 163, 15.
  • Ishibashi S., Schwarz M., Frykman P. K., Herz J. and Russell D. W. (1996) Disruption of cholesterol 7α-hydroxylase gene in mice. I. Post-natal lethality reversed by bile acid and vitamin supplementation. J. Biol. Chem. 271, 1801718023.
  • Khorram O. (1996) DHEA: a hormone with multiple effects. Curr. Opin. Obstet. Gynecol. 8, 351354.
  • Kurose K., Tohkin M., Ushio F. and Fukuhara M. (1998) Cloning and characterization of Syrian hamster testosterone 7α-hydroxylase, CYP2A9. Arch. Biochem. Biophys. 351, 6065.
  • Kurose K., Isozaki E., Tohkin M. and Fukuhara M. (1999) Cloning and expression of a new member of the cyochrome P450, CYP2A15 from Chinese hamster, encoding testosterone 7α-hydroxylase. Arch. Biochem. Biophys. 371, 270276.
  • Labrie F., Belanger A., Luu-The V., Labrie C., Simard J., Cusan L., Gomez J. L. and Candas B. (1998) DHEA and the intracrine formation of androgens and estrogens in peripheral target tissues: its role during aging. Steroids 63, 322328.
  • Labrie F., Luu-The V., Lin S. X., Simard J., Labrie C., El-Alfy M., Pelletier G. and Belanger A. (2000) Intracrinology: role of the family of 17β-hydroxysteroid dehydrogenases in human physiology and disease. J. Mol. Endocrinol. 25, 116.
  • Lafaye P., Chmielewski V., Nato F., Mazie J. C. and Morfin R. (1999) The 7alpha-hydroxysteroids produced in human tonsils enhance the immune response to tetanus toxoid and Bordetella pertussis antigens. Biochim. Biophys. Acta 1472, 222231.
  • Lanthier A. and Patwardhan V. V. (1986) Sex steroids and 5-en-3 beta-hydroxysteroids in specific regions of the human brain and cranial nerves. J. Steroid. Biochem. 25, 445449.
  • Li H., Klein G., Sun P. and Buchan A. M. (2001) Dehydroepiandrosterone (DHEA) reduces neuronal injury in a rat model of global cerebral ischemia. Brain Res. 888, 263266.
  • Li-Hawkins J., Lund E. G., Bronson A. D. and Russell D. W. (2000a) Expression cloning of an oxysterol 7alpha-hydroxylase selective for 24-hydroxycholesterol. J. Biol. Chem. 275, 1654316549.
  • Li-Hawkins J., Lund E. G., Turley S. D. and Russell D. W. (2000b) Disruption of the oxysterol 7α-hydroxylase gene in mice. J. Biol. Chem. 275, 1653616542.
  • Lin H. K., Jez J. M., Schlegel B. P., Peehl D. M., Pachter J. A. and Penning T. M. (1997) Expression and characterization of recombinant type 2 3α-hydroxysteroid dehydrogenase (HSD) from human prostate: demonstration of bifunctional 3α/17β-HSD activity and cellular distribution. Mol. Endocrinol. 11, 19711984.
  • Loria R. M. (1997) Antiglucocorticoid function of androstenetriol. Psychoneuroendocrinology 22, S103S108.
  • Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1952) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275.
  • Lütjohann D., Breuer O., Ahlborg G., Nennesmo I., Sidén Å?., Diczfalusy U. and Björkhem I. (1996) Cholesterol homeostasis in human brain: evidence for an age-dependent flux of 24S-hydroxycholesterol from brain into circulation. Proc. Natl Acad. Sci. USA 93, 97999804.
  • Majewska M. D. (1995) Neuronal actions of dehydroepiandrosterone. Possible roles in brain development, aging, memory, and affect. Ann. NY Acad. Sci. 774, 111120.
  • Martin C., Bean R., Rose K., Habib F. and Seckl J. (2001) CYP7B1 catalyses the 7α-hydroxylation of dehydroepiandrosterone and 25-hydroxycholesterol in rat prostate. Biochem. J. 355, 509515.
  • Mohan P. F. and Cleary M. P. (1992) Studies on nuclear binding of dehydroepiandrosterone in hepatocytes. Steroids 57, 244247.
  • Morfin R. and Courchay G. (1994) Pregnenolone and dehydroepiandrosterone as precursors of native 7-hydroxylated metabolites which increase the immune response in mice. J. Steroid Biochem. Mol. Biol. 50, 91100.
  • Morfin R. and Starka L. (2001) Neurosteroid 7-hydroxylation products in the brain. Int. Rev. Neurobiol. 46, 7995.
  • Nagata K., Matsunaga T., Gillette J., Gelboin H. V. and Gonzales F. J. (1987) Rat testosterone 7α-hydroxylase: isolation, sequence, and expression of cDNA and its developmental regulation and induction by 3-methylcholanthrene. J. Biol. Chem. 262, 27872793.
  • Norlin M., Andersson U., Björkhem I. and Wikvall K. (2000) Oxysterol 7α-hydroxylase activity by cholesterol 7α-hydroxylase (CYP7A). J. Biol. Chem. 275, 3404634053.
  • Padgett D. A. and Loria R. M. (1994) In vitro potentiation of lymphocyte activation by dehydroepiandrosterone, androstenediol, and androstenetriol. J. Immunol. 153, 15441552.
  • Peltoketo H., Luu-The V., Simard J. and Adamski J. (1999) 17β-hydroxysteroid dehydrogenase (HSD)/17-ketosteroid reductase (KSR) family; nomenclature and main characteristics of the 17HSD/KSR enzymes. J. Mol. Endocrinol. 23, 111.
  • De Peretti E. and Forest M. G. (1976) Unconjugated dehydroepiandrosterone plasma levels in normal subjects from birth to adolescence in human: the use of a sensitive radioimmunoassay. J. Clin. Endocrinol. Metab. 43, 991.
  • Rose K., Stapleton G., Dott K., Kieny M. P., Best R., Schwarz M., Russell D. W., Björkhem I., Seckl J. R. and Lathe R. (1997) Cyp7b, a novel brain cytochrome P450, catalyses the synthesis of neurosteroids 7α-hydroxy dehydroepiandrosterone and 7α-hydroxy pregnenolone. Proc. Natl Acad. Sci. USA 94, 49254930.
  • Rose K., Allan A., Gauldie S., Stapleton G., Dobbie L., Dott K., Martin C., Wang L., Hedlund E., Seckl J. R., Gustafsson J. A. and Lathe R. (2001) Neurosteroid hydroxylase CYP7B: vivid reporter activity in dentate gyrus of gene-targeted mice and abolition of a widespread pathway of steroid and oxysterol hydroxylation. J. Biol. Chem. 276, 2393723944.
  • Russell D. W. (2000) Oxysterol biosynthetic enzymes. Biochim. Biophys. Acta 1529, 126135.
  • Schwarz M., Lund E. G., Setchell K. D. R., Kayden H. J., Zerwekh J. E., Björkhem I., Herz J. and Russell D. W. (1996) Disruption of cholesterol 7α-hydroxylase gene in mice. II. Bile acid deficiency is overcome by induction of oxysterol 7α-hydroxylase. J. Biol. Chem. 271, 1802418031.
  • Schwarz M., Lund E. G., Lathe R., Björkhem I. and Russell D. W. (1997) Identification and characterization of a mouse oxysterol 7α-hydroxylase cDNA. J. Biol. Chem. 272, 2399524001.
  • Setchell K. D., Schwarz M., O'Connell N. C., Lund E. G., Davis D. L., Lathe R., Thompson H. R., Weslie Tyson R., Sokol R. J. and Russell D. W. (1998) Identification of a new inborn error in bile acid synthesis: mutation of the oxysterol 7α-hydroxylase gene causes severe neonatal liver disease. J. Clin. Invest. 102, 16901703.
  • Steckelbroeck S., Stoffel-Wagner B., Reichelt R., Schramm J., Bidlingmaier F., Siekmann L. and Klingmüller D. (1999) Characterization of 17β-hydroxysteroid dehydrogenase activity in the human brain. J. Neuroendocrinol. 6, 457464.
  • Steckelbroeck S., Watzka M., Stoffel-Wagner B., Hans V. H. J., Redel L., Clusmann H., Elger C. E., Bidlingmaier F. and Klingmüller D. (2001) Expression of 17β-hydroxysteroid dehydrogenase type 5 mRNA in the human temporal lobe. Mol. Cell. Endocrinol. 171, 165168.
  • Stoffel-Wagner B., Watzka M., Steckelbroeck S., Schramm J., Bidlingmaier F. and Klingmüller D. (1999) Expression of 17β-hydroxysteroid dehydrogenase types 1, 2, 3 and 4 in the human temporal lobe. J. Endocrinol. 160, 119126.
  • Watson R. R., Huls A., Araghinikuam M. and Chung S. (1996) Dehydroepiandrosterone and diseases of aging. Drugs Aging 9, 274291.
  • Waxman D. J., Lapenson D. P., Nagata K. and Conlon H. D. (1990) Participation of two structurally related enzymes in rat hepatic microsomal androstenedione 7α-hydroxylation. Biochem. J. 265, 187194.
  • Williams J. R. (2000) The effects of dehydroepiandrosterone on carcinogenesis, obesity, the immune system, and aging. Lipids 35, 325331.
  • Wolf O. T. and Kirschbaum C. (1999) Actions of dehydroepiandrosterone and its sulfate in the central nervous system: effects on cognition and emotion in animals and humans. Brain Res. Brain Res. Rev. 30, 264288.
  • Wu Z., Martin K. O., Javitt N. B. and Chiang J. Y. (1999) Structure and functions of human oxysterol 7α-hydroxylase cDNAs and gene CYP7B1. J. Lipid Res. 40, 21952203.