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HUMAN LIVER S9 FRACTIONS: METABOLISM OF DEHYDROEPIANDROSTERONE, EPIANDROSTERONE, AND RELATED 7-HYDROXYLATED DERIVATIVES

Laboratoire de Biotechnologie, Conservatoire National des Arts et Métiers, Paris, France

(Received November 15, 2004; accepted January 11, 2005)

	Abstract

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Dehydroepiandrosterone (DHEA) and 3ß-hydroxy-5-androstan-17-one (epiandrosterone, EpiA) are both precursors for 7- and 7ß-hydroxylated metabolites in the human brain. These 7-hydroxylated derivatives were shown to exert anti-glucocorticoid and neuroprotective effects. When these steroids are administered per os to humans, the first organ encountered is the liver, where extensive metabolism takes place. The objective of this work was to assess the cofactor dependence and metabolism of DHEA, EpiA, and their 7-hydroxylated derivatives in S9 fractions of human liver, using a radiolabeled steroid substrate for quantification and gas chromatography-mass spectrometry for identification. The best transformation yields were obtained with NADPH and were larger in female than in male. Results showed that both DHEA and EpiA mainly transformed into their 17ß-hydroxylated derivatives, 7- or 16-hydroxylated metabolites under NAD(P)H conditions, and 5-androstane-3,17-dione for EpiA under NAD(P)⁺ conditions. In turn, 7-hydroxy-DHEA and 7ß-hydroxy-DHEA were partly transformed into each other via a 7-oxo-DHEA intermediate and were reduced into the 17ß-hydroxy derivative, respectively. The same type of transformations occurred for 7-hydroxy-EpiA and 7ß-hydroxy-EpiA, except that no 7-oxo-EpiA intermediate was obtained. These findings determine the presence of enzymes responsible for the 7- and 16-hydroxylation in the human liver, the 11ß-hydroxysteroid dehydrogenase type 1 responsible for the oxidoreduction of the 7-hydroxylated substrates, and the 17ß-hydroxysteroid dehydrogenase responsible for the reduction of 17-oxo-steroids into 17ß-hydroxysteroids.

Investigations of DHEA metabolism in mouse brain tissue provided evidence for the NADPH-dependent formation of almost equal quantities of both 7-hydroxylated and 7ß-hydroxylated transformation products (Doostzadeh et al., 1997; Doostzadeh and Morfin, 1997). No P450 was found to be responsible for 7ß-hydroxy-DHEA production. Investigations with knockout mice for CYP7B1 showed that neither 7-hydroxy-DHEA nor 7ß-hydroxy-DHEA was produced (Lathe, 2002), which implied that the CYP7B1 was a requisite for their production. The question arising on the process responsible for 7ß-hydroxy-DHEA has been tentatively addressed recently with data showing that in liver microsomes, the 11ß-hydroxysteroid dehydrogenases (11ß-HSDs) and unknown enzymes may be involved in an oxidoreduction process transforming 7-hydroxy-DHEA into 7-oxo-DHEA and 7ß-hydroxy-DHEA (Robinzon et al., 2003; Kim et al., 2004a).

Neuroprotection exerted by 7-hydroxy-DHEA was first suggested after work with rat hippocampal cell cultures and comparison with aromatization products (Jellinck et al., 2001). More recently, a test for that hypothesis was developed on a model of hypoxia-induced neurodegeneration in organotypic rat hippocampal slice cultures (Pringle et al., 1997) and on an in vivo model of cerebral ischemia obtained in rats by four-vessel occlusion, which tested several 7-hydroxylated steroids (Pringle et al., 2003). It was then clearly shown that DHEA, EpiA, 7-keto-EpiA, 5-androstane-3ß,7ß,17ß-triol, and estradiol had no neuroprotective effects, whereas 7-hydroxy-DHEA, 7-hydroxy-EpiA, and 7ß-hydroxy-EpiA prevented neuronal damage at 24 h posthypoxia (Pringle et al., 2003). The best results obtained in vivo were with 7ß-hydroxy-EpiA (0.03 mg/kg injected in the femoral vein), which suggested that native 7ß-hydroxysteroids may exert a neuroprotective function (Pringle et al., 2003; Dudas et al., 2004).

Possible pharmacological use per os of any of the neurosteroids described (i.e., DHEA, 7-hydroxy-DHEA, 7ß-hydroxy-DHEA, EpiA, 7-hydroxy-EpiA, 7ß-hydroxy-EpiA) implies that metabolism in the liver will determine drug transformation and subsequent availability of products to the brain. Our goals were to study the yields of these transformations in human liver S9 fractions and to identify the metabolites produced. To achieve this, we produced the labeled steroid substrates, incubated them under various conditions with human liver S9 preparations, and then identified the metabolites by gas chromatography/mass spectrometry.

	Materials and Methods

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Steroids and Reagents. [4-¹⁴C]DHEA (55.5 mCi/mmol) and [4-¹⁴C]5-dihydrotestosterone (53.6 mCi/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Paris, France). [4-¹⁴C]7-hydroxy-DHEA, [4-¹⁴C]-EpiA, [4-¹⁴C]7ß-hydroxy-DHEA, [4-¹⁴C]7-hydroxy-EpiA, and [4-¹⁴C]7ß-hydroxy-EpiA were produced as previously described (Chalbot et al., 2002). DHEA, EpiA, 5-androstene-3ß,17ß-diol, 5-androstane-3ß,17ß-diol, 5-androstane-3,17ß-diol, 17ß-hydroxy-5-androstan-3-one, testosterone, 4-androstene-3,17-dione, 5-androstane-3,17-dione, 17ß-estradiol, estrone, NADH, NAD⁺, NADP⁺, glucose 6-phosphate, glucose-6-phosphate dehydrogenase, and buffer salts were obtained from Sigma-Aldrich (L'Isle d'Abeau Chesnes, France). The steroid references 7ß-hydroxy-EpiA, 7-hydroxy-EpiA, 6-hydroxy-EpiA, and 7-oxo-EpiA were produced first from EpiA after incubation with the Rhizopus nigricans strain and purification according to Chambers et al. (1973). A custom chemical synthesis by Roowin S.A. (Paris, France) provided milligram quantities of chemically pure 7-hydroxy-DHEA, 7ß-hydroxy-DHEA, 7-hydroxy-EpiA, and 7ß-hydroxy-EpiA. 5-Androstane-3ß,7,17ß-triol and 5-androstane-3ß,7ß,17ß-triol were obtained after a KBH₄ reduction of their respective 17-oxo precursors. 7-Oxo-DHEA, 16-hydroxy-DHEA, and 3ß,16-dihydroxy-5-androstan-17-one were obtained from Steraloids (Newport, RI). Both 5-androstene-3ß,7,17ß-triol and 5-androstene-3ß,7ß,17ß-triol were obtained after a KBH₄ reduction of 7-oxo-DHEA. 3ß,17ß-Dihydroxy-5-androstan-7-one and 7-oxo-EpiA were gifts from Dr. E. R. H. Jones (Oxford University, Oxford, UK). All solvents (Merck, Darmstadt, Germany) were of reagent grade.

Human Liver S9 Fractions. Both male and female human liver S9 pools transported in liquid nitrogen were from In Vitro Technologies Inc. (Baltimore, MD) and kept at -80°C before use. The female S9 pool derived from 10 donors with a 22 mg/ml protein content and a catalytic 7-ethoxycoumarin O-deethylase activity of 59 pmol/mg protein/min. The male S9 pool derived from 15 donors with a 21 mg/ml protein content and with a catalytic 7-ethoxycoumarin O-deethylase activity of 105 pmol/mg protein/min. These values correlated well with functional P450 activities and those reported in primary cultures of human hepatocytes obtained from different liver samples (Serralta et al., 2003).

Incubation Conditions. Incubation of 4-¹⁴C-labeled steroid substrates was carried out in 1 ml total volume with human liver S9 fractions supplemented with various cofactors. Briefly, the isotopically diluted 4-¹⁴C-labeled steroid substrate (20 nmol, 50,000 dpm) in ethanol was dried under vacuum at the bottom of 10-ml borosilicate glass tubes prior to the addition of the thawed human liver S9 fraction (0.75 mg of proteins) and either a 10 mM Tris-HCl buffer (pH 7.4) containing 1 mM EDTA or a 66.7 mM phosphate buffer (pH 7.4) containing 1 mM EDTA, followed by a 0.25-ml solution in a buffer of one of the following: 1 mg of NADH, 1 mg of NADP⁺, 1 mg of NAD⁺, or a 0.65 mM concentration of the NADPH-regenerating system in 2% NaHCO₃ containing NADP⁺, 7.09 mM glucose 6-phosphate, and 1.5 U/ml glucose-6-phosphate dehydrogenase. Identical conditions were used for control incubations with either 10-min boiled or unsupplemented S9 fractions. The tubes were left open during the course of incubation with shaking at 37°C for 20 min. Incubations were stopped after the addition of 0.5 ml of acetone followed by 4 ml of ethyl acetate. The extraction process continued with 4 ml of ethyl acetate and was repeated three times. Counting of the aqueous phase and dpm computation were carried out with an Amersham Biosciences AB (Uppsala, Sweden) 1209 rack beta liquid scintillation counter fitted with external standard equipment and automatic background subtraction (PerkinElmer, France).

Steroid Metabolite Separation and Quantification. Ethyl acetate extracts recovered from incubations with 4-¹⁴C-labeled steroids were analyzed by thin-layer chromatography (TLC) on ready to use silica-60-coated glass plates (Merck) developed once either in ethyl acetate or in benzene/ethanol (9:1 v/v). In these systems, the metabolites of each steroid substrate tested were clearly separated, except for the 7-hydroxy-EpiA/7ß-hydroxy-EpiA mixture. Radio-steroids were located on the plates by autoradiography, recovered by scrapping with a razor blade, and eluted from the gel with methanol/ethyl acetate (1:9 v/v). Autoradiography of the thin-layer chromatograms with BioMax Light X-ray film (Eastman Kodak, Rochester, NY) was performed to locate the radio-steroids on TLC silica gel plates. Quantitative scanning of the plates was carried out with use of a Berthold automatic TLC-linear analyzer (PerkinElmer, France). High performance liquid chromatography (HPLC) was used in response to the poor TLC separation of the 7-hydroxy-EpiA/7ß-hydroxy-EpiA mixture. The Shimadzu LC-6A HPLC apparatus was fitted with a 100-µl injection loop and a 26-cm C₁₈-coated silica (5-µm) column (Ultra-base; Societé Françoise Chromato Colonne) from Interchim (Montluçon, France) that was maintained at 30°C. Elution was carried out at 0.7 ml · min^-1 with methanol/water (6:4 v/v) and collection of 0.25-ml portions split for GC/MS identification and for quantification by counting.

Steroid Metabolite Identification (GC/MS Analysis). R_f values after TLC and retention times after HPLC were compared with those of authentic standards, and each major steroid metabolite was identified by GC/MS analysis with an Agilent Technologies (Massy, France) system including a 6890N network GC apparatus coupled with a 5973 network mass selective detector. Trimethylsilyl (TMS) ether derivatives of authentic steroid references and unidentified steroid metabolites were prepared just before analysis by reacting the dried steroids in 30 µl of pyridine with 20 µl of bis-silyl-trifluoroacetic acid containing 1% trimethylchlorosilane (Pierce Chemical, Rockford, IL) at 60°C for 60 min. TMS derivatives were dried and taken up in toluene for injection on an HP-5MS fused silica capillary column (30 m, 0.25-mm i.d.) coated at 5% with a phenylmethylsiloxane phase (0.25-µm thickness). The oven temperature was programmed from 80°C for 1 min and then with a temperature increment of 30°C/min, up to 240°C. After 8 min at 240°C, the temperature was increased again at 30°C/min up to 300°C and then maintained up to the end of the run. The column was directly coupled to the quadrupole mass spectrometer ionization chamber, where the source was set at 230°C and the energy of bombarding electrons was at 70 eV. The mass abundance was determined two times per second by scanning from m/z 50 to m/z 800. The identity of each of the major steroid metabolites produced by the human liver S9 fractions was assessed by comparison of their retention time and mass spectra with those of the 25 authentic reference compounds. The assessment used the match-up function on the built-in library in the MSD Chemstation software D.01.00 (Agilent Technologies).

	Results

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Preliminary experiments were carried out with [4-¹⁴C]DHEA or [4-¹⁴C]EpiA and 0.75 mg of protein from human liver S9 fractions with use of either a 10 mM Tris-HCl buffer (pH 7.4) or a 66.7 mM phosphate buffer (pH 7.4), both containing 1 mM EDTA, and in the presence of the NADPH-regenerating system, or NADH, NAD⁺, and NADP⁺. The best yields were obtained with the phosphate buffer that was then selected for all other incubations. In these conditions, the control incubations carried out with 10-min boiled S9 fractions or in the absence of cofactors yielded no transformation products for any of the steroid substrates used.

DHEA Metabolism. After GC/MS comparison, the metabolites produced were identified with 7-hydroxy-DHEA, 7ß-hydroxy-DHEA, 7-oxo-DHEA, 16-hydroxy-DHEA, and 5-androstene-3ß,17ß-diol authentic steroids (Table 1). One minor metabolite was not separated from 7-oxo-DHEA after TLC; however, after GC under a TMS derivative form of the mixture, it could not be formally identified. Fragments in its mass spectrum were those of a monohydroxylated DHEA derivative, thus leading to its qualification as n-hydroxy-DHEA (Table 1). The liver S9 fractions from both male and female donors transformed DHEA mainly under reductive cofactor supplementation. The largest yields in metabolism concerned 16-hydroxy-DHEA production that was obtained after NADPH supplementation. For 5-androstene-3ß,17ß-diol production, NADH was the cofactor required. Both oxidized cofactors gave poor yields in transformation products or none at all (Table 2). Reductive transformation yields were the largest with female liver S9 fractions, except for 5-androstene-3ß,17ß-diol production, where male liver S9 fractions gave better yields (Table 2). This implied that the liver contents with P450s responsible for DHEA hydroxylation were greater in females than in males, whereas the male liver contained more of the NADH-dependent 17ß-hydroxysteroid oxidoreductase than in the female liver. The female liver S9 fractions were used for the studies of 7- and 7ß-hydroxy-DHEA metabolism because of the equivalent DHEA metabolism in both sexes and higher yields obtained in females.

View this table: [in this window] [in a new window]   TABLE 1 Identification of the steroid metabolites produced after incubation of 20 µM DHEA, 7-hydroxy-DHEA, and 7ß-hydroxy-DHEA with human liver S9 fractions

View this table: [in this window] [in a new window]   TABLE 2 Percentage transformation yields of 20 µM DHEA, 7-hydroxy-DHEA, and 7ß-hydroxy-DHEA by human liver S9 fractions in the presence of reduced or oxidized cofactors All incubations were carried out with 0.75 mg of protein in 0.67 M phosphate buffer containing 1 mM EDTA at 37°C for 20 min. Only human female liver S9 fractions were used for 7- and 7ß-hydroxy-DHEA incubations. Data are the means ± S.D. obtained from triplicate experiments.

7-Hydroxy-DHEA Metabolism. The three metabolites produced by female liver S9 fractions were identified with 7ß-hydroxy-DHEA, 7-oxo-DHEA, and 5-androstene-3ß,7,17ß-triol authentic steroids (Table 1). The largest yields of 7ß-hydroxy-DHEA were obtained under NADPH conditions, whereas those of 7-oxo-DHEA required NADP⁺ (Table 2). This suggests that a NADPH-driven oxidoreduction system is responsible for the production of 7ß-hydroxy-DHEA. The production of 5-androstene-3ß,7,17ß-triol required the action of a 17ß-hydroxysteroid oxidoreductase. This enzyme was almost as active with NADP(H) as with NAD(H), which indicates either that the 7-hydroxy-DHEA substrate could be reduced by both cofactors or that two isoforms of the same enzyme were present with near equivalent activities.

7ß-Hydroxy-DHEA Metabolism. Only two metabolites were produced by female liver S9 fractions under NADPH supplementation, and these were identified with 7-oxo-DHEA and 5-androstene-3ß,7ß,17ß-triol authentic steroids (Table 1). No trace of 7-hydroxy-DHEA was detected, thus implying that the 7-oxo-DHEA produced could not be reduced back into the 7-epimer. As for 7-hydroxy-DHEA, the production of 5-androstene-3ß,7ß,17ß-triol required the action of a 17ß-hydroxysteroid oxidoreductase that was active under NADPH conditions (Table 2).

EpiA Metabolism. After a GC/MS comparison, the metabolites produced were identified with 7ß-hydroxy-EpiA, 16-hydroxy-EpiA, 5-androstane-3ß,17ß-diol, and 5-androstane-3,17-dione authentic steroids (Table 3). One minor metabolite was not formally identified because of the lack of appropriate reference and it was not investigated further (Table 3). The liver S9 fractions from both male and female donors transformed EpiA mainly under reductive conditions. The largest yields in metabolism were obtained after NADPH supplementation, except for 5-androstane-3ß,17ß-diol and 5-androstane-3,17-dione, where NADH and NAD⁺ were the required cofactors, respectively (Table 4). No trace of 7-hydroxy-EpiA was obtained, in contrast to low but significant amounts of 7ß-hydroxy-EpiA. In the female, both 16-hydroxy-EpiA and 5-androstane-3ß,17ß-diol were the major metabolites produced under NADPH and NADH supplementation, respectively. The same cofactor dependence occurred under oxidative conditions resulting in a larger production of 5-androstane-3,17-dione in the female than in the male. This suggested that a 3ß-hydroxysteroid oxidoreductase was present in larger quantities in the female liver as opposed to the male. The female liver S9 fractions were used for the studies of 7- and 7ß-hydroxy-EpiA metabolism because of the equivalent EpiA metabolic patterns in both sexes and the larger yields obtained in females,

View this table: [in this window] [in a new window]   TABLE 3 Identification of the steroid metabolites produced after incubation of 20 µM EpiA, 7-hydroxy-EpiA, and 7ß-hydroxy-EpiA with human liver S9 fractions

View this table: [in this window] [in a new window]   TABLE 4 Percentage transformation yields of 20 µM EpiA, 7-hydroxy-EpiA, and 7ß-hydroxy-EpiA by human liver S9 fractions in the presence of reduced or oxidized cofactors All incubations were carried out with 0.75 mg of protein in 0.067 M phosphate buffer containing 1 mM EDTA at 37°C for 20 min. Only human female liver S9 fractions were used for 7- and 7ß-hydroxy-EpiA incubations. Data are the means ± S.D. obtained from triplicate experiments.

7-Hydroxy-EpiA Metabolism. Neither TLC nor HPLC permitted the separation of the two metabolites produced after incubation with female liver S9 fractions (Table 4). After a TMS derivative formation, the two metabolites were separated by GC and identified with 7ß-hydroxy-EpiA and 5-androstane-3ß,7,17ß-triol authentic steroids (Table 3). The 7ß-hydroxy-EpiA/5-androstane-3ß,7,17ß-triol ratio obtained after GC/MS separation indicated that 7ß-hydroxy-EpiA only was produced in the presence of NADP⁺, whereas increasing amounts of 5-androstane-3ß,7,17ß-triol appeared under NADPH, NAD⁺, and NADH conditions. No trace of 7-oxo-EpiA was detected; therefore, the putative oxidoreduction of 7-hydroxy-EpiA into 7ß-hydroxy-EpiA could only be suspected. Reductive conditions were necessary for a significant production of 5-androstane-3ß,7,17ß-triol by the two NADPH-dependent and NADH-dependent 17ß-hydroxysteroid oxidoreductases.

7ß-Hydroxy-EpiA Metabolism. Two metabolites were obtained after incubation with female liver S9 fractions and were identified with 7-hydroxy-EpiA and 5-androstane-3ß,7ß,17ß-triol authentic steroids by GC/MS as shown in Table 3. Significant and major yields of 7-hydroxy-EpiA were obtained under reductive and oxidative conditions, which led us to suspect an oxidoreductive mechanism (Table 4). The highest amounts of 5-androstane-3ß,7ß,17ß-triol were produced under NADH supplementation, thus inferring the presence of a NADH-dependent 17ß-hydroxysteroid oxidoreductase.

	Discussion

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We used human liver S9 fractions that contained both hepatic microsomes and cytosol with active steroid-metabolizing enzymes. The age of the donors was not provided. Compared with microsomes and cytosol, S9 fractions offer a more complete although lower representation of the steroid metabolism, since they contain both phase I and phase II activity (Brandon et al., 2003). Research into DHEA metabolism helped to link our data with those of others and consequently validate our approach for the studies of EpiA and 7-hydroxysteroid metabolism. The supplementation of S9 fractions with cofactors was necessary (Brandon et al., 2003) and allowed the sorting out of enzymes through their reductive or oxidative activities and dependence upon NAD(H) or NADP(H). The use of a NADPH-regenerating system helped with the hydroxylation process carried out by the microsomal cytochromes P450, which are well known in the liver. Thus, the 7-hydroxylation of DHEA was already shown in human liver (Wu et al., 1999; Fitzpatrick et al., 2001; Miller et al., 2004) and described as resulting from catalysis by CYP3A4 (Fitzpatrick et al., 2001; Miller et al., 2004) and CYP7B1 (Wu et al., 1999). Since CYP7B1 carries out the 7-hydroxylation on most of the 3ß-hydroxysteroid substrates (Rose et al., 1997), it was surprising to find no trace of 7-hydroxy-EpiA in EpiA digests. We have reported that other human tissue preparations 7-hydroxylated EpiA (Lafaye et al., 1999) and found recently that the human CYP7B1 expressed in yeast microsomes carried out the 7-hydroxylation of EpiA with a K_M higher than that of DHEA (Kim et al., 2004b). The fact that 7ß-hydroxy-EpiA was produced instead of 7-hydroxy-EpiA suggests that one liver P450 carried out the 7ß-hydroxylation of EpiA. At present, there is no documentation to support a P450-mediated 7ß-hydroxylation of EpiA. However, the 7ß-hydroxylation of DHEA by CYP3A4 is documented (Stevens et al., 2003; Miller et al., 2004), and this P450 may also carry out the 7ß-hydroxylation of EpiA in the liver. We also observed a 7/7ß interconversion. This was reported as resulting from an oxidoreduction process catalyzed by 11ß-hydroxysteroid dehydrogenase isoforms using both 7-hydroxy-DHEA and 7ß-hydroxy-DHEA via the 7-oxo-DHEA (Robinzon et al., 2003). Our data confirm such a paradigm, with DHEA yielding at least equivalent levels of 7- and 7ß-hydroxy-DHEA produced, together with large amounts of 7-oxo-DHEA. In turn, 7-hydroxy-DHEA and 7ß-hydroxy-DHEA were both precursors for 7-oxo-DHEA. Concerning 7-hydroxy-EpiA and 7ß-hydroxy-EpiA, their interconversion was obtained without evidence for 7-oxo-EpiA production. It is then possible that either the 11ß-hydroxysteroid dehydrogenase-catalyzed oxidoreduction process reported for 7-hydroxy-DHEA (Robinzon et al., 2003) may extend to the 7-hydroxylated EpiA substrates, or that an unknown epimerase is involved in the interconversion process.

Our findings of significant amounts of 16-hydroxy-DHEA and 16-hydroxy-EpiA in DHEA and EpiA digests imply the presence of CYP3A4 and CYP3A5 in liver S9 fractions. These two P450s were described as responsible for DHEA 16-hydroxylation (Niwa et al., 1998; Miller et al., 2004) rather than CYP3A7, which is found in human endometrium, placenta, and fetal liver (Miller et al., 2004). Even though the adult human liver carries out the 16-hydroxylation of DHEA (Miller et al., 2004), no direct evidence is available for the 16-hydroxylation of EpiA. We found a significantly larger production of 16-hydroxy-DHEA and 16-hydroxy-EpiA in females than in males, thus implying sex differences in the P450s responsible. Provided that EpiA 16-hydroxylation is carried out by CYP3A4, this finding is in accordance with the higher levels of CYP3A4 found in the human female liver (Wolbold et al., 2003). In contrast, no unknown metabolite that could be 16-hydroxylated was found in digests of 7- and 7ß-hydroxy-DHEA or 7- and 7ß-hydroxy-EpiA, thus inferring that the 7-hydroxylated derivatives of DHEA and EpiA were not substrates for the 16-hydroxylase.

The human liver contains several isoforms of the 17ß-hydroxysteroid oxidoreductase that differ in their dependence for the NAD(H) or NADP(H) cofactors (Blomquist, 1995). In this study, all of the substrates tested were 17-oxo-steroids and readily transformed into their 17ß-hydroxy derivatives under the reductive conditions using either NADPH or NADH. This is in contrast with the results obtained with liver microsomes (Fitzpatrick et al., 2001; Miller et al., 2004), where no 17ß-reduction of DHEA was obtained. Since the liver S9 fractions contain both microsomes and cytosol, we may suspect the 17ß-reducing enzyme to be cytosolic. Thus, male and female livers contained both a NADP(H)-dependent and a NAD(H)-dependent 17ß-hydroxysteroid oxidoreductase almost equivalently active on the DHEA substrate. In contrast, EpiA metabolism showed none of the NADP(H)-dependent 17ß-hydroxysteroid oxidoreductase activity in the female, but a NAD(H)-dependent 17ß-hydroxysteroid oxidoreductase activity was significantly larger in the female than in the male liver. Whether these differences result from different affinities of the steroid substrates for the 17ß-hydroxysteroid oxidoreductases or from different 17ß-hydroxysteroid oxidoreductase isoforms cannot be stated from the present results.

We found that EpiA was oxidized at the 3ß-position. This is in contrast to DHEA, which was unchanged, in agreement with reports using human liver microsomes (Fitzpatrick et al., 2001; Miller et al., 2004). DHEA oxidation into 4-androstene-3,17-dione implies a two-step mechanism catalyzed by the NAD-dependent 3ß-hydroxysteroid dehydrogenase/isomerase that is commonly found in steroidogenic tissues rather than in the liver. In contrast, the presence in human liver homogenates of a microsomal 3ß-hydroxysteroid oxidoreductase has been reported (Pirog and Collins, 1999).

The present data obtained with the 7-hydroxylated EpiA and 7-hydroxylated DHEA metabolism in liver are of importance when the neuroprotective action of these steroids is considered. The putative use of 7ß-hydroxy-EpiA as a neuroprotective agent is presently sought, and it is of importance to know whether, once administered per os, the liver enzymes will dramatically decrease the blood-borne quantities brought up to the brain. Our data show that more than 90% of the 7ß-hydroxy-EpiA incubated are left intact, and that both the 5-androstane-3ß,7ß,17ß-triol and the 7-hydroxy-EpiA produced may be transformed back into 7ß-hydroxy-EpiA. The same findings apply to 7ß-hydroxy-DHEA. One shortcoming of our present data concerns the putative action of phase II enzymes that would produce water-soluble conjugated derivatives. Recovery yields after ethyl acetate extraction of the digests were between 96 and 99%, and the possible steroid metabolites in the water phase were not examined. Yet, the formation of conjugates requires the addition of specific exogenous cofactors (Brandon et al., 2003), and this was not done in the present study. Nevertheless, the present findings are indicative of the metabolites produced in both sexes, even if transposable to neither whole homogenates nor an in vivo situation.

Our findings about the metabolism of DHEA, EpiA, and 7-hydroxylated derivatives by human liver S9 fractions are summarized in Fig. 1. The main originalities in these findings are that the 7-hydroxylated derivatives of DHEA and EpiA are not substrates for cytochrome P450-hydroxylating enzymes in human liver, and that both 7- and 7ß-epimers are converted into each other. The putative involvement of the microsomal 11ß-hydroxysteroid dehydrogenases through an oxidoreductive process involving the production of a 7-oxo intermediate is appealing and justifies further investigations with use of the purified human enzymes.

View larger version (26K): [in this window] [in a new window]   FIG. 1. Summary of the metabolites identified after DHEA (A) or EpiA (B) incubations with human liver S9 fractions. All structures were identified by GC/MS except those indicated with question marks. The liver enzymes responsible for the production of metabolites are: 1, CYP7B1 or CYP3A4; 1', CYP3A4; 2, CYP3A4 or CYP3A5; 3, unknown hydroxylating enzyme; 4, 17ß-hydroxysteroid dehydrogenase; 5, 11ß-HSD1; 6, unknown oxidoreductase; 7, 3ß-hydroxysteroid dehydrogenase.

	Acknowledgments

 
We thank Patrice Rool (Roowin S.A., Paris, France) for the gift of the 7-hydroxylated DHEA and EpiA derivatives.

	Footnotes

 
This work was supported by a grant from Hunter-Fleming Inc. (UK). This work is a part of the thesis of Sonia Chalbot.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.104.003004.

ABBREVIATIONS: DHEA, dehydroepiandrosterone; P450, cytochrome P450; EpiA, epiandrosterone; TLC, thin-layer chromatography; HPLC, high pressure liquid chromatography; GC/MS, gas chromatography-mass spectrometry; 11ß-HSD1, 11ß-hydroxysteroid dehydrogenase type 1; TMS, trimethylsilyl.

Address correspondence to: Professor Robert Morfin, Biotechnologie CNAM, 2 rue Conté, 75003 Paris, France. E-mail: morfin{at}cnam.fr

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