Characterization of 17α-hydroxysteroid dehydrogenase activity (17α-HSD) and its involvement in the biosynthesis of epitestosterone
© Bellemare et al; licensee BioMed Central Ltd. 2005
Received: 02 February 2005
Accepted: 14 July 2005
Published: 14 July 2005
Epi-testosterone (epiT) is the 17α-epimer of testosterone. It has been found at similar level as testosterone in human biological fluids. This steroid has thus been used as a natural internal standard for assessing testosterone abuse in sports. EpiT has been also shown to accumulate in mammary cyst fluid and in human prostate. It was found to possess antiandrogenic activity as well as neuroprotective effects. So far, the exact pathway leading to the formation of epiT has not been elucidated.
In this report, we describe the isolation and characterization of the enzyme 17α-hydroxysteroid dehydrogenase. The name is given according to its most potent activity. Using cells stably expressing the enzyme, we show that 17α-HSD catalyzes efficienty the transformation of 4-androstenedione (4-dione), dehydroepiandrosterone (DHEA), 5α-androstane-3,17-dione (5α-dione) and androsterone (ADT) into their corresponding 17α-hydroxy-steroids : epiT, 5-androstene-3β,17α-diol (epi5diol), 5α-androstane-17α-ol-3-one (epiDHT) and 5α-androstane-3α,17α-diol (epi3α-diol), respectively. Similar to other members of the aldo-keto reductase family that possess the ability to reduce the keto-group into hydroxyl-group at different position on the steroid nucleus, 17α-HSD could also catalyze the transformation of DHT, 5α-dione, and 5α-pregnane-3,20-dione (DHP) into 3α-diol, ADT and 5α-pregnane-3α-ol-20-one (allopregnanolone) through its less potent 3α-HSD activity. We also have over-expressed the 17α-HSD in Escherichia coli and have purified it by affinity chromatography. The purified enzyme exhibits the same catalytic properties that have been observed with cultured HEK-293 stably transfected cells. Using quantitative Realtime-PCR to study tissue distribution of this enzyme in the mouse, we observed that it is expressed at very high levels in the kidney.
The present study permits to clarify the biosynthesis pathway of epiT. It also offers the opportunity to study gene regulation and function of this enzyme. Further study in human will allow a better comprehension about the use of epiT in drug abuse testing; it will also help to clarify the importance of its accumulation in breast cyst fluid and prostate, as well as its potential role as natural antiandrogen.
Epitestosterone (17α-hydroxy-4-androstene-3-one) is an epimer of testosterone (T). Its concentration in the urine is used as reference substance in the control of T abuse . EpiT was identified for the first time as an androgen metabolite produced by rabbit liver slices . It has been also observed that slices of rabbit, guinea pig and dog liver, mare's ovary, ox and sheep blood as well as guinea pig kidney, ovary and testis possess the ability to produce epiT from T and 4-dione . In the mouse, the kidney is a major site of epiT formation, while the production in the liver is negligible . In castrated male bovine, it has been observed that blood and liver both possess a high ability to convert T into epiT . No interconversion of T and epiT has been observed in testes of bulls, rabbits or rats, even though it has been found that testes is a source of endogenous epiT in these species .
In young boys, the concentration of epiT is higher than T, however it declines in adulthood to a epiT/T ratio of approximately 1 . In hyperplasic prostate, epiT concentration is comparable to that of 4-dione, which represents about twice the amount of T and half the concentration of dihydrotestosterone (DHT) . The excretion of epiT in urine is slightly lower than that of T [8–11]. Plasma concentrations of epiT decline with age and were established at approximately 2.5 nmol/l in adult men and 1.2 nmol/l in women. Since epiT does not originate from endogenous T, and because the ratio of urinary T to epiT in adults is almost constant, this ratio has been used as a basis for the detection of exogenously administered T : the median ratio in normal healthy men is about 1, while being significantly elevated in case of testosterone abuse. Urinary T/epiT ratio from 1 to 6 is considered normal by the International Olympic Committee, while one displaying a ratio greater than 6 is suspected of anabolic steroid use . In order to apply this assay, it is important to assume that the biosynthesis of epitestosterone does not originate from testosterone, and that both epimers undergo similar clearance. It is also important to assume that racial or individual variations do not affect this ratio. Because of these important assumptions, parameters that may influence this ratio and possibly lead to false positive results have been intensively debated since the introduction of the T/epiT ratio in doping analysis . The lack of knowledge about the gene encoding the enzyme responsible for the formation of epiT has made the identification of appropriate parameters difficult.
In humans, the interconversion of T and epiT is negligible . Using labeled-T, it has been shown that epiT does not originate from T. Studying the 16-ene-synthetase reaction in human testicular homogenates, Weusten et al.  hypothesized that 5-androstene-3β,17α-diol (epi5-diol) and 5,16-androstadien-3β-ol are synthesized from pregnenolone in a single step through a 16-ene-synthase, and then that epi5-diol is further converted to epiT by 3β-hydroxysteroid dehydrogenase (3β-HSD). On the other hand, a recent study showed that administration of 4-dione to healthy male subjects increases the urinary excretion rates of epiT, thus suggesting that epiT could be biosynthesized from 4-dione . The authors suggested that a likely candidate to convert 4-dione to epiT would be 17α-hydroxysteroid dehydrogenase (17α-HSD). This enzyme has been purified from liver and kidney tissue from rabbits and hamsters [17–19]. Accordingly, in this report, we describe the isolation and characterization of a cDNA encoding an enzyme exhibiting a strong 17α-HSD acivity, efficiently catalyzing the conversion of 4-dione into epiT.
Characterization of mouse 17α-HSD cDNA and gene
Identification of epiT by HPLC
Substrate specificity of the mouse 17α-HSD activity
Kinetic constants of 17α-HSD for various substrates
Vmax / Km
Tissue distribution of mouse 17α-HSD
Quantification of mRNA expression levels of 17α-HSD and 17β-HSD5 in various mouse tissues using Realtime PCR
17α-HSD (number of copies/μg RNA ± SEM)
17β-HSD5 (number of copies/μg RNA ± SEM)
130 ± 92
692 ± 251
572 ± 992
2986 ± 2591
0 ± 0
12036 ± 356
53445 ± 11410
3714 ± 1960
2344 ± 6
332 ± 73
15369159 ± 2701597
14995082 ± 987906
22226386 ± 1068282
17083578 ± 928740
45536 ± 9194
102586 ± 35774
21610 ± 1369
7466 ± 438
22791 ± 4358
8777 ± 5673
0 ± 0
0 ± 0
0 ± 0
6097 ± 2642
2029 ± 40
3790 ± 134
407 ± 705
10943 ± 7200
0 ± 0
0 ± 0
11858 ± 4062
8539 ± 4606
13860 ± 2184
6299 ± 1049
256686 ± 37628
17438 ± 21120
0 ± 0
0 ± 0
0 ± 0
6821 ± 4402
6333 ± 490
0 ± 0
0 ± 0
1095 ± 1896
2681 ± 261
14551 ± 12605
1980 ± 120
7208 ± 6397
0 ± 0
8328 ± 3819
6516 ± 337
14419 ± 12591
0 ± 0
5666 ± 3188
The present report describes the isolation and characterization of a cDNA sequence encoding the enzyme 17α-HSD. We have shown that this enzyme converts mainly 17-ketosteroids into 17α-hydroxysteroids : 4-dione to epiT, ADT to epi3α-diol, 5α-dione to epiDHT and DHEA to epi5-diol. Since the enzyme is able to catalyze 4 distincts 17-ketosteroids into its 17α-hydroxy-compounds, and because all the four products have shown identitical profile on HPLC and TLC with commercial products, the results, thus, represent a strong evidence that the activity catalyzed by this specific enzyme is a 17-ketoreductase activity producing 17α-hydroxysteroids.
17α-HSD belongs to the aldo-keto reductase family and shares 77 % amino acid with the mouse type 5 17β-HSD, an enzyme catalyzing the transformation of 4-dione into T . To our knowledge, this is the first example of two highly homologous enzymes belonging to the same gene family, and catalyzing the formation of two distinct epimers from a same substrate. This will represent an interesting model to study the mechanism of the 17α- and 17β- stereospecificity. In addition to the difference in substrate specificity, mouse 17α-HSD and 17β-HSD5 show distinct and specific mRNA tissue distribution : 17α-HSD is highly expressed in kidney while 17β-HSD5 is abundant in the liver. This is in agreement with previous reports showing that mouse liver does not produce epiT .
We have previously shown that mouse type 5 17β-HSD is specifically expressed in the liver , while human type 5 17β-HSD is more widely expressed . It is noteworthy that 17α-HSD and type 5 17β-HSD belongs to aldo-keto reductase family. Members of this family share very high homology although they catalyze different activities and are expressed in different tissues; for example, mouse type 5 17β-HSD shares 77, 88 and 86 % identity with mouse 17α-HSD, 3α-HSD and 20α-HSD respectively. Therefore, previous studies based on hybridation experiments such as northern blot analysis could not distinguish different members of the aldo-keto-reductase family. Using Realtime-PCR with specific primers, as described in the present manuscript, mouse type 5 17β-HSD could be specifically identified, distinctly from 17α-HSD.
The present first report of 17α-HSD will help to better understand the physiological role of epiT. It offers a tool to further study at molecular level the role of 17α-HSD in human, espectially for T abuse testing in sports using epiT as a control. It also permits to investigate the importance of epitestosterone in some previously reported interesting phenomenons such as its accumulation in mammary cyst fluid  and in human prostate [7, 26], as well as its potential neuroprotective effects , and natural anti-androgenic effects .
Isolation of mouse 17α-HSD
A cDNA fragment of a coding region of mouse 17α-HSD (AKR1C21) was amplified by polymerase chain reaction (PCR) from a mouse spleen cDNA and the oligoprimer pair (5'-ggg-gtc-gac-ttt-gaa-gag-gga-cac-ata-atg-a-3' and 5'-ggg-ggt-acc-acc-cat-agg-ctt-ttc-agg-aga-3') derived from the DNA sequence NM_029901 from GenBank database. Mouse spleen cDNA was obtained by reverse transcription of 20 μg of mouse spleen total RNA using 400 U SuperScript II reverse transcriptase (Invitrogen, Burlington, Ontario, Canada) and oligo-d(T)24 as primer in a reaction buffer containing 50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT and 0.5 mM dNTPs. The resulting cDNA fragments were subcloned into a pCMVneo expression vector (pCMVneo-m17α-HSD) which was subsequently used to produce a stably transfected HEK-293 cell line. Plasmid DNA was prepared using the Qiagen Mega Kit (Qiagen, Chatsworth, CA, USA). Sequence of the pCMVneo-m17α-HSD was determined using an ABI 3730/XL automatic sequencer, to verify the identity of the amplified sequence.
Stable expression in HEK-293 cells
Stable transfection of pCMVneo-m17α-HSD into HEK-293 cells was performed as described previously . Briefly, HEK-293 cells were cultured in 6-well falcon flasks to approximately 3 × 105 cells/well in Minimum Essential Medium (MEM) (Invitrogen) supplemented with 10% (vol/vol) FCS (Wisent, Saint-Bruno, Québec, Canada) at 37°C under a 95% air- 5% CO2 humidified atmosphere. Five μg of pCMVneo-m17α-HSD was transfected using Exgen 500 reagent (Fermentas, Burlington, Ontario, Canada). After 6 h incubation at 37°C, the transfection medium was removed and 2 ml of MEM were added. Cells were further cultured for 48 h, then transferred into 10 cm Petri dishes and cultured in MEM containing 700 μg/ml of G-418 (Invitrogen) in order to inhibit the growth of non-transfected cells. The medium containing G-418 was changed every two days until resistant colonies were observed.
Overexpression and purification of mouse 17α-HSD
The cDNA encoding mouse 17α-HSD was subcloned into a pGEX vector (Amhersham Biosciences, Baie d'Urfé, Québec, Canada) and expressed in Escherichia coli BL21(DE3) pLysS as a fusion protein with glutathione-S-transferase (GST). The fusion protein was isolated using a Glutathione-Sepharose 4B column, as described by the manufacturer. The purified 17α-HSD enzyme was separated from GST by digestion with thrombin. 17α-HSD is not adsorbed on the DEAE column and is recuperated in the flow-through fraction, while GST and fusion protein remain on the column. With this method we obtain about 10 mg of a purified enzyme preparation per 100 ml of cell culture. Analysis of samples obtained during the purification process was performed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), as described before . The broad range molecular weight standards was purchased from Bio-Rad (Missisauga, Ontario, Canada).
Assay of enzymatic activity
Enzymatic activities of 17α-HSD were determined using both purified enzyme and the cultured HEK-293 cells stably transfected with pCMVneo-m17α-HSD, as previously described . Briefly, 3 μg of purified 17α-HSD were incubated with 10 mM NADPH, 0.1 μM of 4-dione in phosphate saline buffer, 50 mM, pH 7.3, for 20 minutes. For the intact cells, 0.1 μM of the [14C]-labeled steroid (PerkinElmer, Boston, Massachussetts, USA) was added to freshly changed culture medium in a 6-well culture plate. Non-transfected HEK-293 cells were used as control of the background. After incubation, the steroids were extracted with 2 ml of ether. The organic phases were pooled and evaporated to dryness. The steroids were then solubilized in 50 μl of dichloromethane, applied to Silica gel 60 thin layer chromatography (TLC) plates (Merck, Darmstad, Germany). To obtain a better separation and identification of metabolites, different solvent systems were used. Metabolites of substrates 4-dione, DHEA and 5α-dione were separated in chloroform : ether (9:1), while DHP, DHT and ADT products were separated in the toluene : acetone (4:1) solvent system. Substrates and metabolites were identified by comparison with reference steroids, revealed by autoradiography and quantified using the PhosphorImager System (Molecular Dynamics, Sunnyvale, California, USA). The enzymatic reaction was carried out using the condition in which the activity varies linearly with the enzyme concentration and incubation time, indicating that the cofactor concentration produced by the cells is in excess; the reverse reaction was consequently prevented. In our conditions, this linearity was observed at even more than 60% transformation. Determination of the kinetic parameters was done by Lineweaver-Burk graph analysis using Enzfitter software.
Identification of epitestosterone by High Performance Liquid Chromatography (HPLC)
14C-Labeled steroids were analyzed using Waters NovaPak reverse-phase C18 HPLC column (3.9 × 150 mm, 4 μm). The mobile phase was MeOH/H2O/THF (26 : 56 : 18 v/v), with a flow rate of 0.7 ml.min-1. Radioactivity was monitored in the eluent using Beckman 171 HPLC Radioactivity Monitoring System. Unlabelled steroids (4-dione, DHEA, 5α-dione, ADT, T, 5-diol, DHT, 3α-diol, epiT, epi5-diol, epiDHT and epi3α-diol) were obtained from Steraloids (Newport, Rhode Island, USA) and used as standards.
Tissue collection and RNA preparation
Total RNA of indicated tissues was isolated using Trizol Reagent (Invitrogen, Burlington, Ontario) as described by the manufacturer. Twenty μg of total RNA was converted to cDNA by incubation at 42°C for 1 h with 400 U SuperScript II reverse transcriptase (Invitrogen), using oligo-d(T)24 as primer in a reaction buffer containing 50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT and 0.5 mM dNTPs. The tissues were collected in C57BL6 mice at 12-15 weeks of age obtained from Charles River, Inc. (Saint-Constant, Québec, Canada). The mice were housed individually in vinyl cages. The photoperiod was 12 h of light and 12 h of darkness (lights on at 07:15 h). Certified rodent food (Lab Rodent Diet) and tap water were provided ad libitum. The experiment was conducted in an animal facility approved by the Canadian Council on Animal Care (CCAC) and the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The study was performed in accordance with the CCAC Guide for Care and Use of Experimental Animals. The collected organs were rapidly trimmed, snap-frozen in liquid nitrogen and stored at -80°C until RNA extraction.
Tissue distribution of 17α-HSD mRNA using RealTime PCR
- epi5-diol 5-androstene-3α:
- DHP 5α-pregnane-3:
- epi3α-diol 5α-androstane-3α:
17α-diol, 5α-pregnane-3α-ol-20-one allopregnanolone
- 3α-diol 5α-androstane-3α:
- 5-diol 5-androstene-3β:
polymerase chain reaction
We would like to thank Canadian Institutes of Health Research (CIHR) for financial support. Lucille Lacoste and Mélanie Robitaille are acknowledged for their skillful technical assistance.
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