The discovery of tetrahydrogestrinone (THG) abuse by several elite athletes led the U.S. Congress to declare it a controlled substance, although conclusive evidence of its anabolic/androgenic activity is lacking. We determined whether THG affects myogenic differentiation and androgen receptor (AR)-mediated signaling, whether it binds to AR, and whether it has androgenic and anabolic effects in vivo. Accordingly, we measured the dissociation constant for THG with a fluorescence anisotropy assay using recombinant AR-ligand binding domain. The AR nuclear translocation and myogenic activity of androstenedione were evaluated in mesenchymal, multipotent C3H10T1/2 cells. We performed molecular modeling of the THG:AR interaction. The androgenic/anabolic activity was evaluated in orchidectomized rats. THG bound to AR with an affinity similar to that of dihydrotestosterone. In multipotent C3H10T1/2 cells, THG upregulated AR expression, induced AR nuclear translocation, dose dependently increased the area of myosin heavy chain type II-positive myotubes, and up-regulated myogenic determination and myosin heavy chain type II protein expression. The interaction between AR and the A ring of THG was similar to that between AR and the A ring of dihydrotestosterone, but the C17 and C18 substituents in THG had a unique stabilizing interaction with AR. THG administration prevented the castration-induced atrophy of levator ani, prostate gland, and seminal vesicles and loss of fat-free mass in orchidectomized rats. We conclude that THG is an anabolic steroid that binds to AR, activates AR-mediated signaling, promotes myogenesis in mesenchymal multipotent cells, and has anabolic and androgenic activity in vivo. This mechanism-based approach should be useful for rapid screening of anabolic/androgenic agents.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
ALTHOUGH THE USE of androgenic steroids is banned in sports, chemical modifications of the steroidal structure have made possible the production of novel androgenic steroids that are not named on the banned list (1, 2, 3, 4, 5, 6). Athletes who have been caught using such drugs and their counsels have challenged sports authorities based on the absence of experimental data demonstrating the androgenic/anabolic properties of these new designer steroids. The discovery of the abuse of tetrahydrogestrinone (THG) by a number of elite athletes in 2003 exemplified this dilemma facing sports (1). Unlike other anabolic steroids abused by athletes, which are typically pharmaceuticals intended for veterinary or human use, THG had neither been approved nor marketed for any clinical indication and was previously undetectable in sports doping control tests (1). Since then, the U.S. Congress has added THG to the Anabolic Steroid Control Act of 2004 (2). Here, we present in vivo and in vitro data that conclusively establish the myogenic and androgenic effects of THG.
The anabolic effects of androgens are mediated via the androgen receptor (AR), a 110 kDa protein with three functional domains: the transactivation, DNA binding, and ligand binding (LBD) domains (7, 8, 9, 10, 11, 12, 13, 14, 15). Most of the unliganded AR is localized in the cytoplasmic compartment of target cells, in which it is sequestered as a multiprotein complex with heat-shock proteins and immunophilins. During ligand binding, the receptor dissociates from the multiprotein complex, recruits coactivators, and translocates to the nucleus. Subsequently, it modulates the expression of androgen-responsive genes that regulate cell fate determination. We have shown that androgens promote myogenic commitment and differentiation of mesenchymal multipotent cells (16, 17). The promyogenic effects of androgens in mesenchymal multipotent cells are associated with up-regulation of myogenic determination transcription factor (MyoD), myogenin, and myosin heavy chain II (MHC-II) (16, 17). We quantified the biologic effects of THG on several steps in the myogenic differentiation of mesenchymal multipotent cells (C3H10T1/2); we determined its binding affinity to AR and its ability to induce translocation of AR into the nucleus and to induce myogenic differentiation in C3H10T1/2 cells. We also performed molecular modeling of the THG:AR interaction and compared it with the dihydrotestosterone (DHT):AR interaction. These molecular modeling studies provided important information about the structural determinants of THG:AR interaction.
Levator ani muscle mass has been used widely as a marker of anabolic activity (18, 19), whereas prostate and seminal vesicle weights have been used as markers of androgenic activity (20). Accordingly, we determined the effects of graded doses of THG and testosterone enanthate (TE) on levator ani, prostate, and seminal vesicle weights in castrated male rats.
Materials and Methods
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
THG was synthesized by catalytic hydrogenation of gestrinone as reported previously (1). Briefly, catalyst [Palladium on activated carbon (Pd/C), 0.5 mg/ml] was suspended in methanol in an ice bath. The solution was purged with hydrogen for 5 min before adding a solution of gestrinone in methanol (2 mg/ml). The resulting mixture was subsequently purged for 20 min with hydrogen and filtered through a 0.2 µm filter for liquid chromatography-mass spectrometry analysis, and the reaction was monitored by HPLC. The products were filtered though Celite bed, and the filtrate was dried under nitrogen. THG was purified by HPLC and characterized by mass spectrometry and nuclear magnetic resonance spectra obtained on a Bruker (Billerica, MA) Advance 600 MHz nuclear magnetic resonance spectrometer. The final material used for in vitro and in vivo experiments was more than 99% pure. For in vitro experiments of ligand affinity and promyogenic potential of THG, the stock solutions were prepared in ethanol. The resulting concentration of ethanol was less than 1% in all of the experiments. For in vivo experiments with castrated rats, THG suspension was formulated in sesame oil (150 µg/100 µl) for intramuscular injection.
Ligand affinity measurements
Dissociation constant for THG was determined using fluorescence anisotropy measurement of competitive displacement of fluorescent androgen (FA) analog (Fluormone Al green; Invitrogen, San Diego, CA) from LBD of AR (Invitrogen). THG, DHT, and -4-androstene-3,17-dione (4-androstenedione) were titrated into 50 nM AR-LBD solution containing 2 nM FA and 2 mM dithiothreitol. The polarization values of AR-LBD-bound FA were measured at graded ligand concentrations using Genios Pro (Tecan Austria, Grodig, Austria) (ex = 485 nm; em = 535nm) to obtain IC50, the concentration that results in a half-maximal shift in polarization value (17, 21). Measurements were conducted in triplicates, and parallel experiments were performed under similar conditions to quantify relative binding affinities of THG, 4-androstenedione, and DHT.
Molecular modeling
The three-dimensional (3D) structure of rat AR with DHT (Protein Data Bank file 1I137) was used as a template for constructing the 3D model of AR interaction with THG. The LBD of rat AR is identical to human AR. Using the Bioplymer option in the Accelrys Insight II software package (Accelrys, San Diego, CA), DHT was changed to THG. The AR-THG complex was minimized for 100,000 iterations with Discover3 using consistent valence force field and a distance-dependent dielectric constant of 2 to approximate for water in the protein (22).
In vitro experiments of promyogenic activity
C3H10T1/2 cells, grown in DMEM with 10% fetal bovine serum at 37 C in the presence of 5% CO2, were treated with 20 µM 5'-azacytidine for 3 d and seeded at 70% confluence in six-well plates or chamber slides (10, 11). Cells were incubated with medium alone, THG, or DHT without or with bicalutamide for 24 h for AR translocation studies, 4 d for assessment of MyoD, and 12 d for assessment of myotubes and MHC-II expression.
For immunohistochemical analyses, cells grown in chamber slides were fixed in 2% paraformaldehyde, quenched with H2O2, blocked with horse or goat serum, and incubated with antibody for MHC or AR. Detection was based on secondary biotinylated antibody (1:200), followed by addition of streptavidin-horseradish peroxidase ABC complex (1:100) and 3,3 diaminobenzidine. In negative controls, the primary antibody was omitted. The effects of THG and DHT on myogenesis were assessed quantitatively by measuring the area covered by MHC+ myotubes, using ImagePro software (Media Cybernetics, Silver Spring, MD); the area of MHC+ myotubes was averaged over 10 fields.
For Western blot analysis, cell lysates (50–100 µg) were electrophoresed on 7.5% polyacrylamide gels. After transfer, the membranes were incubated with 1:200 mouse monoclonal anti-MHC (slow; Vector Laboratories, Burlingame, CA), 1:500 anti-AR (Santa Cruz Biotechnology, Santa Cruz, CA), 1:500 anti-MyoD (Santa Cruz Biotechnology), or 1:10000 anti-glyceraldehyde-3-phosphate dehydrogenase (Chemicon, Temecula, CA) antibody. The washed membranes were incubated with secondary antibody (1:1000) linked to horseradish peroxidase. Immunoreactive bands were visualized by using ECL detection system (Chemicon).
In vivo experiments of androgenic and anabolic activity
Forty-two male, Sprague Dawley rats (50 d old and 200 g) obtained from Charles River Laboratories (Wilmington, MA) and were housed in under controlled temperature (22 C), humidity (40%), and light (12-h light, 12-h dark cycle) conditions in a specific pathogen-free vivarium according to Institutional Animal Care and Use Committee guidelines. The animals were permitted ad libitum access to standard laboratory chow and water. The animals were allowed to acclimate for 7 d in the facility before the start of the experiment. The orchidectomies were performed under ketamine (40–60 mg/kg) and xylazine (3–5 mg/kg) anesthesia.
The animals were randomly assigned to one of six groups: sham-operated treated with sesame oil injection daily (alternating hindlimb); orchidectomized, treated with sesame oil injection daily; orchidectomized, treated with 75 µg THG daily; orchidectomized, treated with 150 µg THG daily; orchidectomized, treated with 75 µg of TE daily; orchidectomized, treated with 150 µg TE daily. THG and TE were formulated in sesame oil and injected intramuscularly in the hindlimbs daily. The control groups (sham-operated and orchidectomized) received sesame oil injections. Treatment duration was 10 d. Fat-free mass (FFM) was evaluated by dual-energy x-ray absorptiometry (DEXA) on Hologic (Bedford, MA) Quad 4500 Scanner, using software that has been validated previously for rat body composition assessment (23, 24). Lean body mass was calculated by subtracting bone mineral content from FFM. At the conclusion of the experiment, rats were killed by decapitation after deep sedation with ketamine and xylazine. Seminal vesicles, prostate, and levator ani muscles were carefully removed free of adhering fat and immediately weighted.
Results
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
THG binds LBD of AR with an affinity similar to that of DHT
We evaluated the affinity of THG for the AR using a fluorescence polarization assay (17, 21) in which the displacement of a fluorescent androgen (FA) analog from the purified AR-LBD by THG was determined. The relative binding affinities for THG, 4-androstendione, and DHT were 8.4 ± 1.2, 646 ± 22, and 9.8 ± 1.4 nM, respectively (Fig. 1A).
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FIG. 1. A, THG binds AR with an affinity similar to DHT. Dissociation constants for THG, DHT, and 4-androstenedione were calculated using fluorescence anisotropy measurement of competitive displacement of FA analog from AR-LBD. Panel I displays the polarization curve, as a function of LBD with fixed concentration of the FA (2 nM). Panel II shows the competitive displacement by THG, DHT, and 4-androstenedione (AD); Kd values for THG, DHT, and 4-androstenedione, calculated from the isotherms, were 8.4 ± 1.2, 9.8 ± 1.4, and 646 ± 22 nM, respectively. B, Both the AR/DHT crystal structure and AR/THG 3D model have similar stabilizing interactions between the steroid 3-keto group and Gln-711 and Arg-752. In contrast, there are important differences in the interaction between the AR and the D rings of DHT and THG. The AR/THG model shows a rotation of Trp-741 to interact more closely with the C18 ethyl substituent and with Met-895, which also stabilizes the C18 ethyl substituent. The C17 substituent on THG has van der Waals interactions with Leu-701, Leu-704 and Leu-770.
A molecular model of THG:AR interaction (Fig. 1B)
The finding that THG has either equal or slightly higher affinity for AR than DHT was unexpected because THG contains substituents at C17 and C18 that could potentially have unfavorable steric interactions. To identify the amino acid residues that interact with THG, we constructed a 3D structural model of AR interaction with THG. Interestingly, the D ring, an important determinant of steroid specificity (22, 26, 27, 28, 29), displays unique stabilizing interactions with AR. THG has an extra methyl group at C18 and a 17-ethyl substituent that are lacking in DHT, testosterone, or R1881 (17-methyltrienolone). The 3D model shows that the LBD of AR accommodates the 17-ethyl substituent with stabilizing van der Waals interactions between THG and AR. Cß on Leu-701 and Leu-704 are 3.95 and 3.6 A, respectively from THG, and C2 on Leu-880 is 3.45 A from THG (Fig. 1B). Moreover, two of these leucine residues have stabilizing interactions with each other. Thus, C2 on Leu-880 is 3.7 A from C2 of Leu-701.
The 3D model also shows that the C18 substituent on THG has a unique stabilizing interaction with Trp-741, which has rotated from its orientation in the AR-DHT complex, in which it interacts with the C19 methyl group. In the AR-THG complex, C2 on Trp-741 is 4 A from THG. Moreover, Met-895 S is closer to Trp-741 and C on Met-895 is 4 A from THG, which is 3.55 A from O on Thr-877.
THG induces nuclear translocation of AR
The initial step in AR signaling is ligand-induced nuclear translocation of the receptor (7, 8, 9, 10, 11, 12, 13, 14, 15, 19). To determine whether THG-bound receptor acquires the transcriptionally active conformation to translocate into the nucleus, multipotent C3H10T1/2 cells were treated with graded THG doses in subconfluent cultures. The spatial distribution of AR was determined by using immunocytochemical staining (Fig. 2). AR staining was mostly cytoplasmic in control wells treated with medium alone; coincubation with THG or DHT caused AR to translocate into the nucleus (Fig. 2). THG-induced AR nuclear translocation was attenuated by coincubation with a 10-fold excess of an AR antagonist bicalutamide, indicating that this was an AR-mediated process and also providing evidence of the specificity of this effect.
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FIG. 2. THG and DHT cause translocation of AR to the nucleus of C3H10T1/2 mesenchymal, multipotent cells. C3H10T1/2 cells were incubated for 24 h with medium alone, DHT (30 nM), THG (30 nM), THG (30 nM) plus bicalutamide (300 nM), fixed with 4% paraformaldehyde, and probed for AR, using anti-AR antibody. Nuclear translocation of AR in the presence of THG and DHT denotes activation of androgenic signaling. Reduced nuclear staining by THG in presence of the AR antagonist bicalutamide (Casodex) signifies competitive specificity of THG for AR-LBD. Magnification, x400. Scale bar, 35 µm.
THG promotes myogenic differentiation of mesenchymal, multipotent C3H10T1/2 cells
We have shown previously that C3H10T1/2 cells express AR protein and that androgens induce the expression of AR in these cells (16, 17). Incubation with graded concentrations of THG unregulated AR dose dependently.
C3H10T1/2 cells have been used widely as a model to investigate modulation of lineage determination (30, 31, 32). Androgens, such as testosterone, DHT, and 4-androstenedione, cause preferential commitment and differentiation of C3H10T1/2 multipotent cells into myogenic lineage and up-regulate the transcriptional markers in myogenic differentiation (16, 17). Incubation of C3H10T1/2 cells with graded THG concentrations (1–30 nM) led to a dose-dependent increase in MHC+ myotubes, quantified using image analysis (Fig. 3B).
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FIG. 3. A, THG modulates androgen response element-dependent gene expression. THG treatment to C3H10T1/2 cells causes dose-dependent increase in AR (d 1), myogenic commitment marker MyoD (d 4), and terminal myogenic differentiation marker MHC-II (d 12). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal control for loading (a representative blot is included in the figure). B, The top panel displays representative wells containing C3H10T1/2 cells treated for 12 d with medium alone (control), 5 nM DHT, or graded concentrations of THG, and analyzed for MHC expression by immunocytochemical staining, using an anti-MHC antibody. Magnification, 200x. Scale bar, 20 µm. The area covered by MHC+ myotubes was quantitated by image analysis software (bottom panel). C, Control. Data are mean ± SEM (n = 10 for each concentration).
THG up-regulated dose dependently MyoD and MHC-II protein expression (Fig. 3A). The effects of 30 nM THG were not significantly different from those of 30 nM DHT. Thus, THG promotes the differentiation of mesenchymal, multipotent cells into the myogenic lineage with potency not dissimilar from DHT.
THG has anabolic and androgenic activity in vivo in the castrated male rat model
We evaluated the androgenic and anabolic activity of THG in a castrated rat model, using levator ani muscle mass and whole-body FFM measured by DEXA as markers for anabolic activity and prostate and seminal vesicle weights as markers for androgenic activity. Orchidectomized rats had significantly lower FFM (P < 0.05) than sham-operated controls (Fig. 4A). The FFM of orchidectomized rats treated with either 75 or 150 µg dose of THG or TE did not differ significantly from that of sham-operated controls but was significantly greater than that of orchidectomized, placebo-treated rats (Fig. 4A).
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FIG. 4. A, Anabolic effects of THG in vivo. Sprague Dawley rats divided into six groups were injected (im) with sesame oil (castrate, sham) or castrated and injected with steroids suspensions in sesame oil (TE: TE 1 at 75 µg, TE 2 at 150 µg; THG: THG 1 at 75 µg, THG 2 at 150 µg) for 10 d. Overall body composition analysis was performed on d 10 by DEXA. Data are expressed as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 with respect to the castrate group. B, In vivo androgenic effects of THG administration. Tissues from rats in the respective groups (described in A) were weighed after 10 d of treatment. Data are expressed as mean ± SEM. **, P < 0.01; ***, P < 0.001 with respect to castrate group.
Orchidectomized rats had significantly lower levator ani, prostate, and seminal vesicle weights compared with sham-operated controls; supplementation with THG or TE restored the levator ani, prostate, and seminal vesicle weights to levels observed in eugonadal controls. The levator ani, prostate, and seminal vesicle weights were significantly higher in THG- and TE-supplemented groups compared with castrated controls (Fig. 4, A and B).
Discussion
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
THG, a derivative of gestrinone, possesses structural and functional similarities with potent androgens. THG associates with the LBD of AR with an affinity similar to that of DHT (33, 35), induces AR-nuclear translocation, and promotes myogenic differentiation of mesenchymal multipotent cells. THG-mediated nuclear translocation of AR is attenuated by bicalutamide, an AR antagonist, suggesting that THG specifically competes for the AR binding pocket occupancy. THG exerts in vitro promyogenic effects that are similar in potency to DHT. In a castrated rat model, THG has demonstrable anabolic activity because it prevents the castration-induced loss of FFM and levator ani muscle mass (18). THG also prevents the atrophy of prostate and seminal vesicles. Thus, THG displays both androgenic and anabolic properties in a manner similar to TE. Therefore, THG is a bona fide androgen with anabolic properties that meets the essential criteria for an anabolic steroid established by the Controlled Substance Act (2).
We found the AR binding affinity of THG to be similar to that of DHT. Slight differences in AR binding affinity reported by Labrie et al. (35) might be related to the differences in methods used to assess binding affinities and the species differences in AR. Also, the binding affinities estimated by using the LBD of AR might differ from those derived by using the full-length receptor. Using a yeast-based assay, Death et al. (35) have reported that THG is a potent androgen with a transactivation potency that is 10 times that of testosterone. However, we do not know whether this yeast-based system expresses the repertoire of coregulators that is present in the mammalian androgen-responsive tissues. THG activates many of the same genes activated by DHT in the levator ani (35). However, it is possible that THG might exert additional effects that are mediated through the progesterone receptor.
In orchidectomized rats, the anabolic potency of THG in the levator ani assay and its androgenic potency in preventing castration-induced changes in prostate and seminal vesicle weights appear to be similar to TE. While this manuscript was in preparation, Labrie et al. (35) reported that THG possesses only 20% of the potency of DHT in stimulating prostate, seminal vesicle, and levator ani muscle weight in the mouse. Because we used TE, a direct comparison of our data with those reported by Labrie et al. is not possible. Also, the observed differences in in vivo potency may be related to the vehicle used for administering THG and DHT, because the hydrophobicity of the vehicle can greatly affect the pharmacokinetics and bioavailability of the compound. We recognize that an absolute quantitative comparison of the in vivo potencies of THG and TE cannot be made with the available data, because the pharmacokinetics and bioavailability of THG in rats and mice are unknown. The 24-h profile of circulating THG concentrations depends not only on the administered dose but also on the vehicle used; thus, the mode of administration, hydrophobicity of the vehicle used, and the treatment regimen could affect greatly the observed in vivo activity.
THG is structurally related to gestrinone, a 19-nor progestin; it also has some resemblance to trenbolone. Our molecular modeling studies revealed that the D ring of THG, an important determinant of steroid specificity (22, 26, 27, 28, 29), displays unique stabilizing interactions with AR. The interaction between AR and the A ring of THG was similar to that between AR and the A ring of DHT, but both of the unique C17 and C18 substituents in THG had stabilizing interactions with the AR. These data suggest that other D-ring modifications might confer higher affinity and/or anabolic/androgenic potency because of these stabilizing interactions.
Although steroid abuse by professional athletes and recreational bodybuilders has been widely recognized since the 1950s, the synthesis and dissemination of novel designer steroids such as THG has added new complexity to the detection and regulation of these compounds (1, 2, 3, 4, 5, 6). The past year has witnessed a deluge of newspaper reports of widespread use of steroids in major professional sports. As Handelsman has pointed out in an insightful commentary (6), new designer steroids that are not named on the list of compounds banned by sports or controlled by the U.S. government are being introduced at a rate that exceeds the ability of the regulatory agencies to generate evidence of their anabolic/androgenic activity. This has posed a considerable challenge to the ability of the oversight agencies to regulate the distribution or penalize the use of these new designer steroids. Because some of the compounds have not been approved for human use, human testing of these compounds without substantial preclinical toxicology is not possible. We propose the combined use of the in vitro assays described in this manuscript to provide an effective and rapid screening method for demonstrating the androgenic/anabolic activity of these compounds. Compounds demonstrated to have AR binding and promyogenic activity in the multipotent cell assay can be tested further for in vivo activity in castrated rat model. This mechanism-based, analytical strategy should facilitate the efforts of regulatory agencies to regulate the sales of new designer steroids.
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