The androgenic anabolic steroid tetrahydrogestrinone produces dioxin-like effects via the aryl hydrocarbon receptor.

For a long time, athletes have used androgenic anabolic steroids (AASs) in an inappropriate and veiled manner with the aim of improving exercise performance or for cosmetic purposes. Abuse of AASs triggers adverse effects such as hepatocarcinogenesis, heart attacks, and aggressive behavior. However, AAS-induced toxicity is not completely understood at the molecular level. In the present study, we showed, by performing a dioxin response element (DRE)-luciferase reporter gene assay, that tetrahydrogestrinone (THG), a popular and potent androgen receptor agonist, has dioxin-like effects. In addition, we showed that THG increased cytochrome P-450 1A1 (CYP1A1) mRNA and protein levels, and enzyme activity. The gene encoding CYP1A1 is involved in phase 1 xenobiotic metabolism and a target gene of the aryl hydrocarbon receptor (AhR). Using the AhR antagonist CH-223191, we also examined whether the effects of THG on DRE activation depended on AhR. Our results suggest that synthetic anabolic steroids may have dioxin-like side effects that can disturb endocrine systems and may cause other side effects including cancer through AhR.

Searching for in silico predicted metabolites and designer modifications of (cortico)steroids in urine by high-resolution liquid chromatography/time-of-flight mass spectrometry.

Glucocorticosteroids are a restricted class of substances and appear on the 'in-competition' prohibited list of the World Anti-Doping Agency (WADA). Analysis of glucocorticosteroids is complicated since they show significant phase 1 and 2 metabolism in the human body and are excreted into urine in concentrations in the microg/L range. Full scan, high-resolution time-of-flight mass spectrometry analysis generates information on all ionisable components in urine, including known and unknown metabolites of steroids and even designer modifications of anabolic steroids. However, evaluation of the data obtained can be difficult and time-consuming because of the need to differentiate between endogenous components and compounds of interest. MetaboLynx, a spectral and chromatographic search program, was modified for the determination of in silico predicted metabolites of glucocorticosteroids and designer modifications of anabolic steroids in human urine. Spiked urine samples were successfully screened for known components in a targeted approach and for unknown species in a non-targeted approach using data filtering to limit potential false-positives. A simplified combined approach of targeted and untargeted screening was used for the detection of metabolites and designer modifications of existing compounds. This approach proved successful and showed its strength in the detection of tetrahydrogestrinone (THG), a designer modification of gestrinone. THG was positively detected in a spiked urine sample and correctly identified as a twofold hydrogenation of gestrinone. The developed screening method can easily be adapted to specific needs and it is envisaged that a similar approach would be amendable to the discovery of metabolites or designer modifications of other compounds of interest.

Detection and pharmacokinetics of tetrahydrogestrinone in horses.

The anti-doping rules of national and international sport federations ban any use of tetrahydrogestrinone (THG) in human as well as in horse sports. Initiated by the THG doping scandals in human sports a method for the detection of 3-keto-4,9,11-triene steroids in horse blood and urine was developed. The method comprises the isolation of the analytes by a combination of solid phase and liquid-liquid extraction after hydrolysis and solvolysis of the steroid conjugates. The concentrations of THG in blood and urine samples were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A THG excretion study on horses was conducted to verify the method capability for the analysis of postadministration urine samples. In addition, blood samples were collected to allow for determination of the pharmacokinetics of THG in horses. Following the administration of a single oral dose of 25 microg THG per kg bodyweight to 10 horses, samples were collected at appropriate intervals. The plasma levels of THG reached maximal concentrations of 1.5-4.8 ng/mL. Twenty-four hours after the administration plasma levels returned to baseline. In urine, THG was detectable for 36 h. Urinary peak concentrations of total THG ranged from 16 to 206 ng/mL. For the 10 horses tested, the mean plasma clearance of THG was 2250 mL/h/kg and the plasma elimination half-life was 1.9 h.

Tetrahydrogestrinone analysis and designer steroids revisited.

Anabolic steroids are the main abused class of prohibited substances in doping control. These steroids are associated with enhancement of muscular mass and aggressiveness, resulting in increased performance. Chromatography and MS have a key role among methods developed to detect anabolic steroids in doping control laboratories. However, the classical analytical approach fails in detection of the so-called 'designer steroids'. This review focuses on the rise of tetrahydrogestrinone, a drug that became synonymous with designer steroids. The reasons why classical methods fail in tetrahydrogestrinone detection are discussed and how the detection was implemented is shown. Alternative strategies for detection of new drugs designed to cheat current analytical methodology are highlighted. Concern for the abuse of veterinary designer drugs and supplements is also acknowledged.

Screening of in vitro synthesised metabolites of 4,9,11-trien-3-one steroids by liquid chromatography mass spectrometry.

The aim of the work was to develop a flexible in vitro synthesis procedure, which can be applied in order to study and predict the metabolic patterns of new derivatives of anabolic androgenic steroids (AAS) with respect to most prominent target compounds for doping control purposes. Microsomal and S9 fraction of human liver preparations were used as a source of metabolising enzymes and the co-substrates of the synthesis mixture were selected to favour phase-I metabolic reactions and glucuronidation as phase-II conjugation reactions. Model compounds within the study were 4,9,11-trien-3-one steroids, structural derivatives of gestrinone and trenbolone, which both are included in the list of prohibited compounds in sports by the World Anti-Doping Agency (WADA). The correlation between in vitro metabolism of human microsomes and in vivo excretion studies in human was compared with gestrinone and subsequently, the applicability of the in vitro model for prediction of AAS metabolic pathways for new doping agents was evaluated. All the AAS examined within this study were successfully metabolised using the developed in vitro model, hydroxylation, reduction and glucuronide conjugation being the most prominent reaction pathways. Hydroxylated and glucuronide-conjugated metabolites of in vivo experiment with gestrinone were the same metabolites formed in the enzyme-driven process, thus showing good in vitro-in vivo correlation. Liquid chromatographic-mass spectrometric and tandem mass spectrometric methods were developed, relying on the positive polarity of electrospray ionisation, which also allowed the direct detection of intact glucuronide-conjugated AAS metabolites. Due to charge delocalisation and high proton affinity, the developed method was proven effective in the analysis of AAS metabolites bearing extensive conjugated double bond systems in their structures.

Detection of anabolic steroid abuse using a yeast transactivation system.

The classical analytical method for detection of anabolic steroid abuse is gas chromatography followed by mass spectrometry (GC/MS). However, even molecules with a chemical structure typical for this class of substances, are sometimes not identified in routine screening by GC/MS when their precise chemical structure is still unknown. A supplementary approach to identify anabolic steroid abuse could be a structure-independent identification of anabolic steroids based on their biological activity. To test the suitability of such a system, we have analyzed the yeast androgen receptor (AR) reporter gene system to identify anabolic steroids in human urine samples. Analysis of different anabolic steroids dissolved in buffer demonstrated that the yeast reporter gene system is able to detect a variety of different anabolic steroids and their metabolites with high specificity, including the so-called 'designer steroid' tetrahydrogestrinone. In contrast, other non-androgenic steroids, like glucocordicoids, progestins, mineralocordicoids and estrogens had a low potency to stimulate transactivation. To test whether the system would also allow the detection of androgens in urine, experiments with spiked urine samples were performed. The androgen reporter gene in yeast responds very sensitive to 5alpha-dihydrotestosterone (DHT), even at high urine concentrations. To examine whether the test system would also be able to detect anabolic steroids in the urine of anabolic steroid abusers, anonymous urine samples previously characterized by GCMS were analyzed with the reporter gene assay. Even when the concentration of the anabolic metabolites was comparatively low in some positive samples it was possible to identify the majority of positive samples by their biological activity. In conclusion, our results demonstrate that the yeast reporter gene system detects anabolic steroids and corresponding metabolites with high sensitivity even in urine of anabolic steroid abusing athletes. Therefore we believe that this system can be developed towards a powerful (pre) screening tool for the established doping tests. The system is easy to handle, robust, cost-efficient and needs no high-tech equipment. But most importantly, a biological test system does not require knowledge of the chemical structure of androgenic substances and therefore suitable to detect previously unidentified substances, especially those of the class of so-called designer steroids.

Induction of micronuclei in V79 cells by the anabolic doping steroids tetrahydrogestrinone and trenbolone.

The synthetic steroid tetrahydrogestrinone is a new "designer drug" and was recently detected to be illegally used in sports. It is chemically closely related to trenbolone that is known as an animal growth promoter. The potencies of trenbolone, tetrahydrogestrinone and testosterone to induce micronuclei in V79 cells in vitro were determined. CREST analysis was employed to differentiate between aneugenic or clastogenic mechanisms. Cytotoxicity and an influence on the cell cycle were assessed in parallel. Incubations with testosterone, at concentrations between 3 and 300 microM, failed to induce micronuclei. By contrast, tetrahydrogestrinone and trenbolone increased the rate of micronuclei significantly, up to a doubling of the micronuclei rate of untreated controls. Tetrahydrogestrinone and trenbolone displayed a bell-shaped dose-response curve, with maximal effects observed at 3 and 30 microM, respectively. The micronuclei induced by tetrahydrogestrinone and trenbolone were predominantly kinetochor (CREST) positive, pointing to an aneugenic mode of action. This may be related to the specific structure of both molecules with a system of activated double bonds. As the genotoxic effect of tetrahydrogestrinone at a chromosomal level appears at a low concentration range, it cannot be ruled out that tetrahydrogestrinone presents a genotoxic hazard on a chromosomal level under conditions of its current misuse in sports.

Structural characterization of the human androgen receptor ligand-binding domain complexed with EM5744, a rationally designed steroidal ligand bearing a bulky chain directed toward helix 12.

Antiandrogens are commonly used to treat androgen-dependent disorders. The currently used drugs unfortunately possess very weak affinity for the human AR (hAR), thus indicating the need to develop new high-affinity steroidal antiandrogens. Our compounds are specially designed to impede repositioning of the mobile carboxyl-terminal helix 12, which blocks the ligand-dependent transactivation function (AF-2) located in the AR ligand-binding domain (ARLBD). Using crystal structures of the hARLBD, we first found that H12 could be directly reached from the ligand-binding pocket (LBP) by a chain positioned on the C18 atom of an androgen steroid nucleus. A set of 5alpha-dihydrotestosterone-derived molecules bearing various C18 chains were thus synthesized and tested for their capacity to bind hAR and act as antagonists. Although most of those having very high affinity for hAR were agonists, several very potent antagonists were obtained, confirming the structural importance of the C18 chain. To understand the role of the C18 chain in their agonistic/antagonistic properties, the structure of the hARLBD complexed with one of these agonists, EM5744, was determined at a 1.65-A resolution. We have identified new interactions involving Gln(738), Met(742), and His(874) that explain both the high affinity of this compound and the inability of its bulky chain to prevent the repositioning of H12. This structural information will be helpful to refine the structure of the chains placed on the C18 atom to obtain efficient H12-directed steroidal antiandrogens.

Preparation of antibodies for the designer steroid tetrahydrogestrinone and development of an enzyme-linked immunosorbent assay for human urine analysis.

A highly sensitive enzyme-linked immunosorbent assay (ELISA) has been developed for tetrahydrogestrinone (THG), the new designer anabolic steroid responsible for the well-known Balco scandal announced in year 2003. Antibodies have been raised against 18a-homo-pregna-4,9,11-trien-17beta-ol-3-carboxymethyl oxime coupled to horseshoe crab hemocyanin. The hapten has been synthesized from gestrinone by controlled reduction of the triple bond of the ethinyl group at position C-17, without affecting the double bonds of the steroidal rings, followed by reaction of the keto group at C-3 with (carboxymethoxy)amine hemihydrochloride to form the oxime bond. The antisera obtained has been used in combination with 18a-homo-pregna-4,9,11-trien-17beta-ol-20-yn-3-carboxymethyl oxime, a hapten derivative of gestrinone, coupled to bovine serum albumin to establish a competitive ELISA. Under the conditions used, THG can be detected in buffer with a limit of detection (LOD) of 0.045 +/- 0.015 microg L(-1) (N = 9). The assay is very selective since other steroids assessed are not recognized. Preliminary experiments performed with human urine samples demonstrate that the assay can be applied to the analysis of these samples after a simple sample treatment method reaching a LOD of 0.25 +/- 0.14 microg L(-1). Accuracy is very good as demonstrated by the excellent correlation obtained when analyzing blind spiked urine samples (slope 0.93, R2 = 0.992).

Multi-detection of corticosteroids in sports doping and veterinary control using high-resolution liquid chromatography/time-of-flight mass spectrometry.

A liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) method was developed using the latest high-resolution LC column technology, the ultra performance liquid chromatography (UPLC), and electrospray ionization (ESI) in the positive ion mode. Gradient UPLC separation conditions were optimized for a group of 22 analytes comprising 17 glucocorticosteroids, specific designer steroids such as tetrahydrogestrinone (THG) and specific beta2-agonists such as formoterol. The UPLC/TOFMS separation obtained required 5.5 min only for all the substances tested. Even the critical pair of dexamethasone and betamethasone isomers was almost completely resolved. Thanks to the over 10,000 full-width at half maximum (FWHM) mass resolution and high mass accuracy features of TOFMS 50 mDa window accurate mass chromatograms could be reconstructed for the individual analytes. Sensitive screening in human and calf urine samples fortified at the glucocorticosteroids minimum required performance limit (MRPL) of 30 microg L(-1) (human urine, sports doping) and 2 microg L(-1) (calf urine, veterinary control) could be obtained. The potential of UPLC/TOFMS for confirmatory analysis was shown by determining the accurate mass of all compounds and fragment ions upon in-source collision-induced dissociation (CID) at different energies. The exact mass measurement errors for all glucocorticosteroids were found to be within 6 ppm. Considering veterinary control, limits of detection (LOD) and limits of quantification (LOQ) were determined for most of the analytes in calf urine and found to range from 0.1 to 3.3 and from 0.4 to 4.4 microg L(-1), respectively. The method can be easily extended with other banned substances of interest, as demonstrated by the addition of 21 beta2-agonists to the original analyte mixture in urine, without causing any interferences.

Analysis of synthetic 19-norsteroids trenbolone, tetrahydrogestrinone and gestrinone by gas chromatography-mass spectrometry.

Tetrahydrogestrinone, gestrinone and trenbolone are synthetic 19-norsteroids with androgenic properties sharing a labile conjugated ketotrienyl motif. Their LC-MS analyses tend to overcome classical derivatization problems, a shortcoming to the use of GC-MS. Therefore, alternative derivatization procedures were evaluated. The procedure with methoxylamine: pyridine followed by TMSImid: MSTFA gave the best results. This is attributed to the stability of the MO-TMS derivatives hindering the formation of artifacts and tautomerism. A full method is presented including SPE, hydrolysis and liquid-liquid extraction. It was possible to confirm the analytes below 2 ng/mL in urine, being the method robust and cost effective also for screening proposes.

Dehydroepiandrosterone (DHEA) is an anabolic steroid like dihydrotestosterone (DHT), the most potent natural androgen, and tetrahydrogestrinone (THG).

We have recently taken advantage of the unique power of DNA microarrays to compare the genomic expression profile of tetrahydrogestrinone (THG) with that of dihydrotestosterone (DHT), the most potent natural androgen, thus clearly demonstrating that THG is an anabolic steroid. In 2004, the U.S. Controlled Substances Act has been modified to include androstenedione (4-dione) as an anabolic steroid. However, despite the common knowledge that dehydroepiandrosterone (DHEA) is the precursor of testosterone, DHEA has been excluded from the list of anabolic steroids. We thus used the same DNA microarray technology to analyze the expression profile of practically all the 30,000 genes of the mouse genome modulated by DHEA and DHT in classical androgen-sensitive tissues. Daily subcutaneous injections of DHT (0.1mg) or DHEA (3mg) for 1 month in gonadectomized C57BL6/129 SV mice increased ventral prostate, dorsal prostate, seminal vesicle and preputial gland weight (p<0.01 for all tissues). As early as 24h after single injection of the two steroids, 878, 2681 and 14 probe sets were commonly stimulated or inhibited (p<0.01, change> or =30%), in the prostate (ventral+dorsal), seminal vesicles and preputial glands, respectively, compared to tissues from gonadectomized control animals. After 7 days of daily treatment with DHEA and DHT, 629, 919 and 562 probe sets were commonly modulated in the same tissues while after 27 days of treatment, 1195, 5127 and 2883 probe sets were modulated, respectively. In analogy with the data obtained with THG, the present microarray data provide an extremely precise and unquestionable genomic signature and proof of the androgenic/anabolic activity of DHEA. Such data add to the literature showing that DHEA is transformed into androgens in the human peripheral tissues as well as in laboratory animal species, including the monkey, thus exerting potent androgenic/anabolic activity. The present microarray approach to identify anabolic compounds is applicable to all potential androgenic/anabolic compounds.

Comparison of crystal structures of human androgen receptor ligand-binding domain complexed with various agonists reveals molecular determinants responsible for binding affinity.

Androgens exert their effects by binding to the highly specific androgen receptor (AR). In addition to natural potent androgens, AR binds a variety of synthetic agonist or antagonist molecules with different affinities. To identify molecular determinants responsible for this selectivity, we have determined the crystal structure of the human androgen receptor ligand-binding domain (hARLBD) in complex with two natural androgens, testosterone (Testo) and dihydrotestosterone (DHT), and with an androgenic steroid used in sport doping, tetrahydrogestrinone (THG), at 1.64, 1.90, and 1.75 A resolution, respectively. Comparison of these structures first highlights the flexibility of several residues buried in the ligand-binding pocket that can accommodate a variety of ligand structures. As expected, the ligand structure itself (dimension, presence, and position of unsaturated bonds that influence the geometry of the steroidal nucleus or the electronic properties of the neighboring atoms, etc.) determines the number of interactions it can make with the hARLBD. Indeed, THG--which possesses the highest affinity--establishes more van der Waals contacts with the receptor than the other steroids, whereas the geometry of the atoms forming electrostatic interactions at both extremities of the steroid nucleus seems mainly responsible for the higher affinity measured experimentally for DHT over Testo. Moreover, estimation of the ligand-receptor interaction energy through modeling confirms that even minor modifications in ligand structure have a great impact on the strength of these interactions. Our crystallographic data combined with those obtained by modeling will be helpful in the design of novel molecules with stronger affinity for the AR.

Urine testing for designer steroids by liquid chromatography with androgen bioassay detection and electrospray quadrupole time-of-flight mass spectrometry identification.

New anabolic steroids show up occasionally in sports doping and in veterinary control. The discovery of these designer steroids is facilitated by findings of illicit preparations, thus allowing bioactivity testing, structure elucidation using NMR and mass spectrometry, and final incorporation in urine testing. However, as long as these preparations remain undiscovered, new designer steroids are not screened for in routine sports doping or veterinary control urine tests since the established GC/MS and LC/MS/MS methods are set up for the monitoring of a few selected ions or MS/MS transitions of known substances only. In this study, the feasibility of androgen bioactivity testing and mass spectrometric identification is being investigated for trace analysis of designer steroids in urine. Following enzymatic deconjugation and a generic solid-phase extraction, the samples are analyzed by gradient LC with effluent splitting toward two identical 96-well fraction collectors. One well plate is used for androgen bioactivity detection using a novel robust yeast reporter gene bioassay yielding a biogram featuring a 20-s time resolution. The bioactive wells direct the identification efforts to the corresponding well numbers in the duplicate plate. These are subjected to high-resolution LC using a short column packed with 1.7-microm C18 material and coupled with electrospray quadrupole time-of-flight mass spectrometry (LC/QTOFMS) with accurate mass measurement. Element compositions are calculated and used to interrogate electronic substance databases. The feasibility of this approach for doping control is demonstrated via the screening of human urine samples spiked with the designer anabolic steroid tetrahydrogestrinone. Application of the proposed methodology, complementary to the established targeted urine screening for known anabolics, will increase the chance of finding unknown emerging designer steroids, rather then being solely dependent on findings of the illicit preparations themselves.

Tetrahydrogestrinone is a potent but unselective binding steroid and affects glucocorticoid signalling in the liver.

Tetrahydrogestrinone (THG) is a steroid recently identified to be misused as doping agent. However, the knowledge on functions of this substance in humans or animal models is rather limited. Therefore, it was our aim to further characterize the pharmacological profile of THG and identify potential adverse side effects. THG was synthesized, the purity was confirmed and its biological activity was tested. The potency of THG to transactivate AR dependent reporter gene expression was two orders of magnitude lower compared to dihydrotestosterone. THG binds with high affinity but unselective to the androgen (AR), progesterone (PR), glucocorticoid (GR) and mineralocorticoid (MR) receptor. Treatment of orchiectomised rats with THG resulted in a stimulation of prostate, seminal vesicle and levator ani muscle, indicating androgenic and anabolic properties. In the liver THG, in contrast to testosteronepropionate (TP), down regulates the expression of the GR dependent tyrosine aminotransferase gene (TAT). In summary, our results demonstrate that THG is not a specific AR agonist. THG exhibits a high binding affinity to all tested steroid hormone receptors and binds with highest affinity to the GR. Our in vivo data are indicative of an anabolic and androgenic potency of THG, but the repression of TAT demonstrates that THG also interferes with the glucocorticoid hormone system. Therefore, it is conceivable that an intake will result in adverse side effects.

Cardiovascular toxicities of performance-enhancing substances in sports.

Athletes commonly use drugs and dietary supplements to improve athletic performance or to assist with weight loss. Some of these substances are obtainable by prescription or by illegal means; others are marketed as supplements, vitamins, or minerals. Nutritional supplements are protected from Food and Drug Administration regulation by the 1994 US Dietary Supplement Health and Education Act, and manufacturers are not required to demonstrate proof of efficacy or safety. Furthermore, the Food and Drug Administration lacks a regulatory body to evaluate such products for purity. Existing scientific data, which consist of case reports and clinical observations, describe serious cardiovascular adverse effects from use of performance-enhancing substances, including sudden death. Although mounting evidence led to the recent ban of ephedra (ma huang), other performance-enhancing substances continue to be used frequently at all levels, from elementary school children to professional athletes. Thus, although the potential for cardiovascular injury is great, few appropriately designed studies have been conducted to assess the benefits and risks of using performance-enhancing substances. We performed an exhaustive OVID MEDLINE search to Identify all existing scientific data, review articles, case reports, and clinical observations that address this subject. In this review, we examine the current evidence regarding cardiovascular risk for persons using anabolic-androgenic steroids including 2 synthetic substances, tetrahydrogestrinone and androstenedione (andro), stimulants such as ephedra, and nonsteroidal agents such as recombinant human erythropoietin, human growth hormone, creatine, and beta-hydroxy-beta-methylbutyrate.

Tetrahydrogestrinone: the discovery of a designer steroid.

The use of steroids and other pharmaceuticals to gain a competitive edge in athletics has been present in the sports world for a long time. Over the past several years, scientific advances in the detection of sports doping agents and improved collaboration between sports organizations have enhanced the monitoring of fair athletic play. Many have suspected the illegal development of designer steroids by rogue scientists to avoid detection by the standard sports doping drug screen. In 2003, the Olympic Analytical Laboratory at the University of California, Los Angeles discovered the first designer steroid, tetrahydrogestrinone (THG), by using liquid chromatography with tandem mass spectrometry. Over the past year, the THG story continues to shock the sports world with its potential to discredit or terminate several high-profile athletic careers. While confirming the existence of designer steroids is credit to the sports antidoping movement, antidoping agencies will need to continue to invest in research and depend on honest athletic participants to maintain fairness and safety in sports.

Tetrahydrogestrinone is an androgenic steroid that stimulates androgen receptor-mediated, myogenic differentiation in C3H10T1/2 multipotent mesenchymal cells and promotes muscle accretion in orchidectomized male rats.

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.