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Heart Research Institute (A.K.D.), Camperdown, Sydney, New South Wales 2050, Australia; University of Sydney (A.K.D., K.C.Y.M., D.J.H.), Sydney, New South Wales 2006, Australia; Australian Sports Drug Testing Laboratory (R.K.), Australian Government Analytical Laboratories, Pymble, New South Wales 2073, Australia; and ANZAC Research Institute (D.J.H.), Sydney, New South Wales 2139, Australia
Address all correspondence and requests for reprints to: Professor D. J. Handelsman, ANZAC Research Institute, Sydney, New South Wales 2139, Australia. E-mail: djh{at}anzac.edu.au.
Abstract
Tetrahydrogestrinone (THG) was recently identified as a novel steroid used illicitly to improve athletic performance. Although its structure is closely related to gestrinone, a 19-nor progestin, and resembles that of trenbolone, THG was never marketed, so information on its hormonal properties is not known. In this study, we demonstrate that THG is a highly potent androgen and progestin in a yeast-based in vitro bioassay system expressing human androgen and progesterone receptors. It has no estrogenic activity and no antagonism for any of the three steroid receptor classes.
TETRAHYDROGESTRINONE (THG) WAS recently identified as a novel, never marketed steroid used illicitly to enhance performance in elite sports. Its existence was identified by sports doping authorities alerted to the presence of an illicit substance whose molecular structure was established by mass spectrometry and allied methods to be THG, a derivative of gestrinone, a seldom-used progestin marketed to treat endometriosis (1). Subsequently, a mass spectrometry-based method for detection of THG was developed by the International Olympic Committee/World Anti-Doping Agency -accredited laboratory in Los Angeles, California, and distributed to other accredited laboratories. This effective screening and confirmation testing methodology allowed the urine from several athletes to be reported positive, indicating THG use. With the recently described discovery of norbolethone (2), THG is the second steroid identified from use in sports doping that was never marketed. As a result, the hormonal and toxicological profile of THG is not known. The structure of THG resembles trenbolone, a veterinary androgen banned for use in sport and also for use in animals sold for food within the European Union, but has never been evaluated for human use. THG differs from gestrinone by reduction of the 17-ethynyl group to an ethyl group, but the main ring structure is unmodified. Predictions of biological effects of such structural modifications of a steroid, however, require empirical verification. Due to the circumstances of its illicit use and its structural similarity to well-known androgens, we investigated its biological interactions with human androgen receptor (AR), progesterone receptor (PR), and estrogen receptor (ER) stably transformed in a yeast-based in vitro bioassay (3).
Materials and Methods
Materials
THG was obtained as an ISO-certified reference material of 95% purity and was a gift from the United States Anti-Doping Agency to the Australian Sports Drug Testing Laboratory (ASDTL; Pymble, NSW, Australia). Other hormones were obtained from ASDTL and Steraloids, Inc. (Newport, RI), and stock solutions were dissolved in methanol.
Plasmids and reporter gene constructs
The full-length human PR cDNA plasmid and the PRE-ß-galactosidase reporter plasmid were kindly provided by Professor D. P. McDonnell (Duke University Medical Center, Durham, NC). Yeast strain YPH500 (MAT
, ura3–52, lys2–801, ade2–101, trp1-
63, his3-200, leu2-1) was cotransformed with both plasmids by standard alkali transformation (Alkali cation yeast transformation kit, BIO101 systems, Qbiogene Inc., Carlsbad, CA). Cotransformant yeast strains were selected by tryptophan and uracil auxotrophy. Prof. McDonnell also kindly provided two yeast strains: 1) YPH500 transformed with YEPE22 and YRpE2, and 2) YPH500 transformed with YEPAR and YPpG2.
Androgen, progesterone, and ER bioassays
For AR and ER bioassays, yeast from early-mid-log phase growth were diluted (to OD600 = 0.03) in selective medium (CSM-leu-ura) plus 50 µM CuSO4 to induce receptor production. For the PR bioassay, yeast were diluted (to OD600 = 1.0) in selective medium (CSM-trp-ura). Diluted yeast were aliquoted into 24-well culture plates (500 µl/well) and 5-µl doses of steroid in methanol were added. For antagonism experiments, 5 µl of testosterone (5 x 10–9 M) or progesterone (5 x 10–9 M) and 5 µl of THG (1 x 10–7 M) were added. Each assay included testosterone (AR), progesterone (PR), or estradiol (ER) standard curve as well as a vehicle (0.1% methanol) control. Multiwell plates were incubated at 30 C with shaking for either overnight (AR and ER) or 4 h (PR). After incubation, the yeast culture samples were washed in assay buffer, lysed, and extract assayed for ß-galactosidase by a standard method (4). Dose-responses were fitted to a four parameter sigmoid curve using nonlinear regression option in Sigmaplot version 8 (SPSS, Inc., Chicago, IL).
Results
THG agonist activity in the AR bioassay was compared with gestrinone, trenbolone, and nandrolone over the range of 10–7 M to 10–12 M (Fig. 1
). No cellular toxicity was observed for any steroids. THG was a potent activator of AR transactivation with an ED50 of 0.5 nmol/liter, an order of magnitude lower than the ED50 of the positive androgen control, nandrolone; its parent compound, gestrinone; or the structurally similar compound, trenbolone (Table 1).
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) with 7-fold higher molar potency than progesterone (Table 1
). By contrast, gestrinone and trenbolone have much weaker progestin activity than progesterone.
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Discussion
Using a rapid and sensitive in vitro bioassay for androgen, progesterone, and estrogen activity based on yeast cells expressing human AR, PR, and ER, respectively, the present study shows that THG is a potent androgen and progestin, but lacks estrogenic activity or any sex steroid antagonism. In this in vitro system, the potency of THG as an androgen and progestin is remarkably high. In the androgen bioassay, it has higher potency than the two comparator androgens, nandrolone, the most widely abused androgen in sports doping, and trenbolone, among the most potent synthetic androgens known. Similarly, as a progestin it exceeds not only the potency of progesterone but also that of its parent steroid, gestrinone. Although such in vitro bioassay potency demonstrates the bioactivity of THG, its in vivo potency also depends upon the steroid’s circulating half-life, which is largely determined by its binding to SHBG, both of which remain undefined. The biological effects of THG may be related to those of its parent compound gestrinone, a seldom prescribed 19-nor progestin used mostly to treat endometriosis (5) but also uterine myoma, meningiomas, and mastalgia (6, 7, 8). Gestrinone binds to AR and PR (9, 10) and demonstrates androgenic effects including acne, weight gain, voice change, hirsutism, as well as increased hemoglobin and bone density in treated women (11, 12, 13, 14). It is unlikely that THG is an in vivo metabolite of gestrinone (15, 16).
As an illicit designer androgen (2), THG raises important questions of safety and of the detection of other custom synthesized androgens derived from marketed sex steroids. Because THG has not undergone toxicology evaluation, its safety remains undefined, which is particularly disturbing for a potent steroid sold without medical scrutiny. Its potent androgen and progestin effects in concert would be expected to profoundly suppress the hypothalamo-pituitary gonadal axis, leading to reduced spermatogenesis, testicular atrophy, and infertility in men as well as androgen withdrawal effects upon cessation of the exogenous androgen (17). At high doses, behavioral disturbances including hypomania may occur in some users (18). In women, THG could cause virilization and could lead in children to premature epiphyseal closure and reduced final height. The 17 alkylated side-chain makes hepatotoxicity likely (19). An important issue for this nonproprietary steroid is the risk of toxic contaminants and impurities arising from the unknown production processes. Only further evaluation of the hormonal and toxicological properties of pure THG as well as its street variety can allay these concerns.
The potent progestin activity of THG may also interfere markedly in progesterone effects in the female reproductive system (e.g. menstrual disturbances, anovulation, infertility) as well as exerting less well understood nonreproductive effects mediated via PR in men and women, which widen the scope of PR-mediated effects.
The overlap of AR and PR activity is not surprising, given the origin of many synthetic progestins as derivatives of 19-nor testosterone (nandrolone) as well as the evolutionary similarity of the AR and PR, the most closely homologous of the classical nuclear steroid receptors.
In conclusion, we show unequivocally using an in vitro yeast cell bioassay for steroid hormones that THG, a newly discovered illicit designer steroid used by athletes to enhance performance, is a potent androgen and progestin. THG had no effect on ER action and showed no antagonistic effects against any of the sex steroid receptors. Further studies are required to characterize the full spectrum of hormonal and toxicological properties of THG. In addition the first discovery of an illicit designer androgen that has never been evaluated or marketed raises concerns about the possibility of a range of novel androgens being produced from marketed progestins and other synthetic sex steroids.
Acknowledgments
We are grateful to Professor Donald P. McDonnell for the gift of plasmids YEphPR-B, YRpG2, and transformed yeast strains YPH500 transformed with YEPE22 and YRpE2, and YPH500 transformed with YEPAR and YPpG2. We also thank the United States Anti-Doping Agency for commissioning the synthesis of THG by the National Analytical Reference Laboratory and supplying this reference compound to all International Olympic Committee/World Anti-Doping Agency-accredited laboratories for study.
Footnotes
Abbreviations: AR, Androgen receptor; ER, estrogen receptor; PR, progesterone receptor; THG, tetrahydrogestrinone.
Received January 8, 2004.
Accepted March 1, 2004.
References
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