Androgens are the class of sex steroids responsible for male sexual characteristics, including increased muscle mass and decreased fat mass. Illicit use of androgen doping can be an attractive option for those looking to enhance sporting performance and/or physical appearance. The use of in vitro bioassays to detect androgens, especially designer or proandrogens, is becoming increasingly important in combating androgen doping associated with nutritional supplements. The nutritional sports supplement market has grown rapidly throughout the past decade. Many of these supplements contain androgens, designer androgens or proandrogens. Many designer or proandrogens cannot be detected by the standard highly-sensitive screening methods such as gas chromatography-mass spectrometry because their chemical structure is unknown. However, in vitro androgen bioassays can detect designer and proandrogens as these assays are not reliant on knowing the chemical structure but instead are based on androgen receptor activation. For these reasons, it may be advantageous to use routine androgen bioassay screening of nutraceutical samples to help curb the increasing problem of androgen doping.
References
[1]
Barroso, O.; Mazzoni, I.; Rabin, O. Hormone abuse in sports: The antidoping perspective. Asian J. Androl. 2008, 10, 391–402.
[2]
Palacios, S. Androgens and female sexual function. Maturitas 2007, 57, 61–65.
[3]
Yesalis, C.E. Anabolic Steroids in Sport and Exercise, 2nd ed. ed.; Human Kinetics Publishers: Champaign, IL, USA, 2000.
[4]
Hartgens, F.; Kuipers, H. Effects of androgenic-anabolic steroids in athletes. Sports Med. 2004, 34, 513–554.
[5]
Mauras, N.; Hayes, V.; Welch, S.; Rina, A.; Helgeson, K.; Dokler, M.; Veldhuis, J.D.; Urban, R.J. Testosterone deficiency in young men: Marked alterations in whole body protein kinetics, strength, and adiposity. J. Endocrinol. Metab. 1998, 83, 1886–1892.
[6]
Bhasin, S.; Woodhouse, L.; Casaburi, R.; Singh, A.B.; Bhasin, D.; Berman, N.; Chen, X.; Yarasheski, K.E.; Magliano, L.; Dzekov, C.; et al. Testosterone dose-response relationships in healthy young men. Am. J. Physiol. Endocrinol. Metab. 2001, 281, 1172–1181.
[7]
Kadi, F.; Schjerling, P.; Andersen, L.L.; Charifi, N.; Madsen, J.L.; Christensen, L.R.; Andersen, J.L. The effects of heavy resistance training and detraining on satellite cells in human skeletal muscles. J. Physiol. 2004, 558, 1005–1012.
[8]
Casaburi, R. Rationale for anabolic therapy to facilitate rehabilitation in chronic obstructive pulmonary disease. Baillière's Clin. Endocrinol. Metab. 1998, 12, 407–418.
[9]
Demling, R.H.; Orgill, D.P. The anticatabolic and wound healing effects of the testosterone analog oxandrolone after severe burn injury. J. Crit. Care 2000, 15, 12–17.
[10]
Dobs, A.S. Androgen therapy in AIDS wasting. Baillière's Clin. Endocrinol. Metab. 1998, 12, 379–390.
[11]
Soliman, A.T.; Khadir, M.M.A.; Asfour, M. Testosterone treatment in adolescent boys with constitutional delay of growth and development. Metabolism 1995, 44, 1013–1015.
[12]
Srinivas-Shankar, U.; Sharma, D. Testosterone treatment in elderly men. Adv. Ther. 2009, 26, 25–39.
[13]
Fitch, K.D. Androgenic-anabolic steroids and the Olympic Games. Asian J. Androl. 2008, 10, 384–390.
[14]
Pallesen, S.; Josendal, O.; Johnson, B.-H.; Larsen, S.; Molde, H. Anabolic steroid use in high school students. Subst. Use Misuse 2006, 41, 1705–1717.
[15]
Brower, K.J.; Eliopulos, G.A.; Blow, F.C.; Catlin, D.H.; Beresford, T.P. Evidence for physical and psychological dependence on anabolic androgenic steroids in eight weight lifters. Am. J. Psychiatry 1990, 147, 510–512.
[16]
Turillazzi, E.; Perilli, G.; di Paolo, M.; Neri, M.; Riezo, I.; Fineschi, V. Side effects of AAS abuse: An overview. Mini Rev. Med. Chem. 2011, 11, 374–389.
[17]
Kicman, A.T. Pharmacology of anabolic steroids. Br. J. Pharmacol. 2008, 154, 502–521.
[18]
Crowley, R.; Fitzgerald, L.H. The impact of cGMP compliance on consumer confidence in dietary supplement products. Toxicology 2006, 221, 9–16.
[19]
Maughan, R.J.; Depiesse, F.; Geyer, H. The use of dietary supplements by athletes. J. Sport Sci. 2007, 25, S103–S113.
[20]
Geyer, H.; Parr, M.K.; Koehler, K.; Mareck, U.; Schanzer, W.; Thevis, M. Nutritional supplements cross-contaminated and faked with doping substances. J. Mass Spectrom. 2008, 43, 892–902.
[21]
Geyer, H.; Parr, M.K.; Mareck, U.; Reinhart, U.; Schrader, Y.; Schanzer, W. Analysis of non-hormonal nutritional supplements for anabolic-androgenic-steroids—Results of an international study. Int. J. Sports Med. 2004, 25, 124–129.
[22]
Plotan, M.; Elliott, C.T.; Scippo, M.L.; Muller, M.; Antignac, J.P.; Malone, E.; Bovee, T.F.H.; Mitchell, S.; Connolly, L. The application of reporter gene assays for the detection of endocrine disruptors in sports supplements. Anal. Chim. Acta 2011, 700, 34–40.
[23]
Geyer, H.; Bredehoft, M.; Mareck, U.; Parr, M.; Schanzer, W. High doses of the anabolic steroid metandienone found in dietary supplements. Euro. J. Sport Sci. 2003, 3, 1–5.
[24]
Parr, M.K.; Geyer, H.; Hoffmann, B.; Kohler, K.; Mareck, U.; Schanzer, W. High amounts of 17-methylated anabolic-androgenic steroids in effervescent tablets on the dietary supplement market. Biomed. Chromatogr. 2007, 21, 164–168.
[25]
Toorians, A.W.F.T.; Bovee, T.F.H.; de Rooy, J.; Stolker, L.A.A.M.; Hoogenboom, R.L.A.P. Gynaecomastia linked to the intake of a herbal supplement fortified with diethylstilbestrol. Food Addit. Contam. 2010, 27, 917–925.
[26]
Van de Wijngaart, D.J.; Dubbink, H.J.; van Royen, M.E.; Trapman, J.; Jenster, G. Androgen receptor coregulators: Recruitment via the coactivator binding groove. Mol. Cell. Endocrinol. 2012, 352, 57–69.
[27]
Cutress, M.L.; Whitaker, H.C.; Mills, I.G.; Stewart, M.; Neal, D.E. Structural basis for the nuclear import of the human androgen receptor. J. Cell Sci. 2008, 121, 957–968.
Wang, Q.; Li, W.; Liu, X.S.; Carroll, J.S.; Janne, O.A.; Keeton, E.K.; Chinnaiyan, A.M.; Pienta, K.J.; Brown, M. A hierarchical network of transcription factors governs androgen receptor-dependent prostate cancer growth. Mol. Cell 2007, 27, 380–392.
[30]
World Anti-Doping Agency. Available online: http://www.wada-ama.org/Documents/World_Anti-Doping_Program/WADP-IS-Laboratories/Technical_Documents/WADA_TD2004EAAS_Reporting_Evaluation_Testosterone_Epitestosterone_TE_Ratio_EN.pdf (accessed on 7 September 2012).
[31]
Scippo, M.L.; van de Weerdt, C.; Willemsen, P.; Francois, J.M.; Rentier-Delrue, F.; Muller, M.; Martial, J.A.; Maghuin-Rogister, G. Detection of illegal growth promoters in biological samples using receptor binding assays. Anal. Chim. Acta. 2002, 473, 135–141.
[32]
Scippo, M.L.; Argiris, C.; van de Weerdt, C.; Muller, M.; Willemsen, P.; Martial, J.; Maghuin-Rogister, G. Recombinant human estrogen, androgen and progesterone receptors for detection of potential endocrine disruptors. Anal. Bioanal. Chem. 2004, 378, 664–669.
[33]
Freyberger, A.; Weimer, M.; Tran, H.-S.; Ahr, H.-J. Assessment of a recombinant androgen receptor binding assay: Initial steps towards validation. Reprod. Toxicol. 2010, 30, 2–8.
[34]
Friedel, A.; Geyer, H.; Kamber, M.; Laudenbach-Leschowsky, U.; Schanzer, W.; Thevis, M.; Vollmer, G.; Zierau, O.; Diel, P. 17β-hydroxy-5alpha-androst-1-en-3-one (1-testosterone) is a potent androgen with anabolic properties. Toxicol. Lett. 2006, 165, 149–155.
[35]
Attardi, B.J.; Page, S.T.; Hild, S.A.; Coss, C.C. Mechanism of action of bolandiol (19-nortestosterone-3β, 17β-diol), a unique anabolic steroid with androgenic, estrogenic, and progestational activities. J. Steroid Biochem. Mol. Biol. 2010, 118, 151–161.
[36]
Catlin, D.H.; Ahrens, B.D.; Kucherova, Y. Detection of norbolethone, an anabolic steroid never marketed, in athletes' urine. Rapid Commun. Mass Spectrom. 2002, 16, 1273–1275.
[37]
Death, A.K.; McGrath, K.C.Y.; Kazlauskas, R.; Handelsman, D.J. Tetrahydrogestrinone is a potent androgen and progestin. J. Clin. Endocrinol. Metab. 2004, 89, 2498–2500.
[38]
Nielen, M.W.F.; Bovee, T.F.H.; van Engelen, M.C.; Rutgers, P.; Hamers, A.R.M.; Rhijn, J.H.A.; Hoogenboom, L.R.A.P. Urine testing for designer steroids by liquid chromatography with androgen bioassay detection and electrospray quadrupole time-of-flight mass spectrometry identification. Anal. Chem. 2006, 78, 424–431.
[39]
McRobb, L.; Handelsman, D.J.; Kaslauskis, R.; Wilkinson, S.; McLeod, M.D.; Heather, A.K. Structure-activity relationships of synthetic progestins in a yeast-based in vitro androgen bioassay. J. Steroid Biochem. Mol. Biol. 2008, 110, 39–47.
[40]
Kennel, P.F.; Pallen, C.T.; Bars, R.G. Evaluation of the rodent Hershberger assay using three reference endocrine disrupters (androgen and antiandrogens). Reprod. Toxicol. 2004, 18, 63–73.
[41]
Luscio, C.; Magi, E.; di Carro, M.; Suter, M.J.F.; Vermeirssen, E.L.M. Combining passive samples and biomonitors to evaluate endocrine disrupting compounds in a wastewater treatment plant by LC/MS/MS and bioassay analysers. Environ. Pollut. 2009, 157, 2716–2721.
[42]
Cacciatore, G.; Eisenberg, S.W.F.; Situ, C.; Mooney, M.H.; Delahaut, P.; Klarenbeek, S.; Huet, A.; Bergwerff, A.A.; Elliott, C.T. Effect of growth-promoting 17β-estradiol, 19-nortestosterone and dexamethasone on circulating levels of nine potential biomarker candidates in veal calves. Anal. Chim. Acta 2009, 637, 351–359.
[43]
Giantin, M.; Gallina, G.; Pegolo, S.; Lopparelli, R.M.; Sandron, C.; Zancanella, V.; Nebbia, C.; Favretto, D.; Capolongo, F.; Montesissa, C.; et al. Primary hepatocytes as an useful bioassay to characterize metabolism and bioactivity of illicit steroids in cattle. Toxicol. In Vitro 2012, 26, 1224–1232.
[44]
Draisci, R.; Merlanti, R.; Ferretti, G.; Fantozzi, L.; Ferranti, C.; Capolongo, F.; Segato, S.; Montesissa, C. Excretion profile of boldenone in urine of veal calves fed two different milk replacers. Anal. Chim. Acta 2007, 586, 171–176.
[45]
Bovee, T.F.H.; Bor, G.; Heskamp, H.H.; Lasaroms, J.J.P.; Sanders, M.B.; Nielen, M.W.F. Validation and application of a yeast bioassay for screening androgenic activity in calf urine and feed. Anal. Chim. Acta 2009, 637, 225–234.
[46]
Rijk, J.C.W.; Ashwin, H.; van Kuijk, S.J.A.; Groot, M.J.; Heskamp, H.H.; Bovee, T.F.H.; Nielen, M.W.F. Bioassay based screening of steroid derivatives in animal feed and supplements. Anal. Chim. Acta 2011, 700, 183–188.
[47]
Soto, A.M.; Sonnenschein, C.; Chung, K.L.; Fernandez, M.F.; Olea, N.; Serrano, F.O. The E-SCREEN assay as a tool to identify estrogens: An update on estrogenic environmental pollutants. Environ. Health Perspect. 1995, 103, 113–122.
[48]
Connolly, L.; Ropstad, E.; Verhaegen, S. In vitro bioassays for the study of endocrine-disrupting food additives and contaminants. Trends Anal. Chem. 2011, 30, 227–237.
[49]
Becue, I.; Bovee, T.F.H.; van Poucke, C.; Groot, M.J.; Nielen, M.W.F.; van Peteghem, C. Applicability of a yeast bioassay in the detection of steroid esters in hair. Anal. Bioanal. Chem. 2011, 399, 1031–1039.
[50]
Bovee, T.F.H.; Lommerse, J.P.M.; Peijnenburg, A.A.C.M.; Fernandes, E.A.; Nielen, M.W.F. A new highly androgen specific yeast biosensor, enabling optimisation of (Q)SAR model approaches. J. Steroid Biochem. Mol. Biol. 2008, 108, 121–131.
[51]
Bovee, T.F.H.; Helsdingen, R.J.R.; Hamers, A.R.M.; van Duursen, M.B.M.; Nielen, M.W.F.; Hoogenboom, R.L.A.P. A new highly specific and robust yeast androgen bioassay for the detection of agonists and antagonists. Anal. Bioanal. Chem. 2007, 389, 1549–1558.
[52]
Akram, O.N.; Bursill, C.; Desai, R.; Heather, A.K.; Kazlauskas, R.; Handelsman, D.J.; Lambert, G. Evaluation of androgenic activity of nutraceutical-derived steroids using mammalian and yeast in vitro androgen bioassays. Anal. Chem. 2011, 83, 2065–2074.
[53]
Death, A.K.; McGrath, K.C.Y.; Handelsman, D.J. Valproate is an anti-androgen and anti-progestin. Steroids 2005, 70, 946–953.
[54]
Leskinen, P.A.; Michelini, E.B.; Picard, D.C.; Karp, M.A.; Virta, M. Bioluminescent yeast assays for detecting estrogenic and androgenic activity in different matrices. Chemosphere 2005, 61, 259–266.
[55]
Sotoca, A.M.; Bovee, T.F.H.; Brand, W.; Velikova, N.; Boeren, S.; Murk, A.J.; Vervoort, J.; Rietjens, I.M.C.M. Superinduction of estrogen receptor mediated gene expression in Luciferase based reporter gene assays is mediated by a post-transcriptional mechanism. J. Steroid Biochem. Mol. Biol. 2010, 122, 204–211.
[56]
Bovee, T.F.H.; Pikkemaat, M.G. Bioactivity-based screening of antibiotics and hormones. J. Chromatrogr. A 2009, 1216, 8035–8050.
[57]
Roy, P.; Franks, S.; Read, M.; Huhtaniemi, I.T. Determination of androgen bioactivity in human serum samples using a recombinant cell based in vitro bioassay. J. Steroid Biochem. Mol. Biol. 2006, 101, 68–77.
[58]
Houtman, C.J.; Sterk, S.S.; van de Heijning, M.P.M.; Brouwer, A.; Stephany, R.W.; van der Burg, B.; Sonneveld, E. Detection of anabolic androgenic steroid abuse in doping control using mammalian reporter gene bioassays. Anal. Chim. Acta 2009, 637, 247–258.
[59]
Araki, N.; Ohno, K.; Takeyoshi, M.; Lida, M. Evaluation of a rapid in vitro androgen receptor transcriptional activation assay using AR-EcoScreen？ cells. Toxicol. In Vitro 2009, 19, 335–352.
[60]
Lee, H.J.; Lee, Y.S.; Kwon, H.B.; Lee, K. Novel yeast bioassay system for detection of androgenic and antiandrogenic compounds. Toxicol. In Vitro 2003, 17, 237–244.
[61]
Beck, V.; Reiter, E.; Jungbauer, A. Androgen receptor transactivation assay using green fluorescent protein as a reporter. Anal. Biochem. 2008, 373, 263–271.
[62]
Sonneveld, E.; Jansen, H.J.; Riteco, J.A.C.; Brouwer, A.; van der Burg, B. Development of androgen- and estrogen-responsive bioassays, members of a panel of human cell line-based highly selective steroid-responsive bioassays. Toxicol. Sci. 2005, 83, 136–148.
[63]
Michelini, E.; Magliulo, M.; Leskinen, P.; Virta, M.; Karp, M.; Roda, A. Recombinant cell-based bioluminescence assay for androgen bioactivity determination in clinical samples. Clin. Chem. 2005, 51, 1995–1998.
[64]
Rijk, J.C.W.; Bovee, T.F.H.; Peijnenburg, A.A.C.M.; Groot, M.J.; Rietjens, I.M.C.M.; Nielen, M.W.F. Bovine liver slices: A multifunctional in vitro model to study the prohormones dehydroepiandrosterone (DHEA). Toxicol. In Vitro 2012, 26, 1014–1021.
[65]
Rijk, J.C.W.; Bovee, T.F.H.; Groot, M.J.; Peijnenburg, A.A.C.M.; Nielen, M.W.F. Evidence of the indirect hormonal activity of prohormones using liver S9 metabolic bioactivation and an androgen bioassay. Anal. Bioanal. Chem. 2008, 392, 417–425.
