The Androgen Receptor and Its Use in Biological Assays: Looking Toward Effect-Based Testing and Its Applications

Journal of Analytical Toxicology, Vol. 35, November/December 2011 The Androgen Receptor and Its Use in Biological Assays: Looking Toward Effect-Based Testing and Its Applications Amy B. Cadwallader1,*, Carol S. Lim2, Douglas E. Rollins3, and Francesco Botrè1,4 1Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti, 1, 00197 Rome, Italy; 2University of Utah, Department of Pharmaceutics and Pharmaceutical Chemistry, 421 Wakara Way, Room 318, Salt Lake City, Utah 84108; 3Center for Human Toxicology, University of Utah Department of Pharmacology and Toxicology, 417 Wakara Way, Suite 2111, Salt Lake City, Utah 84108; and 4Dipartimento Tecnologie e Management, “Sapienza” Università di Roma, Via del Castro Laurenziano 9, 00161 Rome, Italy Abstract Steroid abuse is a growing problem among amateur and professional athletes. Because of an inundation of newly and illegally synthesized steroids with minor structural modifications and other designer steroid receptor modulators, there is a need to develop new methods of detection which do not require prior knowledge of the abused steroid structure. The number of designer steroids currently being abused is unknown because detection methods in general are only identifying substances with a known structure. The detection of doping is moving away from merely checking for exposure to prohibited substance toward detecting an effect of prohibited substances, as biological assays can do. Cellbased biological assays are the next generation of assays which should be utilized by antidoping laboratories; they can detect androgenic anabolic steroid and other human androgen receptor (hAR) ligand presence without knowledge of their structure and assess the relative biological activity of these compounds. This review summarizes the hAR and its action and discusses its relevance to sports doping and its use in biological assays. Introduction The impetus to gain an edge in competitive sporting events has existed for as long as the sports themselves. Today, not only do athletes strive to be the best in their chosen sports, but there are also large financial incentives and outside pressures to succeed associated with the international sporting industry; these reasons have lead to a constant increase in the use of performance enhancing drugs (1). Despite centuries of reports of using substances to enhance athletic performance, systematic testing of athletes for the use of performance enhancing drugs began only in 1968 (1,2). Since that time, a list of banned sub- * Author to whom correspondence should be addressed. Email: . 594 stances and procedures has been maintained and constantly updated by the International Olympic Committee (IOC) and the World Anti-doping Agency (WADA). The compounds and methods included on the list are those that can be used by athletes to provide an unfair advantage (3). Substances on the prohibited list include anabolic androgenic steroids, glucocorticosteroids, peptide hormones and their modulators, hormone antagonists and their modulators, stimulants, β2agonists, narcotics, alcohol, β-blockers, cannabinoids, and diuretics and masking agents (3). Anabolic androgenic steroids (AAS) and other anabolic agents are by far the most widely abused substances included on the prohibited substances list, accounting for approximately 65% of all positive samples (both adverse and atypical findings) in 2009 (the most recent year for which official data are available) (4). The current regulations, instead of curtailing the use of AAS, have led to their clandestine production and the black market synthesis and sale of structurally unique synthetic steroids as well as other nonsteroidal compounds that modulate steroid receptors to increase endogenous anabolic processes. These compounds are produced so abusers can evade detection and identification of these substances with current analytical procedures. The term AAS refers to testosterone and its derivatives and analogues and SARMS which bind to the human androgen receptor (hAR). Endogenous AAS primary role is the maintenance of male sexual organs (androgenic effects); activation of the hAR by AAS may also result in an increase in muscle mass and strength (anabolic effects). Clinically, AAS are used for the treatment hypogonadism, impotence, and muscle wasting disorders; they are also abused by athletes for their anabolic properties. Major problems with the abuse of endogenous AAS, such as testosterone or dihydrotestosterone, are their high metabolism and serious side effects (5–7). Synthetic AAS are manufactured to reduce metabolism and increase potency (6,8). AAS are also synthesized to circumvent typical detection Downloaded from https://academic.oup.com/jat/article-abstract/35/9/594/853010 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission. by guest on 28 May 2018 Journal of Analytical Toxicology, Vol. 35, November/December 2011 methods such as mass spectrometry (MS). Minor structural modifications of a steroid can render it undetectable via conventional means yet allow it to maintain its anabolic potential, as was the case with tetrahydrogestrinone (THG) (9). The 2004 scandal in which a supposed “undetectable steroid”, later identified as THG, was discovered has brought the problem of detecting AAS and other steroid abuse to light (9–13). The number of designer steroids currently being abused is unknown because detection methods are only identifying substances with a known structure. Current techniques for the detection of sports doping, such as gas chromatography (GC)–MS, rely on prior knowledge of the structure of the steroid. These target methods are used in anti-doping laboratories to detect the presence of low concentrations of known prohibited substances. However, because new steroids and synthetic compounds are made to evade conventional testing methods while retaining desired anabolic activity, new assays need to be developed to detect excess levels of these substances (11,12). Some research developments have recently been made to overcome some of the pitfalls of known target analysis; these methods involve more sophisticated use of MS technology, including full-scan liquid chromatography (LC)– and GC–electrospray ionization orthogonal acceleration time-of-flight MS, full scan LC–time-of-flight MS, and precursor ion scanning after LC–electrospray-tandem MS (14– 16). Although very beneficial, it is still possible that these methods may miss newly developed compounds. The next generation of detection methods, as the field moves away from checking for exposure to prohibited substance toward detecting an effect of prohibited substances, will not require knowledge of the exact structure of the compound and will employ biologically based assays utilizing the hAR and other steroid receptors. Biological assays also have other applications beyond the identification of steroid receptor ligand (...truncated)