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. Cell-
based 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.
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 per-
formance 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-
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 ath-
letes to provide an unfair advantage (3). Substances on the
prohibited list include anabolic androgenic steroids, gluco-
corticosteroids, peptide hormones and their modulators, hor-
mone antagonists and their modulators, stimulants, β2-
agonists, 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 avail-
able) (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 syn-
thetic steroids as well as other nonsteroidal compounds that
modulate steroid receptors to increase endogenous anabolic
processes. These compoundsare produced soabusers can evade
detection and identification of these substances with current
The term AAS refers to testosterone and its derivatives and
analogues and SARMS which bind to the human androgen re-
ceptor (hAR). Endogenous AAS primary role is the mainte-
nance 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
TheAndrogen Receptor and Its Use in BiologicalAssays:
LookingToward Effect-BasedTesting and ItsApplications
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; and4Dipartimento Tecnologie e Management,“Sapienza” Università di Roma, Via del Castro
Laurenziano 9, 00161 Rome, Italy
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
Journal of AnalyticalToxicology,Vol. 35, November/December 2011
* Author to whom correspondence should be addressed. Email: Amy.Cadwallader@gmail.com.
Journal of AnalyticalToxicology,Vol. 35, November/December 2011
methods such as mass spectrometry (MS). Minor structural
modifications of a steroid can render it undetectable via con-
ventional 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 iden-
tified as THG, was discovered has brought the problem of de-
tecting AAS and other steroid abuse to light (9–13). The
number of designer steroids currently being abused is un-
known because detection methods are only identifying sub-
stances 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 concen-
trations of known prohibited substances. However, because
new steroids and synthetic compounds are made to evade con-
ventional testing methods while retaining desired anabolic ac-
tivity, new assays need to be developed to detect excess levels of
these substances (11,12). Some research developments have re-
cently 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 pre-
cursor 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 gen-
eration of detection methods, as the field moves away from
an effect of prohibited substances, will not require knowledge
of the exact structure of the compound and will employ bio-
logically based assays utilizing the hAR and other steroid re-
ceptors. Biological assays also have other applications beyond
the identification of steroid receptor ligands for antidoping
laboratories: they can be used alongside MS to determine the
structure of new ligands, they can be utilized to determine
the relative biological activity of steroid receptor ligands, and
they can be coupled with microsomal metabolism studies to as-
sess the biological activity of compound metabolites. The ob-
jective of this paper is to briefly review the androgen receptor
and its action and discuss current assays being developed using
Steroid Hormone Receptors
Steroid hormone receptors (SHRs) are members of the
steroid and nuclear receptor superfamily (17). This super-
family has over 100 members, only 5 of which are SHRs: es-
trogen, androgen, glucocorticoid, progesterone, and miner-
alocorticoid (18). SHRs are located in the cytosol and in the
nucleus of target cells and also on the plasma membrane. They
are typically cytoplasmic and nuclear transcription factors and
after ligand binding initiate signal transduction which leads to
changes in gene expression.
Similar to the other SHRs, the hAR functions as a tran-
scription factor and is typically regulated by specific steroid lig-
ands, such androgens and selective androgen receptor modu-
lators (SARMs). The hAR cDNA (GenBank ID NM_000044) is
approximately 2.8 kilobases and the eight exons code for 919
amino acids (approximately 112 kDa) (19–21). The hAR has a
characteristic structure consisting of several domains: two ac-
tivation functions (AF1 and AF5) in the N-terminal domain
(NTD), DNA-binding domain (DBD) which contains the dimer-
ization domain, a nuclear localization signal (NLS), hinge re-
gion, and a carboxy-terminal ligand-binding domain (LBD)
which contains a third activation function domain (AF2)
(17,21–22) (Figure 1). AF1 and AF2 are modulatory regions
thatareinvolvedinaccessory protein bindingdependentonthe
conformation of the receptor after ligand binding. AF5 operates
Figure 1. Structure of hAR gene. Domains of the gene and receptor; NTD, N-terminal domain;AF1/2/5, activation functions 1, 2, and 5; NLS, nuclear localization
signal; DBD, DNA binding domain; and LBD, ligand binding domain. Modified from Gao et al. (21).
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