Anabolic steroidAndrogens were first discovered in Since then, testosterone and many other anabolic steroids have been successfully synthesized. There would be a forty-five year period between the discovery of the first androgen and scientists locating the androgen receptor in muscle. Due to anabolic steroids androgen receptor lack of knowledge regarding the androgen receptor in the muscle, scientists anabolic steroids androgen receptor how anabolic fluticasone propionate drug class worked in muscle tissue for many years. Some would question whether they even worked at all.
Which muscles have the most androgen receptors ? - Page 2
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 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 1 , 2. The compounds and methods included on the list are those that can be used by athletes to provide an unfair advantage 3. 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. 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 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 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 , 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 — 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 ligands for antidoping laboratories: The objective of this paper is to briefly review the androgen receptor and its action and discuss current assays being developed using the hAR. Steroid hormone receptors SHRs are members of the steroid and nuclear receptor superfamily This superfamily has over members, only 5 of which are SHRs: 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 transcription factor and is typically regulated by specific steroid ligands, such androgens and selective androgen receptor modulators SARMs. The hAR has a characteristic structure consisting of several domains: AFl and AF2 are modulatory regions that are involved in accessory protein binding dependent on the conformation of the receptor after ligand binding.
AF5 operates in a ligand-independent manner Structure of hAR gene. Modified from Gao et al. The hAR undergoes what has been called the two-step model of steroid action. In this model, inactive steroid receptors referred to as untransformed are associated with several chaperone proteins including the heat shock proteins hsp90, hsp70, and hsp53, as well as p23 and immunophilins via interactions in the LBD 24 — The dissociation of some of these chaperone proteins results in a transformed steroid receptor with exposed DBD which contains the dimerization domain and LBD.
Transformed receptors remain bound to hsp90, as hsp90 is required for proper LBD conformation for ligand binding. Transformed steroid receptors become activated when ligands bind and cause the loss of hsp Additionally, the structure of the NTD changes upon ligand binding 26 exposing a flexible region for the recruitment and assembly of coregulator proteins and transcriptional machinery.
The conformational changes which occur in this region of the hAR may serve as the primary mediator of the cell and gene specific effects of androgens With the loss of hsp90, the ligand-dependent NLS is exposed note that steroid receptors can dimerize while transformed or activated 24 — 25 , 27 ; the ligand-dependent NLS causes translocation of the receptor to the nucleus.
When inside the nucleus, translocated receptor dimers recruit transcriptional binding partners coregulators. This receptor dimer-coregulator complex binds to hormone promoters in DNA, associates with basal transcriptional machinery, and initiates transcription of various genes involved in anabolic processes Figure 2 Structure of hAR protein.
Modified from Nettles and Greene AREs are characterized by six-nucleotide half-site consensus sequences spaced by three random nucleotides in the promoter region of target genes: There are also several hormone response elements HREs which can bind to several or all steroid receptors, including the mouse mammary tumor virus promoter MMTV , and some glucocorticoid response elements GREs 31 , Steroid receptor export from the nucleus occurs at the nuclear pore complex and is facilitated via the RanGTP-dependent CRMl system 28 , It should be noted that in the absence of ligand, steroid receptors are in a steady state between the untransformed and transformed transformed receptors may or may not be active and associated with hsp90 states and constantly shuttling into and out of the nucleus though, in the absence a ligand, hAR remains in a steady state in which most of the receptor is cytosolic 24 , Association of hsp90 is also reversible via an ATP-dependent mechanism The hAR is expressed at varying levels in all major physiological systems including the central nervous, endocrine, metabolic, gastrointestinal, immune, reproductive, cardiovascular, and respiratory systems.
Most tissues have low-to-moderate expression levels; however, there are high expression levels in the epididymis, ovary, uterus, prostate, vas deferens, adrenal gland, kidney, and skeletal muscle Several different cancers, cardiovascular defects, neurological conditions, immune diseases, reproductive conditions, and psychiatric disorders have been associated with hAR dysfunction.
Human satellite cells are stem cells involved in the repair and maintenance of skeletal muscle and have been proposed as the primary site of the anabolic action of AAS 36 — However, the exact mechanism of action of AAS in these cells is still poorly understood. It has been proposed that AAS act through an hAR-mediated mechanism and induce the commitment of these cells into a myogenic lineage. The results of this mechanism in skeletal muscle are hypertrophy of both type I and type II muscle fibers but not an increase in the number of fibers 36 , 38 — 43 , an increase in the number of myonuclei and satellite cells resulting in an increase in the number available for conversion to skeletal muscle fibers 38 , 44 — 46 , and an upregulation of the number of hARs in the cells 39 , 44 — Coregulators of hAR are proteins that are recruited by the receptor and either enhance coactivators or reduce corepressors hAR mediated transactivation.
Coregulators act at DNA response elements in the promoter region of target genes to facilitate DNA binding, chromatin remodeling, or the recruitment of general transcription factors The position of helix 12 changes depending on which ligand is bound to the SHR. In the case of antagonist binding, corepressor proteins can bind and in the case of agonist binding, coactivator proteins can bind 49 — Coactivator recruitment is required for ligand activated SHR mediated transactivation Changes in the amount of free coactivator available for binding, expression level of coactivators, and tissue availability of coactivators can affect transcriptional activity of receptors Several families and types of coactivators and corepressors exist; there are specific coregulator proteins for each SHR as well as several that are shared by some or all SHRs 55 , Numerous families of coactivator proteins have been described and several coactivators have been identified as binding to and enhancing the ligand-inducing transcriptional activation of hAR Heemers and Tindall 48 extensively review the large number of hAR coregulator proteins.
Coregulators indeed play a significant role in activation of hAR, and the interplay of coactivators and coregulators contribute to the fine-tuning of hAR activity. The primary role of androgen mediated hAR signaling is the proper development and function of male reproductive organs as well as muscle maintenance. The physiologic effects of endogenous androgens are both androgenic and anabolic; AAS vary in their balance of androgenic to anabolic effects.
Androgenic effects include the growth, development, and maintenance of primary genitalia and genital tract and secondary sexual characteristics in men, the early stages of breast and pubertal development in girls adrenarche , spermatogenesis promotion, neuroendocrine regulation of gonadotropin secretion, and libido stimulation. Anabolic effects include stimulation of nitrogen retention, increased protein synthesis and production, increased lean body mass, increased body and muscle growth, skeletal growth, and epiphysis closure of long bones during puberty.
There are also some metabolic and hematologic actions of AAS including erythropoiesis, decreased synthesis of several clotting factors, increased sebum production in skin, decreased synthesis of HDL cholesterol, increased synthesis of LDL cholesterol, androgenic alopecia male pattern baldness , and increased bone density 57 , Testosterone is primarily synthesized in Leydig cells of the testes in men and the adrenal cortex, liver, and ovary in women.
Although some of the precursors in the synthetic pathway of testosterone, such as androstenedione, have weak agonist activity, testosterone and DHT are the primary endogenous androgens Figure 3. Abridged pathway of AAS and cortisol biosynthesis. Steroid synthesis begins with cholesterol. Modified from Schimmer and Parker Testosterone is metabolized in target tissues and in the liver. Testosterone can also be metabolized by aromatase enzymes to estradiol Figure 3. In addition to these metabolic pathways, TEST can be inactivated in the liver through reduction and oxidation followed by glucuronidation and renal excretion 21 , 57 — In addition to the endogenous AAS, there are several synthetic testosterone derivatives which are used clinically.
The therapeutic uses of androgens include hormone replacement therapy HRT in primary or secondary hypogonadism in men, induction of puberty in delayed sexual maturation of boys, osteoporosis in males, HRT in female menopause, endometriosis, treatment of anemia, treatment of hereditary angioedema, and the stimulation weight gain after surgery, infection, and AIDS. Because of aromatization, AAS also possess some estrogenic effects such as gynecomastia.
Common adverse effects include acne, scalp hair loss, obstructive sleep apnea, hirsutism, mild voice deepening, and edema. Serious adverse effects of AAS use and abuse are both physiological and psychiatric and include hepatotoxicity cholestatic hepatitis and jaundice, adenomas, carcinoma, peliosis hepatis , premature bone maturation and epiphyseal closure in adolescents , increased risk of cardiovascular disease, testicular atrophy, oligospermia, and prostatic hyperplasia or carcinoma 43 , 57 , The hAR in skeletal muscle is typically saturated with physiologic concentrations of circulating testosterone 6 , 8 ; however, those who use anabolic agents administer supraphysiologic doses times the physiological dose or more and multiple steroids on a daily basis.
Evidence suggests that at the supraphysiologic concentrations some steroids are competitive antagonists at the human glucocorticoid receptor hGR complementary to their agonist activity at hAR 60 — These actions can lead to increased nitrogen retention and protein production mediated by hAR, decreased protein catabolism mediated by the hGR 8 , and ultimately a net gain in muscle mass. Because of their anabolic effects, AAS are commonly abused by athletes 5 , 6 , 8 , Many synthetic AAS have been developed in an attempt to alleviate some of the adverse androgenic and estrogenic effects while enhancing the anabolic properties.
Most designer androgens originate from —s pharmaceutical industry androgen discovery and synthesis programs largely geared at identifying a purely anabolic steroid and oral contraceptives In , Julius A. Vida published a comprehensive book which discusses metabolic factors, structure activity relationships, and the therapeutic action of androgens. He compiles approximately androgens and anabolic agents and documents the relatively small changes in chemical structure which can bring about sharp changes in potency Recently, this textbook has re-emerged in bodybuilding circles and is discussed on website forums.