Asian J Androl 2008; 10 (3):351–363 DOI: 10.1111/j.1745-7262.2008.00407.x .Review . The physiological and pharmacological basis for the ergogenic effects of androgens in elite sports Karen Choong, Kishore M. Lakshman, Shalender Bhasin Boston University School of Medicine, Section of Endocrinology, Diabetes, and Nutrition, Boston Medical Center, Boston, MA 02118, USA Abstract Androgen doping in power sports is undeniably rampant worldwide. There is strong evidence that androgen administration in men increases skeletal muscle mass, maximal voluntary strength and muscle power. However, we do not have good experimental evidence to support the presumption that androgen administration improves physical function or athletic performance. Androgens do not increase specific force or whole body endurance measures. The anabolic effects of testosterone on the skeletal muscle are mediated through androgen receptor signaling. Testoster- one promotes myogenic differentiation of multipotent mesenchymal stem cells and inhibits their differentiation into the adipogenic lineage. Testosterone binding to androgen receptor induces a conformational change in androgen receptor protein, causing it to associate with beta-catenin and TCF-4 and activate downstream Wnt target genes thus promot- ing myogenic differentiation. The adverse effects of androgens among athletes and recreational bodybuilders are under reported and include acne, deleterious changes in the cardiovascular risk factors, including a marked decrease in plasma high-density lipoproteins (HDL) cholesterol level, suppression of spermatogenesis resulting in infertility, increase in liver enzymes, hepatic neoplasms, mood and behavioral disturbances, and long term suppression of the endogenous hypothalamic-pituitary-gonadal axis. Androgens are often used in combination with other drugs which may have serious adverse events of their own. In spite of effective methods for detecting androgen doping, the policies for screening of athletes are highly variable in different countries and organizations and even existing policies are not uniformly enforced. (Asian J Androl 2008 May; 10: 351–363) Keywords: testosterone; dihydrotestosterone; muscle mass; mechanisms of androgen action; androgen doping; mesenchymal stem cells; detection of androgen doping 1 Introduction the USA, and in all professional sports—not just base- ball—for the past two decade, were hardly surprised George J. Mitchell, a former US Senator, in a re- by these revelations. The use of performance enhanc- cent report on the use of illegal performance-enhanc- ing agents in sports is not a new phenomenon; docu- ing drugs in professional baseball, acknowledged per- mentation exists of the use of a variety of potions, plants, vasive use of androgenic-anabolic steroids by Major animal extracts as far back as the original Olympiads in League Baseball players in the USA; the list of those ancient Greece. Long before the isolation and synthe- linked to steroid use included many well known names sis of testosterone in the 1930s, Brown-Sequard and in baseball. The keen observers of the professional sports later Zoth and Pregl had recognized that contents of the scene, who have been sounding the alarm over the wide- testicular extracts could improve physical and mental spread use of ergogenic drugs worldwide—not just in energy, and muscle strength [1–4]. Shortly after suc- cessful synthesis of testosterone, Boje [5] suggested Correspondence to: Prof. Shalender Bhasin, Boston University that sex hormones might enhance physical performance. School of Medicine, Section of Endocrinology, Diabetes, and The Germans are alleged to have administered anabolic Nutrition, Boston Medical Center, Boston, MA 02118, USA. Tel: +1-617-414-2951 Fax: +1-617-638-8217 -androgenic steroids to soldiers going into combat [6]. E-mail: [email protected] Although it has been alleged that some German athletes Received 2008-01-02 Accepted 2008-01-08 were given testosterone in preparation for the 1936 Berlin T©e l2:0 +088,6 A-2s1ia-n5 4J9ou2r-n2a8l2 o4f; AFnadxro: l+og8y6,- 2S1IM-5M49 a2n-d2 8S2J5T;U S. Ahalln rgighhatsi, rCesherinvead. .351. Androgen doping in sports Olympics [6], the most cited account of systematic use One of the most alarming finding of this survey was of androgens in elite sports is that of the Soviet weight that approximately 70% of lifetime users of anabolic lifting team in the 1952 and 1956 Olympics. In 1954, steroids met criteria for an alcohol use disorder [17]. at the weight lifting championships in Vienna, Dr John Thus, college athletes who abuse androgens are at in- Ziegler, a physician associated with the US Weight Lifting creased risk for other risky health behaviors. Team, learned about the use of androgens by the Rus- sian weight lifters [6, 7]. Zeigler returned to the United 2 Patterns of androgen abuse by athletes and States and experimented with testosterone on himself and recreational body builders other weight lifters in the York Babel Club [7]. When Ciba Pharmaceuticals introduced Dianabol (methandro- Nandrolone, testosterone, stanozolol, methandienone, stenolone) in 1958, he began to experiment with this and methenolol are the most frequently abused androgens new drug [6, 7]. The use of androgens that was lim- [19–21]. Intramuscular formulations of androgens are ited initially to strength-intensive sports, spread gradu- used far more frequently than oral formulations [19]. ally over the ensuing decades to other sports and to Combinations of androgens are used more frequently recreational body building [6, 7]. The media lime light than single agents [19, 21]. Typically, athletes use two surrounding the detection of androgen use by elite ath- or more androgens in progressively increasing doses over letes such as Ben Johnson, Lyle Alzado, Mark Maguire, a period of several weeks in a practice known as “stacking”. Barry Bonds, Floyd Landis, and Marion Jones has only The doses of testosterone or other androgens used by added to the allure of performance enhancing drugs. athletes are substantially larger than those prescribed Admittedly, the exact prevalence of androgens use for the treatment of androgen deficiency. In one sur- by athletes is difficult to determine because the data vey [19], 50% of androgen users reported using at least rely on self-reports and many users understandably do 500 mg of testosterone weekly or an equivalent dose of not admit to the use of these drugs. But even surveys another androgen; in another survey [21], almost one based on self-report have found high rates of andro- fourth of androgen users used 1 000 mg testosterone gens use among professional athletes and Olympians weekly or an equivalent dose of other androgens. Cy- [7, 8]. Yesalis estimated that approximately one million cling of androgens refers to the intermittent use of an- Americans had used androgens sometime in their lives drogens in which weeks of androgen use are followed [9]. Four to six percent of high school boys and one to by periods of drug holiday; this practice is based on the two percent of high school girls admit to using andro- unproven premise that cyclic prevents desensitization gens at least once [10–13]. The androgen use among to massive doses of androgen. girls also has increased slightly during the past decade In addition to the use of androgens, athletes also although the overall use rates are substantially lower in abuse other drugs to purportedly enhance muscle- women than in men [7, 10]. building, muscle shaping, or athletic performance [19]. The abuse of performance-enhancing drugs is not These accessory drugs include stimulants, such as limited to the USA; similar high prevalence rates of an- amphetamine, clenbuterol, ephedrine, and thyroxine, other drogen use have been reported in surveys conducted in anabolic agents such as growth hormone, IGF-1, and other countries [14, 15]. The most egregious example of insulin, and drugs perceived to reduce adverse effects state-sponsored anabolic steroid doping was uncovered such as hCG, aromatase inhibitors or estrogen antago- in the former German Democratic Republic after the nists [19]. The potential adverse effects of some acces- fall of the communist government in 1990 [16]; classi- sory drugs may be more serious than those of androgens. fied documents revealed a secret state program from 1966 to improve national athletic performance using an- 3 Do androgens improve athletic performance? drogens with complicity of the sports medicine physicians. Surprising as it might seem in light of the wide- A recent study of the use of anabolic steroids among spread abuse of androgens, the evidence demonstra- US college students found that the overall prevalence ting improvements in athletic performance after andro- of androgen use was 1% or less [17]. The number of gen administration is sparse and weak. There is strong students reporting past-year use of androgens increased evidence that androgens increase skeletal muscle mass, among men from 1993 (0.36%) to 2001 (0.90%) [17] maximal voluntary strength, and leg power [22–27]; and has decreased slightly since then [18]. The life- even this assertion was debated rancorously for almost time and past-year steroid use were associated with being five decades. Much of the controversy stemmed from male, participation in intercollegiate athletics, and risky the well recognized difficulties in conducting placebo- behaviors, including cigarette smoking, illicit drug use, controlled, randomized, masked trials in athletes [27, drinking and driving, and alcohol use disorders [17]. 28]. It is not surprising that many of the earlier andro- .352. http://www.asiaandro.com; [email protected] Asian J Androl 2008; 10 (3):351–363 gen trials were neither randomized nor blinded. Some [25, 26] and that the androgen-induced gains in skeletal studies included competitive athletes whose adherence muscle mass and muscle strength are correlated with the to rigid research protocols is always suspect [27, 28]. administered dose and the circulating concentrations The protein and energy intake was not standardized; in (Figure 1) [23, 24, 29, 30]. Thus, administration of re- some studies, the participants continued to ingest pro- placement doses of testosterone to healthy, hypogonadal tein supplements ad lib. The exercise stimulus was not men [31–35] and of supraphysiologic doses to eugonadal standardized and, therefore, the effects of resistance men [22, 23] increases lean body mass, muscle size and exercise could not be separated from those of andro- strength (Figure 2). Systematic reviews of randomized gen administration. clinical trials have confirmed that testosterone therapy However, a growing body of data over the past de- is associated with greater gains in lean body mass and cade has established that androgens increase muscle mass grip strength than placebo in older men with low or low normal testosterone levels [25]. Similarly, test- Figure 2. Changes from baseline values in fat-free mass, triceps and quadriceps cross-sectional areas, and 1-repetition strength in the bench-press and squat exercises. Healthy young men were ran- domly assigned to receive either placebo or 600 mg testosterone Figure 1. Linear regression of the testosterone enanthate (TE) dose enanthate (TE) intramuscularly weekly with or without a stan- and change in total body fat-free mass (A), appendicular fat-free dardized program of resistance exercise training. The P values mass (B) and thigh muscle volume (C). TE dose was strongly and shown are for the comparison between the change indicated and a significantly correlated with changes from baseline in total body fat- change of zero. *P < 0.05 for the comparison between the change free mass, appendicular fat-free mass, and thigh muscle volume indicated and that in either no-exercise group; †P < 0.05 for the (Pearson product-moment correlation coefficients of r = 0.79–0.81, comparison between the change indicated and that in the group P < 0.0001 for each model), although there was substantial heteroge- assigned to placebo with no exercise; and ‡P < 0.05 for the compari- neity in individual responses to testosterone administration. Repro- son against the changes in all three other groups. Reproduced with duced with permission from Woodhouse et al. [30]. permission from Bhasin et al. [22]. Tel: +86-21-5492-2824; Fax: +86-21-5492-2825; Shanghai, China .353. Androgen doping in sports osterone therapy in HIV-infected men with weight loss reaction time by improving neuromuscular transmis- and in men with chronic obstructive lung disease pro- sion [39, 40]. Improved reaction time in conjunction motes greater gains in lean body mass and muscle with increased strength and power could potentially strength than placebo. There is considerable inter-sub- explain the perceived improvements in athletic perfor- ject variability in the anabolic response to androgen mance by baseball players, although the evidence to administration. A large part of the variance in anabolic support these hypotheses is lacking. response can be explained by the circulating androgen The use of androgens by legendary sprinters like concentrations; however, polymorphisms in the Ben Johnson is even more difficult to explain. Among polyglutamine and polyglycine tract length in androgen sprint runners, androgen-induced gains in body weight receptor protein, testosterone metabolism, and other might potentially increase the amount of work done in unknown genetic factors may contribute to this vari- carrying that body weight against gravity and resistance ance [30]. across the race track. Thus weight gain might be viewed The effects of testosterone on muscle performance as potentially deleterious to performance. The improved are domain specific; testosterone administration improves reaction time, the psychological edge gained because muscle strength and power, but does not affect spe- of the motivational effects of androgens, and the ability cific force or muscle fatigability. The gains in maximal to train harder, have been cited as possible explanations voluntary strength during testosterone administration are without verifiable evidence. highly correlated with increments in muscle mass; tes- tosterone does not improve the contractile property of 4 Mechanisms of anabolic effects of androgens the skeletal muscle. In contrast, resistance exercise training increases muscle mass as well as specific force. Testosterone-induced increase in skeletal muscle Androgens have not been shown to improve measures mass is associated with dose-dependent increase in of whole body endurance, such as VO2max and lactate cross-sectional area of both type I and type II muscle threshold. fibers [41]. Testosterone administration does not af- Based on their demonstrable effects on maximal fect the absolute number or the relative proportion of voluntary muscle strength, androgens would be ex- type I and type II muscle fibers [41]. Testosterone pected to improve performance in events such as power administration increases the numbers of myonuclei and lifting in which performance is dependent upon muscle satellite cells [42], muscle progenitor cells that reside in strength. Therefore, not surprisingly, high rates of an- a unique niche adjacent to the muscle fiber. Androgen drogen use have been reported among power lifters. receptors are expressed in the satellite cells and other Body builders use androgens to increase skeletal muscle stem-like cells in the interstitium of the skeletal muscle mass and decrease fat mass, which provides greater fibers and in some myonuclei of the myofibers [43]. definition to the muscles. However, the use of andro- A growing body of evidence supports the hypothesis gens by athletes participating in endurance events such that androgens promote the differentiation of mesenchy- as long distance running and bicycling is not easily com- mal multipotent stem cells into the myogenic lineage and prehensible because androgens have not been shown to inhibit their differentiation into the adipogenic lineage improve endurance measures [36, 37]. It is possible [44–46]. Thus, in cultures of mesenchymal multipotent that the androgen-induced increments in hemoglobin may C3H10T1/2 cells, androgens upregulate markers of improve the oxygen carrying capacity of blood [38]. myogenic differentiation, such as MyoD and myosin The speculation that androgens might allow the ath- heavy chain II and downregulate markers of adipogenic letes to train harder by improving the regenerative re- differentiation, such as PPAR-gamma and C/EBP-alpha sponse of the skeletal muscle to injury has not been [46]. These effects of testosterone and DHT on tested rigorously. Others have suggested that andro- myogenesis are mediated through the classical andro- gens might increase “motivation” and “aggression”, gen receptor-mediated signaling and are blocked by which may be advantageous in competitive sports. bicalutamide, an androgen receptor antagonist [46]. The widespread use of androgens by baseball play- Upon binding to its cognate ligand, androgen re- ers and sprint runners also is not easily explained by the ceptor undergoes conformational change and associ- available data on the effects of androgens. The ability ates with its co-activator beta-catenin [45]. The an- to hit a home run against a ball traveling at speeds ap- drogen receptor-beta catenin complex moves into the proaching 100 mph requires extraordinary degree of nucleus, forms a complex with LEF/TCF-4, and acti- hand-eye coordination—the ability to locate the ball in a vates a number of Wnt target genes, including follistatin specific coordinate of space and to place the bat in that [45]. The signal from androgen receptor is cross-com- precise coordinate with considerable strength and municated to the TGF-beta pathway through beta- power. There is some evidence that androgens decrease catenin and TCF-4. Beta-catenin and follistatin play an .354. http://www.asiaandro.com; [email protected] Asian J Androl 2008; 10 (3):351–363 essential role in mediating the effects of testosterone aromatizable or not. Thus, orally administered, 17-al- on myogenic differentiation [45]. pha-alylated, nonaromatizable androgens produce Testosterone also has been reported to promote satel- greater reductions in plasma HDL cholesterol levels than lite cell entry into the cell cycle [47–51]. Additionally, parenterally administered testosterone. Orally-admini- testosterone and DHT inhibit the differentiation of stered, 17-alpha alkylated androgens also have been preadipocytes into adipocytes [45]. Androgens also associated with insulin resistance and glucose intoler- stimulate fractional muscle protein synthesis and to in- ance [70]. Androgen use has been associated with in- crease the efficiency of reutilization of amino acids by creases in hematocrit, homocysteine levels, blood pres- the skeletal muscle [32, 52–54]. The effects of testo- sure and peripheral arterial resistance, and left ventricular sterone on muscle protein degradation need further hypertrophy and diastolic dysfunction [71–81]. investigation. However, it is not clear whether myocardial hypertro- phy reported in power lifters is the result of resistance 5 Potential adverse effects of androgen use exercise or androgen use. In a cross-sectional investi- gation [82], power athletes who had used androgens Because of the variability in the dose, frequency, showed subclinical impairment of both systolic and di- duration, and the type of drugs used, systematic inves- astolic myocardial function that was correlated with the tigations of the adverse effects of androgens in athletes dosage and duration of androgen use. Also, one con- and recreational body builders have been difficult to trolled trial in healthy volunteers [83] and other conduct. These analyses are further complicated by uncontrolled, open-label studies in weight lifters, have the concurrent use of accessory drugs. The low fre- not found significant changes in left ventricular mass quency of serious adverse effects reported with andro- or function with androgen use [84]. The long term gen use is surprising; it is likely that the adverse effects effects of androgen abuse on the risk of prostate and are under-reported. Furthermore, the accuracy of self- cardiovascular disease are unknown. reported drug use is difficult to verify. The anecdotal reports of “roid rage” among an- Adverse events associated with androgen use in- drogen users have received much attention in lay press. clude deleterious changes in the cardiovascular risk However, in placebo-controlled trials, testosterone ad- factors, including a marked decrease in plasma high- ministration has not been associated with a statistically density lipoproteins (HDL) cholesterol level [55] and significant increase in anger scores or measures of ag- changes in clotting factors [56], suppression of sper- gressive behaviors [63, 85–90]. It is possible that the matogenesis resulting in infertility, increase in liver self-reporting questionnaires lacked the sensitivity to enzymes, hepatic neoplasms, and mood and behavioral detect small but significant changes in aggression. In disturbances [57–63]. Elevations of liver enzymes, he- controlled trials, a small number of subjects have demon- patic neoplasms, and peliosis hepatic and even hepatic strated marked increases in aggression measures with rupture have been reported with the use of oral, 17- the use of supraphysiologic doses of testosterone, while alpha alkylated androgens [57, 58, 64], but not with a majority of participants show little or no change, lead- parenterally administered testosterone or its esters [65]. ing to speculation that high doses of androgens might Acne and premature hair loss can occur with androgen provoke rage reactions in a subset of individuals with use. Women using large dose of androgens are at risk pre-existing psychopathology. Kouri et al. [88] reported for menstrual irregularities, infertility, and virilizing side that administration of supraphysiologic doses (600 mg effects, including hirsutism, deepening of voice, changes weekly) of testosterone enanthate to healthy, young men in body habitus, and clitoral enlargement; some of these was associated with a significant increase in aggressive virilizing adverse effects may be irreversible. responses to provocation than placebo administration. A number of deaths due to unexpected coronary Testosterone doses that approximated the replacement and cerebrovasuclar thrombotic events among andro- doses or were slightly above the replacement dose did gen users have been reported [66–68], but these re- not produce significant changes in aggressive response ports are largely anecdotal and do not establish a cause in this experimental setting [88]. and effect relationship. In a stunning report that has A wide range of psychiatric side effects, including received surprisingly little attention, Finnish world class increased aggression and hostility, and mood distur- power lifters suspected of AAS intake during their sports bances (e.g. depression, hypomania, and psychosis) career experienced five times higher mortality than age- have been reported among androgen users [91]. De- matched controls [69]. The findings of this small study pendence and withdrawal effects (such as depression) need further confirmation. The changes in plasma lipids occur in a small number of steroid users. Dissatisfac- vary depending on the dose, the route of administration tion with the body and low self-esteem is common among (oral or parenteral), and whether the androgen is androgen users and may predispose these individuals to Tel: +86-21-5492-2824; Fax: +86-21-5492-2825; Shanghai, China .355. Androgen doping in sports the abuse of muscle building drugs [91]. Both increased Thirty four laboratories around the world have been and decreased sexual desire and function have been re- accredited by the International Olympic Committee to ported [61]. perform doping tests. Traditional radioimmunoassay Breast tenderness and breast enlargement (“bitch techniques were used initially to detect androgens in tits” in street parlance) are frequently associated with the urine specimens. However, since 1981, the ac- the use of aromatizable androgens. It is not uncom- credited laboratories have used either gas chromatog- mon for athletes to use an aromatase inhibitor or an raphy-mass spectrometry (GC-MS) or in some in- estrogen antagonist in combination with androgens to stances liquid chromatography mass spectrometry (LC- prevent breast enlargement. MS) to detect androgen or their metabolites that show The long-term suppression of the hypothalamic-pi- poor gas chromatographic properties or are tempera- tuitary-testicular axis with its attendant risk of depen- ture labile [100]. Also, during the past ten years, the dence and continued use of androgens is a serious com- introduction of the high resolution mass spectrometry plication of androgen use that has not been widely (HRMS) and tandem mass spectrometry (MS/MS) has appreciated. Androgen administration suppresses en- further improved the sensitivity of androgen steroid dogenous testosterone and sperm production by sup- detection techniques. Derivatization of samples is of- pressing the hypothalamic-pituitary-testicular axis [92, ten used to improve the sensitivity of the gas chroma- 93]. Men using androgens may experience subfertility tography [101]. Thus, silylation reaction converts the or infertility [94]. The recovery of the hypothalamic- polar groups such as hydroxyl and keto groups to less pituitary axis after discontinuation of the exogenous polar trimethylsilyl ethers and improves the signal to androgen, may take weeks to months, depending on noise ratio [101]. the dose and duration of prior androgen use [95–98]. For detection of testosterone abuse, the analysis During the period immediately after discontinuation of of testosterone to epitestosterone ratio in conjunction androgen use when circulating testosterone levels are with isotope ratio combustion mass spectrometry is low, the users experience symptoms of androgen used [102–109]. Urinary testosterone to epitestosterone deficiency, including loss of sexual desire and function, ratio typically is less than 6 and is constant in any lack of energy, depressed mood, and hot flushes. Some individual. There are genetic differences in testosterone patients may find these withdrawal symptoms difficult to epitestosterone ratio. Administration of exogenous to tolerate and may revert back to using androgens, testosterone increases the urinary excretion of testoster- thus perpetuating the vicious cycle of abuse, withdrawal one glucoronide and increases the testosterone to symptoms, and dependence [96–98]. Others may re- epitestosterone ratio. Testosterone to epitestosterone sort to off-label use of aromatase inhibitors or hCG ratio greater than 4 is viewed suspiciously. Ratios greater obtained illicitly based on the presumption that these than 4 need evaluation of previous urine samples or agents accelerate the recovery of the hypothalamic-pi- additional urine samples obtained after a time interval. tuitary-testicular axis, although there is no evidence to If the high ratio is due to genetic variation, then all support this premise and it is possible that the use of samples obtained from the subject would show the high hCG may delay the ultimate recovery of the hypotha- ratio. A high testosterone to epitestosterone ratio that is lamic-pituitary-gonadal axis. higher than that observed in previous samples is viewed Self administration of intramuscular injections in- as a positive test. creases the risk of infection, muscle abscess, and even If the results of the testosterone to epitestosterone sepsis [20]. Transmission of HIV infection has been re- ratio test are abnormal and suggest exogenous testoster- ported among anabolic steroid users presumably because one use, then additional confirmation by using gas chro- of needle sharing or the use of improperly sterilized needles matography combustion isotope ratio mass spectro- and syringes. metry is required [101, 104]. This method is based on Excessive muscle hypertrophy without commen- the measurement of 13C/12C isotope ratio in testos- surate adaptations in the associated tendons and con- terone. In nature, 1.1% of carbon exists as 13C. Syn- nective tissues may predispose athletes using andro- thetic androgens are synthesized from plant sterols di- gens to the risk of tendon injury and rupture and un- osgenin and stigmasterol that have less 13C than their usual stress on joints [99]. endogenous homologs. Therefore, synthetic A vast majority of androgen users also abuse addi- testosterone, in a manner similar to other synthetic or- tional drugs [19]. Some of these additional drugs of ganic compounds, has lower 13C to 12C ratio than a abuse, such as cocaine, amphetamine, and ephedra may reference gas standard. During the course of the GC be associated with potentially serious complications. combustion isotope ratio mass spectrometry, the steroids are separated by gas chromatography and oxidized to 6 Detection of illicit androgen use carbon dioxide in a combustion chamber. The ratio of .356. http://www.asiaandro.com; [email protected] Asian J Androl 2008; 10 (3):351–363 13CO (m/e 45) and 12CO (m/e 44) is monitored in an Administration of 100 mg androstenedione orally 2 2 isotope ratio mass spectrometer, and the δ value is cal- daily is associated with little or no change in circulat- culated (δ value refers to the decrease in 13C relative to ing testosterone concentrations, while administration the reference gas with a standardized 13C to 12C ratio) of 300-mg dose produces only modest increments in [110]. A negative δ value along with a high testoster- testosterone area-under-the-curve. However, Jasuja et one to epitestosterone ratio suggests exogenous test- al. [115] demonstrated that 500-mg androstenedione ad- osterone administration. ministered thrice daily for 12-weeks to hypogonadal The procedures for the collection and transporta- men increased serum testosterone and free testoster- tion of samples for doping tests follow strict rules that one concentrations into the eugonadal range, and in- have been established by the individual sports organiza- creased fat-free mass and muscle strength. Similarly, tions [101]. Typically, each urine sample, collected in women, administration of 100-mg androstenedione under direct visual oversight of an accredited supervisor, significantly increased serum testosterone concentra- is divided in to two parts (A and B samples) and trans- tions above the physiologic range for women [116]. ported to the testing laboratory using strict “chain of In female hyenas and several other mammalian species, custody” procedures. If A sample is deemed positive, circulating concentrations of androstenedione are then B sample is analyzed in the presence of the athlete higher than those in male members of these species or an authorized representative of the athlete. If B and are associated with virilization of external genita- sample is also positive, then doping with an androgen is lia and increased aggression [117]. Jasuja et al. [115] confirmed, and the sports organization can impose pu- demonstrated that androstenedione binds androgen nitive sanctions [101]. receptor albeit with a substantially lower binding af- Some controversy has erupted recently over the finity than testosterone, and that it promotes myogenic large number of positive tests for nandrolone. Small differentiation in a mesenchymal, multipotent cell line. quantities of nandrolone, 17beta-hydroxy-19-nor-4- Thus, androstenedione meets all the criteria for an ana- androsten-3-one, and its metabolite 19-norandrosterone, bolic steroid: it has structural resemblance to testoste- are excreted in the urine naturally in men. The Interna- rone, it binds androgen receptor, and it promotes myo- tional Olympic Committee has established a threshold genic differentiation in vitro and when administered in level of 2 ng/mL for 19-norandrosterone. Levels higher sufficiently high doses, it increases muscle mass than this threshold have been reported in some indi- [115]. Based on these data, the US Congress recently viduals eating a high meat diet in conjunction with in- classified androstenedione as an anabolic steroid and tense resistance exercise [111] and in individuals ingest- banned its over the counter sales. ing dietary supplements such as delta4-androstenedi- Androstenedione administration produces substan- one [112]. tial increments in serum estradiol and estrone concen- trations [116, 118–121]. Most of the orally adminis- 7 The abuse of androgen precursors and designer tered androstenedione is inactivated during its androgens presystemic metabolism as indicated by a marked in- crease in its urinary metabolites, including testosterone 7.1 δ-4-androstenedione glucuronide with only a small increase in serum testo- δ-4-Androstenedione is a precursor of testosterone sterone [122]. that is converted by the enzyme 17beta hydroxy-ste- The over the counter preparations of androstenedi- roid dehydrogenase to testosterone. Androstenedione one have not been subject to the rigorous quality con- witnessed a brief period of rapid growth in sales fol- trol required of the FDA-approved pharmaceuticals [123, lowing Mark McGuire’s admission of its use during an 124]. Substantial variability has been observed in an- extraordinary season replete with 68 home runs. Un- drostenedione content of different preparations and der the Dietary Supplement Health and Education Act among different batches from the same manufacturer passed by the US Congress, for many years, andros- [112, 125]. Some batches of over the counter andros- tenedione was sold over the counter as a dietary supple- tenedione have been found to contain one or more banned ment [113–114]. Unlike other androgens, whose sales androgens such as nandrolone; thus, ingestion of an- were regulated within the dictates of Anabolic Steroid drostenedione may result in the doping tests becoming Control Act, androstenedione’s sales had not been sub- positive [112]. ject to regulatory oversight of Food and Drug Adminis- tration and Drug Enforcement Agency. However, the 7.2 Potential adverse effects of androstenedione US Congress recently added androstenedione to the list The long-term side effects of androstenedione use of banned anabolic steroids and it is no longer sold over are unknown. Short term administration of andros- the counter. tenedione is associated with a significant increase in Tel: +86-21-5492-2824; Fax: +86-21-5492-2825; Shanghai, China .357. Androgen doping in sports estradiol levels. The long-term consequences of the One randomized trial has reported greater improvements marked increase in estrogen levels in men taking an- in bone mineral density in older men and women re- drostenedione are unknown. In men, the increases in ceiving 50 mg DHEA daily than with placebo [145]. serum estrogen concentrations may potentially affect DHEA trials in women with adrenal insufficiency semen quality, increase inflammatory markers, cause have yielded inconsistent results. Arlt et al. [129] used gynecomastia, and induce epigenetic and cytogenetic a double-blind, placebo-controlled, crossover study de- effects on sperm. sign in women with primary or secondary adrenal in- The supplementation of androstenedione decreases sufficiency who received either placebo or 50 mg DHEA HDL levels and increases low-density lipoprotein (LDL)/ daily for 16 weeks each. DHEA administration was HDL ratio. Other adverse effects of androstenedione associated with improvements in scores for depression stem from the potential increase in testosterone levels. and anxiety, sexual function, and circulating osteocalcin These may include adverse effects on plasma lipids, levels, but no significant changes in body composition erythrocytosis, acne, sleep apnea, and increased risk [122]. Other trials of DHEA supplementation in women of detecting prostate events. with adrenal insufficiency failed to confirm the benefi- Given the lack of efficacy data and total absence of cial effects of DHEA on mood, well being or sexual long term safety data, the use of androstenedione is not function that were observed in the Arlt study [130–132]. clinically recommended for any indication, including the DHEA has not been shown to consistently improve body treatment of androgen deficiency in men or women. composition, physical function, or insulin sensitivity. The effects of DHEA administration on cardiovascular 7.3 Dehydroepiandrosterone (DHEA) event rates or cancer incidence rates are unknown. DHEA is a weak androgen by itself, but it is con- In a placebo-controlled trial that used pharmaco- verted in peripheral tissues to testosterone and estradiol. logical doses of DHEA (200 mg daily), modest improve- In addition to being a weak androgen and an androgen ments in lupus outcomes and a greater reduction in dis- precursor, DHEA has been shown to function as a ease flares and disease activity were reported in pa- neurosteroid [126]. tients receiving DHEA than in those receiving placebo DHEA binds androgen receptor with a binding af- [142–144]. The effects of DHEA on bone mineral den- finity that is substantially lower than that of dihydro- sity in patients with SLE have been inconsistent. testosterone (DHT). A separate G-protein coupled mem- Thus, the efficacy of DHEA has not been demon- brane receptor for DHEA has been proposed [127]; strated in any disease state and DHEA use cannot be however, the existence of such a DHEA-specific mem- recommended for any clinical indication at present. brane receptor has not been confirmed. DHEA also has been shown to modulate the activities of N-methyl-D- 7.4 Other androgen precursors and designer steroids aspartate (NMDA) and γ-amino-butyric acid (GABA) Precursors of testosterone (4-androstenediol and receptors [128]. 5-androstenediol in addition to 4-androstenedione and The literature on DHEA is difficult to interpret. DHEA discussed above), dihydrotestosterone (5-alpha- DHEA studies in rodents have limited applicability to androstane-3beta-17 beta-diol, 5-alpha-androstane-3 humans, because rodents have very little endogenous alpha, 17 beta diol, 5-alpha-androstane-3, 17 dione, 5- circulating DHEA. Many DHEA studies reporting ben- alpha-androst-1-ene-3, 17 dione, 17 beta-hydroxy-5- eficial neurotropic and anti-cancer effects, and immune alpha-androst-1-en-3-one, 5-alpha-androst, 1-ene, 17 enhancement were conducted in rodents. beta-diol) or nortestosterone (4-norandrostenedione, 4- The human trials of DHEA have been characterized norandrostenediol, and 5-norandrostenediol) [101] that by heterogeneity of doses, formulations, and study are weakly androgenic by themselves, but that are con- populations. DHEA studies have been conducted in pa- verted in the body to potent androgens, have become tients with adrenal insufficiency [129–133], older men available on the internet. Even a precursor (androsta- and women [134–139], peri- and post-menopausal 1,4-diene-3, 17-dione) of boldenone (17-beta-hydrox- women [140, 141], and in patients with autoimmune yandrosta-1, 4-dien-3-one) has been introduced [101]. disease [142–144]. These trials used doses as high as The androgens abused by athletes had been synthe- 1500 mg daily and as low as 25 mg daily. Most human sized initially for medicinal or veterinary indications. trials used 50 mg DHEA daily for three to six months, However, recent years have witnessed the appearance included small samples, and were of relatively short of designer steroids, such as tetrahydrogestrinone durations. (THG) [146, 147] and madol [148] that were developed A Cochrane review of DHEA trials concluded that solely for abuse [149]. The detection of these novel there was insufficient evidence of beneficial effect of androgens has proven challenging to the testing labora- DHEA on cognition in older men and women [138, 139]. tories because detection methods have not been stan- .358. http://www.asiaandro.com; [email protected] Asian J Androl 2008; 10 (3):351–363 dardized for these new designer androgens. 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