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Steroids. 2010 Jun;75(6):377-89. Epub 2010 Feb 4.
Tissue selectivity and potential clinical applications of trenbolone (17beta-hydroxyestra-4,9,11-trien-3-one): A potent anabolic steroid with reduced androgenic and estrogenic activity.
Yarrow JF, McCoy SC, Borst SE.
Geriatric Research, Education & Clinical Center, VA Medical Center, Gainesville, FL 32608, United States.
1. Introduction
Testosterone, and its more potent metabolites, dihydrotestosterone (DHT) and estradiol (17â-E2), are known to influence the development and maintenance of numerous tissues, including skeletal muscle [1], bone [2], adipose tissue [3], and sex-organs [4]. In males, reduced testosterone (i.e., hypogonadism) induces losses in skeletal muscle mass and bone mineral density (BMD) and increases adiposity [5]. However, administration of testosterone at replacement doses results in only minor improvements in skeletal muscle mass and strength in hypogonadal older men [6]. In contrast, administration of supraphysiological doses of testosterone results in robust increases in skeletal muscle mass and BMD and reductions in adiposity in both humans [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17] and [18] and animals [19], [20] and [21], but also results in a variety of adverse events, of which prostate enlargement and polycythemia appear to be most prevalent [22]. Alternative pharmacological treatments such as selective androgen receptor modulators (SARMs) [23] or combined treatment with high-dose testosterone plus 5á reductase inhibitors (e.g., finasteride or dutasteride) [7], [19], [20] and [24] have been proposed as a means of producing the desired anabolic effects with a lower incidence of adverse effects. Our main objective is to review current research related to the potent anabolic steroid 17â-hydroxyestra-4,9,11-trien-3-one (trenbolone; 17â-TBOH), especially its SARM-like properties that may make it beneficial for the treatment of several clinical conditions [25] and [26]. Additionally, we will offer brief overviews on androgen signaling and on the recent developments with SARMs.
2. Androgen receptor signaling
Classical, or genomic, androgen signaling begins with binding of testosterone or DHT to cytosolic androgen receptors (ARs) and ultimately results in altered gene expression. Most of the familiar effects of androgens on muscle, bone, and male sexual development are genomic effects which require the synthesis of new protein; thus, they may take days to months to manifest. More recently, rapid non-genomic testosterone signaling has been discovered [27] which is mediated by cell-surface, G-protein (GP) coupled receptors [28]. An example of non-genomic signaling is the rapid, testosterone-induced release of calcium (Ca2+) from intracellular stores occurring in mouse IC-21 macrophages [28]. The non-genomic, actions of testosterone are not inhibited by classical androgen blockers such as cyproterone and flutamide, suggesting that the genomic and non-genomic effects of androgens may be quite different. For example, we [29] and others [30] have shown that long-term administration of testosterone to male rats confers cardioprotection against ischemia/reperfusion (IR) injury. However, when testosterone is added in vitro to the working heart preparation, it worsens IR injury [31].
The genomic effects of androgens occur through two distinct pathways. In the first pathway, binding of androgens to cytosolic ARs cause ARs to translocate to the nucleus and bind to chromosomal DNA as homodimers [32] (Fig. 1). The specific regions of DNA that bind ARs are called hormone response elements (HREs) and they regulate the transcription of specific genes, producing androgenic effects. In the second pathway, the actions of androgens are mediated by interaction of ARs with the Wnt/â-catenin pathway. Wnts are a family of secreted glycoproteins that regulate differentiation in a wide variety of tissues. Canonical Wnt/â-catenin signaling involves binding of Wnt to the cell-surface frizzled receptor (FZ), which through its interactions with Axin, Frat-1 and Dsh, results in the inhibition of glycogen synthase kinase-3 (GSK3). GSK3 phosphorylates â-catenin, marking it for proteasomal degradation. As a result, inhibition of GSK3 results in the accumulation of â-catenin in the cytosol and increased translocation of â-catenin into the nucleus, where it interacts with transcriptional regulators to alter gene expression [33]. It is hypothesized that androgen signaling bypasses the canonical Wnt pathway by interacting with downstream Wnt effectors [34] to stimulate the commitment of mesenchymal pluripotent cells to the myogenic line and to inhibit their commitment to the adipogenic line [3]. By this mechanism, androgens increase the number of muscle satellite cells [35]. In culture, androgens have been shown to induce translocation of â-catenin to the nucleus in mouse 3T3 preadipocytes and inhibit their differentiation into mature adipocytes [34]. Wnt/â-catenin signaling has also been implicated in androgen-induced masculinization of external genitalia [4].
Full-size image (42K)
Fig. 1. Androgen receptor signaling pathways. A = androgen, AR = androgen receptor, Ca2+ = calcium, FZ = frizzled receptor, GP = G protein, GSK-3 = glycogen synthase kinase 3, HRE = hormone response element, PLC = phospholipase C, SR = sarcoplasmic reticulum, TCF = T cell factor, LEF = lymphoid enhancer factor 1, Wnt = Wingless-Int.
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The metabolism of testosterone also plays an important role in its actions. Testosterone may be converted to DHT by either 5á reductase isoenzyme [36] and to 17â-E2 by aromatase [37]. Tissues expressing 5á reductase include the prostate, testes, accessory sex organs, beard and scalp, sebaceous glands, liver, brain, and skin [38] and in these tissues the effects of circulating testosterone are amplified by two known mechanisms. First, DHT has an approximate 3-fold greater affinity for the AR than does testosterone [39]. Second conversion of testosterone to DHT prolongs the androgen action because testosterone can be converted to the weaker androgen androstenedione, while DHT maintains a longer presence [36]. Aromatization of testosterone occurs within specific tissues (e.g., bone and brain) and systemically in some species [40] and [41]. In men, significant systemic aromatization also occurs in adipose tissue [42] and because of the resulting elevation of serum 17â-E2, may cause adverse effects (e.g., fluid retention, gynecomastia, and worsening of sleep apnea) in obese men receiving testosterone [43]. Thus, the biological effects associated with testosterone may result from classic AR mediated pathways, interactions with the Wnt/â-catenin pathway, or from AR or ER activation following conversion of testosterone to DHT or 17â-E2, respectively.
3. Recent advances in SARMs
Selective androgen receptors modulators (SARMs) are a class of drugs that have action in some tissues expressing ARs and reduced action in others [44]. One of the key goals in the development of SARMs has been to find orally available agents that have anabolic effects on muscle, bone, and erythropoiesis, but that do not cause prostate enlargement or other common adverse events associated with supraphysiological testosterone administration [23]. The name SARMs is analogous to the older drug class of selective estrogen receptor modulators (SERMs), which act as agonists in some tissues and antagonists in others [45]. SARMs are under development in many major pharmaceutical firms as a potential means of treating the symptoms associated with hypogonadism and various muscle and bone wasting conditions [23] but much of the work defining their mechanism of action and structure-activity relationships has not been published. Much of the pioneering work in developing non-steroidal SARMs was done at Ligand Pharmaceuticals and at the University of Tennessee [46] and [47] where the first generation of SARMs including S1 and S4 were developed [48]; both of which prevent atrophy of the levator ani muscle without significant prostate enlargement in a castrated rat model [49]. The following SARMs are leading candidates in each of the major chemical classes. LGD2226 is quinolinone analog, developed by Ligand Pharmaceuticals [50]. S4 is an aryl proprionamide analog developed by Gao and coworkers [49]. BMS564929 is a hydantoin analog developed by Bristol-Myers Squibb [51]. S40503 is a tetrahydro-quiniline analog developed by Kaken Pharmaceuticals [52]. Each has been shown to have strong anabolic actions in skeletal muscle and bone with partial agonist activity in the prostate [44]. Although, the non-steroidal SARMs are a chemically diverse group of compounds, in many cases, their tissue selectivity may derive from the drug not being a substrate for the 5á reductase and/or aromatase enzymes [53].
Testosterone is not effective when administered orally due to a high liver first-pass effect [54] and is readily 5á reduced or aromatized in a variety of tissues. In contrast, some orally available synthetic testosterone analogs may have SARM-like activity. For example, oxandrolone (17-á methyl-DHT) is not a substrate for aromatase [53]. Similarly, nandrolone (19-nortestosterone) is 5á reduced, but to a relatively weak androgen, dihydronandrolone, and is a poor substrate for aromatase [55]. However, both nandrolone and oxandrolone cause liver toxicity at high doses. Conversely, 17â-TBOH (trenbolone) has a low oral bioavailability (because it is not methylated at the 17á position), but may have SARM-like properties considering that it is not a substrate for 5á reductase and may not be a substrate for aromatase. The toxicity of 17â-TBOH has not been scientifically studied in humans, but anecdotally has been reported to have a low potential for liver toxicity because this drug is generally administered intramuscularly.
4. 17â-TBOH metabolism
17â-acetoxyestra-4,9,11-trien-3-one (17â-TBOH-acetate) is a highly potent anabolic androgenic steroid which is primarily used legally as a growth promoting agent in domestic livestock production within the US [56] either alone, as Finaplix (Intervet, Inc., Millsboro, DE), or in combination with 17â-E2, as Revalor (Intervet, Inc., Millsboro, DE), or 17â-E2-benzoate, as Synovex (Wyeth, Madison, NJ) [57]. Following administration, 17â-TBOH-acetate is rapidly converted to the biologically active steroid 17â-TBOH [58] which, along with its metabolites, are included on the World Anti-Doping Association (WADA) prohibited substance list [59] due to their potential ability to augment skeletal muscle mass and improve athletic performance. As such, a variety of testing methods have been proposed to detect the presence of 17â-TBOH or its metabolites in human urine or blood [60], [61], [62], [63], [64], [65], [66], [67], [68] and [69]; although, evaluation of these methods is beyond the scope of this review.
Most evidence regarding the in vivo mammalian metabolism of 17â-TBOH is derived from studies on livestock and rodents [58], [70] and [71]; however, several studies have evaluated 17â-TBOH metabolism in humans [68] and [72]. Some variation in the in vivo metabolism of 17â-TBOH exists among mammalian species [58] and [68], but the primary metabolites are 17â-hydroxy- and 17-oxo-metabolites of trenbolone in rodents or 17á-hydroxy-metabolites of trenbolone in ruminants [58]. Similarly, in humans, ingested 6,7-3H labeled 17â-TBOH is primarily excreted intact, as 17â-TBOH, as the 17á epimer (epitrenbolone; 17á-TBOH) or as trendione (TBO) (Fig. 2) [68]. In addition, several yet to be identified polar metabolites of 17â-TBOH have also been detected in human urine, albeit in much lower concentrations than the primary metabolites previously listed [68]. 17â-TBOH has a greater affinity for the AR than any of its primary metabolites [39] and [73], suggesting that biotransformation of 17â-TBOH reduces the biological activity of this steroid [26] and [68]. This is in contrast to testosterone, which undergoes irreversible 5á reduction [36] or aromatization [37] and [74] to the more potent DHT and 17â-E2.
Full-size image (53K)
Fig. 2. Structures of trenbolone and it primary metabolites in humans, epitrenbolone and trendione, along with testosterone and its metabolites dihydrotestosterone and estradiol. 17â-TBOH = trenbolone, 17á-TBOH = epitrenbolone, TBO = trendione, DHT = dihydrotestosterone, 17â-E2 = estradiol.
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4.1. 17â-TBOH and 5á reductase
Despite its structural similarities to testosterone, 17â-TBOH does not undergo 5á reduction due to the presence of a 3-oxotriene structure, which prevents A ring reduction (Fig. 2) [58]. In fact, 17â-TBOH undergoes biotransformation to less biologically active androgens [26] and [68], similar to other anabolic androgenic steroids, such as 19-nortestosterone [55]. Indirect evidence indicates this is true, as 17â-TBOH administration has been shown to reduce prostate mass in growing male rodents when compared with control animals [75]. Similarly, 17â-TBOH exerts less pronounced effects than testosterone in androgen-sensitive tissues which express the 5á reductase enzyme including the prostate and accessory sex-organs [25], [26], [36], [76], [77], [78], [79] and [80], despite the fact that 17â-TBOH binds to the human AR [26] and [39], along with ARs of various model species [26], [81] and [82], with approximately three times the affinity of testosterone. Despite its inability to undergo 5á reduction, 17â-TBOH remains highly anabolic, evidenced by equal or greater growth in the levator ani skeletal muscle (an androgen responsive tissue which lacks the 5á reductase enzymes), compared to testosterone [25], [26], [76], [77], [78], [79], [80] and [83]. Thus while testosterone exerts enhanced effects in tissues expressing 5á reductase, 17â-TBOH exerts equal effects in those tissues expressing 5á reductase (e.g., prostate and accessory sex-organs) vs. those not (e.g., skeletal muscle) [36]. Taken together, these reports indicate that 17â-TBOH produces a ratio of anabolic:androgenic effects that may be favorable compared to the effects of testosterone. However, we are unaware of any paper that has reported the effects of 17â-TBOH administration on the growth of androgen-sensitive tissues in humans.
4.2. 17â-TBOH and aromatase
17â-TBOH and other C19 norandrogens [84] are reported to not be substrates for the aromatase enzyme [85] and [86] and to be relatively non-estrogenic [87] and [88]; although some debate exists regarding 19-nortestosterone (a C19 norandrogen) to undergo aromatization and induce estrogenic effects [84] and [89]. In vitro bioassays and cell culture experiments demonstrate that 17â-TBOH and its metabolites have a very low binding affinity for ERs and have low estrogenic activity with approximately 20% of the efficacy of 17â-E2 [87]. Reports also suggest that 17â-TBOH reduces serum 17â-E2 concentrations in vivo [81], [90] and [91], inhibits estrus in ovariectomized estrogen-treated female rats [92], and exerts a variety of anti-estrogenic effects [92], perhaps through hypothalamic feedback inhibition of the production of testosterone (a substrate necessary for endogenous 17â-E2 biosynthesis) [93] and [94]. Additionally, in various fish models, environmental exposure to 17â-TBOH downregulates brain CYP19B (aromatase B) and upregulates gonadal CYP19A (aromatase A) expression in females, but not males [95] and [96], while reducing tissue concentrations and tissue-specific gene expression of vitellogenin (VTG), a protein found in oviparous animals which is positively associated with exposure to estrogenic compounds, in both sexes [81], [88], [93], [94], [95], [96], [97], [98], [99], [100] and [101]; together these may represent compensatory responses resulting from reduced endogenous estrogen concentrations [101]. Conversely, others report that 17â-TBOH either increases [81] and [102] or has no effect [81], [90] and [103] 17â-E2 concentrations in ruminants and oviparous animals.
The mechanism(s) through which 17â-TBOH alters estrogenic activity remain to be elucidated, but may be related to the (1) inhibition of endogenous androgen synthesis [104] and [105] (presumably through pituitary or hypothalamic feedback inhibition [93] and [106]), (2) altered expression or activity of the aromatase enzyme [96] and [107], and/or (3) down-regulation of ERá or ERâ expression [95] X. Zhang, M. Hecker, J.W. Park, A.R. Tompsett, J. Newsted and K. Nakayama et al., Real-time PCR array to study effects of chemicals on the hypothalamic-pituitary-gonadal axis of the Japanese medaka, Aquat Toxicol 88 (2008), pp. 173–182. Abstract | Article | PDF (797 K) | View Record in Scopus | Cited By in Scopus (12)[95] and [108]; however, they do not appear to be mediated by direct androgen receptor activation as co-treatment with 17â-TBOH and flutamide (an AR antagonist) also results in anti-estrogenic activity in female fathead minnows [81]. To summarize, 17â-TBOH is not a substrate for the aromatase enzyme, but may exert both anti- and pro-estrogenic effects, with the bulk being anti-estrogenic.
5. Mechanisms of body growth
The growth promoting effects of 17â-TBOH administration are well known [56], [109] and [110], as numerous studies have reported that administration of 17â-TBOH or its acetate ester enhance total body growth and skeletal muscle mass in various rodent and livestock models when administered alone [25], [26], [75], [76], [77], [78], [79], [80], [83], [86], [90], [102], [103], [111], [112], [113], [114], [115], [116], [117], [118], [119], [120], [121], [122], [123], [124], [125], [126], [127], [128], [129], [130], [131], [132], [133], [134], [135] and [136] or when administered in combination with 17â-E2 [86], [90], [103], [104], [111], [112], [123], [127], [128], [129], [137], [138], [139], [140], [141], [142], [143], [144], [145], [146], [147], [148], [149], [150], [151], [152], [153], [154], [155], [156], [157], [158], [159], [160], [161], [162], [163], [164], [165], [166], [167], [168], [169], [170], [171], [172], [173], [174], [175], [176] and [177]. Interestingly, several studies have reported that administration of 17â-TBOH in combination with 17â-E2 results in greater body growth and skeletal muscle mass than either steroid alone [103], [112], [127], [128], [171], [172] and [173]; indicating that 17â-E2 enhances the anabolic effects of 17â-TBOH, as others have suggested [109] and [110]. Ultimately, enhanced body mass results from increases in lean mass (predominantly comprised of muscle and bone) and/or fat mass; thus, the known effects of 17â-TBOH on each of these tissues will be discussed. In addition, the effects of 17â-TBOH on erythropoiesis will be briefly discussed because androgens are known to exert potent effects on red blood cell production.
5.1. Effects of 17â-TBOH on skeletal muscle
Skeletal muscle expresses ARs to varying degrees among species [178], [179], [180], [181] and [182]. As such, androgens induce skeletal muscle protein accretion following dimerization of ARs (Fig. 1). Skeletal muscle expresses 5á reductase and dose-dependently converts testosterone to DHT [183], however, our laboratory [19] and [20] and others [24] have recently demonstrated the 5á reduction of testosterone is not required for skeletal muscle maintenance in hypogonadal animals or humans. In addition, skeletal muscle expresses ERs within both sexes of various species [184], [185] and [186] and 17â-E2 administration has been shown to protect against loss of muscle strength in ovariectomized female rodents [187] and [188]; suggesting that aromatization might contribute to the effects of testosterone on skeletal muscle in males.
In ruminants, 17â-TBOH, alone or in combination with 17â-E2, has been shown to increase the cross-sectional area (CSA) of type I, but not type II, skeletal muscle fibers and induce a fiber switch from more glycolytic to more oxidative fibers, indicating an increase in the oxidative capacity of skeletal muscle [152] and [189]. However, the presence of 17â-E2 is not required for 17â-TBOH to augment skeletal muscle mass as demonstrated in rodent models which experience significant growth of the levator ani muscle [25], [26], [76], [77], [78], [79], [80] and [83] and other skeletal muscles [25], [26], [76], [77], [78], [79], [83], [117], [126] and [133] following 17â-TBOH administration, despite lacking the primary source of endogenous 17â-E2. However, not all rodent models experience peripheral (i.e., hindlimb) muscle growth following 17â-TBOH administration [115], [118] and [126]; although, elevated skeletal muscle DNA concentrations are present in muscles that do not increase in mass in response to 17â-TBOH treatment [117]. The inconsistent skeletal muscle response to 17â-TBOH in rodents may occur because certain peripheral rodent skeletal muscles possess a low percentage of AR positive myonuclei (i.e., extensor digitorum longus with 7% AR positive myonuclei) [181]. Conversely, human myonuclei are approximately 50% AR positive [182] and ruminants are highly sensitive to androgen-induced myotropic stimuli due to a high concentrations of ARs in bovine skeletal muscle [178] and [179] and skeletal muscle satellite cells [180]. Similarly, the androgen-sensitive levator ani muscle in rodents contains approximately 74% AR positive myonuclei [181] and thus experiences robust atrophic responses to castration [190] and hypertrophic responses to androgen administration [25], [26], [76], [77], [78], [79], [80] and [83].
The underlying mechanism(s) through which 17â-TBOH enhances skeletal muscle growth have not been completely characterized; although, it is suspected that 17â-TBOH exerts direct anabolic effects on skeletal muscle primarily via AR activation and associated nuclear translocation and transcription or via modulation of the Wnt/â-catenin pathway, similar to other androgens [26] (Fig. 1). In vitro evidence indicates that 17â-TBOH induces translocation of human ARs to the nucleus in a dose-dependent manner and induces gene transcription to at least the same extent as DHT, the most potent endogenous androgen [26]. Further, 17â-TBOH treatment of cultured bovine satellite cells upregulates AR mRNA expression [191], perhaps explaining the observations that administration of 17â-TBOH increases satellite cell activation and proliferation in various species [117], [191] and [192].
Additionally, 17â-TBOH may induce anabolic effects via mechanisms associated with alterations in endogenous growth factor concentrations [193] or the responsiveness of skeletal muscle to such growth factors [117] (Fig. 3). For example, 17â-TBOH alone or in combination with 17â-E2 upregulates insulin-like growth factor (IGF-1) mRNA in a variety of tissues, including the liver and skeletal muscle in vivo [140], [141], [160], [170], [191], [194] and [195] and satellite cells in vitro [140], via distinct androgen- and estrogen receptor mediated mechanisms [180] and [196]; although 17â-TBOH alone (without the addition of 17â-E2) does not appear to alter skeletal muscle IGF-1 mRNA [184]. Ultimately, the upregulation of IGF-1 mRNA translates into increased serum IGF-1 in 17â-TBOH treated animals [138], [140], [148], [150], [160], [165], [170], [192], [194], [197] and [198], which may stimulate satellite cells proliferation and fusion as has been shown in vitro [117]. Interestingly, 17â-TBOH administration also appears to increase the responsiveness of satellite cells to the proliferating and differentiating effects of IGF-1 and fibroblast growth factor [117]. These results are intriguing considering that the inhibition of several of the downstream targets of IGF-1 (e.g., Raf-1/MAPK kinase (MEK)1/2/ERK1/2, or phosphatidylinositol 3-kinase (PI3K)/Akt) suppresses 17â-TBOH induced satellite cell proliferation in culture [180]. Thus, it seems likely that increased growth factor expression resulting from 17â-TBOH administration is one mechanism underlying the anabolic responses to this steroid in skeletal muscle, especially considering that binding of IGF-1 to the type 1 IGF receptor is required for proliferation of satellite cells [180].
Full-size image (67K)
Fig. 3. Potential mechanisms underlying the anabolic effects of trenbolone on skeletal muscle. 17â-TBOH = trenbolone, GR = gluccocorticoid receptor, AR = androgen receptor, IGF-1 = insulin-like growth factor 1, IGF-1R = insulin-like growth factor 1 receptor.
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17â-TBOH may also preserve or increase lean mass via anti-catabolic effects associated with reductions in endogenous glucocorticoid activity [126] and [135] or with the suppression of amino acid degradation within the liver [118], [122], [134], [199] and [200] (Fig. 3). For example, 17â-TBOH administration has been shown to reduce circulating corticosterone concentrations in rodents [115], [118] and [122] and resting cortisol in cattle [150]. In vivo [122] and in vitro [201] evidence indicates that 17â-TBOH works in the adrenals to suppress adrenocorticotropic hormone (ACTH)-stimulated cortisol synthesis and to suppress cortisol release. Further, 17â-TBOH has been shown to reduce the ability of cortisol to bind to skeletal muscle glucocorticoid receptors (GRs) [121] and to down regulate skeletal muscle GR expression [108] and [121]. Thus the multiple anti-glucocorticoid actions induced by 17â-TBOH explain, in part, the 17â-TBOH-mediated increase in total body nitrogen retention [133], [151], [155], [156] and [202] and the reductions in total [80], [129] and [156] and myofibrillar protein degradation in several species [126], [135], [156], [202] and [203]; especially considering that 17â-TBOH reportedly reduces skeletal muscle protein synthesis in male rodents [80] and [133]. As a result of its anti-glucocorticoid actions, 17â-TBOH produces a more robust inhibition of protein degradation than does testosterone, which only slightly reduces protein degradation while increasing protein synthesis [204]. Thus, future research comparing the effectiveness of 17â-TBOH and the endogenous androgens in altering skeletal muscle degradation via the ubiquitin proteasome system or other pathways associated with muscle atrophy [205] is warranted and may further elucidate the anti-catabolic mechanism(s) underlying the potent augmentation of skeletal muscle mass associated with 17â-TBOH.
Tissue selectivity and potential clinical applications of trenbolone (17beta-hydroxyestra-4,9,11-trien-3-one): A potent anabolic steroid with reduced androgenic and estrogenic activity.
Yarrow JF, McCoy SC, Borst SE.
Geriatric Research, Education & Clinical Center, VA Medical Center, Gainesville, FL 32608, United States.
1. Introduction
Testosterone, and its more potent metabolites, dihydrotestosterone (DHT) and estradiol (17â-E2), are known to influence the development and maintenance of numerous tissues, including skeletal muscle [1], bone [2], adipose tissue [3], and sex-organs [4]. In males, reduced testosterone (i.e., hypogonadism) induces losses in skeletal muscle mass and bone mineral density (BMD) and increases adiposity [5]. However, administration of testosterone at replacement doses results in only minor improvements in skeletal muscle mass and strength in hypogonadal older men [6]. In contrast, administration of supraphysiological doses of testosterone results in robust increases in skeletal muscle mass and BMD and reductions in adiposity in both humans [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17] and [18] and animals [19], [20] and [21], but also results in a variety of adverse events, of which prostate enlargement and polycythemia appear to be most prevalent [22]. Alternative pharmacological treatments such as selective androgen receptor modulators (SARMs) [23] or combined treatment with high-dose testosterone plus 5á reductase inhibitors (e.g., finasteride or dutasteride) [7], [19], [20] and [24] have been proposed as a means of producing the desired anabolic effects with a lower incidence of adverse effects. Our main objective is to review current research related to the potent anabolic steroid 17â-hydroxyestra-4,9,11-trien-3-one (trenbolone; 17â-TBOH), especially its SARM-like properties that may make it beneficial for the treatment of several clinical conditions [25] and [26]. Additionally, we will offer brief overviews on androgen signaling and on the recent developments with SARMs.
2. Androgen receptor signaling
Classical, or genomic, androgen signaling begins with binding of testosterone or DHT to cytosolic androgen receptors (ARs) and ultimately results in altered gene expression. Most of the familiar effects of androgens on muscle, bone, and male sexual development are genomic effects which require the synthesis of new protein; thus, they may take days to months to manifest. More recently, rapid non-genomic testosterone signaling has been discovered [27] which is mediated by cell-surface, G-protein (GP) coupled receptors [28]. An example of non-genomic signaling is the rapid, testosterone-induced release of calcium (Ca2+) from intracellular stores occurring in mouse IC-21 macrophages [28]. The non-genomic, actions of testosterone are not inhibited by classical androgen blockers such as cyproterone and flutamide, suggesting that the genomic and non-genomic effects of androgens may be quite different. For example, we [29] and others [30] have shown that long-term administration of testosterone to male rats confers cardioprotection against ischemia/reperfusion (IR) injury. However, when testosterone is added in vitro to the working heart preparation, it worsens IR injury [31].
The genomic effects of androgens occur through two distinct pathways. In the first pathway, binding of androgens to cytosolic ARs cause ARs to translocate to the nucleus and bind to chromosomal DNA as homodimers [32] (Fig. 1). The specific regions of DNA that bind ARs are called hormone response elements (HREs) and they regulate the transcription of specific genes, producing androgenic effects. In the second pathway, the actions of androgens are mediated by interaction of ARs with the Wnt/â-catenin pathway. Wnts are a family of secreted glycoproteins that regulate differentiation in a wide variety of tissues. Canonical Wnt/â-catenin signaling involves binding of Wnt to the cell-surface frizzled receptor (FZ), which through its interactions with Axin, Frat-1 and Dsh, results in the inhibition of glycogen synthase kinase-3 (GSK3). GSK3 phosphorylates â-catenin, marking it for proteasomal degradation. As a result, inhibition of GSK3 results in the accumulation of â-catenin in the cytosol and increased translocation of â-catenin into the nucleus, where it interacts with transcriptional regulators to alter gene expression [33]. It is hypothesized that androgen signaling bypasses the canonical Wnt pathway by interacting with downstream Wnt effectors [34] to stimulate the commitment of mesenchymal pluripotent cells to the myogenic line and to inhibit their commitment to the adipogenic line [3]. By this mechanism, androgens increase the number of muscle satellite cells [35]. In culture, androgens have been shown to induce translocation of â-catenin to the nucleus in mouse 3T3 preadipocytes and inhibit their differentiation into mature adipocytes [34]. Wnt/â-catenin signaling has also been implicated in androgen-induced masculinization of external genitalia [4].
Full-size image (42K)
Fig. 1. Androgen receptor signaling pathways. A = androgen, AR = androgen receptor, Ca2+ = calcium, FZ = frizzled receptor, GP = G protein, GSK-3 = glycogen synthase kinase 3, HRE = hormone response element, PLC = phospholipase C, SR = sarcoplasmic reticulum, TCF = T cell factor, LEF = lymphoid enhancer factor 1, Wnt = Wingless-Int.
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The metabolism of testosterone also plays an important role in its actions. Testosterone may be converted to DHT by either 5á reductase isoenzyme [36] and to 17â-E2 by aromatase [37]. Tissues expressing 5á reductase include the prostate, testes, accessory sex organs, beard and scalp, sebaceous glands, liver, brain, and skin [38] and in these tissues the effects of circulating testosterone are amplified by two known mechanisms. First, DHT has an approximate 3-fold greater affinity for the AR than does testosterone [39]. Second conversion of testosterone to DHT prolongs the androgen action because testosterone can be converted to the weaker androgen androstenedione, while DHT maintains a longer presence [36]. Aromatization of testosterone occurs within specific tissues (e.g., bone and brain) and systemically in some species [40] and [41]. In men, significant systemic aromatization also occurs in adipose tissue [42] and because of the resulting elevation of serum 17â-E2, may cause adverse effects (e.g., fluid retention, gynecomastia, and worsening of sleep apnea) in obese men receiving testosterone [43]. Thus, the biological effects associated with testosterone may result from classic AR mediated pathways, interactions with the Wnt/â-catenin pathway, or from AR or ER activation following conversion of testosterone to DHT or 17â-E2, respectively.
3. Recent advances in SARMs
Selective androgen receptors modulators (SARMs) are a class of drugs that have action in some tissues expressing ARs and reduced action in others [44]. One of the key goals in the development of SARMs has been to find orally available agents that have anabolic effects on muscle, bone, and erythropoiesis, but that do not cause prostate enlargement or other common adverse events associated with supraphysiological testosterone administration [23]. The name SARMs is analogous to the older drug class of selective estrogen receptor modulators (SERMs), which act as agonists in some tissues and antagonists in others [45]. SARMs are under development in many major pharmaceutical firms as a potential means of treating the symptoms associated with hypogonadism and various muscle and bone wasting conditions [23] but much of the work defining their mechanism of action and structure-activity relationships has not been published. Much of the pioneering work in developing non-steroidal SARMs was done at Ligand Pharmaceuticals and at the University of Tennessee [46] and [47] where the first generation of SARMs including S1 and S4 were developed [48]; both of which prevent atrophy of the levator ani muscle without significant prostate enlargement in a castrated rat model [49]. The following SARMs are leading candidates in each of the major chemical classes. LGD2226 is quinolinone analog, developed by Ligand Pharmaceuticals [50]. S4 is an aryl proprionamide analog developed by Gao and coworkers [49]. BMS564929 is a hydantoin analog developed by Bristol-Myers Squibb [51]. S40503 is a tetrahydro-quiniline analog developed by Kaken Pharmaceuticals [52]. Each has been shown to have strong anabolic actions in skeletal muscle and bone with partial agonist activity in the prostate [44]. Although, the non-steroidal SARMs are a chemically diverse group of compounds, in many cases, their tissue selectivity may derive from the drug not being a substrate for the 5á reductase and/or aromatase enzymes [53].
Testosterone is not effective when administered orally due to a high liver first-pass effect [54] and is readily 5á reduced or aromatized in a variety of tissues. In contrast, some orally available synthetic testosterone analogs may have SARM-like activity. For example, oxandrolone (17-á methyl-DHT) is not a substrate for aromatase [53]. Similarly, nandrolone (19-nortestosterone) is 5á reduced, but to a relatively weak androgen, dihydronandrolone, and is a poor substrate for aromatase [55]. However, both nandrolone and oxandrolone cause liver toxicity at high doses. Conversely, 17â-TBOH (trenbolone) has a low oral bioavailability (because it is not methylated at the 17á position), but may have SARM-like properties considering that it is not a substrate for 5á reductase and may not be a substrate for aromatase. The toxicity of 17â-TBOH has not been scientifically studied in humans, but anecdotally has been reported to have a low potential for liver toxicity because this drug is generally administered intramuscularly.
4. 17â-TBOH metabolism
17â-acetoxyestra-4,9,11-trien-3-one (17â-TBOH-acetate) is a highly potent anabolic androgenic steroid which is primarily used legally as a growth promoting agent in domestic livestock production within the US [56] either alone, as Finaplix (Intervet, Inc., Millsboro, DE), or in combination with 17â-E2, as Revalor (Intervet, Inc., Millsboro, DE), or 17â-E2-benzoate, as Synovex (Wyeth, Madison, NJ) [57]. Following administration, 17â-TBOH-acetate is rapidly converted to the biologically active steroid 17â-TBOH [58] which, along with its metabolites, are included on the World Anti-Doping Association (WADA) prohibited substance list [59] due to their potential ability to augment skeletal muscle mass and improve athletic performance. As such, a variety of testing methods have been proposed to detect the presence of 17â-TBOH or its metabolites in human urine or blood [60], [61], [62], [63], [64], [65], [66], [67], [68] and [69]; although, evaluation of these methods is beyond the scope of this review.
Most evidence regarding the in vivo mammalian metabolism of 17â-TBOH is derived from studies on livestock and rodents [58], [70] and [71]; however, several studies have evaluated 17â-TBOH metabolism in humans [68] and [72]. Some variation in the in vivo metabolism of 17â-TBOH exists among mammalian species [58] and [68], but the primary metabolites are 17â-hydroxy- and 17-oxo-metabolites of trenbolone in rodents or 17á-hydroxy-metabolites of trenbolone in ruminants [58]. Similarly, in humans, ingested 6,7-3H labeled 17â-TBOH is primarily excreted intact, as 17â-TBOH, as the 17á epimer (epitrenbolone; 17á-TBOH) or as trendione (TBO) (Fig. 2) [68]. In addition, several yet to be identified polar metabolites of 17â-TBOH have also been detected in human urine, albeit in much lower concentrations than the primary metabolites previously listed [68]. 17â-TBOH has a greater affinity for the AR than any of its primary metabolites [39] and [73], suggesting that biotransformation of 17â-TBOH reduces the biological activity of this steroid [26] and [68]. This is in contrast to testosterone, which undergoes irreversible 5á reduction [36] or aromatization [37] and [74] to the more potent DHT and 17â-E2.
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Fig. 2. Structures of trenbolone and it primary metabolites in humans, epitrenbolone and trendione, along with testosterone and its metabolites dihydrotestosterone and estradiol. 17â-TBOH = trenbolone, 17á-TBOH = epitrenbolone, TBO = trendione, DHT = dihydrotestosterone, 17â-E2 = estradiol.
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4.1. 17â-TBOH and 5á reductase
Despite its structural similarities to testosterone, 17â-TBOH does not undergo 5á reduction due to the presence of a 3-oxotriene structure, which prevents A ring reduction (Fig. 2) [58]. In fact, 17â-TBOH undergoes biotransformation to less biologically active androgens [26] and [68], similar to other anabolic androgenic steroids, such as 19-nortestosterone [55]. Indirect evidence indicates this is true, as 17â-TBOH administration has been shown to reduce prostate mass in growing male rodents when compared with control animals [75]. Similarly, 17â-TBOH exerts less pronounced effects than testosterone in androgen-sensitive tissues which express the 5á reductase enzyme including the prostate and accessory sex-organs [25], [26], [36], [76], [77], [78], [79] and [80], despite the fact that 17â-TBOH binds to the human AR [26] and [39], along with ARs of various model species [26], [81] and [82], with approximately three times the affinity of testosterone. Despite its inability to undergo 5á reduction, 17â-TBOH remains highly anabolic, evidenced by equal or greater growth in the levator ani skeletal muscle (an androgen responsive tissue which lacks the 5á reductase enzymes), compared to testosterone [25], [26], [76], [77], [78], [79], [80] and [83]. Thus while testosterone exerts enhanced effects in tissues expressing 5á reductase, 17â-TBOH exerts equal effects in those tissues expressing 5á reductase (e.g., prostate and accessory sex-organs) vs. those not (e.g., skeletal muscle) [36]. Taken together, these reports indicate that 17â-TBOH produces a ratio of anabolic:androgenic effects that may be favorable compared to the effects of testosterone. However, we are unaware of any paper that has reported the effects of 17â-TBOH administration on the growth of androgen-sensitive tissues in humans.
4.2. 17â-TBOH and aromatase
17â-TBOH and other C19 norandrogens [84] are reported to not be substrates for the aromatase enzyme [85] and [86] and to be relatively non-estrogenic [87] and [88]; although some debate exists regarding 19-nortestosterone (a C19 norandrogen) to undergo aromatization and induce estrogenic effects [84] and [89]. In vitro bioassays and cell culture experiments demonstrate that 17â-TBOH and its metabolites have a very low binding affinity for ERs and have low estrogenic activity with approximately 20% of the efficacy of 17â-E2 [87]. Reports also suggest that 17â-TBOH reduces serum 17â-E2 concentrations in vivo [81], [90] and [91], inhibits estrus in ovariectomized estrogen-treated female rats [92], and exerts a variety of anti-estrogenic effects [92], perhaps through hypothalamic feedback inhibition of the production of testosterone (a substrate necessary for endogenous 17â-E2 biosynthesis) [93] and [94]. Additionally, in various fish models, environmental exposure to 17â-TBOH downregulates brain CYP19B (aromatase B) and upregulates gonadal CYP19A (aromatase A) expression in females, but not males [95] and [96], while reducing tissue concentrations and tissue-specific gene expression of vitellogenin (VTG), a protein found in oviparous animals which is positively associated with exposure to estrogenic compounds, in both sexes [81], [88], [93], [94], [95], [96], [97], [98], [99], [100] and [101]; together these may represent compensatory responses resulting from reduced endogenous estrogen concentrations [101]. Conversely, others report that 17â-TBOH either increases [81] and [102] or has no effect [81], [90] and [103] 17â-E2 concentrations in ruminants and oviparous animals.
The mechanism(s) through which 17â-TBOH alters estrogenic activity remain to be elucidated, but may be related to the (1) inhibition of endogenous androgen synthesis [104] and [105] (presumably through pituitary or hypothalamic feedback inhibition [93] and [106]), (2) altered expression or activity of the aromatase enzyme [96] and [107], and/or (3) down-regulation of ERá or ERâ expression [95] X. Zhang, M. Hecker, J.W. Park, A.R. Tompsett, J. Newsted and K. Nakayama et al., Real-time PCR array to study effects of chemicals on the hypothalamic-pituitary-gonadal axis of the Japanese medaka, Aquat Toxicol 88 (2008), pp. 173–182. Abstract | Article | PDF (797 K) | View Record in Scopus | Cited By in Scopus (12)[95] and [108]; however, they do not appear to be mediated by direct androgen receptor activation as co-treatment with 17â-TBOH and flutamide (an AR antagonist) also results in anti-estrogenic activity in female fathead minnows [81]. To summarize, 17â-TBOH is not a substrate for the aromatase enzyme, but may exert both anti- and pro-estrogenic effects, with the bulk being anti-estrogenic.
5. Mechanisms of body growth
The growth promoting effects of 17â-TBOH administration are well known [56], [109] and [110], as numerous studies have reported that administration of 17â-TBOH or its acetate ester enhance total body growth and skeletal muscle mass in various rodent and livestock models when administered alone [25], [26], [75], [76], [77], [78], [79], [80], [83], [86], [90], [102], [103], [111], [112], [113], [114], [115], [116], [117], [118], [119], [120], [121], [122], [123], [124], [125], [126], [127], [128], [129], [130], [131], [132], [133], [134], [135] and [136] or when administered in combination with 17â-E2 [86], [90], [103], [104], [111], [112], [123], [127], [128], [129], [137], [138], [139], [140], [141], [142], [143], [144], [145], [146], [147], [148], [149], [150], [151], [152], [153], [154], [155], [156], [157], [158], [159], [160], [161], [162], [163], [164], [165], [166], [167], [168], [169], [170], [171], [172], [173], [174], [175], [176] and [177]. Interestingly, several studies have reported that administration of 17â-TBOH in combination with 17â-E2 results in greater body growth and skeletal muscle mass than either steroid alone [103], [112], [127], [128], [171], [172] and [173]; indicating that 17â-E2 enhances the anabolic effects of 17â-TBOH, as others have suggested [109] and [110]. Ultimately, enhanced body mass results from increases in lean mass (predominantly comprised of muscle and bone) and/or fat mass; thus, the known effects of 17â-TBOH on each of these tissues will be discussed. In addition, the effects of 17â-TBOH on erythropoiesis will be briefly discussed because androgens are known to exert potent effects on red blood cell production.
5.1. Effects of 17â-TBOH on skeletal muscle
Skeletal muscle expresses ARs to varying degrees among species [178], [179], [180], [181] and [182]. As such, androgens induce skeletal muscle protein accretion following dimerization of ARs (Fig. 1). Skeletal muscle expresses 5á reductase and dose-dependently converts testosterone to DHT [183], however, our laboratory [19] and [20] and others [24] have recently demonstrated the 5á reduction of testosterone is not required for skeletal muscle maintenance in hypogonadal animals or humans. In addition, skeletal muscle expresses ERs within both sexes of various species [184], [185] and [186] and 17â-E2 administration has been shown to protect against loss of muscle strength in ovariectomized female rodents [187] and [188]; suggesting that aromatization might contribute to the effects of testosterone on skeletal muscle in males.
In ruminants, 17â-TBOH, alone or in combination with 17â-E2, has been shown to increase the cross-sectional area (CSA) of type I, but not type II, skeletal muscle fibers and induce a fiber switch from more glycolytic to more oxidative fibers, indicating an increase in the oxidative capacity of skeletal muscle [152] and [189]. However, the presence of 17â-E2 is not required for 17â-TBOH to augment skeletal muscle mass as demonstrated in rodent models which experience significant growth of the levator ani muscle [25], [26], [76], [77], [78], [79], [80] and [83] and other skeletal muscles [25], [26], [76], [77], [78], [79], [83], [117], [126] and [133] following 17â-TBOH administration, despite lacking the primary source of endogenous 17â-E2. However, not all rodent models experience peripheral (i.e., hindlimb) muscle growth following 17â-TBOH administration [115], [118] and [126]; although, elevated skeletal muscle DNA concentrations are present in muscles that do not increase in mass in response to 17â-TBOH treatment [117]. The inconsistent skeletal muscle response to 17â-TBOH in rodents may occur because certain peripheral rodent skeletal muscles possess a low percentage of AR positive myonuclei (i.e., extensor digitorum longus with 7% AR positive myonuclei) [181]. Conversely, human myonuclei are approximately 50% AR positive [182] and ruminants are highly sensitive to androgen-induced myotropic stimuli due to a high concentrations of ARs in bovine skeletal muscle [178] and [179] and skeletal muscle satellite cells [180]. Similarly, the androgen-sensitive levator ani muscle in rodents contains approximately 74% AR positive myonuclei [181] and thus experiences robust atrophic responses to castration [190] and hypertrophic responses to androgen administration [25], [26], [76], [77], [78], [79], [80] and [83].
The underlying mechanism(s) through which 17â-TBOH enhances skeletal muscle growth have not been completely characterized; although, it is suspected that 17â-TBOH exerts direct anabolic effects on skeletal muscle primarily via AR activation and associated nuclear translocation and transcription or via modulation of the Wnt/â-catenin pathway, similar to other androgens [26] (Fig. 1). In vitro evidence indicates that 17â-TBOH induces translocation of human ARs to the nucleus in a dose-dependent manner and induces gene transcription to at least the same extent as DHT, the most potent endogenous androgen [26]. Further, 17â-TBOH treatment of cultured bovine satellite cells upregulates AR mRNA expression [191], perhaps explaining the observations that administration of 17â-TBOH increases satellite cell activation and proliferation in various species [117], [191] and [192].
Additionally, 17â-TBOH may induce anabolic effects via mechanisms associated with alterations in endogenous growth factor concentrations [193] or the responsiveness of skeletal muscle to such growth factors [117] (Fig. 3). For example, 17â-TBOH alone or in combination with 17â-E2 upregulates insulin-like growth factor (IGF-1) mRNA in a variety of tissues, including the liver and skeletal muscle in vivo [140], [141], [160], [170], [191], [194] and [195] and satellite cells in vitro [140], via distinct androgen- and estrogen receptor mediated mechanisms [180] and [196]; although 17â-TBOH alone (without the addition of 17â-E2) does not appear to alter skeletal muscle IGF-1 mRNA [184]. Ultimately, the upregulation of IGF-1 mRNA translates into increased serum IGF-1 in 17â-TBOH treated animals [138], [140], [148], [150], [160], [165], [170], [192], [194], [197] and [198], which may stimulate satellite cells proliferation and fusion as has been shown in vitro [117]. Interestingly, 17â-TBOH administration also appears to increase the responsiveness of satellite cells to the proliferating and differentiating effects of IGF-1 and fibroblast growth factor [117]. These results are intriguing considering that the inhibition of several of the downstream targets of IGF-1 (e.g., Raf-1/MAPK kinase (MEK)1/2/ERK1/2, or phosphatidylinositol 3-kinase (PI3K)/Akt) suppresses 17â-TBOH induced satellite cell proliferation in culture [180]. Thus, it seems likely that increased growth factor expression resulting from 17â-TBOH administration is one mechanism underlying the anabolic responses to this steroid in skeletal muscle, especially considering that binding of IGF-1 to the type 1 IGF receptor is required for proliferation of satellite cells [180].
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Fig. 3. Potential mechanisms underlying the anabolic effects of trenbolone on skeletal muscle. 17â-TBOH = trenbolone, GR = gluccocorticoid receptor, AR = androgen receptor, IGF-1 = insulin-like growth factor 1, IGF-1R = insulin-like growth factor 1 receptor.
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17â-TBOH may also preserve or increase lean mass via anti-catabolic effects associated with reductions in endogenous glucocorticoid activity [126] and [135] or with the suppression of amino acid degradation within the liver [118], [122], [134], [199] and [200] (Fig. 3). For example, 17â-TBOH administration has been shown to reduce circulating corticosterone concentrations in rodents [115], [118] and [122] and resting cortisol in cattle [150]. In vivo [122] and in vitro [201] evidence indicates that 17â-TBOH works in the adrenals to suppress adrenocorticotropic hormone (ACTH)-stimulated cortisol synthesis and to suppress cortisol release. Further, 17â-TBOH has been shown to reduce the ability of cortisol to bind to skeletal muscle glucocorticoid receptors (GRs) [121] and to down regulate skeletal muscle GR expression [108] and [121]. Thus the multiple anti-glucocorticoid actions induced by 17â-TBOH explain, in part, the 17â-TBOH-mediated increase in total body nitrogen retention [133], [151], [155], [156] and [202] and the reductions in total [80], [129] and [156] and myofibrillar protein degradation in several species [126], [135], [156], [202] and [203]; especially considering that 17â-TBOH reportedly reduces skeletal muscle protein synthesis in male rodents [80] and [133]. As a result of its anti-glucocorticoid actions, 17â-TBOH produces a more robust inhibition of protein degradation than does testosterone, which only slightly reduces protein degradation while increasing protein synthesis [204]. Thus, future research comparing the effectiveness of 17â-TBOH and the endogenous androgens in altering skeletal muscle degradation via the ubiquitin proteasome system or other pathways associated with muscle atrophy [205] is warranted and may further elucidate the anti-catabolic mechanism(s) underlying the potent augmentation of skeletal muscle mass associated with 17â-TBOH.
