Introduction
Anabolic-androgenic steroids (AAS) are among the most dangerous substances commonly used in Western societies. The global lifetime prevalence rate obtained in 2014 was 3.3%, with the rate being four times higher for men than for women [1]. In most countries, access to AASs is unrestricted, either through numerous websites or illegal sales by private individuals, often in sports centres.
Under Polish law, limited possession of such substances in undetermined quantities for personal use is not illegal [2]. As many as 70–80% of AAS users are amateur level athletes, who primarily want to improve their appearance by increasing their muscle mass, or their athletic performance [3]. Worryingly, many AAS users lack adequate medical knowledge and use the substances without any medical supervision. Indeed, more than 90% of users have been found to experience injurious side effects [3].
One of the most potent [4] and commonly-used substances [3] is trenbolone, a steroidal analogue of testosterone. Trenbolone was introduced to the market in the 1970s, when it was used in the agri-food industry as a growth promoter for livestock in the USA [5]. The cattle farmers used it to obtain defatted animal meat containing more muscle tissue. In such cases, trenbolone was administered to livestock as a subcutaneous implant with a slow release time.
Since the 1980s, the use of AASs, and thus trenbolone, has become more popular in among athletes in Western societies. A few decades ago, most users were believed to be professional athletes; however, this has since changed dramatically, with as many as 70–80% of AAS users now being amateur athletes [1, 3, 6]. Furthermore, studies indicate a growing tendency in the use of these substances in developed countries, with most of users commencing “steroid cycles” at a median age of around 20 years [3].
Structure and chemical characteristics
Trenbolone (Fig. 1) is a synthetic analogue of testosterone, belonging to the nandrolone group [7, 8]. Compared to testosterone, this group is characterised by the demethylation of the carbon at the C19 position. This augments the anabolic effect of nandrolones, while also simultaneously limiting their androgenic effect on the body [3, 9].
After entering the mammalian body, trenbolone is rapidly hydrolysed to 17-b-trenbolone (17b-hydroxyestra-4,9,11-trien-3-one) [10]. This metabolite is officially banned in sports and figures on the World Anti-Doping Association (WADA) list [11]. 17b-trenbolone is a potent mammalian androgen receptor (AR) agonist comparable to 5a-dihydrotestosterone (DHT), which is considered the most biologically active metabolite of testosterone [4, 12]. However, due to the presence of a 3-oxotriene structure in its formation [10], it is not a substrate for the enzyme 5-alpha-reductase or aromatase [4]. Due to its lack of aromatisation to estrogens, trenbolone is more androgenic than nandrolone, another potent testosterone analogue [7].
Side effects
Studies indicate that as many as 90% of users experience a wide array of side effects on various organs, which creates a significant issue for public health institutions on a global scale. Its perceived side effects are so serious that it has been placed on the USA register of dangerous substances [13].
Effects on nervous tissue
An important biochemical characteristic of trenbolone is its structure. It contains a sterane core, and is thus classified as a lipophilic compound [7]. It is thus able to penetrate the blood-brain barrier [7, 14] and exert a potential effect on the central nervous system. A number of studies have found AAS [15–17], and hence trenbolone, to influence numerous neurochemical systems and receptors in rodent brains, resulting in a range of somatic symptoms and changes in the behavioural patterns of the studied animals. While it is possible that similar effects might be observed on human neural tissue, these have yet to be confirmed.
One of the most severe impacts of trenbolone is its influence on the cerebral cortex: long-term use of this agent has been shown to reduce its volume in rats, which implies that brain tissue atrophy may take place [18]. It is suspected that there is a link between the induction of pro-apoptotic processes caused by this agent [18-22] and a reduction in viability, as well as an increase in toxicity to central nervous system cells. The induction of apoptosis is believed to be triggered the activation of both caspase 3 (CASP3) and caspase 7 (CASP7) by trenbolone, which can occur even at low doses [18]. Both caspases belong to a group of 12 serine proteases whose function is intrinsically linked to programmed cell death, and are among those known as apoptosis executors [23, 24]. Hence, it is believed that the observed decrease in neuronal abundance in the brain and thus its volume, may be due to the activation of these caspases by trenbolone.
Furthermore, trenbolone use has been associated with decreased mitochondrial activity and elevated blood lactate dehydrogenase (LDH) levels, indicating general damage to cells throughout the body. This was found to be accompanied by chromatin condensation and nuclear fragmentation in the structure of brain cells, which may reflect the damage occurring in the cells and the occurrence of apoptosis [14]. The effects in question depended on the dose and duration of trenbolone use, with even nano-molar concentrations increasing the risk of excitotoxic cell death in mouse cortical cultures [24].
These cytotoxic effects occurring in both animals and cell cultures could possibly be responsible for the behavioural changes observed in users of AASs, such as trenbolone. These included disturbances in parts of the central nervous system, particularly those concerning cognitive function, learning efficiency and visuospatial memory [3]. Furthermore, specific changes were noted in the medial prefrontal cortex (mPFC) region, which plays a key role in the biochemical stress axis in the brain [25, 26]. The region was found to manifest changes in oligodendrocyte differentiation, translating into alterations in the formation of mouse myelin basic protein [26]. These changes have been correlated with characteristic behavioural patterns in mice, such as disorders in establishing social relationships and high levels of anxiety [26]. Similar disturbances are also observed in trenbolone users [27, 28].
A separate issue is the effect of trenbolone on amyloid plaque formation processes. Studies on a rat model found trenbolone use to reduce the amount of the protein presenilin 1 (PS1), involved in the activity of the enzyme g-secretase, which is connected to the formation of abnormal protein-Ab42. The formation of this abnormal protein is directly proportional to the trenbolone dose taken, and the highest amount was observed in the hippocampus [14]. This protein is responsible for the formation of senile plaques and shows strong neurotoxic effects, both from the larger agglomerations of the protein and the smaller oligomers and protofibrils surrounding them. The structures modify the curvature of neuronal cells and distort them, which can affect neurotransmission along their length and disrupt the transmission of neural signals through neuronal synapses [29]. A similar polypeptide has been found to accumulate in other neurodegenerative diseases such as Alzheimer’s disease [29]. The formation of protein deposits can be correlated with symptoms from the hippocampus, which include impaired multiple memory function [30]. Such memory deficits are also observed in trenbolone users, and mainly take the form of deficits in everyday memory [31].
Another observed activity of trenbolone in the central nervous system is its effect on ionotropic N-methyl-D-aspartate receptors (NMDARs), which are heterotetramers consisting of two obligatory GluN1 subunits, and two GluN2 or GluN3 [32, 33]. All subunits can be encoded by different genes and can be formed by alternative splicing. It has been found that these components can combine in as many as 60 combinations that can fit NMDARs [32]. The fact that changes in the composition of the subunits in the receptor can affect the biochemical activity of NMDARs [34, 35] and that the expression of specific subunits is dynamic and can fluctuate with time [36, 37] may be of importance.
Taking testosterone analogues such as trenbolone has been shown to modulate subunit composition and thus decrease the formation of mRNA encoding the GluN2 subunit in the hippocampus and hypothalamus. Moreover, studies on rat models have found usage to be associated with a reduction in the overall amount of NMDARs, particularly in the hypothalamus and hippocampus [15]. In both the hypothalamus and hippocampus, NMDARs have important roles in brain function. In the hypothalamus, for instance, they are responsible for aggression [38], whereas in the hippocampus they are responsible for personal memory, and their disruption has been implicated as a possible cause of epileptic disorders [39].
As mentioned earlier, changing the proportion of individual subunits can affect the function of NMDARs. In the case of the hypothalamus, such alterations may be responsible for the aggressive behaviours reported abundantly in animal research [40]. In human studies, trenbolone users also reported aggression as one of the most perceived side effects [41]. A decrease in the number of receptors in the hippocampus [15] is also likely to affect memory deficits in users; however, these may arise through a different pathway [31].
Gamma-amino butyric acid (GABA) receptors are present in the central nervous system and their function is significantly affected by AASs such as trenbolone. Studies suggest that GABA receptors may be sensitive to natural sex steroids. Their influence may manifest as specific behavioural changes during sexual maturation in users who are experiencing hormonal dysregulation [42, 43]. These hormones lead to changes in the expression and function of GABA receptors, such as modification of neuronal transmission, its allosteric modulation and the activation and sensitisation of receptors [43]. GABA type A (GABAA) receptors in the forebrain, specifically in regions such as the medial preoptic area (mPOA), ventromedial nucleus accumbens (VMN) and medial amygdala (MeA), are thought to influence feelings of anxiety, aggression and sexual desire in mammals [43]; all have been reported to be altered in AAS users [3, 25–28, 41], which may also result from the modification of their receptors by trenbolone.
Effects on muscle tissue
The most commonly-reported motivation for beginning AAS use is the desire to obtain a muscular, proportional and aesthetic physique in the shortest possible time [3]. However, some athletes training for a specific sport may turn to AASs to improve their own performance or enhance their appearance (e.g. professional bodybuilders) [3].
Trenbolone exhibits one of the highest anabolic-androgenic ratios, and is therefore one of the most potent agents for stimulating muscular development [44]. Such an effect is called anabolism, a state in which nitrogen is retained in lean body mass by stimulating the synthesis of new proteins and/or inhibiting the degradation of proteins already present. It promotes an increase in muscle protein synthesis and collagen fibres, and an upsurge in bone metabolism [45].
Muscle hypertrophy, i.e. an increase in the size and thus volume of muscle fibres, is the leading desired effect of trenbolone use. Unfortunately, the exact mechanisms of this effect have not been fully characterised. Studies have observed that hypertrophy occurs through several pathways induced by androgens such as trenbolone. The first of these, known for many years as the genomic, or classical, pathway (Fig. 2), occurs through the anabolic agent binding to the cytosolic AR; in such cases, trenbolone has three times higher affinity than testosterone. This results in the translocation of the agent and the bound AR into the nucleus of the muscle cell, with movement being dependent on the dose of the attached agent. Finally, in the cell nucleus, the androgen and receptor combine with chromosomal DNA, at sites called androgen response elements (AREs), in the form of homodimers. These promote the transcription of specific genes and subsequent translation of proanabolic proteins [4]. This pathway is slower, and its effects can manifest over a longer period of time.
In contrast, the second biochemical/molecular pathway also involves the AR, but the anabolic effects that occur are mediated by the Wnt/ b-catenin and Notch pathways (Fig. 2). Wnt signalling is an extremely important pathway, associated with a set of glycoproteins. Its purpose is to regulate myogenesis, both during embryogenesis and at maturity. It promotes the differentiation of stem cells into myogenic cells and, if required, the repair of damaged muscle tissues [46–48]. First, b-catenin acts as a transcriptional effector. The molecule binds to the transcriptional repressor Tcf2/Lef in the nucleus of the muscle cell, converting it into transcriptional activators that induce proanabolic genes acting as targets of the Wnt pathway. Several studies indicate that the administration of trenbolone increases b-catenin levels and thus Wnt pathway levels [49–51]. This is likely due to the negative effects of trenbolone on glycogen synthase kinase 3 (GSK3), another component of this molecular system. The kinase is related to the Notch pathway, which inhibits the Wnt pathway, and marks b-catenin for intracellular degradation by phosphorylation; in the absence of GSK3, the amount of b-catenin will increase and perform its role as a translation factor [4].
Both the Notch pathway and the Wnt-related pathway share very similar cellular functions in regulating myogenesis; however, Notch pathway is Wnt-related pathway antagonist and additionally promotes satellite cell activation and differentiation [52–55]. Both pathways constitute an integrated molecular system in which each component interacts with the other. The exact interactions between the pathways are not fully understood, but the Numb molecule, which inhibits the Notch pathway and participates in myocyte cell gene recombination, is thought to act as a signalling molecule [55–61].
Nandrolone or trenbolone administration results in increased levels of b-catenin, and thus its greater attachment to the regulatory sites of the Numb gene promoter (at the Tcf site); this causes an increase in the expression of the gene encoding Numb, and thus greater Numb mRNA/protein levels in the nuclear and cytosolic fractions [62–64]. This probably occurs through conformational stabilisation of the Numb protein; this is made possible by reducing the concentration of the MDM2 molecule, related to the p53 protein, and altering the level of the Musashi protein, which is involved in organismal development and the fate determination of individual cells [65]. In summary, trenbolone administration increases the levels of the Numb molecule, thus enhancing the pro-anabolic action of the Wnt pathway by further inhibiting the Notch pathway, an antagonist of the Wnt pathway.
However, recent studies indicate the presence of another pathway through which androgens can potentially act, viz. the non-genomic pathway (Fig. 2), also referred to as the fast pathway, because its action involves a surface receptor associated with the G protein. The pathway is activated by a sudden increase in calcium (Ca2+) levels from intracellular stores. Unfortunately, the pathway is believed to act through testosterone, not trenbolone, and has been described in IC-21 macrophage cells [66, 67]. Even so, it is still possible that this pathway could occur in cells other than macrophages, such as muscle cells, and that they could influence the action of anabolic substances: they may stimulate the binding androgens to AR or influence gene expression [63, 68, 69]. Furthermore, it is possible that by acting on similar structures in the cell, trenbolone could have similar non-genomic activity as testosterone on various cell types. However, more research on this topic is required to confirm this.
Finally, trenbolone can also influence skeletal muscle tissue growth by changes associated with muscle satellite cells (Fig. 2). This is a group of cells located in the niche between the muscle fibre membrane and the surrounding basement membrane, which have the ability to differentiate into myoblasts and form subsequent generations of muscle cells. They are categorised as tissue-targeted unipotent stem cells [70–72]. During laboratory studies, their activation was recorded as early as the first hour after trenbolone application, and this continued for 12 hours. They then entered a resting state, from which they could re-activate under the influence of trenbolone after a further 24-hour period [53]. Their activation is probably driven by ARs, which increase in number after trenbolone treatment, reflected in an increase in their mRNA levels [73, 74]. AR stimulation may induce the expression of mind bomb 1 (Mib1) — ubiquitin ligase and the subsequent trans-endocytosis of the Notch pathway ligands Delta-like 1 (DLL1) and Jagged 1 (JAG1) in the myofibers [75, 76]. These actions inhibit Notch pathway signalling, which is responsible for maintaining satellite cells in a quiescent state. This leads to the activation of satellite cells, during which, the satellite cells become direct precursors of skeletal muscle, i.e. myoblasts. The newly-formed myoblasts then fuse with each other and integrate into adjacent mature muscle cells by fusion, resulting in significant changes in the muscle tissue: an increase in the number of cell nuclei with a corresponding increase in the amount of DNA by up to 60% in some of the muscles studied, an expansion in the total amount of cytoplasm and contractile proteins (actin and myosin). The increase in the amount of actin and myosin results in the muscle cell generating more total force [44].
These changes may potentially be responsible for user reports of significant increases in total muscle strength following use of the drug. The exact molecular mechanisms behind the activation of satellite cells by trenbolone have not been fully explained. However, studies have shown that direct addition of trenbolone to the in vitro cell culture containing satellite cells did not increase their proliferation, differentiation or fusion. Instead, it was found that trenbolone increased the sensitivity of satellite cells to two growth factors, IGF-1 and FGF, which acted as mediators indirectly stimulating the activation and further function of satellite cells [77].
In addition to satellite cell sensitivity, trenbolone is also believed to be involved in the potentiation of various mediators. Notably, various studies report a build-up of IGF-1 mRNA in many tissues after trenbolone treatment, resulting in an increase in blood IGF-1 level [78, 79].
Such an increase may significantly affect sensitised satellite cells and induce their activation and proliferation, potentially resulting in skeletal muscle growth.
Effects on adipose tissue
It was observed that hypogonadal rats, characterised by low levels of testosterone, demonstrated a statistically-significant decrease in muscle mass and increase in adipose tissue, which is analogous to observations of humans [4, 80]. In this study, moderate doses of exogenous testosterone halted the decrease in muscle mass and reduced fat gain, while higher doses resulted in a noticeable increase in muscle mass and a concomitant reduction in body fat compared to control rats without induced hypogonadism [4]. The study yielded extremely satisfactory results regarding the marked inhibition of catabolic effects in the body, and confirmed the validity of hormone replacement therapy in humans with low testosterone levels.
Unfortunately, the mechanisms by which trenbolone affects adipose tissue remain unknown. However, trenbolone is known that trenbolone demonstrates a high affinity for ARs commonly found in the cells that make up adipose tissue e.g. adipocytes, preadipocytes and mesenchymal stem cells [81], and that androgens inhibit the differentiation of preadipocytes and mesenchymal stem cells into mature adipocytes through AR [82]. Such effects may occur through reduced expression of adipogenic genes, such as PPARg and C/EBPa, as well as through increased nuclear translocation of b-catenin and activation of Wnt signalling, promoting the myogenic lineage [76, 83]. All these activities may lead to lipolysis of adipose tissue. In various laboratory studies, rats have been observed to demonstrate a marked loss of subcutaneous adipose tissue, intramuscular fat, retroperitoneal fat and perirenal fat after trenbolone treatment [84–86]. It has also been shown that trenbolone can directly induce lipolytic effects by affecting the liver; presumably, this may occur by increasing the expression of the enoylo-CoA hydratase (ECH) and acyl-CoA dehydrogenase involved in fat metabolism in this organ [87].
Hence, it can be concluded that trenbolone appears to have a lipolytic effect on adipose tissue. These findings could potentially confirm the beneficial fat-burning effects of androgens reported in human cell studies [82]. However, studies are needed to confirm this effect in human groups, particularly with regard to trenbolone, and identify the mechanism responsible.
Effects on male reproductive system
The World Health Organisation (WHO) classifies infertility as the state of not being able to get pregnant, despite regular sexual intercourse (three or four times a week), maintained for more than 12 months, without any preventive measures [88]. Infertility is a serious problem in developed countries, where it is estimated to affect about 10–12% of couples; this would equate to nearly one million couples in Poland. It is highly probable that the number of infertile men and women will increase over time, as a result of delayed decisions about parenthood. The increasing use of AASs by men, and its associated infertility, adds to the problems facing the public health institutions of developed countries in the realities of the modern world.
Infertility in users of AAS, including trenbolone, most commonly manifests as oligo- and azoospermia, as well as abnormalities in sperm mobility and morphology [89]. In addition, significant hormonal deviations can be observed, but decreased libido levels or erectile dysfunction, are typically only reported after ceasing the use of exogenous hormones. Such changes include decreased levels of endogenous testosterone and the pituitary hormones LH and FSH. The symptoms can be defined by the term “functional hypogonadism” [44, 90], which manifests as a significant decrease in endogenous testosterone, testicular atrophy and impaired spermatogenesis [91]. The endocrine abnormalities associated with the condition derive from the negative effects of exogenous androgens on the hypothalamic-pituitary-gonadal axis, as well as their direct action on Leydig cells, which produce testosterone under physiological conditions [91].
A number of studies have also report AAS-induced morphological changes in various cells of the male reproductive system and the male gametes. One such change observed in male rat testes involves structural changes in Leydig cells, and an overall reduction in their number, resulting in a decrease in endogenous testosterone; in contrast, physiological concentration determines optimal spermatogenesis [92, 93]. The impaired spermatogenesis has also been observed as a lack of advanced forms of spermatids (spermatogenesis arrest) and various changes in spermatozoa. One of the most noticeable changes was a decrease in sperm cell motility. Fluorescent in-situ hybridization (FISH) studies have noted changes in cellular ultrastructure in some spermatozoa, as well as an increased frequency of sex chromosome (XY) disomy, indicating a segregation anomaly at the first meiotic division and of chromosome 9 disomy [94, 95]. Last but not least, elevated apoptosis has been noted in sperm cells; when research subjects are administered agents from the nandrolone group, such as trenbolone, the degree of apoptosis increases above level as noted during intensive physical exercise under physiological conditions, which is already elevated from proper intensity of apoptosis, anyway [96].
Some studies were carried out in rats with nandrolone, a compound with a similar mechanism of action and belonging to the same AAS group, so it can be speculated that trenbolone will induce similar changes. In these experiments a decrease in the diameter of seminiferous tubules was noticed, what is associated with the lower height of the seminiferous epithelium and hypospermatogenesis [97]. Moreover, changes in the blood-testis barrier were found, such as the degradation of the tight junction protein 1 (TJP1), deregulation of metalloproteinase 9, metalloproteinase 2 (MMP-2), the tissue inhibitor of MMP-2 and mislocalization of mucin 1 [98]. All these changes can lead to impairment of blood-testis barrier and, as a consequence, disturb the immune integrity of testicles.
In most men, a spontaneous return of semen quality occurs four to twelve months after discontinuation of exogenous anabolic agents [44, 92]. However, in those who do not recover, treatment may be applied as in other forms of hypogonadotropic hypogonadism. Gonadotropins or their analogues are then used. It has been found that the greatest chance of restoring fertility is associated with the use of an LH/hCG-acting preparation, alone or in combination with an FSH-acting preparation. Antiestrogens (aromatase inhibitors and selective estrogen receptor modulators — SERMs) are used to unblock pituitary, irrespective of blood estradiol levels. Reduced inhibiting influence of estrogen on hypothalamus and pituitary gland leads to an increase in both gonadotropins secretion and thus spermatogenesis recovery [99].
With these methods, it has been shown that even prolonged infertility, manifested by oligozoospermia, can be cured up to five years after AAS withdrawal [100]. However, the use of these agents permanently affects semen quality, as indicated in studies of previous AAS users; the treatment results in a higher than normal amount of hypokinetic and abnormal sperm in the semen [44, 100, 101].
Effects on the breast gland
Gynecomastia, which is characterized by development and swelling of breast tissue, is frequent side effect in trenbolone users. Although trenbolone does not convert to estrogen it binds with high affinity to the progesterone receptor [102]. Progesterone appears to be required to form true glandular acini acting in synergy with insulin growth factor 1 (IGF-1) [103]. AAS cause also a reduction in the level of thyreoglobulin (TBG), which binds thyroid hormones in the blood. This implies a reduction in the serum levels of free triiodothyronine (fT3) and thyroxine (fT4) [104]. As a result of a negative return loop this causes an increase in TRH secretion, which stimulates prolactin secretion [105]. Prolactin receptors have been demonstrated in the breast tissue. Moreover, hyperprolactinemia probably plays an indirect role in gynaecomastia, since it causes central hypogonadism and alters the androgen/estrogen ratio [106].
Effects on the immune system
Another interesting issue regarding the use of trenbolone, is its effect on the immune system. So far, relatively few studies have explored the effects of high concentrations of AAS in this regard; however, a relationship has been noted between the immune and endocrine systems in vertebrate organisms [107–109], and it seems certain that AAS use can cause disturbances in hormone levels and general endocrine disruption. As mentioned, these disorders are not isolated from the immune system, and it is important to note that androgens can influence innate and specific immunity. Furthermore, it is known that endogenous steroid sex hormones reduce immunity [110], and the potential harm of the synthetic steroid trenbolone on immunity merits further interest. Men using AAS report decreased immunity during and immediately after a trenbolone “cycle”, such as an increase in the incidence of various colds, viral and bacterial infections and hemiplegia, with recurrences noted after treatment [111, 112].
It was revealed that endogenous testosterone has a suppressive effect on the immune system [113]. This action affected T lymphocytes in particular and included a general reduction in their activity levels. However, the pathway by which this effect occurs was not explained. Instead, it was found that it could occur directly by acting on the AR present on the lymphocytes or by converting testosterone to estrogen and acting through a pathway related to it. In another study, castrated rats demonstrated a 90% increased immune response [114] compared to controls, and this value decreased exponentially after external testosterone was administered.
While these findings provide a general picture of the immunosuppressive role of endogenous, steroidal sex hormones, more detailed information has been obtained regarding their influence on T lymphocytes. T lymphocytes can be divided into three subpopulations based on their function: T cytotoxic (Tc), T helper (Th) and T regulatory (Treg) lymphocytes. Accordingly, the first population is responsible for destroying cells infected by microorganisms (including viruses) and tumor cells and the second supports the immune response through various cytokinins, while the final one regulates the immune response process and, if necessary, inhibits it. It has been presented, that the immune response exponent was the phytohemagglutinin PHA skin test, which yields a classical type IV delayed response: being a myotogen, PHA activates T lymphocytes without the antigen induction necessary in a type IV response [115]. This results in the activation of tissue macrophages by substances secreted by the lymphocytes, and the development of local inflammation on the skin, i.e. delayed cutaneous hypersensitivity (DCH). This reaction is dependent on Th lymphocytes. The study found all applied AASs, including trenbolone, to cause a marked reduction in DCH levels, indicating a reduction in Th lymphocyte activity [116]. The exact metabolic pathways of trenbolone are not known, but it is thought to act both directly through AR, which has been shown to be present on various T-lymphocyte subpopulations [116–118], as well as by modulating hormone secretion by the adrenal cortex or reproductive organs [116].
Trenbolone may also act on T lymphocytes via the activation of membrane receptors caused by increased Ca2+ levels. This would initiate a sequence of molecular events in the cell [119, 120] resulting in the successive phosphorylation of individual tyrosine-based substrates, the membrane linker of T cell activation and Src domains [121, 122] and thus the activation of transcription factors, including nuclear factor of activated T cells (NFAT). The final effect of this pathway is increased expression of transcription factors associated with T cells: T-bet, Gata3, RORt and Foxp3 [123]. These are necessary for the generation of a subpopulation of effector T cells: CD4+ helper T cells. In addition, the activation of NFAT results in increased secretion of various cytokines, e.g. IL-2 and various chemokines, together with the suppression of Treg lymphocytes [120, 124], which may affect the overall immune response in the body.
The use of high levels of trenbolone was found to decrease lysozyme levels significantly in a tissue-specific manner [125]: a significant decrease was observed in trout liver and plasma, while a statistically insignificant decrease was found in the kidney. Interestingly, no significant changes in the expression of the genes encoding this protein were recorded in the two tested organs. However, it is important to note that mRNA and protein expression are not always directly proportional to each other, and in this case, there may have been a simple delay in the expression of this gene connected to a decrease in the level of the enzyme; the timing of the study may not have allowed this to be visualised [126]. Lysozyme is part of the innate immune system and can have a destructive effect on the bacterial cell wall by hydrolysing the glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) [127]. In conclusion, reduced levels of lysozyme may have clinical implications, in terms of more frequent or clinically-intense bacterial infections, but more research is needed to confirm this.
Another component of the innate immune system that is ambiguously affected by trenbolone is the complement system. Its levels fluctuated significantly under the influence of different concentrations of an administered anabolic androgen hormone in trout. As with lysozyme, such changes depended very much on the specific area of the body: a significant decrease in activity was demonstrated in plasma, while the opposite effect occurred in kidneys [125]. As a component of the innate immune system, the complement plays various roles during the immune response, following the sequential activation of a protein cascade. Among other things, it is responsible for initiating the inflammatory process, opsonisation and facilitating phagocytosis, and bacterial cell lysis. While it has been proposed that disturbances in its plasma activity can, like lysozyme, impair the immune response against bacteria, more research is needed to confirm this.
A final aspect of the action of trenbolone that should be mentioned is its effect on genes involved in the development and maturation of different lymphoid cell populations. Administration was associated with a marked decrease in the expression of the RAG-1 and RAG-2 genes, which are essential for the proper functioning of lymphocytes [125]. However, no changes were noted in the total number of lymphocytes and the immunoglobulins produced by them. This may be due to the same reasons as described above for lysozyme.
Summary
The effects of trenbolone are not limited to a single tissue. It acts systemically on the body, and as such, its side effects can manifest as symptoms from different systems (Tab. 1).
|
Tissue type |
Effects of trenbolone |
|
Nervous |
Pro-apoptotic effect on neurons Reduction in brain tissue volume Accumulation of the toxic protein Ab-42 Modulation of the structure and function of GABA and NDMA receptors |
|
Muscle |
Stimulation of skeletal muscle for supra-physiological growth through direct effects on muscle fibres and indirect effects on myosatellite cells and endogenous growth factors |
|
Adipose |
Lipolytic effects resulting in a reduction of fat deposits in various areas of the body |
|
Reproductive |
Induction of functional hypogonadism Disturbances in the structure and number of Leydig cells Disturbances of the structure and number of sperm cells Acceleration of sperm apoptosis processes |
|
Immune |
Decrease in T lymphocyte activity Decrease in plasma lysozyme levels Decrease in plasma complement system activity |
This article focuses only on the most significant consequences of trenbolone use with regard to selected tissue structures, and the morphological and functional changes observed in them. However, various other adverse consequences of trenbolone utilization are included in the literature, with severe acne and gynaecomastia affecting approximately one-third of all users [3], as well as excessive body hair, stretch marks, hypertension and cardiac arrhythmia [4]. The drugs are also subject to contamination, with use frequently resulting in local inflammation at the injection site [128], muscle adhesions and fibrosis, nerve damage or, in extreme cases, necrosis of the injection site (Nicolau syndrome) [129].
In addition, the fact that multiple AASs may be used simultaneously presents a significant problem in their study. The users may report that they use smaller doses of different agents to minimise the negative side-effects that are more likely to occur when one agent is taken at an increased dose. Bodybuilders usually take trenbolone together with various testosterone esters (cypionate, enanthate or propionate) and nandrolone phenylpropionate [3, 4, 44]. This makes it very difficult to precisely determine the AAS responsible for the negative side effects, and the results cannot always be related to the actual conditions under which trenbolone acts in the body.
Additionally, due to the lack of available knowledge on the subject, many of the effects of trenbolone use remain unknown. Therefore, given its prevalence and potential danger, further research is necessary to better understand the effects of trenbolone use on the body, and to expand our currently incomplete knowledge of its functional pathways.
Author contributions
R.B. — conception, drafting the article, preparation of figures, final approval; P.B. — drafting the article, revising, final approval; J.S.-H. — Conception, drafting the article, preparation of figures, final approval.
Funding
The article is funded by Medical University of Lodz: Grant no 503/1-089-03/503-11-001.
Acknowledgments
We thank mgr Edward Lowczowski from the Foreign Language Teaching Center, Medical University of Lodz.
Conflict of interest
Authors declare no conflict of interest.
