Mutation Research/Genetic Toxicology and Environmental Mutagenesis
Low-dose effects and biphasic effect profiles: Is trenbolone a genotoxicant?
Introduction
Over the last two decades, research has documented endocrine-disrupting activities for various environmental pollutants including hormones, pharmaceuticals, pesticides and surfactants [1], [2], [3]. Many of these chemicals are intended for direct human use; others serve as veterinary drugs such as growth promoters and antibiotics and are extensively used in agriculture and released into the environment. Therefore, ecotoxicological studies have frequently focused on investigating the effects of estrogenic compounds on (non-target) higher eukaryotes. As an example, 17α-ethinylestradiol, the active component of contraceptive pills, and nonylphenol, a derivative of industrial chemicals, are among the most widely studied endocrine-disrupting chemicals (EDCs) [4].
In contrast, although very high androgenic activities have been detected, e.g., in sediment samples [5], the effects of androgens have been investigated less intensively. In fact, the potential of androgenic substances to influence sexual development of animals have already been shown at environmentally relevant concentrations by Jegou et al. [6], who reported masculinization of the fathead minnow (Pimephales promelas) in rivers downstream from cattle feedlots. The androgenic activity observed in cattle feedlots could be attributed to exposure to major metabolites (17α-trenbolone, 17β-trenbolone, and triendione) of the highly potent androgen trenbolone acetate, which is used across the US as a growth promoter in cattle [7], [8]. The synthetic androgen 17β-trenbolone is applied to cattle in the form of its ester, 17β-trenbolone acetate. Since this ester is immediately hydrolysed in the blood, subcutaneous implants of 17β-trenbolone-acetate pellets provide a continuous supply of 17β-trenbolone to cattle [9]. In cattle bile, the predominating metabolite of 17β-trenbolone acetate is 17α-trenbolone. In contrast, rat bile contains predominantly 16α-hydroxylation products of 17β-trenbolone and of the 17-keto compound triendione. Thus, species differences in the oxidative metabolism of 17β-trenbolone exist at least between rat and cattle. Human urine contained mostly 17α-trenbolone together with some 17β-trenbolone and triendione, resembling the bovine metabolites more closely than the rat metabolites of 17β-trenbolone acetate [10]. Furthermore, at least five more polar human metabolites were observed, which have not been identified to date [10]. However, if α-trenbolone is incubated with human liver microsomes, it is converted to a significant extent to the strong androgen β-trenbolone [11]. Apart from this, however, little is known about the metabolism and the possible pathways of trenbolone in mammals and, even less so, in other vertebrates or even invertebrates.
In fact, to the best of our knowledge, nothing is known about the metabolism of trenbolone in fish or other aquatic animals that can be directly exposed to runoff from farms using large concentrations of pharmaceutical agents, such as cattle feedlots. Previous studies detected 12% of previously applied 17β-trenbolone acetate in liquid manure and 20% in solid dung after application of implants to heifers [11].
The active compound 17β-trenbolone is a potent anabolic steroid and is known to masculinize fish after exposure via food and water [12]. Concerning its androgenic mode of action, it can be assumed that trenbolone acts like other androgens; however, in contrast to most endogenous androgens, 17β-trenbolone cannot be metabolized to estrogens. Compared with the most active endogenous hormone dihydrotestosterone (receptor binding activity = 100), the affinity of 17β-trenbolone for the androgen receptor is even higher (receptor binding activity = 109). The gestagenic activity may not contribute to the anabolic effect, but must be considered for residue evaluation. The strong growth-promoting activity of trenbolone is based on its anabolic activity as an androgen and its anti-catabolic activity as an anti-glucocorticoid [13].
Given the hormonal nature of 17β-trenbolone and in view of the fact that hormone-like substances have frequently been shown to produce tumours via epigenetic mechanisms, different assays have been conducted to elucidate whether 17β-trenbolone causes genotoxic effects. The genotoxic potential of trenbolone has been discussed in the literature with contradictory results, however, with a trend to the conclusion that trenbolone does not exert genotoxic activity (for review see [14]). However, particularly in fish, only little information is available on its potential genotoxicity. Therefore, the present study was initiated (1) to elucidate if very low doses of trenbolone cause genotoxicity in fish or a fish cell-line and (2) to investigate how different biotransformation capacities influence the genotoxic potential of trenbolone.
Section snippets
Trenbolone
Trenbolone (CAS No. 10161-33-8) is an anabolic steroid hormone with a water solubility between 340 and 380 mg/L without solvent (25 °C; [15]) and a half-life in liquid manure of >250 days [3], [16]. Technical-grade trenbolone was purchased from Steraloids Inc. (Newport, USA).
Cell-culture conditions
RTL-W1 cells originally derived from rainbow trout liver (Oncorhynchus mykiss); [17] were cultured according to Klee et al. [18] in Leibovitz L15 medium (Sigma–Aldrich, Deisenhofen, Germany). Prior to use in the in vitro
Genotoxicity of trenbolone in the micronucleus test with RTL-W1 cells
In a range-finding test, RTL-W1 cells were exposed to concentrations between 0.01 and 32 mg/L trenbolone. At 4 mg/L, the micronucleus frequency was not significantly elevated over controls, whereas between 0.03 and 1 mg/L each concentration showed significant increases. Therefore, in subsequent experiments, RTL-W1 cells were exposed to concentrations of 0.01–0.2 mg/L trenbolone in three independent replicates, whereas concentrations of 0.25–32 mg/L were tested in two replicates. Previous studies had
Genotoxicity of trenbolone in the micronucleus assay
Trenbolone induced micronuclei both in RTL-W1 cells and in primary cell suspensions derived from zebrafish embryos. Nevertheless, the micronucleus assay with freshly isolated zebrafish cells proved less sensitive, with an LOEC (Lowest Observed Effect Concentration) 10-fold higher than the LOEC for RTL-W1 cells (0.25 and 0.025 mg/L, respectively). Most importantly, however, the LOEC of 0.25 mg/L for zebrafish embryo-derived cells was also the only concentration at which a significant increase in
Conclusions
The results of this study provide evidence of non-linear genotoxic and mutagenic activity of trenbolone in a very low concentration range. Since fish and other aquatic organisms are potentially exposed to trenbolone in runoff from cattle feedlots, manure or sewage discharge [16], genotoxic/mutagenic effects by trenbolone in aquatic ecosystems cannot be ruled out. Fish take a top position in the aquatic food web and serve as a food source for man; thus, there may be a risk to both environment
Conflict of interest
There is no conflict of interest of any kind.
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