Trenbolone causes mortality and altered sexual differentiation in Xenopus tropicalis during larval development

Abstract

Trenbolone is an androgen agonist used in cattle production and has been measured in aquatic systems associated with concentrated animal-feeding operations. In this study, the authors characterized the effects of aqueous exposure to 17β-trenbolone during larval Xenopus tropicalis development. Trenbolone exposure resulted in increased mortality of post-Nieuwkoop-Faber stage 58 tadpoles at concentrations ≥100 ng/L. Morphological observations and the timing of this mortality are consistent with hypertrophy of the larynx. Development of nuptial pads, a male secondary sex characteristic, was induced in tadpoles of both sexes at 100 ng/L. Effects on time to complete metamorphosis or body sizes were not observed; however, grow-outs placed in clean media for six weeks were significantly smaller in body size at 78 ng/L. Effects on sex ratios were equivocal, with the first experiment showing a significant shift in sex ratio toward males at 78 ng/L. In the second experiment, no significant effects were observed up to 100 ng/L, although overall sex ratios were similar. Histological assessment of gonads at metamorphosis showed half with normal male phenotypes and half that possessed a mixed-sex phenotype at 100 ng/L. Hypertrophy of the Wolffian ducts was also observed at this concentration. These results indicate that larval 17β-trenbolone exposure results in effects down to 78 ng/L, illustrating potential effects from exposure to androgenic compounds in anurans.

Sex Reversal in Amphibians

“…This substance and its metabolites are widespread in surface water. Exposure to 17β-trenbolone (between 78 and 100 ng/l) during lar-val life could induce male-biased sex ratios at metamorphosis in X. laevis [Olmstead et al, 2012]. This compound also induced female-to-male sex reversal at concentrations as low as 100 ng/l in P. nigromaculatus .…”

“…Pollutants can also influence the genital tract. For instance, in case of exposure during larval life, EE 2 induced hypertrophy of the Müllerian ducts in P. waltl males, and 17β-trenbolone induced Wolffian ducts hypertrophy in X. laevis [Flament et al, 2011;Olmstead et al, 2012]. Taken together, these different actions of various pollutants could account for the amphibian decline.…”

Environmental (anti-)androgenic chemicals affect germinal vesicle breakdown (GVBD) of Xenopus laevis oocytes in vitro

“…Several studies reported the presence of nandrolone in wastewater, and even the level exceeded 60 ng/L (Backe et al, 2011;Sun et al, 2010). Recently, two studies described effects of trenbolone on the growth, survival, and sexual differentiation of X. laevis (Olmstead et al, 2012;Finch et al, 2013). Overall, available information on adverse effects of AAS on amphibians has been very limited.…”

Low concentrations of 17β-trenbolone induce female-to-male reversal and mortality in the frog Pelophylax nigromaculatus

“…In this study, however, 0.1-10 g/L trenbolone was found to induce significant mortality in P. nigromaculatus compared with the solvent control. Olmstead et al (2012) reported that exposure to 17␤-trenbolone resulted in increased mortality in X. tropicalis tadpoles, with a lowest observed effective concentration of 100 ng/L. The authors described that X. tropicalis tadpoles mainly died at the onset of metamorphic climax (Nieuwkoop and Faber stage 58) (Nieuwkoop and Faber, 1994), and floated upside down in water before death, with the presence of air inside the abdomen.…”

“…Thus, environmental androgen trenbolone might pose risks for some of amphibian species as well as for fish. Olmstead et al (2012) investigated for the first time the effects of 17␤-trenbolone on amphibian Xenopus tropicalis, and found a slight shift of sex ratio toward males, histological intersexuality, precocious nuptial pads and hypertrophy of the Wolffian ducts at 78-100 ng/L, showing masculinizing effects of 17␤-trenbolone. Also, significantly high mortality was observed in X. tropicalis at 100 ng/L and higher concentrations.…”