Impacts of the synthetic androgen Trenbolone on gonad differentiation and development - comparisons between three deeply diverged anuran families.

Using a recently developed approach for testing endocrine disruptive chemicals (EDCs) in amphibians, comprising synchronized tadpole exposure plus genetic and histological sexing of metamorphs in a flow-through-system, we tested the effects of 17ß-Trenbolone (Tb), a widely used growth promoter in cattle farming, in three deeply diverged anuran families: the amphibian model species Xenopus laevis (Pipidae) and the non-models Bufo(tes) viridis (Bufonidae) and Hyla arborea (Hylidae). Trenbolone was applied in three environmentally and/or physiologically relevant concentrations (0.027 µg/L (10-10 M), 0.27 µg/L (10-9 M), 2.7 µg/L (10-8 M)). In none of the species, Tb caused sex reversals or masculinization of gonads but had negative species-specific impacts on gonad morphology and differentiation after the completion of metamorphosis, independently of genetic sex. In H. arborea and B. viridis, mounting Tb-concentration correlated positively with anatomical abnormalities at 27 µg/L (10-9 M) and 2.7 µg/L (10-8 M), occurring in X. laevis only at the highest Tb concentration. Despite anatomical aberrations, histologically all gonadal tissues differentiated seemingly normally when examined at the histological level but at various rates. Tb-concentration caused various species-specific mortalities (low in Xenopus, uncertain in Bufo). Our data suggest that deep phylogenetic divergence modifies EDC-vulnerability, as previously demonstrated for Bisphenol A (BPA) and Ethinylestradiol (EE2).

The progestin norethisterone affects thyroid hormone-dependent metamorphosis of Xenopus laevis tadpoles at environmentally relevant concentrations.

Previously, levonorgestrel (LNG) has been shown to be an endocrine disruptor of the amphibian thyroid system. In the present study, we investigated whether anti-thyroidal effects are a common property of progestins other than LNG. Premetamorphic Xenopus laevis tadpoles were exposed to norethisterone (NET) and dienogest DIE (each at 0.1-10nM) and LNG (10nM) until completion of metamorphosis. LNG and NET at all concentrations caused a significant developmental retardation whereas DIE did not impair time to metamorphosis. In LNG and 10nM NET exposed animals, tsh mRNA levels increased considerably later than the developmental delay occurred and thyroid histopathology showed no signs of TSH-hyperstimulation. Instead, thyroid glands from these treatments appeared inactive in producing thyroid hormones. Thyroidal transcript levels of dio2 and dio3 were increased by treatments with LNG and NET at 1nM and 10nM, whereas iyd mRNA was reduced by LNG and 10nM NET. Expression of slc5α5 was not changed by any treatment. Effects of DIE differed from those induced by LNG and NET. No developmental delay was measurable; however, tshß and dio2 mRNAs were increased in pituitary glands of tadpoles exposed to 1.0nM and 10nM DIE. Thyroid histopathology displayed no abnormalities and thyroidal mRNA expression of the genes analyzed (slc5α5, iyd, dio2, dio3) was not changed by DIE. Overall, our results provide evidence that the anti-thyroidal effects already known from LNG are also present in another progestin, namely NET, even at environmentally relevant concentrations. In conclusion we suggest that progestins do not only pose an environmental risk in terms of their impact on reproductive success of aquatic vertebrates, but also with respect to their anti-thyroidal properties affecting amphibian metamorphosis.