Rozenblut-Kościsty B et al. (2019), Impacts of the synthetic androgen Trenbolone on...

XB-ART-56105

Sci Rep January 1, 2019; 9 (1): 9623.

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

Rozenblut-Kościsty B , Ogielska M , Hahn J , Kleemann D , Kossakowski R , Tamschick S , Schöning V , Krüger A , Lutz I , Lymberakis P , Kloas W , Stöck M .

Abstract
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 17β-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).

PubMed ID: 31270347
Article link: Sci Rep
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	Figure 1. Monitoring of concentrations of trenbolone. (a) Recovery of Trenbolone in stock solutions; (b) recovery of Trenbolone in tanks. Note that in (b) in week 5, due to a technical issue during samples processing, data for 10−10 M are missing. Nominal Tb-concentrations in tanks (b) comprised (0.027 µg/L (10−10 M, red), 0.27 µg/L (10−9 M, green), 2.7 µg/L (10−8 M, violet). Controls were free of Tb, apart from one measurement in study week (0.05 ng/L in one tank); see text for technical details on measurements.
	Figure 2. Mortality of tadpoles and metamorphs under three Trenbolone concentrations in percent. Xl: Xenopus laevis, Ha: Hyla arborea, Bv: Bufo viridis, *significant difference between control and treatment group within the same species (2-sided Chi2-tests, p < 0.05).
	Figure 3. Photographs of anatomically normal and small gonads of Hyla arborea. (a) Normal ovary (control), (b) shortened ovary (10−8), (c) normal testis (control), (d) small testis (10−9). Scale bars represent 1 mm, fb – fat body, o – ovary, t – testis.
	Figure 4. Photographs of anatomically normal and small gonads of Bufo viridis. (a) Normal ovary (control), (b) shortened ovary (10−8 M), (c) normal testis (control), (d) small testis (10−9 M). Scale bars represent 1 mm; the red dotted line marks the boundary between Bidder's organ and the gonad. Bo – Bidder’s organ (occurring only in bufonid gonads), fb – fat body, o – ovary, t – testis.
	Figure 5. Photographs of fragmented (discontinuous) (a) ovary (10−8) and (b) testis (10−9) of Bufo viridis, (c) ovary in Hyla arborea (10−9) and (d) ovary in Xenopus laevis (10−8). Scale bars represent 1 mm. Note mesovarium indicated by white arrow head, Bo – Bidder’s organ (occurring only in bufonid gonads), fb – fat body, k – kidney, o – all parts of the ovary are marked, t – testis.
	Figure 6. Frequency of discontinuous gonads of Xenopus laevis (Xl), Hyla arborea (Ha), and Bufo viridis (Bv) after treatment with three concentrations of Trenbolone. *Significant difference between control and treatment group within the same species (2-sided Chi2-tests, p < 0.05).
	Figure 7. Histological sections of sterile ovary in (a) Bufo viridis (10−8) and (b) Xenopus laevis (10−9). Visible intense degeneration of germ cells in B. viridis ovary – o. Scale bars represent 100 µm. Bo – Bidder’s organ (characteristic of bufonid gonads), *ovarian cavity, arrow heads – single oogonia in ovary cortex.
	Figure 8. Anatomical photographs of doubled Bidder’s organ (Bo) in (a) ovary (10−10) and (b) testis (10−10) of Bufo viridis. Scale bars represent 1 mm, fb – fat body, o – ovary, t – testis.
	Figure 9. Histological sections of degenerated Bidder’s organ (Bo) in (a) ovary (10−10) and (b) testis (10−10) of Bufo viridis. Scale bars represent 100 µm, o – ovary, t – testis.
	Figure 10. Frequency of impaired gonads of Xenopus laevis (Xl), Hyla arborea (Ha), and Bufo viridis (Bv) after treatment in various concentrations of Trenbolone. *Significant difference between control and treatment group within the same species (2-sided Chi2-tests, p < 0.05).