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Linking the response of endocrine regulated genes to adverse effects on sex differentiation improves comprehension of aromatase inhibition in a Fish Sexual Development Test

 
: Muth-Köhne, Elke; Westphal-Settele, Kathi; Brückner, Jasmin; Konradi, Sabine; Schiller, Viktoria; Schäfers, Christoph; Teigeler, Matthias; Fenske, Martina

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Aquatic toxicology 176 (2016), pp.116-127
ISSN: 0166-445X
English
Journal Article, Electronic Publication
Fraunhofer IME ()
aromatase inhibition; zebrafish; qPCR; Fadrozole; fish sexual development test (FSDT); adverse outcome pathway (AOP)

Abstract
The Fish Sexual Development Test (FSDT) is a non-reproductive test to assess adverse effects of endocrine disrupting chemicals. With the present study it was intended to evaluate whether gene expression end-points would serve as predictive markers of endocrine disruption in a FSDT. For proof-of-concept, a FSDTaccording to the OECD TG 234 was conducted with the non-steroidal aromatase inhibitor fadrozole (testconcentrations: 10µg/L, 32 µg/L, 100 µg/L) using zebrafish (Danio rerio). Gene expression analyses using quantitative RT-PCR were included at 48 h, 96 h, 28 days and 63 days post fertilization (hpf, dpf). The selection of genes aimed at finding molecular endpoints which could be directly linked to the adverse apical effects of aromatase inhibition. The most prominent effects of fadrozole exposure on the sexual development of zebrafish were a complete sex ratio shift towards males and an acceleration of gonad maturation already at low fadrozole concentrations (10 µg/L). Due to the specific inhibition of the aromatase enzyme (Cyp19) by fadrozole and thus, the conversion of C19-androgens to C18-estrogens, the steroid hormone balance controlling the sex ratio of zebrafish was altered. The resulting key event is the regulation of directly estrogen-responsive genes. Subsequently, gene expression of vitellogenin 1 (vtg1)and of the aromatase cyp19a1b isoform (cyp19a1b), were down-regulated upon fadrozole treatment compared to controls. For example, mRNA levels of vtg1 were down-regulated compared to the controls as early as 48 hpf and 96 hpf. Further regulated genes cumulated in pathways suggested to be controlled by endocrine mechanisms, like the steroid and terpenoid synthesis pathway (e.g. mevalonate (diphospho)decarboxylase (mvd), lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase; lss), methylsterol monooxy-genase 1 (sc4mol)) and in lipid transport/metabolic processes (steroidogenic acute regulatory protein (star),apolipoprotein Eb (apoEb)). Taken together, this study demonstrated that the existing Adverse Outcome Pathway (AOP) for aromatase inhibition in fish can be translated to the life-stage of sexual differentiation. We were further able to identify MoA-specific marker gene expression which can be instrumentalin defining new measurable key events (KE) of existing or new AOPs related to endocrine disruption.

: http://publica.fraunhofer.de/documents/N-391838.html