title: Linking the response of endocrine regulated genes to adverse effects on sex differentiation improves comprehension of aromatase inhibition in a Fish Sexual Development Test.
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 endpoints would serve as predictive markers of endocrine disruption in a FSDT. For proof-of-concept, a FSDT according to the OECD TG 234 was conducted with the non-steroidal aromatase inhibitor fadrozole (test concentrations: 10μg/L, 32μg/L, 100μg/L) using zebrafish (Danio rerio). Gene expression analyses using quantitative RT-PCR were included at 48h, 96h, 28days and 63days 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 (10μg/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 monooxygenase 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 instrumental in defining new measurable key events (KE) of existing or new AOPs related to endocrine disruption.
authors.affiliation: Fraunhofer IME, Department of Ecotoxicology, Auf dem Aberg 1, 57392 Schmallenberg, Germany. Electronic address: elke.muth-koehne@ime.fraunhofer.de.
authors.forename: Elke
authors.initials: E
authors.lastname: Muth-Köhne
authors.affiliation: German Environment Agency (UBA), Woerlitzer Platz 1, 06844 Dessau, Germany.
authors.forename: Kathi
authors.initials: K
authors.lastname: Westphal-Settele
authors.affiliation: German Environment Agency (UBA), Woerlitzer Platz 1, 06844 Dessau, Germany.
authors.forename: Jasmin
authors.initials: J
authors.lastname: Brückner
authors.affiliation: German Environment Agency (UBA), Woerlitzer Platz 1, 06844 Dessau, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Konradi
authors.affiliation: Fraunhofer IME, Attract Group UNIFISH, Forckenbeckstraße 6, 52074 Aachen, Germany.
authors.forename: Viktoria
authors.initials: V
authors.lastname: Schiller
authors.affiliation: Fraunhofer IME, Department of Ecotoxicology, Auf dem Aberg 1, 57392 Schmallenberg, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Schäfers
authors.affiliation: Fraunhofer IME, Department of Ecotoxicology, Auf dem Aberg 1, 57392 Schmallenberg, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Teigeler
authors.affiliation: Fraunhofer IME, Project Group Translational Medicine and Pharmacology TMP, Forckenbeckstraße 6, 52074 Aachen, Germany.
authors.forename: Martina
authors.initials: M
authors.lastname: Fenske
mesh_terms: D000818:Animals; D001141:Aromatase / Q000235:genetics; D047072:Aromatase Inhibitors / Q000633:toxicity*; D052244:Endocrine Disruptors / Q000633:toxicity*; D017316:Fadrozole / Q000633:toxicity*; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006066:Gonads / Q000187:drug effects / Q000254:growth & development; D008297:Male; D012733:Sex Differentiation / Q000187:drug effects* / Q000235:genetics; D012744:Sex Ratio; D046468:Sexual Development / Q000187:drug effects; D014819:Vitellogenins / Q000235:genetics; D014874:Water Pollutants, Chemical / Q000633:toxicity*; D015027:Zebrafish
chemical_list: D047072:Aromatase Inhibitors; D052244:Endocrine Disruptors; D014819:Vitellogenins; D014874:Water Pollutants, Chemical; D001141:Aromatase; D017316:Fadrozole
keywords: Adverse outcome pathway (AOP); Aromatase inhibition; Fadrozole; Fish sexual development test (FSDT); Zebrafish; qPCR