title: Species-specific considerations in using the fish embryo test as an alternative to identify endocrine disruption.
abstract: A number of regulations have been implemented that aim to control the release of potentially adverse endocrine disrupters into the aquatic environment based on evidence from laboratory studies. Currently, such studies rely on testing approaches with adult fish because reliable alternatives have not been validated so far. Fish embryo tests have been proposed as such an alternative, and here we compared two species (medaka and zebrafish) to determine their suitability for the assessment of substances with estrogenic and anti-androgenic activity. Changes in gene expression (in here the phrase gene expression is used synonymously to gene transcription, although it is acknowledged that gene expression is additionally regulated, e.g., by translation and protein stability) patterns between the two species were compared in short term embryo exposure tests (medaka: 7-day post fertilization [dpf]; zebrafish: 48 and 96h post fertilization [hpf]) by using relative quantitative real-time RT-PCR. The tested genes were related to the hypothalamic-gonadal-axis and early steroidogenesis. Test chemicals included 17α-ethinylestradiol and flutamide as estrogenic and anti-androgenic reference compounds, respectively, as well as five additional substances with endocrine activities, namely bisphenol A, genistein, prochloraz, linuron and propanil. Estrogenic responses were comparable in 7-dpf medaka and 48/96-hpf zebrafish embryos and included transcriptional upregulation of aromatase b, vitellogenin 1 as well as steroidogenic genes, suggesting that both species reliably detected exposure to estrogenic compounds. However, anti-androgenic responses differed between the two species, with each species providing specific information concerning the mechanism of anti-androgenic disruption in fish embryos. Although small but significant changes in the expression of selected genes was observed in 48-hpf zebrafish embryos, exposure prolonged to 96hpf was necessary to obtain a response indicative of anti-androgenic activity. In contrast, for medaka clear anti-androgenic response, e.g. transcriptional downregulation of 11β-hydroxylase, 3β-hydroxysteroid-dehydrogenase, gonadotropin-releasing hormone receptor 2, was already observed at the pre-hatch stage. Together, this data suggests that medaka and zebrafish embryos would provide a beneficial alternative testing platform for endocrine disruption that involves additive information on interspecies and exposure time variability when using both species.
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology, 52074 Aachen, Germany. Electronic address: ViktoriaSchiller@gmx.de.
authors.forename: Viktoria
authors.initials: V
authors.lastname: Schiller
authors.affiliation: Toxicology Centre University of Saskatchewan, 44 Campus Drive Saskatoon, Saskatchewan, Canada.
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Toxicology Centre University of Saskatchewan, 44 Campus Drive Saskatoon, Saskatchewan, Canada.
authors.forename: Markus
authors.initials: M
authors.lastname: Hecker
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology, 57392 Schmallenberg, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Schäfers
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology, 52074 Aachen, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology, 52074 Aachen, Germany.
authors.forename: Martina
authors.initials: M
authors.lastname: Fenske
mesh_terms: D000726:Androgen Antagonists; D000818:Animals; D001141:Aromatase / Q000378:metabolism; D001559:Benzhydryl Compounds; D004625:Embryo, Nonmammalian / Q000187:drug effects*; D052244:Endocrine Disruptors / Q000378:metabolism / Q000633:toxicity*; D004703:Endocrine System / Q000378:metabolism; D004970:Estrone / Q000378:metabolism; D004997:Ethinyl Estradiol / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D006066:Gonads / Q000378:metabolism; D009990:Oryzias / Q000196:embryology*; D010636:Phenols; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013045:Species Specificity; D059467:Transcriptome; D014819:Vitellogenins / Q000378:metabolism; D014874:Water Pollutants, Chemical / Q000378:metabolism / Q000633:toxicity*; D015027:Zebrafish / Q000196:embryology*; D000098826:Bisphenol A Compounds
chemical_list: D000726:Androgen Antagonists; D001141:Aromatase; D001559:Benzhydryl Compounds; D052244:Endocrine Disruptors; D004970:Estrone; D004997:Ethinyl Estradiol; D010636:Phenols; D014819:Vitellogenins; D014874:Water Pollutants, Chemical; C006780:bisphenol A; D000098826:Bisphenol A Compounds
keywords: Fish embryo test, Endocrine disruption; Medaka embryo; Quantitative RT-PCR; Zebrafish embryo