Reproductive Toxicology 42 (2013) 210–223 Contents lists available at ScienceDirect Reproductive Toxicology jo ur nal home p age: www. elsevier.com/locate/reprotox Transcriptome alterations in zebrafish embryos after exposure to environmental estrogens and anti-androgens can reveal endocrine disruption Viktoria Schiller a,∗ , Arne Wichmann a , Ralf Kriehuber c , Christoph Schäfers b , Rainer Fischer a , Martina Fenske a a Fraunhofer Institute for Molecular Biology and Applied Ecology, 52074 Aachen, Germany b Fraunhofer Institute for Molecular Biology and Applied Ecology, 57392 Schmallenberg, Germany c Forschungszentrum Jülich GmbH, Department of Safety and Radiation Protection, 52425 Juelich, Germany a r t i c l e i n f o Article history: Received 12 February 2013 Received in revised form 13 August 2013 Accepted 7 September 2013 Available online xxx Keywords: Zebrafish embryo Endocrine disruption Transcriptomics 17-Ethinylestradiol Flutamide Estrogen Anti-androgen a b s t r a c t Exposure to environmental chemicals known as endocrine disruptors (EDs) is in many cases associated with an unpredictable hazard for wildlife and human health. The identification of endocrine disrup- tive properties of chemicals certain to enter the aquatic environment relies on toxicity tests with fish, assessing adverse effects on reproduction and sexual development. The demand for quick, reliable ED assays favored the use of fish embryos as alternative test organisms. We investigated the application of a transcriptomics-based assay for estrogenic and anti-androgenic chemicals with zebrafish embryos. Two reference compounds, 17-ethinylestradiol and flutamide, were tested to evaluate the effects on development and the transcriptome after 48 h-exposures. Comparison of the transcriptome response with other estrogenic and anti-androgenic compounds (genistein, bisphenol A, methylparaben, linuron, prochloraz, propanil) showed commonalities and differences in regulated pathways, enabling us to clas- sify the estrogenic and anti-androgenic potencies. This demonstrates that different mechanism of ED can be assessed already in fish embryos. © 2013 Elsevier Inc. All rights reserved. 1. Introduction Endocrine disruption is of high environmental concern since it affects the reproductive health of aquatic vertebrates. For this reason, endocrine disruption has been in the focus of the hazard assessment of substances occurring in the environment. Since 2007, with the implementation of REACH, every environmental relevant substance produced by the industry has to be tested for its endocrine potential. To this end, regulatory authorities like the US EPA (United States Environmental Protection Agency) or the OECD (Organisation of Economic Co-operation and Development) proposed testing strategies, which also include in vitro and in vivo studies for fish [1,2]. Although promising efforts have been made to promote in vitro testing, the final decision for a substance to be classified as endocrine active relies on in vivo data from tests with adult animals. In order to reduce the number of animals used for, e.g., life-cycle studies, which are regarded as a standard for ∗ Corresponding author at: Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstrasse 6, 52074 Aachen, Germany. Tel.: +49 0241 6085 12272. E-mail addresses: schiller@molbiotech.rwth-aachen.de, ViktoriaSchiller@gmx.de (V. Schiller). assessing reproduction related and thus considered endocrine disrupting effects for fish, alternative test guidelines for short- term screening assays were published by the OECD and the OPPTS (Office of Prevention, Pesticides, and Toxic Substances) in 2009. Although these test guidelines (TG) (i.e., TG OECD 229 and 230, OPPTS 890.135) comply with the 3R concept of animal testing (replacement, reduction and refinement) by reducing the number of animals [3] and minimizing the duration of suffering from several months to 21 days, neither of these tests replaces animals fully. Alternative testing methods to further reduce the number of animals and to replace current standard tests for endocrine disruption, are therefore of urgent need. In this context, the zebrafish embryo emerged as a poten- tial alternative model because according to the revised European directive on the protection of animals used for scientific purposes (Directive 2010/63/EU), fish embryos are excluded from protec- tion. Zebrafish embryos represent whole organisms and offer many advantages including a rapid development (the majority of the organs develop within the first 48 h post fertilization (hpf) of the embryonic phase), small size, transparency and easy handling. However, with regard to endocrine disruption, a disadvantage is that any morphological effects occurring in the embryos due to exposure cannot be associated with endocrine or reproductive 0890-6238/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.reprotox.2013.09.003