Transcriptional regulatory dynamics of the hypothalamic–pituitary–gonadal axis and its peripheral pathways as impacted by the 3-beta HSD inhibitor trilostane in zebrafish (Danio rerio) Rong-Lin Wang a,n , David Bencic a , Jim Lazorchak a , Daniel Villeneuve b , Gerald T. Ankley b a USEPA, Ecological Exposure Research Division, National Exposure Research Laboratory, 26 W Martin Luther King Dr. Cincinnati, OH 45268, USA b USEPA, Mid-Continent Ecology Division, National Health and Environmental Effects Research Laboratory, 6201 Congdon Boulevard, Duluth, MN 55804, USA article info Article history: Received 18 November 2010 Received in revised form 28 January 2011 Accepted 1 May 2011 Available online 12 May 2011 Keywords: Trilostane HPG-axis Transcription networks Biological pathways abstract To study mechanisms underlying generalized effects of 3b hydroxysteroid dehydrogenase (HSD3B) inhibition, reproductively mature zebrafish (Danio rerio) were exposed to trilostane at two dosages for 24, 48, or 96 h and their gonadal RNA samples profiled with Agilent zebrafish microarrays. Trilostane had substantial impact on the transcriptional dynamics of zebrafish, as reflected by a number of differentially expressed genes (DEGs) including transcription factors (TFs), altered TF networks, signaling pathways, and Gene Ontology (GO) biological processes. Changes in gene expression between a treatment and its control were mostly moderate, ranging from 1.3 to 2.0 fold. Expression of genes coding for HSD3B and many of its transcriptional regulators remained unchanged, suggesting transcriptional up-regulation is not a primary compensatory mechanism for HSD3B enzyme inhibition. While some trilostane-responsive TFs appear to share cellular functions linked to endocrine disruption, there are also many other DEGs not directly linked to steroidogenesis. Of the 65 significant TF networks, little similarity, and therefore little cross-talk, existed between them and the hypothalamic–pituitary– gonadal (HPG) axis. The most enriched GO biological processes are regulations of transcription, phosphorylation, and protein kinase activity. Most of the impacted TFs and TF networks are involved in cellular proliferation, differentiation, migration, and apoptosis. While these functions are fairly broad, their underlying TF networks may be useful to development of generalized toxicological screening methods. These findings suggest that trilostane-induced effects on fish endocrine functions are not confined to the HPG-axis alone. Its impact on corticosteroid synthesis could also have contributed to some system wide transcriptional changes in zebrafish observed in this study. Published by Elsevier Inc. 1. Introduction Over the past decade there has been an increasing emphasis on the potential harmful effects of endocrine disrupting chemicals (EDCs) on humans and wildlife (WHO, 2002; Diamanti-Kandarakis et al., 2009). Investigation of the role and function of the hypothalamic–pituitary–gonadal axis (HPG-axis), and more specifi- cally, receptors and enzymes involved in steroidogenesis, is critical to an improved mechanistic understanding of chemical effects on endocrine function (Ankley et al., 2009). While the identities of many HPG-axis components targeted by various chemicals are known, information concerning how these genes/proteins function in a wider biological context remains limited. A better understand- ing of the relationship between the HPG-axis and the transcription factor (TF) networks/signaling pathways it interacts with would facilitate the development of mechanistically based indicators/end- points and enhance the extrapolation of toxic effects across species and chemical structures. This should provide a basis for a more informative and efficient assessment of EDC exposures, adverse effects, and risks (Ankley et al., 2009). Conceptually, perturbing the HPG-axis in a targeted manner over a series of experiments can help reveal its transcriptional regulatory dynamics (Ankley et al., 2009). One potential target for perturbation is 3b hydroxysteroid dehydrogenase (HSD3B, EC 1.1.1.145), a well-characterized enzyme catalyzing key steps in formation of corticosteroids and sex steroids (Simard et al., 2005). The HSD3B gene (family) is conserved across vertebrate species Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/ecoenv Ecotoxicology and Environmental Safety 0147-6513/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.ecoenv.2011.05.001 Abbreviations: Cy3, Cyanine 3; Cy5, Cyanine 5; DEG, differentially expressed gene; GSEA, gene set enrichment analysis; E-GSEA, extended-GSEA; EDC, endo- crine disrupting chemical; FDR, false discovery rate; GO, gene ontology; HPG, hypothalamic–pituitary–gonadal; HSD3B, 3b hydroxysteroid dehydrogenase; IPA, Ingenuity Pathway Analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; TF, transcription factor n Corresponding author. Fax: þ1 513 569 7438. E-mail address: wang.rong-lin@epa.gov (R.-L. Wang). Ecotoxicology and Environmental Safety 74 (2011) 1461–1470