title: Adaptation of a Bioinformatics Microarray Analysis Workflow for a Toxicogenomic Study in Rainbow Trout.
abstract: Sex steroids play a key role in triggering sex differentiation in fish, the use of exogenous hormone treatment leading to partial or complete sex reversal. This phenomenon has attracted attention since the discovery that even low environmental doses of exogenous steroids can adversely affect gonad morphology (ovotestis development) and induce reproductive failure. Modern genomic-based technologies have enhanced opportunities to find out mechanisms of actions (MOA) and identify biomarkers related to the toxic action of a compound. However, high throughput data interpretation relies on statistical analysis, species genomic resources, and bioinformatics tools. The goals of this study are to improve the knowledge of feminisation in fish, by the analysis of molecular responses in the gonads of rainbow trout fry after chronic exposure to several doses (0.01, 0.1, 1 and 10 μg/L) of ethynylestradiol (EE2) and to offer target genes as potential biomarkers of ovotestis development. We successfully adapted a bioinformatics microarray analysis workflow elaborated on human data to a toxicogenomic study using rainbow trout, a fish species lacking accurate functional annotation and genomic resources. The workflow allowed to obtain lists of genes supposed to be enriched in true positive differentially expressed genes (DEGs), which were subjected to over-representation analysis methods (ORA). Several pathways and ontologies, mostly related to cell division and metabolism, sexual reproduction and steroid production, were found significantly enriched in our analyses. Moreover, two sets of potential ovotestis biomarkers were selected using several criteria. The first group displayed specific potential biomarkers belonging to pathways/ontologies highlighted in the experiment. Among them, the early ovarian differentiation gene foxl2a was overexpressed. The second group, which was highly sensitive but not specific, included the DEGs presenting the highest fold change and lowest p-value of the statistical workflow output. The methodology can be generalized to other (non-model) species and various types of microarray platforms.
authors.affiliation: Unit of research in Environmental and Evolutionary Biology (URBE-NARILIS), Laboratory of Ecophysiology and Ecotoxicology, University of Namur, Namur, Belgium.
authors.forename: Sophie
authors.initials: S
authors.lastname: Depiereux
authors.affiliation: Unit of Research in Molecular Biology (URBM-NARILIS), University of Namur, Namur, Belgium.
authors.forename: Bertrand
authors.initials: B
authors.lastname: De Meulder
authors.affiliation: Unit of Research in Molecular Biology (URBM-NARILIS), University of Namur, Namur, Belgium; Sainte-Justine UHC Research Centre, University of Montreal, Montréal (Québec), H3T 1C5, Canada.
authors.forename: Eric
authors.initials: E
authors.lastname: Bareke
authors.affiliation: Unit of Research in Molecular Biology (URBM-NARILIS), University of Namur, Namur, Belgium.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Berger
authors.affiliation: Institut National de la Recherche Agronomique, INRA-LPGP, UPR1037, Campus de Beaulieu, 35042, Rennes, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Le Gac
authors.affiliation: Unit of Research in Molecular Biology (URBM-NARILIS), University of Namur, Namur, Belgium.
authors.forename: Eric
authors.initials: E
authors.lastname: Depiereux
authors.affiliation: Unit of research in Environmental and Evolutionary Biology (URBE-NARILIS), Laboratory of Ecophysiology and Ecotoxicology, University of Namur, Namur, Belgium.
authors.forename: Patrick
authors.initials: P
authors.lastname: Kestemont
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D019295:Computational Biology; D004305:Dose-Response Relationship, Drug; D004967:Estrogens / Q000494:pharmacology*; D004997:Ethinyl Estradiol / Q000494:pharmacology*; D005260:Female; D029941:Fish Proteins / Q000235:genetics* / Q000378:metabolism; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D006066:Gonads / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism; D008297:Male; D053858:Metabolic Networks and Pathways / Q000187:drug effects / Q000235:genetics; D046228:Microarray Analysis; D058977:Molecular Sequence Annotation; D017686:Oncorhynchus mykiss / Q000235:genetics* / Q000254:growth & development; D019849:Sex Determination Processes / Q000187:drug effects* / Q000235:genetics
chemical_list: D015415:Biomarkers; D004967:Estrogens; D029941:Fish Proteins; D051858:Forkhead Transcription Factors; D004997:Ethinyl Estradiol