Sensitive and robust gene expression changes in fish exposed to estrogen – a microarray approach

BackgroundVitellogenin is a well established biomarker for estrogenic exposure in fish. However, effects on gonadal differentiation at concentrations of estrogen not sufficient to give rise to a measurable vitellogenin response suggest that more sensitive biomarkers would be useful. Induction of zona pellucida genes may be more sensitive but their specificities are not as clear. The objective of this study was to find additional sensitive and robust candidate biomarkers of estrogenic exposure.ResultsHepatic mRNA expression profiles were characterized in juvenile rainbow trout exposed to a measured concentration of 0.87 and 10 ng ethinylestradiol/L using a salmonid cDNA microarray. The higher concentration was used to guide the subsequent identification of generally more subtle responses at the low concentration not sufficient to induce vitellogenin. A meta-analysis was performed with data from the present study and three similar microarray studies using different fish species and platforms. Within the generated list of presumably robust responses, several well-known estrogen-regulated genes were identified. Two genes, confirmed by quantitative RT-PCR (qPCR), fulfilled both the criteria of high sensitivity and robustness; the induction of the genes encoding zona pellucida protein 3 and a nucleoside diphosphate kinase (nm23).ConclusionThe cross-species, cross-platform meta-analysis correctly identified several robust responses. This adds confidence to our approach used for identifying candidate biomarkers. Specifically, we propose that analyses of an nm23 gene together with zona pellucida genes may increase the possibilities to detect an exposure to low levels of estrogenic compounds in fish.

[1]  Jurgen Del-Favero,et al.  Expression profiling of endocrine-disrupting compounds using a customized Cyprinus carpio cDNA microarray. , 2006, Toxicological sciences : an official journal of the Society of Toxicology.

[2]  I. Schultz,et al.  Gene expression patterns in rainbow trout, Oncorhynchus mykiss, exposed to a suite of model toxicants. , 2006, Aquatic toxicology.

[3]  J. Sumpter,et al.  Estrogenic Effects of Effluents from Sewage Treatment Works , 1994 .

[4]  Edwin J. Routledge,et al.  Identification of Estrogenic Chemicals in STW Effluent. 1. Chemical Fractionation and in Vitro Biological Screening , 1998 .

[5]  J. Giesy,et al.  Quantification of rainbow trout (Oncorhynchus mykiss) zona radiata and vitellogenin mRNA levels using real-time PCR after in vivo treatment with estradiol-17β or α-zearalenol , 2000, The Journal of Steroid Biochemistry and Molecular Biology.

[6]  Gordon K Smyth,et al.  Statistical Applications in Genetics and Molecular Biology Linear Models and Empirical Bayes Methods for Assessing Differential Expression in Microarray Experiments , 2011 .

[7]  Thomas H Hutchinson,et al.  Ecotoxicogenomics: the challenge of integrating genomics into aquatic and terrestrial ecotoxicology. , 2004, Aquatic toxicology.

[8]  Yi-Hsin Wu,et al.  Activation of antimetastatic Nm23-H1 gene expression by estrogen and its alpha-receptor. , 2002, Endocrinology.

[9]  B. Koop,et al.  Microarray analyses identify molecular biomarkers of Atlantic salmon macrophage and hematopoietic kidney response to Piscirickettsia salmonis infection. , 2004, Physiological genomics.

[10]  Z. Ju,et al.  DNA microarray technology in toxicogenomics of aquatic models: methods and applications. , 2007, Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.

[11]  G. Churchill,et al.  A comparison of cDNA, oligonucleotide, and Affymetrix GeneChip gene expression microarray platforms. , 2004, Journal of biomolecular techniques : JBT.

[12]  L. Moens,et al.  Effects of the antidepressant mianserin in zebrafish: molecular markers of endocrine disruption. , 2006, Chemosphere.

[13]  Bryan Frank,et al.  Independence and reproducibility across microarray platforms , 2005, Nature Methods.

[14]  M. Severgnini,et al.  Strategies for comparing gene expression profiles from different microarray platforms: application to a case-control experiment. , 2006, Analytical Biochemistry.

[15]  Sergio Contrino,et al.  ArrayExpress—a public repository for microarray gene expression data at the EBI , 2004, Nucleic Acids Res..

[16]  J. Parrott,et al.  Life‐cycle exposure of fathead minnows (Pimephales promelas) to an ethinylestradiol concentration below 1 ng/L reduces egg fertilization success and demasculinizes males , 2005, Environmental toxicology.

[17]  D. Larsson,et al.  Seasonal variations of vitelline envelope proteins, vitellogenin, and sex steroids in male and female eelpout (Zoarces viviparus). , 2002, General and comparative endocrinology.

[18]  Margaretha Adolfsson-Erici,et al.  Using NMR metabolomics to identify responses of an environmental estrogen in blood plasma of fish. , 2006, Aquatic toxicology.

[19]  Norio Onikura,et al.  Expression analysis of sex-specific and 17beta-estradiol-responsive genes in the Japanese medaka, Oryzias latipes, using oligonucleotide microarrays. , 2006, Genomics.

[20]  Henrik Holbech,et al.  Gonad development and vitellogenin production in zebrafish (Danio rerio) exposed to ethinylestradiol and methyltestosterone. , 2003, Aquatic toxicology.

[21]  J. Sumpter,et al.  Vitellogenesis as a biomarker for estrogenic contamination of the aquatic environment. , 1995, Environmental health perspectives.

[22]  P. Olsson,et al.  Regulation of Arctic char (Salvelinus alpinus) egg shell proteins and vitellogenin during reproduction and in response to 17beta-estradiol and cortisol. , 2004, General and comparative endocrinology.

[23]  Teresa Lettieri,et al.  Recent Applications of DNA Microarray Technology to Toxicology and Ecotoxicology , 2005, Environmental health perspectives.

[24]  Ceri A. Morris,et al.  Dynamics of estrogen biomarker responses in rainbow trout exposed to 17β‐estradiol and 17α‐ethinylestradiol , 2003 .

[25]  Yi-Hsin Wu,et al.  Activation of Antimetastatic Nm23-H1 Gene Expression by Estrogen and Its α-Receptor. , 2002, Endocrinology.

[26]  Jean YH Yang,et al.  Bioconductor: open software development for computational biology and bioinformatics , 2004, Genome Biology.

[27]  B. Koop,et al.  Fish and chips: Various methodologies demonstrate utility of a 16,006-gene salmonid microarray , 2005, BMC Genomics.

[28]  J. Giesy,et al.  Quantification of rainbow trout (Oncorhynchus mykiss) zona radiata and vitellogenin mRNA levels using real-time PCR after in vivo treatment with estradiol-17β or α-zearalenol , 2000, The Journal of Steroid Biochemistry and Molecular Biology.

[29]  S. Dudoit,et al.  Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. , 2002, Nucleic acids research.

[30]  G J Carr,et al.  Hepatic gene expression profiling using Genechips in zebrafish exposed to 17alpha-ethynylestradiol. , 2006, Aquatic toxicology.

[31]  Dynamics of estrogen biomarker responses in rainbow trout exposed to 17beta-estradiol and 17alpha-ethinylestradiol. , 2003, Environmental toxicology and chemistry.

[32]  David E. Williams,et al.  Toxicogenomic profiling of the hepatic tumor promoters indole-3-carbinol, 17beta-estradiol and beta-naphthoflavone in rainbow trout. , 2006, Toxicological sciences : an official journal of the Society of Toxicology.

[33]  G. Ankley,et al.  Ecotoxicogenomics: linkages between exposure and effects in assessing risks of aquatic contaminants to fish. , 2005, Reproductive toxicology.

[34]  J. Sumpter,et al.  Wild Intersex Roach (Rutilus rutilus) Have Reduced Fertility1 , 2002, Biology of reproduction.

[35]  M. Pettersson,et al.  ETHINYLOESTRADIOL - AN UNDESIRED FISH CONTRACEPTIVE , 1999 .

[36]  J. Rotchell,et al.  Molecular Markers of Endocrine Disruption in Aquatic Organisms , 2003, Journal of toxicology and environmental health. Part B, Critical reviews.

[37]  Edwin J. Routledge,et al.  Identification of Estrogenic Chemicals in STW Effluent. 2. In Vivo Responses in Trout and Roach , 1998 .

[38]  J. McInerney,et al.  Molecular cloning of an Atlantic salmon nucleoside diphosphate kinase cDNA and its pattern of expression during embryogenesis. , 2000, Gene.

[39]  Susan C. Tilton,et al.  Toxicogenomic Profiling of the Hepatic Tumor Promoters Indole-3-Carbinol, 17β-Estradiol and β-Naphthoflavone in Rainbow Trout , 2006 .