Biomarkers for exposure to estrogenic compounds: Gene expression analysis in zebrafish (<i>Danio rerio</i>)

Gene expression analyses in male zebrafish (Danio rerio) were carried out using microarray technique and quantitative polymerase chain reaction. Genes responding to the exposure to 17beta-estradiol, bisphenol A and genistein were identified, among them genes involved in metabolism, reproductional and developmental processes. Threshold levels of 17beta-estradiol (200 ng/L), bisphenol A (2000 microg/L), and genistein (5000 microg/L) for the upregulation of the vtg1 gene in short-time exposures (11 days) were determined by qPCR. 14k microarrays were used to generate complete lists of genes regulated by these estrogenic compounds. For this purpose, liver samples from 10 exposed zebrafish and 10 controls were processed. In this case the expressions of 211 genes were significantly regulated by 17beta-estradiol, 47 by bisphenol A and 231 by genistein. Furthermore, it is shown that fish exposed to 17beta-estradiol and genistein have similarities in their gene expression patterns, whereas bisphenol A apparently affected gene expression in a different way. Only genes coding for egg-yolk precursor protein vitellogenin were found to be regulated by all three compounds, which shows that these genes are the only suitable markers for exposure to different estrogenic compounds. The regulated genes were assigned to gene ontology classes. All three estrogenic compounds regulated genes mainly involved in primary and cellular metabolism, but genistein regulated several genes involved in cell cycle-regulation and bisphenol A several genes involved in protein biosynthesis. Genistein also upregulated the expression of four eggshell proteins, which can be used as biomarkers for exposure to this chemical.