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 17b-estradiol, bisphenol A and genistein were identified, among them genes involved in metabolism, reproductional and developmental processes. Threshold levels of 17b-estradiol (200 ng/L), bisphenol A (2000 lg/L), and genistein (5000 lg/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 17b-estradiol, 47 by bisphenol A and 231 by genistein. Furthermore, it is shown that fish exposed to 17b-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. # 2008 Wiley Periodicals, Inc. Environ Toxicol 23: 15-24, 2008
Biomonitoring is currently performed at two levels, assessing exposure to pollutants and effects monitoring by bioassays. As an example for the first approach, vitellogenin (VTG) in male fish of Abramis brama as an endpoint for estrogen exposure is discussed. However, similar changes of VTG or VTG-like proteins in the hemolymph of mussels could not be detected. Enzyme-linked receptor assays for monitoring estrogenic effects at the molecular level serve as an example for the second category. Applications of the enzyme-linked receptor assay (ELRA) developed in our laboratory are presented. Detection limits of 0.02 μg/l 17β-estradiol were recently achieved with the chemiluminescent format. Although effect monitoring provides information in terms of toxicity equivalents, it is not possible to relate the signals to specific pollutants and their concentrations. For this purpose, chemical analysis is required. New approaches are reported for the direct coupling of bioassays and chemical analysis. This concept is defined as bioresponse-linked instrumental analysis. It combines biomolecular recognition, initiating a biological effect, and chemical analysis. In addition to the classical bioanalytical approaches, new strategies in genomics and proteomics have been developed. This may lead to multimarker approaches opening this area to environmental analytics.
a Zebrafish (Danio rerio) were exposed with 17β-estradiol (E2) and nonylphenol (NP) in a Technische Universitaet different concentrations. Gene expression analysis was carried out by two different apMuenchen, Center of Life and proaches. First, RT-PCR experiments were performed for the examination of expression Food Sciences Weihenstephan, Chair of Cell Biology, levels of the two marker genes vitellogenin and aromatase. This approach showed a Alte Akademie 12, significant increase in the expression of the vitellogenin gene in exposed male fish 85354 Freising, Germany (500 ng/L 17β-estradiol and 250 μg/L nonylphenol). This egg yolk protein is usually not synthesized in male vertebrates. A slight decrease of expression of the aromatase gene was observed in exposed female zebrafish. Aromatase is known to catalyze the conversion of androgens to estrogens. Second, DNA microarray experiments were carried out, which allow the simultaneous examination of the expression levels of a great number of marker genes. The microarray experiments resulted in an up-regulation of vitellogenin up to 850-fold. In addition, several other genes were identified to be up-regulated by estrogens, for example the high mobility group box protein ssrp1 (78-fold) or the chaperonin containing t-complex polypeptide 1, beta subunit cctb (22-fold).
Genexpressionsanalysen nach der Exposition mit östrogenen Wirkstoffen
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