Differential gene expression as a toxicant-sensitive endpoint in zebrafish embryos and larvae

• Published in: Aquatic toxicology 2007-03, Vol.81 (4), p.355-364
• Main Authors: Voelker, Doris, Vess, Christoph, Tillmann, Michaela, Nagel, Roland, Otto, Georg W., Geisler, Robert, Schirmer, Kristin, Scholz, Stefan
• Format: Article
• Language: English
• Subjects: Aniline Compounds - toxicity; Animal, plant and microbial ecology; Animals; Applied ecology; Biological and medical sciences; Chronic toxicity; Danio rerio; DNA Primers; Early life stage test; Ecotoxicology, biological effects of pollution; Embryo test; Embryo, Nonmammalian - drug effects; Embryo, Nonmammalian - metabolism; Endpoint Determination - methods; Freshwater; Fundamental and applied biological sciences. Psychology; Gene Expression Profiling - methods; Gene Expression Regulation - drug effects; General aspects; Larva - drug effects; Larva - metabolism; Microarray; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Toxicity Tests - methods; Zebrafish; Zebrafish - metabolism
• ISSN: 0166-445X; 1879-1514
• DOI: 10.1016/j.aquatox.2006.12.013

The zebrafish ( Danio rerio) embryo toxicity test ( DarT) is under consideration as an alternative to the acute fish toxicity test. Microscopically visible developmental disorders or death are the endpoints used to report on toxicity in DarT. These endpoints are easily observed. They, however, rarely reveal mechanisms leading to a toxic effect and are relatively insensitive compared to chronic toxic effects. We hypothesized that, by using gene expression profiles as an additional endpoint, it may be possible to increase the sensitivity and predictive value of DarT. Therefore, as a proof of principle, we exposed zebrafish embryos to the reference compound 3,4-dichloroaniline (3,4-DCA) and analyzed gene expression patterns with a 14k oligonucleotide array. Important stress response genes not included in the microarray were additionally quantified by reverse transcriptase polymerase chain reaction. Six genes involved in biotransformation ( cyp1a, ahr2), stress response ( nfe212, maft, hmox1) and cell cycle control ( fzr1) were significantly regulated. With the exception of fzr1, these genes proved to be differentially expressed in post hatch life stages as well. The identified genes point toward an aryl hydrocarbon receptor-mediated response. Differential gene expression in embryos exposed for 48 h was observed at 3,4-DCA concentrations as low as 0.78 μM, which is more than 10-fold below the concentrations that elicited visible toxic effects. Upon exposure for 5 days, differential expression was detected at concentrations as low as 0.22 μM of 3,4-DCA, which was close to the lowest observed effect concentration (0.11 μM) in the 30-day early life stage test. This study therefore indicates that gene expression analysis in DarT is able to reveal mechanistic information and may also be exploited for the development of replacement methods for chronic fish tests.