Gene expression analysis in zebrafish embryos: A potential approach to predict effect concentrations in the fish early life stage test

Abstract

Based on the hypothesis that analysis of gene expression could be used to predict chronic fish toxicity, the zebrafish (Danio rerio) embryo test (DarT), developed as a replacement method for the acute fish test, was expanded to a gene expression D. rerio embryo test (Gene-DarT). The effects of 14 substances on lethal and sublethal endpoints of the DarT and on expression of potential marker genes were investigated: the aryl hydrocarbon receptor 2, cytochrome P450 1A (cypla), heat shock protein 70, fizzy-related protein 1, the transcription factors v-maf musculoaponeurotic fibrosarcoma oncogene family protein g (avian) 1 and NF-E2-p45-related factor, and heme oxygenase 1 (hmox1). After exposure of zebrafish embryos for 48 h, differential gene expression was evaluated using reverse transcriptase-polymerase chain reaction, gel electrophoresis, and densitometric analysis of the gels. All tested compounds significantly affected the expression of at least one potential marker gene, with cyp1a and hmox1 being most sensitive. Lowest-observed-effect concentrations (LOECs) for gene expression were below concentrations resulting in 10% lethal effects in the DarT. For 10 (3,4- and 3,5-dichloroaniline, 1,4-dichlorobenzene, 2,4-dinitrophenol, atrazine, parathion-ethyl, chlorotoluron, genistein, 4-nitroquinoline-1-oxide, and cadmium) out of the 14 tested substances, LOEC values derived with the Gene-DarT differ by a factor of less than 10 from LOEC values of fish early life stage tests with zebrafish. For pentachloroaniline and pentachlorobenzene, the Gene-DarT showed a 23- and 153-fold higher sensitivity, respectively, while for lindane, it showed a 13-fold lower sensitivity. For ivermectin, the Gene-DarT was by a factor of more than 1,000 less sensitive than the acute fish test. The results of the present study indicate that gene expression analysis in zebrafish embryos could principally be used to predict effect concentrations in the fish early life stage test.

The zebrafish embryo model in environmental risk assessment—applications beyond acute toxicity testing

“…Analysis of embryos exposed to 3,4-dichloroaniline by microarrays and RT-PCR of selected candidate genes revealed changes in genes primarily involved in biotransformation and stress response (Voelker et al 2007). The identified genes-for instance cyp1a, hmox1-were also induced by 13 further test compounds (Weil et al 2008). A bar code-like response was observed in zebrafish exposed to 11 different chemicals (Yang et al 2007).…”

“…The molecular markers may respond more sensitively than endpoints of the conventional embryo test and might be used to predict chronic toxicity. The project Gene-DarT (gene expression D. rerio embryo test) aims at the identification of such sensitive marker genes and it has indeed been found that certain gene responses in the zebrafish embryo are exerted in the same range of concentration as toxic responses in a chronic fish toxicity test (fish early life stage test, Voelker et al 2007;Weil et al 2008). By inclusion of additional marker genes into GeneDarT, such as identified by Yang et al (2007) may be possible to correlate gene responses with chronic toxicity and to establish a prediction model.…”

Molecular and phenotypic responses of Japanese medaka (Oryzias latipes) early life stages to environmental concentrations of cadmium in sediment

“…(Weil et al 2009;Hsu et al 2010) after exposure of fish embryos to Cd. However, little is known about the effects of Cd store in the sediment at a molecular level in fish early life stages (ELS) and the possible link between gene transcription disturbances and phenotypic impairments.…”

The oxidized state of the nanocomposite Carbo-Iron® causes no adverse effects on growth, survival and differential gene expression in zebrafish

“…After 3, 8, 24, 32, and 48 h, lethal and sublethal effects (edemas, deformations, lack of spontaneous movement) were evaluated as described in Weil et al (2009) and dead embryos were removed. Oxygen content, pH and temperature were measured in all test vessels at the beginning (0 h) and end of the exposure (48 h).…”