Vitellogenin 1 mRNA as an early molecular biomarker for endocrine disruption in developing zebrafish (Danio rerio)

Contemporary ecotoxicology is faced with the challenge of mechanistic understanding, a prerequisite for advanced risk assessment where acute toxicity is not the main issue. To achieve this, bioassay systems that are fast and biologically integrating and that detect a multitude of effects on a molecular level are needed. We present here the concept of such a novel test system that is built on the Danio rerio teratogenicity (DarT) assay but is extended in time and is based on testing molecular effects in the subacute toxicity range, named MolDarT. As proof of principle, we show the use of measuring vitellogenin 1 gene (vtg1) mRNA levels as a molecular marker for estrogenicity in developing zebrafish, a first module of MolDarT. Fertilized zebrafish eggs were exposed to 100, 1,000, and 2,000 ng/L (6.75 nM) 17 alpha-ethinylestradiol (EE2), and total RNA was isolated every 24 h up to 120 h postfertilization (hpf). Abundance of vtg1 mRNA was detected using reverse transcription real-time polymerase chain reaction and normalized to beta-actin mRNA abundance. Between 48 and 120 hpf, beta-actin mRNA levels were constant, making this gene a suitable reference gene for normalization. A significant up-regulation of vtg1 expression was detected at 48 hpf for 1,000 and 2,000 ng/L EE2. At 72, 96, and 120 hpf, vtg1 was significantly induced for all EE2 concentrations. Expression of vtg1 was also measured in unexposed developing zebrafish. At 24 hpf and at all later time points, zebrafish embryos contained vtg1 transcripts. These findings show that vtg1 is regularly expressed in developing zebrafish and that it is inducible by EE2. We propose the use of vtg1 as molecular target for estrogenicity in the MolDarT.