Muncke Jane, Eggen Rik I L
Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Duebendorf, Switzerland.
Environ Toxicol Chem. 2006 Oct;25(10):2734-41. doi: 10.1897/05-683r.1.
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.
当代生态毒理学面临着机理理解方面的挑战，这是高级风险评估的先决条件，而急性毒性并非主要问题。要实现这一点，就需要快速且具有生物整合性、能在分子水平检测多种效应的生物测定系统。我们在此提出一种新型测试系统的概念，它基于斑马鱼致畸性（DarT）测定法构建，但在时间上有所扩展，且基于亚急性毒性范围内分子效应的测试，名为MolDarT。作为原理验证，我们展示了使用测量卵黄蛋白原1基因（vtg1）mRNA水平作为发育中斑马鱼雌激素活性的分子标记，这是MolDarT的第一个模块。将受精的斑马鱼卵暴露于100、1000和2000 ng/L（6.75 nM）的17α-乙炔雌二醇（EE2）中，在受精后（hpf）长达120小时内，每24小时分离一次总RNA。使用逆转录实时聚合酶链反应检测vtg1 mRNA的丰度，并将其标准化为β-肌动蛋白mRNA的丰度。在48至120 hpf之间，β-肌动蛋白mRNA水平保持恒定，使该基因成为用于标准化的合适参考基因。在48 hpf时，检测到1000和2000 ng/L EE2导致vtg1表达显著上调。在72、96和120 hpf时，所有EE2浓度下vtg1均被显著诱导。还在未暴露的发育中的斑马鱼中测量了vtg1的表达。在24 hpf及所有后续时间点，斑马鱼胚胎均含有vtg1转录本。这些发现表明vtg1在发育中的斑马鱼中正常表达，且可被EE2诱导。我们提议在MolDarT中使用vtg1作为雌激素活性的分子靶点。
