Liedtke Anja, Muncke Jane, Rüfenacht Karin, Eggen Rik I L
Eawag, Swiss Federal Institute of Aquatic Science and Technology, Environmental Toxicology, Ueberlandstrasse 133, 8600, Dübendorf, Switzerland.
Environ Toxicol. 2008 Feb;23(1):59-67. doi: 10.1002/tox.20305.
Molecular effect detection is a useful approach for ecotoxicological screening of chemicals. We show here the application of the molecular DarT (MolDarT), where the expression of selected target genes is detected in short-term (120 h) exposed developing zebrafish (Danio rerio), thus allowing subacute multi-effect compound screening. The genes metallothionein 2 (mt2), cytochrome P450 1A1 (cyp1a1), and recombination activation gene 1 (rag1) are used as endpoints that describe detoxification/metal toxicity (mt2), detoxification/PAH toxicity (cyp1a1), and acquired immune system disruption (rag1). Each gene's developmental expression was studied in unexposed zebrafish during 4 to 120 h past fertilization (hpf), and all three genes were found to be expressed at 120 hpf. Furthermore, mt2 transcripts were present at high levels at 4 hpf, indicating a maternal transfer. For positive toxicity controls, freshly fertilized zebrafish eggs were exposed for 120 hpf to ZnSO(4), 1,5-dimethylnaphthalene (DMN) and CdCl(2). Exposure to 100 and 200 microM ZnSO(4) significantly induced mt2; 10 microM DMN and 20 microM DMN resulted in significantly increased cyp1a1 abundance; and 5 and 10 microM CdCl(2) significantly reduced rag1 expression levels. Furthermore, we analysed these target genes for their expression in zebrafish eggs from a previous exposure study. The eggs were exposed for 120 hpf to the environmental pollutants estradiol (E2), ethinylestradiol (EE2), nonylphenol (NP), atrazine, cyproconazol, and bisphenol A (BPA) and found differential expression of the three genes. Exposure to the (xeno-)estrogenic compound NP (0.75 microM) significantly lowered mt2 expression. This study shows the potential of short-term in vivo multi-effect screenings within one single subacute exposure using the MolDarT.
分子效应检测是化学物质生态毒理学筛选的一种有用方法。我们在此展示了分子达尔文技术（MolDarT）的应用，即在短期（120小时）暴露的发育中的斑马鱼（Danio rerio）中检测选定目标基因的表达，从而实现亚急性多效应化合物筛选。金属硫蛋白2（mt2）、细胞色素P450 1A1（cyp1a1）和重组激活基因1（rag1）基因被用作描述解毒/金属毒性（mt2）、解毒/多环芳烃毒性（cyp1a1）和获得性免疫系统破坏（rag1）的终点指标。在受精后4至120小时（hpf）对未暴露的斑马鱼研究了每个基因的发育表达，发现所有三个基因在120 hpf时均有表达。此外，mt2转录本在4 hpf时大量存在，表明存在母体传递。对于阳性毒性对照，将刚受精的斑马鱼卵暴露于硫酸锌（ZnSO₄）、1,5 - 二甲基萘（DMN）和氯化镉（CdCl₂）中120 hpf。暴露于100和200微摩尔的硫酸锌显著诱导了mt2；10微摩尔和20微摩尔的DMN导致cyp1a1丰度显著增加；5和10微摩尔的氯化镉显著降低了rag1表达水平。此外，我们分析了这些目标基因在先前暴露研究的斑马鱼卵中的表达。将卵暴露于环境污染物雌二醇（E2）、乙炔雌二醇（EE2）、壬基酚（NP）、阿特拉津、环丙唑醇和双酚A（BPA）中120 hpf，发现这三个基因存在差异表达。暴露于（外源性）雌激素化合物NP（0.75微摩尔）显著降低了mt2表达。这项研究展示了使用MolDarT在单次亚急性暴露内进行短期体内多效应筛选的潜力。
