Molecular multi-effect screening of environmental pollutants using the MolDarT

Abstract

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.