J Vet Sci.  2008 Dec;9(4):351-357. 10.4142/jvs.2008.9.4.351.

In vivo alternative testing with zebrafish in ecotoxicology

• Seok SH
• Baek MW
• Lee HY
• Kim DJ
• Na YR
• Noh KJ
• Park SH
• Lee HK
• Lee BH
• Park JH

• KMID: 1104909
• DOI: http://doi.org/10.4142/jvs.2008.9.4.351

Abstract

Although rodents have previously been used in ecotoxicological studies, they are expensive, time-consuming, and are limited by strict legal restrictions. The present study used a zebrafish (Danio rerio) model and generated data that was useful for extrapolating toxicant effects in this system to that of humans. Here we treated embryos of the naive-type as well as a transiently transfected zebrafish liver cell line carrying a plasmid (phAhREEGFP), for comparing toxicity levels with the well-known aryl hydrocarbon receptor (AhR)-binding toxicants: 3,3',4,4',5-pentachlorobiphenyl (PCB126), 2,3,7,8-tetrachlorodibenzo-p-dioxin, and 3-methylcholanthrene. These toxicants induced a concentration-dependent increase in morphological disruption, indicating toxicity at early life-stages. The transient transgenic zebrafish liver cell line was sensitive enough to these toxicants to express the CYP1A1 regulated enhanced green fluorescent protein. The findings of this study demonstrated that the zebrafish in vivo model might allow for extremely rapid and reproducible toxicological profiling of early life-stage embryo development. We have also shown that the transient transgenic zebrafish liver cell line can be used for research on AhR mechanism studies.

Keyword

aryl hydrocarbon receptor; enhanced green fluorescent protein; zebrafish

MeSH Terms

Animals
Benz(a)Anthracenes/toxicity
Cell Line
Green Fluorescent Proteins
Hepatocytes/cytology/physiology
Larva/drug effects/growth & development
Lethal Dose 50
Polychlorinated Biphenyls/toxicity
Tetrachlorodibenzodioxin/toxicity
Water Pollutants, Chemical/*adverse effects
Zebrafish/*physiology

Figure

• 

  Fig. 1 PCB126, TCDD, and 3-MC induced dysmorphogenesis in developing zebrafish. Embryos were immediately exposed to 10 nM PCB126 (A), 100 nM PCB126 (B, C) or 10 nM TCDD (E), 30 nM TCDD (F, G, H) or 10 µM 3-MC (I, J) or their vehicle DMSO (0.01%) (D) for 96 hr. Pericardial edema (arrows), swollen yolk sac and trunk abnormalities (A) were characterized by PCB126 toxicity. Also, PCB126-exposed zebrafish exhibited contorted tail and other tail malformations [EWE-DT238] (B, inset represents DMSO (0.01%) exposed zebrafish) and vessel irregularity (C). TCDD caused an increased incidence of trunk abnormalities, such as spinal lordosis (E). Spinal lordosis was more severe where a high concentration of TCDD was administered to the zebrafish (F). Swollen and discontinuous yolk sac was observed with TCDD exposure (G, inset represents DMSO (0.01%) exposed zebrafish). Brain hemorrhage (1), somite irregularity (2), elongated and unlooped heart (3), pericardial edema (4), no swim bladder inflation (5), swollen yolk sac (6), and lower jaw shortening (7) (arrows) were observed with TCDD exposure (H, inset represents DMSO (0.01%) exposed zebrafish). 3-MC caused pericardial edema (arrows), and swollen yolk sac (I). Also 3-MC exposed zebrafish exhibited swollen yolk sac (1), pericardial sac edema (2), elongated and unlooped heart (3), and lower jaw shortening (4) (arrows) (J). The scale bar (1 cm) in (A); the scale bar (250 µm) in (B) applies also to (G, H); the scale bar (200 µm) in (C); the scale bar (1 cm) in (D) applies also to (E, F); the scale bar (1 cm) in (I); the scale bar (250 µm) in (J).

• 

  Fig. 2 Human AhR-regulated reporter construct. phAhRE-EGFP was constructed by fusing a portion of the 5' regulatory region of the human cytochrome P4501A1 (CYP1A1) to the cDNA sequence of jellyfish GFP.

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  Fig. 3 EGFP expression in human AhR promoter following exposure to PCB126, TCDD, or 3-MC. ZFL cells transfected with phAhRE-EGFP and treated with 500 nM PCB126 (A) and 10 µM PCB126 (B), 20 nM TCDD (C) and 200 nM TCDD (D), 1 µM 3-MC (E) and 10 µM 3-MC (F), or their vehicle DMSO (G). Cell morphology was observed under a confocal microscope. The image merges the differential interference contrast image with the EGFP expression fluorescence image.

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