Zebrafish: An Animal Model for Toxicological Studies

Chaojie Zhang1, Catherine Willett1, Trisha Fremgen2

1 Phylonix Pharmaceuticals, Cambridge, Massachusetts, 2 Northeastern University, Boston, Massachusetts
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 1.7
DOI:  10.1002/0471140856.tx0107s17
Online Posting Date:  November, 2003
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Abstract

Zebrafish (Danio rerio) has been extensively studied and well described for environmental toxicity studies. Molecular biology and genetics have recently been used to elucidate the underlying mechanisms of toxicity in zebrafish and to predict effects in mammals. The versatile zebrafish is now incorporated in many areas of toxicological programs for assessing human risk and for preclinical drug discovery and screening.

Keywords: zebrafish; animal model; toxicity; developmental toxicity; drug testing

     
 
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Table of Contents

  • Zebrafish as an Animal Model
  • Zebrafish Developmental and Genetic Studies
  • Toxicological Studies
  • Toxicity Testing of Therapeutics: A Comprehensive Approach Using Zebrafish
  • Organ Toxicity
  • Toxicity‐Related Cell Death
  • Summary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

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Figures

  •   FigureFigure 1.7.1 Zebrafish development occurs rapidly. By 1 hpf (top left), two cell divisions have occurred. Gastrulation begins at 6 hpf (top center). By 24 hpf (top right), the organ primordia have been laid down and the brain has significantly developed. The heart is beating and a rudimentary circulatory loop through the main body axis has been established. By 48 hpf (middle panel), pigment has begun to form; the body axis has elongated. By 120 hpf (bottom panel), organ morphogenesis is complete and most organs are functioning. The swim bladder has inflated and the embryo can swim and eat on its own. At this point the embryo is considered a larva.
  •   FigureFigure 1.7.2 Comparative LC50 values. LC50 values obtained with zebrafish (expressed as mg/liter in Table ) are compared with the corresponding LD50 values (mg/lg) obtained with mammals, in logarithmic scale. A graphic representation of the data was developed; the line represents the best fit between the two sets of values (slope = 1.13). LD50 values for mammals were obtained from the NIH TOXNET database, NCI, and others.
  •   FigureFigure 1.7.3 CYP 1A1 expression in zebrafish embryos. Zebrafish embryos (96 hpf) were treated with 0.1% DMSO (−) or 10 µM tacrine, 50 µM phenytoin, 10 µM ibuprofen, or 10 µM doxorubicin (+) for 4 hr. RNAs were isolated, and RT‐PCR was used to analyze expression of both CYP 1A1 and GAPDH, a housekeeping gene. CYP 1A1 was up‐regulated by tacrine and phenytoin treatment and down‐regulated by ibuprofen and doxorubicin treatment.
  •   FigureFigure 1.7.4 Visual assessment of liver and kidney organ toxicity. (A) Zebrafish embryos (24 hpf) treated for 5 days were used to determine the effects of dexamethasone on liver toxicity. The embryos were fixed with paraformaldehyde, incubated with streptavidin‐peroxidase, and stained with a chromogenic dye to specifically label biotinylated enzymes in the liver and gut. Arrows indicate the position of the liver. Top, untreated embryo; bottom, embryo treated with 100 µM dexamethasone for 5 days. E, eye; G, gastrointestine; T, tail. Scale bar = 1 mm. (B) Zebrafish embryos (48 hpf) were fixed with methanol and formalin and stained with a mouse antibody against sodium/potassium ATPase. Black arrow indicates the position of the pronephric duct; white arrow indicates the nephron. Top, untreated embryo; bottom, embryo treated with 5 µM brefeldin A for 24 hr.
  •   FigureFigure 1.7.5 Quantification of liver toxicity after dexamethasone treatment: generation of dose response. Larva 144 hpf show a dose response to 1 to 100 µM dexamethasone on liver size detected and quantified by peroxidase and stained with a soluble dye. Ten zebrafish were analyzed for each concentration. The absorbance was detected at 405 nm. Values are expressed as a percentage of control (% Control) ± standard deviation.
  •   FigureFigure 1.7.6 Morphological characteristics for developmental index assessment. Comparison of zebrafish (120 hpf) untreated (A) and treated (B) with 5 µM brefeldin A. Treated zebrafish shows heart edema (black arrow), necrotic liver (white arrow), undeveloped intestine (large arrow), underdeveloped jaw (gray arrow), and lack of swim bladder (arrowhead).
  •   FigureFigure 1.7.7 Dose‐response curves for developmental toxicity of flavopiridol (A) and TCDD (B). Developmental malformation (diamonds) and mortality (squares) are shown.
  •   FigureFigure 1.7.8 Neomycin‐induced liver apoptosis in zebrafish. Zebrafish embryos (96 hpf) were stained with acridine orange. (A) Untreated zebrafish. (B) Zebrafish treated with 2.5 µg/ml neomycin for 24 hr. Left panels show light micrographs and right panels, fluorescent micrographs. Neomycin induced apoptosis in the liver (arrow in lower right panel). Intestine (GI) is fluorescent in both treated and untreated zebrafish due to ingestion of dye. Abbreviations: L, liver; E, ear; SB, swim bladder.

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Literature Cited

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