Use of Medaka in Toxicity Testing

Stephanie Padilla1, John Cowden1, David E. Hinton2, Bonny Yuen2, Sheran Law2, Seth W. Kullman3, Rodney Johnson4, Ronald C. Hardman2, Kevin Flynn4, Doris W.T. Au5

1 United States Environmental Protection Agency, Research Triangle Park, North Carolina, 2 Nicholas School of the Environment and Earth Sciences, Duke University, Durham, North Carolina, 3 North Carolina State University, Raleigh, North Carolina, 4 U.S. Environmental Protection Agency, Duluth, Minnesota, 5 City University of Hong Kong, Hong Kong, China
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 1.10
DOI:  10.1002/0471140856.tx0110s39
Online Posting Date:  February, 2009
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Abstract

Small aquarium fishes are increasingly used as animal models, and one of these, the Japanese Medaka (Oryzias latipes), is frequently utilized for toxicity testing. While these vertebrates have many similarities with their terrestrial counterparts, there are differences that must be considered if these organisms are to be used to their highest potential. Commonly, testing may employ either the developing embryo or adults; both are easy to use and work with. To illustrate the utility and breadth of toxicity testing possible using medaka fish, we present protocols for assessing neurotoxicity in developing embryos, evaluating toxicant effects on sexual phenotype after treatment with endocrine‐disrupting chemicals by sexual genotyping, and measuring hepatotoxicity in adult fish after treatment with a model hepatotoxicant. The methods run the gamut from immunohistology through PCR to basic histological techniques. Curr. Protoc. Toxicol. 39:1.10.1‐1.10.36. © 2009 by John Wiley & Sons, Inc.

Keywords: medaka; neurotoxicity; hepatotoxicity; endocrine disruption

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Maintaining and Handling Adult Medaka
  • Basic Protocol 2: Breeding Medaka and Collecting Embryos
  • Basic Protocol 3: Embryonic Development of Medaka
  • Support Protocol 1: Chemical Exposure of Embryo and Adult Medaka
  • Experimental Neurotoxicology Techniques for Medaka
  • Support Protocol 2: Pronase Dechorionation of Medaka Embryos
  • Basic Protocol 4: Whole‐Mount Antibody Staining of Cranial Nerves in Hatched Medaka Fry
  • Determining Sexual Genotype in Medaka After Endocrine Disruption
  • Basic Protocol 5: PCR/Gel Electrophoresis Protocol for Determining Sexual Genotype
  • Alternate Protocol 1: High‐Throughput Quantitative Real‐Time PCR Protocol for Determining Genotypic Sex
  • Methods for Detecting Toxic Alterations in the Intrahepatic Biliary Passageways of Medaka
  • Basic Protocol 6: Anesthesia, Sacrifice, Fixation, and Paraffin Embedment of Medaka Fry
  • Basic Protocol 7: Immunohistochemistry for Cell Proliferation (PCNA)
  • Basic Protocol 8: Immunohistochemistry for Apoptosis (ApopTag)
  • Basic Protocol 9: Immunocytochemistry for Pan‐Cytokeratins (AE1/AE3)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Maintaining and Handling Adult Medaka

  Materials
  • Water for aquaria: prepare treated water from a public water supply by removing the chemicals added during water disinfection, using a reverse osmosis system (readily available in many different sizes and relatively inexpensive)
  • Japanese Medaka fish, male and female (e.g., Carolina Biological Supply)
  • Tropical fish food (e.g., Otohime B, Ashby Aquatics) or purified casein‐based diet (DeKoven et al., )
  • Test materials (toxicants)
  • 5‐gallon or 10‐gallon freshwater aquaria with flow‐through system for water exchange, temperature controlled

Basic Protocol 2: Breeding Medaka and Collecting Embryos

  Materials
  • Medaka colony of mixed sexes (see protocol 1)
  • Embryo rearing medium (1× ERM; see recipe) or marine water (see recipe)
  • Test materials (toxicants)
  • Dip‐net
  • Shallow dish containing aquarium water
  • Wide‐mouth pipet or forceps
  • Paper towels
  • 20‐ml vials or 96‐well microtiter plates

Basic Protocol 3: Embryonic Development of Medaka

  Materials
  • Stage 20 or older medaka embryos (see Basic Protocols protocol 22 and protocol 33)
  • Embryo rearing medium (1× ERM; see recipe)
  • 1% (w/v) pronase solution (see recipe)
  • 50% 1× ERM (see recipe)/50% phosphate‐buffered saline (PBS; appendix 2A)
  • PBS
  • 4% (w/v) paraformaldehyde: prepared by diluting 16% (w/v) paraformaldehyde for Zamboni's fixative (see recipe) with PBS
  • 6‐well, microtiter plates
  • 34°C water bath
  • Test tube rocker or rotator (e.g., MaxiRotator, LabLine)

Support Protocol 1: Chemical Exposure of Embryo and Adult Medaka

  Materials
  • Medaka fry, hatched (stage 39; see protocol 3)
  • Zamboni's fixative (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1% (w/v) trypsin in PBS, freshly prepared
  • 4% (w/v) paraformaldehyde: prepared by diluting 16% (w/v) paraformaldehyde for Zamboni's fixative (see recipe) with PBS
  • High‐Triton PBS (see recipe)
  • Low‐Triton PBS (see recipe)
  • Primary antibody: mouse anti‐acetylated α‐tubulin (Sigma)
  • Secondary antibody: fluorophore‐conjugated anti‐mouse antibody (e.g., Alexa Fluor 555 donkey anti‐mouse IgG, Invitrogen)
  • Fluorescence mounting medium, e.g., glycerol or Vectashield (Vector Labs)
  • 2‐ml clear plastic tubes with caps
  • Test tube rotator or rocker (e.g., 4631 MaxiRotator, LabLine)
  • Glass depression slides
  • Coverslips
  • Fluorescence microscope

Support Protocol 2: Pronase Dechorionation of Medaka Embryos

  Materials
  • 10× PCR buffer, without MgCl 2 (Sigma, cat. no. P2317)
  • 25 mM MgCl 2
  • 10 mM (each nucleotide) dNTP mix
  • PG17.19: 5′‐GAA CCA CAG CTT GAA GAC CCC GCT GA‐3′ (Inui et al., )
  • PG17.20: 5′‐GCA TCT GCT GGT ACT GCT GGT AGT TG‐3′ (Inui et al., )
  • JumpStart Taq DNA polymerase (Sigma, cat. no. D4184)
  • DNA sample from fish: prepared using a DNA preparation kit (e.g., DNeasy Kits, Qiagen)
  • DEPC‐treated water (e.g., see unit 2.9)
  • NuSieve 3:1 agarose (Cambrex, cat. no. 50091)
  • 1× TBE buffer (unit 2.2)
  • 10 mg/ml ethidium bromide stock solution
  • PCR tubes, appropriate to thermocycler
  • Thermocycler (e.g., PTC‐200, MJ Research)
  • UV transilluminator
  • Additional reagents and equipment for performing agarose gel electrophoresis (unit 2.2)

Basic Protocol 4: Whole‐Mount Antibody Staining of Cranial Nerves in Hatched Medaka Fry

  Materials
  • Taqman Universal PCR Master Mix (Applied Biosystems, cat. no. 4304437)
  • 18S rRNA Taqman Assay Reagent (Applied Biosystems, cat. no. 4319413E)
  • DMY HTP forward primer (5′‐TTC TGC TGG AAA GAC‐3′)
  • DMY HTP reverse primer (5′‐TCT CTG GCG GAC CAT GAT‐3′)
  • DMY HTP probe (5′‐FAM‐CCA GTG CTT CAA ATG CGA GCA‐BHQ‐3′)
  • RNase‐free water (e.g., see unit 2.9)
  • DNA sample from fish: prepared using a DNA preparation kit (e.g., DNeasy Kits, Qiagen)
  • Primer design software (e.g., Primer Express, Applied Biosystems)
  • 96‐well optical plates, prechilled
  • Refrigerated centrifuge with rotor adapted for 96‐well plates, 4°C
  • Real‐time thermocycler (e.g., model 7500, Applied Biosystems)

Basic Protocol 5: PCR/Gel Electrophoresis Protocol for Determining Sexual Genotype

  Materials
  • Medaka ( protocol 1), treated with toxicant ( protocol 3) and nontreated or vehicle‐treated controls
  • Ethyl 3‐aminobenzoate methanesulfonate (MS‐222; Sigma‐Aldrich)
  • GPHS fixative (see recipe)
  • 70%, 85%, 95%, and 100% (v/v) ethanol
  • Xylene
  • Paraffin (Paraplast Plus, EMS)
  • Plastic Pasteur pipets
  • Dissecting tools, including scalpel and curved forceps
  • Glass vials of appropriate size, with caps
  • Test tube rotator or rocker (e.g., 4631 MaxiRotator, LabLine)
  • Tissue molds and embedding rings (EMS)
  • Rotary microtome (e.g., MICROM, International GmbH)
  • Poly‐L‐lysine‐coated glass slides (EMS)

Alternate Protocol 1: High‐Throughput Quantitative Real‐Time PCR Protocol for Determining Genotypic Sex

  Materials
  • Paraffin sections of toxicant‐treated and control medaka on a poly‐L‐lysine glass slide ( protocol 9)
  • Xylene
  • 70%, 95%, and 100% (v/v) ethanol
  • 10 mM citrate buffer, pH 6
  • 3% (v/v) hydrogen peroxide in phosphate‐buffered saline (PBS; appendix 2A)
  • 1× PBS
  • 10% (v/v) normal goat serum, diluted in PBS containing 0.1% (v/v) Tween 20
  • Primary antibody: mouse anti‐PCNA antibody (Dako, cat. no. M0879), diluted 500‐fold in antibody diluent (Dako, cat. no. S0809)
  • Secondary antibody: goat anti‐mouse‐HRP conjugated immunoglobulins (DakoCytomation Envision+ System‐HRP; Dako, cat. no. K4006)
  • Peroxidase substrate: 3,3′‐diaminobenzidine solution (DAB, Dako)
  • Harris' hematoxylin
  • Permount (Fisher Scientific)
  • Microwave oven
  • Humidified chamber of appropriate size (home made or commercially available)
  • Coverslips
  • Light microscope

Basic Protocol 6: Anesthesia, Sacrifice, Fixation, and Paraffin Embedment of Medaka Fry

  Materials
  • Paraffin sections of toxicant‐treated and control medaka on poly‐L‐lysine coated slide ( protocol 9)
  • Xylene
  • 70%, 95%, and 100% ethanol
  • 10 mM citrate buffer, pH 6.0
  • 3% (v/v) hydrogen peroxide/phosphate‐buffered saline (PBS; appendix 2A)
  • PBS
  • 10% (v/v) normal goat serum, diluted in PBS containing 0.1% (v/v) Tween 20
  • Apop Tag Plus Peroxidase In Situ Apoptosis Detection Kit (Millipore) containing:
    • Equilibration buffer
    • TdT (terminal deoxyribonucleotide transferase)
    • Stop/wash buffer
    • Anti‐digoxigenin conjugate
    • DAB substrate solution
  • Harris' hematoxylin
  • Permount (Fisher Scientific)
  • Boiling water bath
  • Room temperature and 37°C humidified chamber of appropriate size (home made or commercially available)
  • Coverslips
  • Light microscope

Basic Protocol 7: Immunohistochemistry for Cell Proliferation (PCNA)

  Materials
  • Paraffin section of toxicant‐treated and control medaka mounted on poly‐L‐lysine coated slide ( protocol 9)
  • Xylene
  • 70%, 95%, and 100% (v/v) ethanol
  • 10 mM citrate buffer, pH 6.0
  • 3% (v/v) hydrogen peroxide in phosphate‐buffered saline (PBS; appendix 2A)
  • PBS
  • 10% (v/v) normal goat serum, diluted in PBS containing 0.1% (v/v) Tween 20
  • Primary antibody: mouse anti‐AE1/AE3 antibody (Zymed, cat. no. 08‐0132), diluted 200‐fold in antibody diluent (Dako, cat. no. S0809)
  • Secondary antibody: goat anti‐mouse‐HRP conjugated immunoglobulins (DakoCytomation Envision+ System‐HRP; Dako, cat. no. K4006)
  • Peroxidase substrate: 3,3′‐diaminobenzidine (DAB, Dako)
  • Harris' hematoxylin
  • Permount (Fisher Scientific)
  • Boiling water bath
  • Room temperature and 37°C humidified chambers (home made or commercially available)
  • Coverslips
  • Light microscope
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Literature Cited

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