The Use of Fish‐Derived Cell Lines for Investigation of Environmental Contaminants: An Update Following OECD's Fish Toxicity Testing Framework No. 171

Vivian R. Dayeh1, Niels C. Bols1, Katrin Tanneberger2, Kristin Schirmer2, Lucy E. J. Lee3

1 Department of Biology, University of Waterloo, Waterloo, Ontario, Canada, 2 Department of Environmental Toxicology, Eawag: Swiss Federal Institute of Aquatic Science and Technology, Dübendorf, Switzerland, 3 Department of Biology, University of the Fraser Valley, Abbotsford, British Columbia, Canada
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 1.5
DOI:  10.1002/0471140856.tx0105s56
Online Posting Date:  May, 2013
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Abstract

Protocols for evaluating chemical toxicity at the cellular level using fish cell lines are described in this unit. Routine methodologies for growing salmonid cell lines, and using them in aquatic toxicology studies that support the mandate of the Organization for Economic Co‐operation and Development (OECD) to reduce the use of whole animals in toxicity testing, are presented. Rapid, simple, cost‐effective tests evaluating viability of cells with three indicator dyes per sample provides a broad overview of the sensitivity of cells to chemical contaminants. This fluorometric assay involves: (1) alamar blue for metabolic activity, (2) CFDA‐AM for membrane integrity, and (3) neutral red for lysosomal function. These protocols are conveniently performed in semi‐unison within the same multiwell plates and read at three different wavelengths. Detailed step‐by‐step descriptions of the assays, parameters to consider, troubleshooting, and guidelines for data interpretation are provided as essential tools for investigating environmental aquatic contaminants at the cellular level. Curr. Protoc. Toxicol. 56:1.5.1‐1.5.20. © 2013 by John Wiley & Sons, Inc.

Keywords: fish cell lines; in vitro toxicity; fluorescent indicator dyes; alamar Blue; CFDA‐AM; neutral red; toxicity test; cell viability

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Evaluation of Toxicity in Fish‐Derived Cell Lines Using Alamar Blue to Assess Metabolic Activity
  • Alternate Protocol 1: Evaluation of Toxicity in Fish‐Derived Cell Lines Using CFDA‐AM to Assess Membrane Integrity
  • Alternate Protocol 2: Evaluation of Toxicity in Fish‐Derived Cell Lines Using Neutral Red to Assess Lysosomal Activity
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Evaluation of Toxicity in Fish‐Derived Cell Lines Using Alamar Blue to Assess Metabolic Activity

  Materials
  • 70% ethanol solution
  • Confluent culture of RTgill‐W1 cells (ATCC #CRL‐2523) in a 75‐cm2 flask
  • 0.53 mM versene (EDTA; Invitrogen/Life Technologies) diluted 1:5000 (1× 0.2 g tetrasodium EDTA/liter in PBS)
  • Trypsin solution (see recipe) or 0.25% trypsin solution in PBS without Ca2+, Mg2+, or phenol red (Biowest, Biochrom, or Invitrogen/Life Technologies) or TrypLE (Invitrogen/Life Technologies; see notes below)
  • Leibovitz's (L‐15) complete medium containing FBS (see recipe)
  • Test compounds (toxicants), stock solutions
  • DMSO
  • L‐15/ex solution (see recipe)
  • Alamar blue (Immunocorp, Invitrogen/Life Technologies)
  • Laminar flow hood, either horizontal or vertical
  • Inverted phase‐contrast microscope
  • Vacuum aspirator
  • 15‐ml or 50‐ml centrifuge tubes, sterile
  • 24‐, 48‐ or 96‐well tissue‐culture treated microwell plate
  • 75‐cm2 tissue culture flask
  • Incubator (see Critical Parameters and Troubleshooting)
  • Catch basin (a plastic container at least slightly larger than the size of the microwell plate)
  • Positive‐displacement digital microdispenser (e.g., Nichiryo model 800) or adjustable‐volume micropipet (e.g., Eppendorf Reference 0.1 to 2.5 µl)
  • Glass pipet tips for digital microdispenser (Nichiryo) or plastic micropipet tips (e.g., Eppendorf)
  • Multichannel pipettor (e.g., Eppendorf Research plus, 100 to 1000 µl, or 30 to 300 µl)
  • Parafilm
  • Cover foil (non‐breathable; Nunc)
  • Radiation exposure chamber containing UV‐A and/or UV‐B fluorescent lamps (Southern New England Ultraviolet) and a fan
  • Spectroradiometer (e.g., InstaSpec II photodiode array spectroradiometer, Oriel)
  • Transformer to modulate UV intensity
  • Fluorometric microwell plate reader
  • Additional reagents and equipment for counting cells ( appendix 3B)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and aseptic technique should be used accordingly. Work >6‐in. from the front of the vertical laminar flow hood, as the sterile zone begins there.NOTE: EDTA and trypsin steps can be replaced with TrypLE, a recombinant form of trypsin available from Invitrogen that works without the EDTA rinse step and which is a much gentler dissociating solution.

Alternate Protocol 1: Evaluation of Toxicity in Fish‐Derived Cell Lines Using CFDA‐AM to Assess Membrane Integrity

  • 4 mM 5‐carboxyfluorescein diacetate acetoxymethyl ester (CFDA‐AM, see recipe)
  • RTgill‐W1 cells in a 24‐well, 48‐well or 96‐well plate exposed to toxicants (see Basic Protocol, steps 1 to 23)

Alternate Protocol 2: Evaluation of Toxicity in Fish‐Derived Cell Lines Using Neutral Red to Assess Lysosomal Activity

  • Neutral red (NR) solution (0.33% w/v in DPBS; Sigma Aldrich, or see recipe for stock solution in Reagents and Solutions)
  • RTgill‐W1 cells in a 24‐well, 48‐well or 96‐well plate exposed to toxicants (see Basic Protocol, steps 1 to 23)
  • Neutral red fixative solution (see recipe)
  • Neutral red extraction solution (see recipe)
  • Orbital shaker
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Figures

Videos

Literature Cited

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   Clemons, J.H., van den Heuvel, M.R., Stegeman, J.J., Dixon, D.G., and Bols, N.C. 1994. A comparison of toxic equivalent factors for selected dioxin and furan congeners derived using fish and mammalian liver cell lines. Can. J. Fish. Aquat. Sci. 51:1577‐1584.
   Dayeh, V.R., Schirmer, K., and Bols, N.C. 2009. Ammonia‐containing industrial effluents lethal to rainbow trout vacuolization and neutral red uptake in the rainbow trout gill cell line, RTgill‐W1. ATLA 37:77‐87.
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   Lee, L.E.J., Dayeh, V.R., Schirmer, K., and Bols, N.C. 2009. Applications and potential uses of fish gill cell lines: Examples with RTgill‐W1. In Vitro Cell Dev. Biol. Anim. 45:127‐134.
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   OECD. 2012. Fish Testing Framework. Series on Testing and Assessment. No 171. http://search.oecd.org/officialdocuments/displaydocumentpdf/?cote=ENV/JM/MONO(2012)16&doclanguage=en.
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   Schirmer, K., Chan, A.G.J., Greenberg, B.M., Dixon, D.G., and Bols, N.C. 1997. Methodology for demonstrating and measuring the photocytotoxicity of fluoranthene to fish cells in culture. Toxicol. In Vitro 11:107‐119.
   Schirmer, K., Greenberg, B., Dixon, D.G., and Bols, N.C. 1998a. Ability of 16 priority PAHs to be directly cytotoxic to a cell line from the rainbow trout gill. Toxicology 127:129‐141.
   Schirmer, K., Chan, A.G.J., Greenberg, B.M., Dixon, D.G., and Bols, N.C. 1998b. Ability of 16 priority PAHs to be photocytotoxic to a cell line from the rainbow trout gill. Toxicology 127:143‐155.
   Schirmer, K., Chan, A.G.J., and Bols, N.C. 2000. Transitory metabolic disruption and cytotoxicity elicited by benzo[a]pyrene in two cell lines from rainbow trout liver. J. Biochem. Mol. Toxicol. 14:262‐276.
   Schnell, S., Kawano, A., Porte, C., Lee, L.E.J., and Bols, N.C. 2009. Effects of ibuprofen on the viability and proliferation of rainbow trout liver cell lines and potential problems and interactions in effect assessment. Environ. Toxicol. 24:157‐165.
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   Segner, H., Behrens, A., Joyce, E.M., Schirmer, K., and Bols, N.C. 2000. Transient induction of 7‐ethoxyresorufin‐o‐deethylase (EROD) activity by medium change in the rainbow trout liver cell line, RTL‐W1. Mar. Environ. Res. 50:489‐493.
   Shaw, A.J. 1994. Defining cell viability and cytotoxicity. A.T.L.A. 22:124‐126.
   Smith, K.E.C., Oostingh, G.J., and Mayer, P. 2010. Passive dosing for producing defined and constant exposure of hydrophobic organic compounds during in vitro toxicity tests. Chem. Res. Toxicol. 23:55‐65.
   Tanneberger, K., Rico‐Rico, A., Kramer, N.I., Busser, F.J.M., Hermens, J.L.M., and Schirmer, K. 2010. Effects of solvents and dosing procedure on chemical toxicity in cell‐based in vitro assays. Environ. Sci. Technol. 44:4775‐4781.
   Tanneberger, K., Knöbel, M., Busser, F.J.M., Sinnige, T.L., Hermens, J.L.M., and Schirmer, K. 2013. Predicting fish acute toxicity using a fish gill cell line‐based toxicity assay. Environ. Sci. Technol. 47:1110‐1119.
   Yu, K.O., Fisher, J.W., Burton, G.A., and Tillitt, D.E. 1997. Carrier effects of dosing the H4IIE cells with 3,3′,4,4′‐tetrachlorobiphenyl (PCB 77) in dimethyl sulfoxide or isooctane. Chemosphere 35:895‐904.
   Zhang, S.Z., Lipsky, M.M., Trump, B.F., and Hsu, I.C. 1990. Neutral red (NR) assay for cell viability and xenobiotic‐induced cytotoxicity in primary cultures of human and rat hepatocytes. Cell. Biol. Toxicol. 6:210‐234.
Key References
   Bols, N.C. and Lee, L.E.J. 1994. Cell lines: Availability, propagation and isolation. In Biochemistry and Molecular Biology of Fishes, vol. 3. (P.W. Hochachka and T.P. Mommsen, eds.) pp. 145‐159. Elsevier Science, Amsterdam.
  An overview of protocols for obtaining and growing fish cell lines.
   Ganassin, R.C., Schirmer, K., and Bols, N.C. 2000. Cell and tissue culture. In The Laboratory Fish (G.K. Ostrander, ed.) pp. 631‐651. Academic Press, San Diego.
  Gives technical protocols for using fish cell lines in toxicology.
   Babich, H and Borenfreund, E. 1991. Cytotoxicity and genotoxicity assays with cultured fish cells: A review. Toxicol. In Vitro 5:91‐100.
  The above four articles provide a broad overview on use of fish cell lines in toxicology and ecotoxicology.
   Bols, N.C., Dayeh, V.R., Lee, L.E.J. and Schirmer, K. 2005. Use of fish cell lines in toxicology of fish. In Biochemistry and Molecular Biology of Fishes‐Environmental Toxicology. Vol. 6. (T.W. Moon and T. P. Mommsen, eds) pp. 43‐84. Elsevier Science, Amsterdam.
   Fent, K. 2001. Fish cell lines as versatile tools in ecotoxicology: Assessment of cytotoxicity, cytochrome P4501A induction potential and estrogenic activity of chemicals and environmental samples. Toxicol. In Vitro 15:477‐488.
   Schirmer, K. 2006. Proposal to improve vertebrate cell cultures to establish them as substitutes for the regulatory testing of chemicals and effluents using fish. Toxicology 224:163‐183.
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