Assessment of Mitochondrial Toxicity in HepG2 Cells Cultured in High‐Glucose‐ or Galactose‐Containing Media

Rachel Swiss1, Yvonne Will2

1 Compound Safety Prediction, World Wide Medicinal Chemistry, Pfizer, Inc., Groton, Connecticut, 2 Exploratory Safety Differentiation, Pfizer, Inc, Groton, Connecticut
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.20
DOI:  10.1002/0471140856.tx0220s49
Online Posting Date:  August, 2011
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Abstract

Drug‐induced mitochondrial toxicity has been recognized as contributing to a variety of organ toxicities, such as liver, heart, kidney, and CNS, and has been found to contribute to drug attrition and black box warnings. Here, we describe a cell‐based assay that can detect direct drug‐induced mitochondrial toxicity, providing protocols for screening in 96‐ and 384‐well format. Cultured cells grown in glucose media produce their ATP by glycolysis, largely bypassing the mitochondria, and hence are fairly resistant to drugs that affect mitochondrial function. However, when growing the same cells in media supplemented with galactose as opposed to glucose, they are forced to produce ATP through oxidative phosphorylation, which then makes them vulnerable to mitochondrial insult. By measuring viability of cells grown in either glucose‐ or galactose‐supplemented media, direct mitochondrial impairment can be detected. Curr. Protoc. Toxicol. 49:2.20.1‐2.20.14. © 2011 by John Wiley & Sons, Inc.

Keywords: mitochondria; HepG2; toxicity; high glucose; galactose; ATP depletion; mitochondrial toxicity

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: ATP Viability Measurement in a 96‐Well Plate Format
  • Basic Protocol 2: ATP Viability Measurement in a 384‐Well Plate Format
  • Basic Protocol 3: HepG2 Cell Maintenance and Splitting
  • Support Protocols: Assay Considerations
  • Support Protocol 1: Choosing an Appropriate Cell Line
  • Support Protocol 2: Compound Storage Conditions
  • Support Protocol 3: Coating Untreated Tissue Culture Plates or Flasks with Collagen
  • Support Protocol 4: Other Endpoints for Assessing Mitochondrial Toxicity
  • Support Protocol 5: Determining Cell Number for Plate Format
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: ATP Viability Measurement in a 96‐Well Plate Format

  Materials
  • HepG2 cells or other appropriate cell line
  • Compounds to be tested
  • Dimethylsulfoxide (DMSO)
  • Control compounds (Sigma‐Aldrich):
    • Rotenone, dissolved in DMSO, 100 µM and 10 mM stock
    • Nefazadone, dissolved in DMSO, 30 mM stock
    • Tacrine, dissolved in DMSO, 30 mM stock
    • Tamoxifen, dissolved in DMSO, 30 mM stock
  • Triton X‐100 diluted 1:10 (v/v) in phosphate‐buffered saline (PBS; appendix 2A)
  • Glucose medium (see recipe)
  • Galactose medium (see recipe)
  • Promega CellTiter‐Glo kit (or equivalent)
  • 96‐well tissue culture plate with collagen coating (see protocol 6)
  • Standard 96‐well microtiter plates
  • Deep‐well (1‐ml well volume) 96‐well plates
  • Swinging‐bucket centrifuge with microtiter plate carrier
  • Plate sealers
  • Luminescence plate reader

Basic Protocol 2: ATP Viability Measurement in a 384‐Well Plate Format

  Materials
  • HepG2 cells or other appropriate cell line
  • Compounds to be tested
  • Dimethylsulfoxide (DMSO)
  • Control compounds (Sigma‐Aldrich):
    • Rotenone, dissolved in DMSO, 100 µM and 10 mM stock
    • Nefazadone, dissolved in DMSO, 30 mM stock
    • Tacrine, dissolved in DMSO, 30 mM stock
    • Tamoxifen, dissolved in DMSO, 30 mM stock
  • Triton X‐100 diluted 1:10 (v/v) in phosphate‐buffered saline (PBS; appendix 2A)
  • Glucose medium (see recipe)
  • Galactose medium (see recipe)
  • Promega CellTiter‐Glo kit (or equivalent)
  • 384‐well tissue culture plate with collagen coating (see protocol 6)
  • Aspirating device for 384‐well plate
  • 384‐well plate, polypropylene
  • 384‐well plate, deep well
  • Platform shaker
  • Luminescence plate reader
  • Computer running Microsoft Excel

Basic Protocol 3: HepG2 Cell Maintenance and Splitting

  Materials
  • Flasks of HepG2 cells
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 0.25% trypsin/EDTA (Invitrogen, cat no. 25200)
  • Glucose medium (see recipe)
  • Galactose medium (see recipe)
  • 15‐ml conical tubes
  • 150‐cm2 collagen‐coated (see protocol 6) tissue culture flask
  • Additional reagents and equipment for counting cells ( appendix 3B)

Support Protocol 1: Choosing an Appropriate Cell Line

  Materials
  • Collagen type I, from rat tail tendon (BD Biosciences)
  • 0.02 N acetic acid
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Untreated tissue culture plate or flask
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Figures

Videos

Literature Cited

   Marroquin, L.D., Hynes, J., Dykens, J.A., Jamieson, J.D., and Will, Y. 2007. Circumventing the Crabtree Effect: Replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants. Toxicol. Sci. 97:539‐547.
   Warburg, O., Geissler, A.W., and Lorenz, S. 1967. On growth of cancer cells in media in which glucose is replaced by galactose. Hoppe Seylers Z. Physiol. Chem. 348:1686‐1687.
Internet Resources
   http://www.bdbiosciences.com/external_files/dl/doc/manuals/live/web_enabled/354236_pug.pdf
  Manual for Rat Tail Collagen, Type 1 (BD Biosciences).
   http://www.clontech.com/images/pt/PT3947‐1.pdf
  Clontech Laboratories Protocol Manual—LDH Cytotoxicity Detection Kit.
  http://www.promega.com/resources/protocols/technical‐bulletins/0/celltiter‐glo‐luminescent‐cell‐viability‐assay‐protocol/
  Promega Technical Bulletin—CellTiter‐Glo Luminescent Cell Viability Assay.
   https://e‐labdoc.roche.com/LFR_PublicDocs/ras/11465015001_en_17.0.pdf
  Roche Protocol Manual—Cell Proliferation Kit II (XTT).
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