High‐Throughput Assays for Assessing Mitochondrial Dysfunction Caused by Compounds that Impair mtDNA‐Encoded Protein Levels in Eukaryotic Cells

Sashi Nadanaciva1, James Murray2, Casey Wilson2, David F. Gebhard3, Yvonne Will1

1 Compound Safety Prediction, Worldwide Medicinal Chemistry, Pfizer Inc., Groton, Connecticut, 2 MitoSciences Inc., Eugene, Oregon, 3 Primary Pharmacology, Research Center of Emphasis, Pfizer Inc., Groton, Connecticut
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 3.11
DOI:  10.1002/0471140856.tx0311s48
Online Posting Date:  May, 2011
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Abstract

Compounds that impair the synthesis of either mitochondrial DNA (mtNDA) or mtDNA‐encoded proteins reduce the levels of 13 proteins essential for oxidative phosphorylation, leading to a decrease in mitochondrial ATP production. Toxicity caused by these compounds is seldom identified in 24 to 72 hr cytotoxicity assays due to the low turnover rates of both mtDNA and mtDNA‐encoded proteins. Here, we describe three high‐throughput screening assays that detect compounds that affect mtDNA‐encoded protein levels. All three assays measure the levels of two proteins, one a mtDNA‐encoded protein synthesized on mitochondrial ribosomes and the other, a nuclear DNA‐encoded protein synthesized on cytosolic ribosomes. The first assay measures the levels of these two proteins by quantitative image analysis and requires a high‐content imaging system. The second assay is an in‐cell immunoassay that utilizes infrared dyes for detection of the two proteins and, thus, requires a LI‐COR Odyssey system. The third assay is an in‐cell immunoassay that utilizes colorimetric detection of the two proteins and requires an absorbance microplate reader. Curr. Protoc. Toxicol. 48:3.11.1‐3.11.17. © 2011 by John Wiley & Sons, Inc.

Keywords: mitochondrial; ribosome; antibiotic; anti‐viral; high‐content screening

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: High‐Content Imaging Assay for Detecting Compounds that Affect mtDNA‐Encoded Protein Levels
  • Basic Protocol 2: Infrared In‐Cell Immunoassay for Detecting Compounds that Affect mtDNA‐Encoded Protein Levels
  • Alternate Protocol 1: Colorimetric In‐Cell ELISA for Detecting Compounds that Affect mtDNA‐Encoded Protein Levels
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: High‐Content Imaging Assay for Detecting Compounds that Affect mtDNA‐Encoded Protein Levels

  Materials
  • Cells to be tested (e.g., THLE‐2 cells, ATCC, #CRL‐2706)
  • Cell culture medium (see recipe)
  • Test compounds
  • Dimethyl sulfoxide (DMSO), pure
  • Phosphate‐buffered saline (PBS; Sigma, cat. no. D8537)
  • 4% (w/v) paraformaldehyde in PBS (see recipe)
  • 0.1% Triton X‐100 in PBS (see recipe)
  • 10% goat serum in PBS (see recipe)
  • Primary antibody solution I (see recipe)
  • 1% goat serum in PBS (see recipe)
  • Secondary antibody solution I (see recipe)
  • 4′,6‐diamidino‐2‐phenylindole dilactate (DAPI) solution (see recipe)
  • 8‐channel or 12‐channel aspirator
  • 12‐channel pipettor (2 to 20 µl)
  • 12‐channel pipettor (20 to 300 µl)
  • Collagen‐coated black‐walled clear‐bottom 96‐well plates (Becton Dickinson, cat. no. 356700)
  • 37°C, 5% CO 2, 95% humidified incubator
  • Light microscope
  • 96‐well plates with lids for diluting compounds
  • Parafilm
  • An automated fluorescence microscopic imaging system (e.g., Thermo Fisher Scientific Cellomics ArrayScan VTI High‐Content Screening Reader)
  • Excel
  • GraphPad Prism 5

Basic Protocol 2: Infrared In‐Cell Immunoassay for Detecting Compounds that Affect mtDNA‐Encoded Protein Levels

  Materials
  • Cells to be tested (e.g., HepG2 cells, ATCC, #HB‐8065)
  • Cell culture medium (see recipe)
  • Test compounds
  • Dimethyl sulfoxide (DMSO), pure
  • 4% paraformaldehyde in PBS (see recipe)
  • Phosphate‐buffered saline (PBS; Sigma, cat. no. D8537)
  • 0.1% Triton X‐100 in PBS (see recipe)
  • 20% blocking buffer in PBS (see recipe)
  • Primary antibody solution II (see recipe)
  • Secondary antibody solution II (see recipe)
  • 0.05% Tween‐20 in PBS (see recipe)
  • 70% ethanol
  • 0.3% (w/v) Janus Green stain in H 2O (see recipe)
  • Deionized water
  • 0.5 M HCl
  • 8‐channel or 12‐channel aspirator
  • 12‐channel pipettor (2 to 20 µl)
  • 12‐channel pipettor (20 to 300 µl)
  • Collagen‐coated black‐walled clear‐bottom 96‐well plates
  • 37°C, 5% CO 2, 95% humidified incubator
  • 96‐well plates with lids for diluting compounds
  • Parafilm
  • A LI‐COR Odyssey near‐infrared imaging system
  • Excel or GraphPad

Alternate Protocol 1: Colorimetric In‐Cell ELISA for Detecting Compounds that Affect mtDNA‐Encoded Protein Levels

  • Secondary antibody solution III for colorimetric detection: in place of fluorescent infrared‐labeled secondary antibodies, enzyme‐linked secondary antibodies are used.
  • Alkaline phosphatase (AP) development solution (12.5 mM pNPP, 0.1 M diethanolamine, 1 mM MgCl 2, pH 10)
  • Horseradish peroxidase (HRP) development solution (TMB solution)
  • An absorbance microplate reader (since this protocol describes a colorimetric assay, a near‐infrared imaging system is not required)
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Figures

Videos

Literature Cited

Literature Cited
   Birkus, G., Hitchcock, M.J., and Cihlar, T. 2002. Assessment of mitochondrial toxicity in human cells treated with tenofovir: Comparison with other nucleoside reverse transcriptase inhibitors. Antimicrob. Agents Chemother. 46:716‐723.
   Böttger, E.C. 2007. Antimicrobial agents targeting the ribosome: The issue of selectivity and toxicity ‐ lessons to be learned. Cell. Mol. Life Sci. 64:791‐795.
   Dykens, J.A., Marroquin, L.D., and Will, Y. 2007. Strategies to reduce late‐stage drug attrition due to mitochondrial toxicity. Expert Rev. Mol. Diagn. 7:161‐175.
   Gross, N.J., Getz, G.S., and Rabinowitz, M. 1969. Apparent turnover of mitochondrial deoxyribonucleic acid and mitochondrial phospholipids in the tissues of the rat. J. Biol. Chem. 244:1552‐1562.
   Hare, J.F. and Hodges, R. 1982. Turnover of mitochondrial inner membrane proteins in hepatoma monolayer cultures. J. Biol. Chem. 257:3575‐3580.
   Lewis, W., Day, B.J., and Copeland, W.C. 2003. Mitochondrial toxicity of NRTI antiviral drugs: An integrated cellular perspective. Nat. Rev. Drug Discov. 2:812‐822.
   Lundholt, B.K., Scudder, K.M., and Pagliaro, L. 2003. A simple technique for reducing edge effect in cell‐based assays. J. Biomol. Screen. 8:566‐570.
   McKee, E.E., Ferguson, M., Bentley, A.T., and Marks, T.A. 2006. Inhibition of mammalian mitochondrial protein synthesis by oxazolidinones. Antimicrob. Agents Chemother. 50:2042‐2049.
   Nadanaciva, S., Dillman, K., Gebhard, D.F., Shrikhande, A., and Will, Y. 2010. High‐content screening for compounds that affect mtDNA‐encoded protein levels in eukaryotic cells. J. Biomol. Screen. 15:937‐934
   Nagiec, E.E., Wu, L., Swaney, S.M., Chosay, J.G., Ross, D.E., Brieland, J.K., and Leach, K.L. 2005. Oxazolidinones inhibit cellular proliferation via inhibition of mitochondrial protein synthesis. Antimicrob. Agents Chemother. 49:3896‐3902.
   Pan‐Zhou, X.R., Cui, L., Zhou, X.J., Sommadossi, J.P., and Darley‐Usmar, V.M. 2000. Differential effects of antiretroviral nucleoside analogs on mitochondrial function in HepG2 cells. Antimicrob. Agents Chemother. 44:496‐503.
   Scheffler, I. 2008. Basic molecular biology of mitochondrial replication. In Drug‐Induced Mitochondrial Dysfunction (J.A. Dykens and Y. Will, eds.) pp. 37‐70. John Wiley & Sons, Hoboken, N.J.
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