Unit

UNIT 3.11 High-Throughput Assays for Assessing Mitochondrial Dysfunction Caused by Compounds that Impair mtDNA-Encoded Protein Levels in Eukaryotic Cells

  1. Sashi Nadanaciva1,
  2. James Murray2,
  3. Casey Wilson2,
  4. David F. Gebhard3,
  5. Yvonne Will1

Published Online: 1 MAY 2011

DOI: 10.1002/0471140856.tx0311s48

Current Protocols in Toxicology

Current Protocols in Toxicology

How to Cite

Nadanaciva, S., Murray, J., Wilson, C., Gebhard, D. F. and Will, Y. 2011. High-Throughput Assays for Assessing Mitochondrial Dysfunction Caused by Compounds that Impair mtDNA-Encoded Protein Levels in Eukaryotic Cells. Current Protocols in Toxicology. 48:3.11:3.11.1–3.11.17.

Author Information

  1. 1

    Compound Safety Prediction, Worldwide Medicinal Chemistry, Pfizer Inc., Groton, Connecticut

  2. 2

    MitoSciences Inc., Eugene, Oregon

  3. 3

    Primary Pharmacology, Research Center of Emphasis, Pfizer Inc., Groton, Connecticut

Publication History

  1. Published Online: 1 MAY 2011
  2. Published Print: MAY 2011

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