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ToxSci Advance Access originally published online on April 25, 2006
Toxicological Sciences 2006 92(1):186-200; doi:10.1093/toxsci/kfj208
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© The Author 2006. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Investigation of Drug-Induced Mitochondrial Toxicity Using Fluorescence-Based Oxygen-Sensitive Probes

James Hynes*,1, Lisa D. Marroquin{dagger},1, Vladimir I. Ogurtsov*, Katerina N. Christiansen{dagger}, Gregory J. Stevens{dagger}, Dmitri B. Papkovsky* and Yvonne Will{dagger},2

* Luxcel Biosciences Ltd., G.17, Lee Maltings, Cork, Ireland; and {dagger} Pfizer Global R&D, Safety Sciences, 10646 Science Centre Drive, San Diego, California 92121

Received January 23, 2006; accepted April 19, 2006

Mitochondrial dysfunction is a common mechanism of drug-induced toxicity. Early identification of new chemical entities (NCEs) that perturb mitochondrial function is of significant importance to avoid attrition in later stages of drug development. One of the most informative ways of assessing mitochondrial dysfunction is by measuring mitochondrial oxygen consumption. However, the conventional polarographic method of measuring oxygen consumption is not amenable to high sample throughput or automation. We present an alternative, low-bulk, high-throughput approach to the analysis of isolated-mitochondrial oxygen consumption using luminescent oxygen-sensitive probes. These probes are dispensable and are analyzed in standard microtitre plates on a fluorescence plate reader. Respiratory substrate and adenosine diphosphate (ADP) dependencies of mitochondrial oxygen consumption were assessed using the fluorescence-based method, and results compared favourably to conventional polarographic analysis. To assess assay performance, the method was then applied to the analysis of a panel of classical modulators of oxidative phosphorylation. The effect of uncoupler concentration was analyzed in detail to identify factors which would be important in applying this method to large scale NCE screening and mechanistic investigations. Results demonstrate that the 96-well format can accommodate up to ~ 200 compounds/day at a single concentration or alternatively IC50 values can be generated for ~ 25 compounds. Throughput may be increased by moving to a 384-well plate format.

Key Words: mitochondria; respiration; polarimetry; fluorescence; oxygen-sensitive probes; oxygen-dependent enzymes; HTS; toxicity; drug safety.


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