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

Abstract

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 approximately 200 compounds/day at a single concentration or alternatively IC(50) values can be generated for approximately 25 compounds. Throughput may be increased by moving to a 384-well plate format.

Peroxiredoxin-6 protects against mitochondrial dysfunction and liver injury during ischemia-reperfusion in mice

“…Hepatocyte mitochondria were isolated as previously outlined with minor modifications (9,18,43). All steps were carried out at 4°C.…”

Decreasedin vitroMitochondrial Function is Associated with Enhanced Brain Metabolism, Blood Flow, and Memory in Surfl-Deficient Mice

“…11 Briefly, mitochondria suspended in ROS buffer with 0.3% bovine serum albumin were used and glutamate (5 mmol/L)/malate (5 mmol/L) was the respiratory substrate. 12 State 3 respiration was induced with ADP (0.3 mmol/L).…”

Sensing intracellular oxygen using near-infrared phosphorescent probes and live-cell fluorescence imaging

“…High-resolution fluorescent imaging facilitates measuring changes in oxygen within individual cells, whereas the microplate-based systems are only applicable to large populations of cells (10 3 cells/well or greater in a sealed compartment) with end-point parameter readout (14). Furthermore, the kinetics of local changes in intracellular oxygen are much faster than the formation of global oxygen gradients in bulk sample, which eliminates the need for exclusion of ambient oxygen and sealing test samples (1).…”

“…A similar principle has been applied to develop simple formats for measuring biological oxygen consumption in microtiter plates on a fluorescent plate reader using phosphorescent solid-state oxygen sensors (18,27) and water-soluble probes (11). These systems, which can measure oxygen consumption noninvasively, on a microscale and with high sample throughput, have been demonstrated with bacteria (18), isolated mitochondria (14), cell lines (12), and small organisms (26). All these, however, rely on measurement "extracellular" oxygen in relatively large samples.…”