James Hynes

Analysis of mitochondrial function using phosphorescent oxygen-sensitive probes

Mitochondrial dysfunction has been associated with a variety of currently marketed therapeutics and has also been implicated in many disease states. Alterations in the rate of oxygen consumption are an informative indicator of mitochondrial dysfunction, but the use of such assays has been limited by the constraints of traditional measurement approaches. Here, we present a high-throughput, fluorescence-based methodology for the analysis of mitochondrial oxygen consumption using a phosphorescent oxygen-sensitive probe, standard microtitre plates and plate reader detection. The protocol describes the isolation of mitochondria from animal tissue, initial establishment and optimization of the oxygen consumption assay, subsequent screening of compounds for mitochondrial toxicity (uncoupling and inhibition), data analysis and generation of dose-response curves. It allows dozens of compounds (or hundreds of assay points) to be analyzed in a single day, and can be further up-scaled, automated and adapted for other enzyme- and cell-based screening applications.

Effect of the Multitargeted Tyrosine Kinase Inhibitors Imatinib, Dasatinib, Sunitinib, and Sorafenib on Mitochondrial Function in Isolated Rat Heart Mitochondria and H9c2 Cells

Cardiovascular disease has recently been suggested to be a significant complication of cancer treatment with several kinase inhibitors. In some cases, the mechanisms leading to cardiotoxicity are postulated to include mitochondrial dysfunction, either as a primary or secondary effect. Detecting direct effects on mitochondrial function, such as uncoupling of oxidative phosphorylation or inhibition of electron transport chain components, as well as identifying targets within the mitochondrial electron transport chain, can be accomplished in vitro. Here, we examined the effects of the tyrosine kinase inhibitor drugs imatinib, dasatinib, sunitinib, and sorafenib on ATP content in H9c2 cells grown under conditions where cells are either glycolytically or aerobically poised. Furthermore, we measured respiratory capacity of isolated rat heart mitochondria in the presence of the four kinase inhibitors and examined their effect on each of the oxidative phosphorylation complexes. Of the four kinase inhibitors examined, only sorafenib directly impaired mitochondrial function at clinically relevant concentrations, potentially contributing to the cytotoxic effect of the drug. For the other three kinase inhibitors lacking direct mitochondrial effects, altered kinase and other signaling pathways, are a more reasonable explanation for potential toxicity.

Mitochondrial respiration protects against oxygen-associated DNA damage

Oxygen is not only required for oxidative phosphorylation but also serves as the essential substrate for the formation of reactive oxygen species (ROS), which is implicated in ageing and tumorigenesis. Although the mitochondrion is known for its bioenergetic function, the symbiotic theory originally proposed that it provided protection against the toxicity of increasing oxygen in the primordial atmosphere. Using human cells lacking Synthesis of Cytochrome c Oxidase 2 (SCO2-/-), we have tested the oxygen toxicity hypothesis. These cells are oxidative phosphorylation defective and glycolysis dependent; they exhibit increased viability under hypoxia and feature an inverted growth response to oxygen compared with wild-type cells. SCO2-/- cells have increased intracellular oxygen and nicotinamide adenine dinucleotide (NADH) levels, which result in increased ROS and oxidative DNA damage. Using this isogenic cell line, we have revealed the genotoxicity of ambient oxygen. Our study highlights the importance of mitochondrial respiration both for bioenergetic benefits and for maintaining genomic stability in an oxygen-rich environment.

Fluorescence-Based Cell Viability Screening Assays Using Water-Soluble Oxygen Probes

A simple luminescence-based assay for screening the viability of mammalian cells is described, based on the monitoring of cell respiration by means of a phosphorescent water-soluble oxygen probe that responds to changes in the concentration of dissolved oxygen by changing its emission intensity and lifetime. The probe was added at low concentrations (0.3 μM to 0.5 nM) to each sample containing a culture of cells in the wells of a standard 96-well plate. Analysis of oxygen consumption was initiated by applying a layer of mineral oil on top of each sample followed by monitoring of the phosphorescent signal on a prompt or time-resolved fluorescence plate reader. Rates of oxygen uptake could be determined on the basis of kinetic changes of the phosphorescence (initial slopes) and correlated with cell numbers (105 to 107 cells/mL for FL5.12 lymphoblastic cell line), cell viability, or drug/effector action using appropriate control samples. The assay is cell noninvasive, more simple, robust, and cost-effective than existing microplate-based cell viability assays; is compatible with existing instrumentation; and allows for high-throughput analysis of cell viability. (Journal of Biomolecular Screening 2003:264-272)

Phosphorescent oxygen-sensitive materials for biological applications

A number of macromolecular probes employing different carriers and a number of microparticular probes employing different oxygen sensitive dyes were fabricated, giving a panel of oxygen sensitive probes. The photophysical and oxygen sensing properties of these probes were examined comparatively. The probes were used successfully to monitor cellular oxygen uptake and their ability to overcome a number of problems associated with oxygen sensing in biological samples was assessed. Macromolecular probes proved sufficient in a number of cases, particularly where spectral solutions can resolve the interferences. However where physical interactions cause interference the added protection of the polymer in the particle based probes was required.

A high-throughput dual parameter assay for assessing drug-induced mitochondrial dysfunction provides additional predictivity over two established mitochondrial toxicity assays

Mitochondrial toxicity is a major reason for safety-related compound attrition and post-market drug withdrawals, highlighting the necessity for higher-throughput screens that can identify this mechanism of toxicity during the early stages of drug discovery. Here, we present the validation of a 384-well dual parameter plate-based assay capable of measuring oxygen consumption and extracellular acidification in intact cells simultaneously. The assay showed good reproducibility and robustness and is suitable for use with both suspension cells and adherent cells. To determine if the assay provides additional value in detecting mitochondrial toxicity over existing platforms, 200 commercially available drugs were tested in the assay using HL60 suspension cells as well as in two conventional mitochondrial toxicity assays: an oxygen consumption assay that uses isolated mitochondria and a cell-based assay that uses HepG2 cells grown in glucose and galactose media. The combination of the dual parameter assay and the isolated mitochondrial oxygen consumption assay identified more compounds that caused mitochondrial impairment than any other combination of the three assays or each of the three assays on its own. Furthermore, novel information was obtained from the dual parameter assay on drugs not previously reported to cause mitochondrial impairment.

Respirometric Screening Technology for ADME-Tox studies

Analysis of mitochondrial dysfunction is of particular importance in drug development, as it has been implicated in many common diseases and therapeutic treatments. Here, the markers of mitochondrial function and toxicity are reviewed, as well as current methods of assessment, with particular emphasis on oxygen respirometry. Fluorescence-based Respirometric Screening Technology (RST) allows convenient high-throughput analysis of oxygen consumption by cells, isolated mitochondria, enzymes, tissues and organisms, and is, therefore, of high value for such studies and general absorption, distribution, metabolism and excretion and toxicology studies. Various RST assay formats are described and specific applications are discussed. Consideration is also given to the future potential of this analytical approach.

Analysis of activity and inhibition of oxygen-dependent enzymes by optical respirometry on the LightCycler system.

There is currently a need for a method capable of measuring the activity and inhibition of biologically relevant oxygenases in a format that enables the convenient, fast, and cost-efficient generation of dose-response information. Here we describe a low-volume luminescence-based assay for the measurement of such oxygen-dependent enzymes. The assay employs a photoluminescent oxygen-sensitive probe and glass capillary microcuvettes measured on the Roche LightCycler detection platform. Three discrete types of oxygen probe were evaluated for this application: (i) solid-state coatings, (ii) soluble macromolecular MitoXpress probe, both phosphorescent porphyrin-based, and (iii) a luminescent Ir(III)-based nanoparticle probe. Measurement parameters were optimised and subsequently applied to the analysis of three biologically relevant oxygenases, namely cytochrome P450 (CYP), monoamine oxygenase (MAO), and cyclooxygenase (COX). CYP enzymes are central players in drug detoxification while specific inhibitors of MAO and COX are important for therapeutic intervention and treatment of neurological and inflammatory diseases, respectively. To determine assay utility, oxygen consumption catalysed by all three enzyme types was measured and the effect of specific inhibitors determined. The panel included the MAO-A/B inhibitors clorgyline, toloxatone, deprenyl, and the COX-1/2 inhibitors niflumic acid, nimesulide, SC-560, ketoprofen, and phenylbutazone. IC(50) values were then compared with literature values. The measurement methodology described allows the low-volume analysis of biologically relevant oxygenases and displays the requisite sensitivity and throughput to facilitate routine analysis. It is also applicable to other O(2)-dependent enzymes and enzymatic systems.

Fluorescent pH and Oxygen Probes of the Assessment of Mitochondrial Toxicity in Isolated Mitochondria and Whole Cells

Oxygen consumption measurements provide information on the activity of the mitochondrial electron transport chain and such measurements can be carried out on both isolated mitochondria and whole cells. Additional information is available on glycolytic metabolism by assessing extracellular acidification. The protocols in this unit describe how recently developed fluorescent pH‐ and O2‐sensitive probes can be used as tools for the assessment of mitochondrial toxicity and metabolic function in standard microtiter plates. In addition, protocols for the isolation of mitochondria from different tissue sources are provided. Curr. Protoc. Toxicol. 40:2.16.1‐2.16.22.

Phosphorescent oxygen-sensitive probes

O2-sensitive probes based on phosphorescent metalloporphyrins.- O2 analysis on a fluorescence spectrometer or plate reader.- O2 Imaging in biological specimens.