title: A systematic assessment of mitochondrial function identified novel signatures for drug-induced mitochondrial disruption in cells.
abstract: Mitochondrial perturbation has been recognized as a contributing factor to various drug-induced organ toxicities. To address this issue, we developed a high-throughput flow cytometry-based mitochondrial signaling assay to systematically investigate mitochondrial/cellular parameters known to be directly impacted by mitochondrial dysfunction: mitochondrial membrane potential (MMP), mitochondrial reactive oxygen species (ROS), intracellular reduced glutathione (GSH) level, and cell viability. Modulation of these parameters by a training set of compounds, comprised of established mitochondrial poisons and 60 marketed drugs (30 nM to 1mM), was tested in HL-60 cells (a human pro-myelocytic leukemia cell line) cultured in either glucose-supplemented (GSM) or glucose-free (containing galactose/glutamine; GFM) RPMI-1640 media. Post-hoc bio-informatic analyses of IC50 or EC50 values for all parameters tested revealed that MMP depolarization in HL-60 cells cultured in GSM was the most reliable parameter for determining mitochondrial dysfunction in these cells. Disruptors of mitochondrial function depolarized MMP at concentrations lower than those that caused loss of cell viability, especially in cells cultured in GSM; cellular GSH levels correlated more closely to loss of viability in vitro. Some mitochondrial respiratory chain inhibitors increased mitochondrial ROS generation; however, measuring an increase in ROS alone was not sufficient to identify mitochondrial disruptors. Furthermore, hierarchical cluster analysis of all measured parameters provided confirmation that MMP depletion, without loss of cell viability, was the key signature for identifying mitochondrial disruptors. Subsequent classification of compounds based on ratios of IC50s of cell viability:MMP determined that this parameter is the most critical indicator of mitochondrial health in cells and provides a powerful tool to predict whether novel small molecule entities possess this liability.
authors.affiliation: Department of Comparative Biology and Safety Sciences, Amgen, Amgen Court West 1201, Seattle, Washington 98119.
authors.forename: Nianyu
authors.initials: N
authors.lastname: Li
authors.affiliation: MitoSciences Inc., Eugene, Oregon 97403.
authors.forename: Elisa
authors.initials: E
authors.lastname: Oquendo
authors.affiliation: MitoSciences Inc., Eugene, Oregon 97403.
authors.forename: Roderick A
authors.initials: RA
authors.lastname: Capaldi
authors.affiliation: Purdue University Cytometry Laboratories, Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana 47907.
authors.forename: J Paul
authors.initials: JP
authors.lastname: Robinson
authors.affiliation: Department of Comparative Biology and Safety Sciences, Amgen, Amgen Court West 1201, Seattle, Washington 98119.
authors.forename: Yudong D
authors.initials: YD
authors.lastname: He
authors.affiliation: Department of Comparative Biology and Safety Sciences, Amgen, 1 Amgen Center Dr, Thousand Oaks, California 91320-1799.
authors.forename: Hisham K
authors.initials: HK
authors.lastname: Hamadeh
authors.affiliation: Department of Comparative Biology and Safety Sciences, Amgen, 1 Amgen Center Dr, Thousand Oaks, California 91320-1799.
authors.forename: Cynthia A
authors.initials: CA
authors.lastname: Afshari
authors.affiliation: Department of Comparative Biology and Safety Sciences, Amgen, 1 Amgen Center Dr, Thousand Oaks, California 91320-1799.
authors.forename: Ruth
authors.initials: R
authors.lastname: Lightfoot-Dunn
authors.affiliation: Department of Comparative Biology and Safety Sciences, Amgen, Amgen Court West 1201, Seattle, Washington 98119 padman@amgen.com.
authors.forename: Padma Kumar
authors.initials: PK
authors.lastname: Narayanan
mesh_terms: D018929:Cell Culture Techniques; D002470:Cell Survival / Q000187:drug effects; D005434:Flow Cytometry; D005978:Glutathione / Q000378:metabolism*; D018922:HL-60 Cells; D015386:Hazardous Substances / Q000737:chemistry / Q000633:toxicity*; D057166:High-Throughput Screening Assays; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D015386:Hazardous Substances; D017382:Reactive Oxygen Species; D005978:Glutathione
keywords: chemicals; drugs; hazard identification; mitochondria; mitochondrial membrane potential; reactive oxygen species; redox; screening