Gyda C. Beeson

A high-throughput respirometric assay for mitochondrial biogenesis and toxicity

Mitochondria are a common target of toxicity for drugs and other chemicals and result in decreased aerobic metabolism and cell death. In contrast, mitochondrial biogenesis restores cell vitality, and there is a need for new agents to induce biogenesis. Current cell-based models of mitochondrial biogenesis or toxicity are inadequate because cultured cell lines are highly glycolytic with minimal aerobic metabolism and altered mitochondrial physiology. In addition, there are no high-throughput real-time assays that assess mitochondrial function. We adapted primary cultures of renal proximal tubular cells (RPTCs) that exhibit in vivo levels of aerobic metabolism, are not glycolytic, and retain higher levels of differentiated functions and used the Seahorse Bioscience analyzer to measure mitochondrial function in real time in multiwell plates. Using uncoupled respiration as a marker of electron transport chain (ETC) integrity, the nephrotoxicants cisplatin, HgCl(2), and gentamicin exhibited mitochondrial toxicity prior to decreases in basal respiration and cell death. Conversely, using FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone)-uncoupled respiration as a marker of maximal ETC activity, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), SRT1720, resveratrol, daidzein, and metformin produced mitochondrial biogenesis in RPTCs. The merger of the RPTC model and multiwell respirometry results in a single high-throughput assay to measure mitochondrial biogenesis and toxicity and nephrotoxic potential.

Assessment of drug-induced mitochondrial dysfunction via altered cellular respiration and acidification measured in a 96-well platform.

High-throughput applicable screens for identifying drug-induced mitochondrial impairment are necessary in the pharmaceutical industry. Hence, we evaluated the XF96 Extracellular Flux Analyzer, a 96-well platform that measures changes in the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of cells. The sensitivity of the platform was bench-marked with known modulators of oxidative phosphorylation and glycolysis. Sixteen therapeutic agents were screened in HepG2 cells for mitochondrial effects. Four of these compounds, thiazolidinediones, were also tested in primary feline cardiomyocytes for cell-type specific effects. We show that the XF96 platform is a robust, sensitive system for analyzing drug-induced mitochondrial impairment in whole cells. We identified changes in cellular respiration and acidification upon addition of therapeutic agents reported to have a mitochondrial effect. Furthermore, we show that respiration and acidification changes upon addition of the thiazoldinediones were cell-type specific, with the rank order of mitochondrial impairment in whole cells being in accord with the known adverse effects of these drugs.

The β2-Adrenoceptor Agonist Formoterol Stimulates Mitochondrial Biogenesis

Mitochondrial dysfunction is a common mediator of disease and organ injury. Although recent studies show that inducing mitochondrial biogenesis (MB) stimulates cell repair and regeneration, only a limited number of chemicals are known to induce MB. To examine the impact of the β-adrenoceptor (β-AR) signaling pathway on MB, primary renal proximal tubule cells (RPTC) and adult feline cardiomyocytes were exposed for 24 h to multiple β-AR agonists: isoproterenol (nonselective β-AR agonist), (±)-(R*,R*)-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy] acetic acid sodium hydrate (BRL 37344) (selective β3-AR agonist), and formoterol (selective β2-AR agonist). The Seahorse Biosciences (North Billerica, MA) extracellular flux analyzer was used to quantify carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)-uncoupled oxygen consumption rate (OCR), a marker of maximal electron transport chain activity. Isoproterenol and BRL 37244 did not alter mitochondrial respiration at any of the concentrations examined. Formoterol exposure resulted in increases in both FCCP-uncoupled OCR and mitochondrial DNA (mtDNA) copy number. The effect of formoterol on OCR in RPTC was inhibited by the β-AR antagonist propranolol and the β2-AR inverse agonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol hydrochloride (ICI-118,551). Mice exposed to formoterol for 24 or 72 h exhibited increases in kidney and heart mtDNA copy number, peroxisome proliferator-activated receptor γ coactivator 1α, and multiple genes involved in the mitochondrial electron transport chain (F0 subunit 6 of transmembrane F-type ATP synthase, NADH dehydrogenase subunit 1, NADH dehydrogenase subunit 6, and NADH dehydrogenase [ubiquinone] 1β subcomplex subunit 8). Cheminformatic modeling, virtual chemical library screening, and experimental validation identified nisoxetine from the Sigma Library of Pharmacologically Active Compounds and two compounds from the ChemBridge DIVERSet that increased mitochondrial respiratory capacity. These data provide compelling evidence for the use and development of β2-AR ligands for therapeutic MB.

LCL124, a Cationic Analog of Ceramide, Selectively Induces Pancreatic Cancer Cell Death by Accumulating in Mitochondria

Treatment of pancreatic cancer that cannot be surgically resected currently relies on minimally beneficial cytotoxic chemotherapy with gemcitabine. As the fourth leading cause of cancer-related death in the United States with dismal survival statistics, pancreatic cancer demands new and more effective treatment approaches. Resistance to gemcitabine is nearly universal and appears to involve defects in the intrinsic/mitochondrial apoptotic pathway. The bioactive sphingolipid ceramide is a critical mediator of apoptosis initiated by a number of therapeutic modalities. It is noteworthy that insufficient ceramide accumulation has been linked to gemcitabine resistance in multiple cancer types, including pancreatic cancer. Taking advantage of the fact that cancer cells frequently have more negatively charged mitochondria, we investigated a means to circumvent resistance to gemcitabine by targeting delivery of a cationic ceramide (l-t-C6-CCPS [LCL124: ((2S,3S,4E)-2-N-[6′-(1″-pyridinium)-hexanoyl-sphingosine bromide)]) to cancer cell mitochondria. LCL124 was effective in initiating apoptosis by causing mitochondrial depolarization in pancreatic cancer cells but demonstrated significantly less activity against nonmalignant pancreatic ductal epithelial cells. Furthermore, we demonstrate that the mitochondrial membrane potentials of the cancer cells were more negative than nonmalignant cells and that dissipation of this potential abrogated cell killing by LCL124, establishing that the effectiveness of this compound is potential-dependent. LCL124 selectively accumulated in and inhibited the growth of xenografts in vivo, confirming the tumor selectivity and therapeutic potential of cationic ceramides in pancreatic cancer. It is noteworthy that gemcitabine-resistant pancreatic cancer cells became more sensitive to subsequent treatment with LCL124, suggesting that this compound may be a uniquely suited to overcome gemcitabine resistance in pancreatic cancer.

Systematic Study of Mitochondrial Toxicity of Environmental Chemicals Using Quantitative High Throughput Screening

A goal of the Tox21 program is to transit toxicity testing from traditional in vivo models to in vitro assays that assess how chemicals affect cellular responses and toxicity pathways. A critical contribution of the NIH Chemical Genomics center (NCGC) to the Tox21 program is the implementation of a quantitative high throughput screening (qHTS) approach, using cell- and biochemical-based assays to generate toxicological profiles for thousands of environmental compounds. Here, we evaluated the effect of chemical compounds on mitochondrial membrane potential in HepG2 cells by screening a library of 1,408 compounds provided by the National Toxicology Program (NTP) in a qHTS platform. Compounds were screened over 14 concentrations, and results showed that 91 and 88 compounds disrupted mitochondrial membrane potential after treatment for 1 or 5 h, respectively. Seventy-six compounds active at both time points were clustered by structural similarity, producing 11 clusters and 23 singletons. Thirty-eight compounds covering most of the active chemical space were more extensively evaluated. Thirty-six of the 38 compounds were confirmed to disrupt mitochondrial membrane potential using a fluorescence plate reader, and 35 were confirmed using a high content imaging approach. Among the 38 compounds, 4 and 6 induced LDH release, a measure of cytotoxicity, at 1 or 5 h, respectively. Compounds were further assessed for mechanism of action (MOA) by measuring changes in oxygen consumption rate, which enabled the identification of 20 compounds as uncouplers. This comprehensive approach allows for the evaluation of thousands of environmental chemicals for mitochondrial toxicity and identification of possible MOAs.

Mitochondria-Targeted Catalase Reverts the Neurotoxicity of hSOD1G93A Astrocytes without Extending the Survival of ALS-Linked Mutant hSOD1 Mice

Dominant mutations in the Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons. The molecular mechanism underlying the toxic gain-of-function of mutant hSOD1s remains uncertain. Several lines of evidence suggest that toxicity to motor neurons requires damage to non-neuronal cells. In line with this observation, primary astrocytes isolated from mutant hSOD1 over-expressing rodents induce motor neuron death in co-culture. Mitochondrial alterations have been documented in both neuronal and glial cells from ALS patients as well as in ALS-animal models. In addition, mitochondrial dysfunction and increased oxidative stress have been linked to the toxicity of mutant hSOD1 in astrocytes and neurons. In mutant SOD1-linked ALS, mitochondrial alterations may be partially due to the increased association of mutant SOD1 with the outer membrane and intermembrane space of the mitochondria, where it can affect several critical aspects of mitochondrial function. We have previously shown that decreasing glutathione levels, which is crucial for peroxide detoxification in the mitochondria, significantly accelerates motor neuron death in hSOD1G93A mice. Here we employed a catalase targeted to the mitochondria to investigate the effect of increased mitochondrial peroxide detoxification capacity in models of mutant hSOD1-mediated motor neuron death. The over-expression of mitochondria-targeted catalase improved mitochondrial antioxidant defenses and mitochondrial function in hSOD1G93A astrocyte cultures. It also reverted the toxicity of hSOD1G93A-expressing astrocytes towards co-cultured motor neurons, however ALS-animals did not develop the disease later or survive longer. Hence, while increased oxidative stress and mitochondrial dysfunction have been extensively documented in ALS, these results suggest that preventing peroxide-mediated mitochondrial damage alone is not sufficient to delay the disease.

High-throughput respirometric assay identifies predictive toxicophore of mitochondrial injury

Many environmental chemicals and drugs negatively affect human health through deleterious effects on mitochondrial function. Currently there is no chemical library of mitochondrial toxicants, and no reliable methods for predicting mitochondrial toxicity. We hypothesized that discrete toxicophores defined by distinct chemical entities can identify previously unidentified mitochondrial toxicants. We used a respirometric assay to screen 1760 compounds (5 μM) from the LOPAC and ChemBridge DIVERSet libraries. Thirty-one of the assayed compounds decreased uncoupled respiration, a stress test for mitochondrial dysfunction, prior to a decrease in cell viability and reduced the oxygen consumption rate in isolated mitochondria. The mitochondrial toxicants were grouped by chemical similarity and two clusters containing four compounds each were identified. Cheminformatic analysis of one of the clusters identified previously uncharacterized mitochondrial toxicants from the ChemBridge DIVERSet. This approach will enable the identification of mitochondrial toxicants and advance the prediction of mitochondrial toxicity for both drug discovery and risk assessment.

Promoting Thiol Expression Increases the Durability of Antitumor T-cell Functions

Ex vivo-expanded CD8(+) T cells used for adoptive immunotherapy generally acquire an effector memory-like phenotype (TEM cells). With regard to therapeutic applications, two undesired features of this phenotype in vivo are limited persistence and reduced antitumor efficacy, relative to CD8(+) T cells with a central memory-like phenotype (TCM cells). Furthermore, there is incomplete knowledge about all the differences between TEM and TCM cells that may influence tumor treatment outcomes. Given that TCM cells survive relatively longer in oxidative tumor microenvironments, we investigated the hypothesis that TCM cells possess relatively greater antioxidative capacity than TEM cells. Here, we report that TCM cells exhibit a relative increase compared with TEM cells in the expression of cell surface thiols, a key target of cellular redox controls, along with other antioxidant molecules. Increased expression of redox regulators in TCM cells inversely correlated with the generation of reactive oxygen and nitrogen species, proliferative capacity, and glycolytic enzyme levels. Notably, T-cell receptor-transduced T cells pretreated with thiol donors, such as N-acetyl cysteine or rapamycin, upregulated thiol levels and antioxidant genes. A comparison of antitumor CD8(+) T-cell populations on the basis of surface thiol expression showed that thiol-high cells persisted longer in vivo and exerted superior tumor control. Our results suggest that higher levels of reduced cell surface thiols are a key characteristic of T cells that can control tumor growth and that profiling this biomarker may have benefits to adoptive T-cell immunotherapy protocols.

Assessment of ToxCast Phase II for Mitochondrial Liabilities Using a High-Throughput Respirometric Assay

Previous high-throughput screens to identify mitochondrial toxicants used immortalized cell lines and focused on changes in mitochondrial membrane potential, which may not be sufficient and do not identify different types of mitochondrial dysfunction. Primary cultures of renal proximal tubule cells (RPTC) were examined with the Seahorse Extracellular Flux Analyzer to screen 676 compounds (5 μM; 1 h) from the ToxCast Phase II library for mitochondrial toxicants. Of the 676 compounds, 19 were classified as cytotoxicants, 376 were electron transport chain (ETC) inhibitors, and 5 were uncouplers. The remaining 276 compounds were examined after a 5-h exposure to identify slower acting mitochondrial toxicants. This experiment identified 3 cytotoxicants, 110 ETC inhibitors, and 163 compounds with no effect. A subset of the ToxCast Phase II library was also examined in immortalized human renal cells (HK2) to determine differences in susceptibility to mitochondrial toxicity. Of the 131 RPTC ETC inhibitors tested, only 14 were ETC inhibitors in HK2 cells. Of the 5 RPTC uncouplers, 1 compound was an uncoupler in HK2 cells. These results demonstrate that 73% (491/676) of the compounds in the ToxCast Phase II library compounds exhibit RPTC mitochondrial toxicity, overwhelmingly ETC inhibition. In contrast, renal HK2 cells are markedly less sensitive and only identified 6% of the compounds as mitochondrial toxicants. We suggest caution is needed when studying mitochondrial toxicity in immortalized cell lines. This information will provide mechanisms and chemical-based criteria for assessing and predicting mitochondrial liabilities of new drugs, consumer products, and environmental agents.

Integrin-Dependent Akt1 Activation Regulates PGC-1 Expression and Fatty Acid Oxidation

Background: Poly-N-acetyl glucosamine nanofibers derived from a marine diatom have been used to increase cutaneous wound healing. These nanofibers exert their activity by specifically activating integrins, which makes them a useful tool for dissecting integrin-mediated pathways. We have shown that short-fiber poly-N-acetyl glucosamine nanofiber (sNAG) treatment of endothelial cells results in increased cell motility and metabolic rate in the absence of increased cell proliferation. Results: Using a Seahorse Bioanalyzer to measure oxygen consumption in real time, we show that sNAG treatment increases oxygen consumption rates, correlated with an integrin-dependent activation of Akt1. Akt1 activation leads to an increase in the expression of the transcriptional coactivator, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). This is not due to increased mitochondrial biogenesis, but is associated with an increase in the expression of pyruvate dehydrogenase kinase 4 (PDK4), suggesting regulation of fatty acid oxidation. Blockade of fatty acid oxidation with etomoxir, an O-carnitine palmitoyltransferase-1 inhibitor, blocks the sNAG-dependent increased oxygen consumption. 3H-palmitate uptake experiments indicate a PDK4-dependent increase in fatty acid oxidation, which is required for nanofiber-induced cell motility. Conclusions: Our findings imply a linear pathway whereby an integrin-dependent activation of Akt1 leads to increased PGC-1α and PDK4 expression resulting in increased energy production by fatty acid oxidation.