Multiplex Detection of Oxidative Phosphorylation as a Mechanistic Indicator for Drug Induced Mitochondrial Toxicity

Summary

o The MIILIPLEX MAG Human OXPHOS 6-Plex Panel enables multiplex simultaneous detection of respiratory chain complexes a mechanistic indicator for mitochondrial toxicity.

o The OXPHOS quantification profile correlated well with pre-established complex activity and cellular oxygen consumption data (data not shown).

o The MIILIPLEX MAG Human OXPHOS 6-Plex Panel detected mitochondrial toxicity induced by antibacterial and antiviral drugs, and a new class of anti diabetes drugs, the glitazones.

o The data suggest that the MIILIPLEX MAG Human OXPHOS

6-Plex Panel is suitable as a novel safety screen tool to identify potential off-target effects on mitochondrial respiratory chain complexes for new antiviral, antibiotic and other drug molecule leads.

Results

Methods Instrument: Luminex 200 reader with xPONENT data acquisition software; BioTek ELX405 (catalog # 40-015) plate washer for magnetic beads. Microspheres: Superparamagnetic MagPlex® microspheres were purchased from Luminex. Capture antibodies were covalently coupled to the carboxylate-modified microsphere beads using 1-ethyl-3-(3-dimethylaminopropyl)-carbadiimide hydrochloride (EDC) and N-hydroxy-sulfosuccimide (Sulfo-NHS) according to the manufacturer’s instructions. Assay Procedure: Each multiplex assay is performed in a solid flat-bottom 96-well plate. Customer can also use Millipore MultiScreen HTSTM, BV, 96-well filter plate as an alternative if using Millipore vacuum pump (catalog # WP6111560) and Millipore MultiScreen® RESIST vacuum manifold (catalog # MAVM0960R). Wells of the 96-well plate are blocked with assay buffer, followed by incubation of samples/positive control in lysis buffer and beads for 2h at room temperature. After washing, biotinylated detection antibodies were added and incubated for 1h at room temperature. After washing, Streptavidin-Phycoerytrin conjugate was added and incubated for 30 min. After washing, plate was read with the Luminex instrument. Samples: HepG2 hepatocellular carcinoma cells were cultured to 95% confluency then treated with either 20 µM of chloramphenicol for 6 days, or 20 µM of ddC (2'-3'-dideoxycytidine) for 6 days, during which the cells were split in conditioned media if necessary. In addition, HepG2 cells were cultured and treated with two members of the thiazolidinediones anti-diabetes drugs, rosiglitazone (50 µM for 2 days) and troglitazone (20 µM for 2 days). DMSO mock-treated cells were used as control. At the end of the treatment, cell lysate was harvested. Equal amounts of cell lysate were analyzed in triplicate with the Human OXPHOS Panel (6-Plex) to determine the quantity of each analyte. Drug induced mitochondrial toxicity was evaluated by normalizing the quantity of each analyte to mock-treated cells as a percentage.

Introduction Drug-induced mitochondrial toxicity is a problem that is receiving growing recognition. Many, widely prescribed therapeutics have "off-target" effects on mitochondria that impair function through several mechanisms. Growing evidence shows that mitochondrial malfunction is linked to several chronic diseases, such as diabetes, cancer and neurodegenerative disorders. Oxidative phosphorylation (OXPHOS) produces more than 95% of the conserved cellular energy in the form of ATP under normal conditions. This process involves 5 different protein complexes, NADH –ubiquinone oxidoreductase (Complex I), succinate ubiquinone oxidoreductase (Complex II), ubiquinone cytochrome c oxidoreductase (complex III), cytochrome c oxidase (Complex IV) and the ATP synthase (Complex V). Nicotinamide nucleotide transhydrogenase (NNT), a nuclear-encoded mitochondrial protein is involved in detoxification of reactive oxygen species (ROS). Many drug classes cause mitochondrial toxicity: • Inhibitors of the electron transport chain, e.g., fibrates. statins, glitazones (thiazolidinediones). • Inhibitors of mitochondrial protein synthesis, e.g. antibiotics • Depletion of mtDNA: Nuceloside analogs, e.g., acido- thymidine (Retrovir) , dideoxycytidine; ddC (Zalcitabine), Abacavir (Ziagen). To reduce late-stage drug attrition due to mitochondrial toxicity, there is a growing need for assay tools that allow convenient measurement of mitochondrial key proteins in the early stage of drug development process. Recently, based on the Luminex® xMAP® Technology, we developed the Mitochondrial Toxicity Oxidative Phosphorylation 6-plex Panel using the MILLIPLEX MAG magnetic bead format which features ease of automation and good performance for all analytes. We established the accuracy, reproducibility and insights provided by this assay in the studies of the safety profile of various classes of drugs on HepG2 cells: dideoxycytidine (ddC) (antiretroviral drug), chloramphenicol (antibiotic), and gliatazones (anti-diabetes drugs).

Human OXPHOS 6-Plex Control Curve

The M logo is a trademark of Merck KGaA, Darmstadt, Germany. © 2011 Millipore Corporation. All rights reserved. www.millipore.com

Mitochondria

Human OXPHOS Panel: Control Curve

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Wei Zheng1, Lothar Goretzki1, Gabriela Korzus1, Jue Wang1, James Murray2, Rod Capaldi2

1EMD Millipore, San Diego, CA; 2Mitosciences, Eugene, OR

Chloramphenicol Causes Mitochondrial Toxicity in HepG2 Cells

Figure 2. Significant reduction of Complex I and IV of OXPHOS in antibiotics induced mitochondrial toxicity.. Equal amount of cell lysates were analyzed in triplicates with the MILLIPLEX MAG Human OXPHOS 6-Plex Panel (left graph) the ELISAs (right graph) to determine the quantity of each analyte within OXPHOS. Drug induced mitochondrial toxicity was evaluated by normalizing the quantity of each analyte to mock-treated cells as a percentage. The data from the OXPHOS panel are consistent with the rrespective ELISAs as well as those from activity assay and the cellular oxygen consumption study (data not shown). The results indicate that the antibiotic chloramphenicol clearly induced mitochondrial toxicity as demonstrated by significant reduction of Complex I and IV while Complex II, III, V and NNT remained relatively stable.

Figure 1. Postive Control Curves. Lyophilized Control (HepG2 lysate) was calibrated to the known concentration for each analyte from highly purified human heart.

The mammalian mitochondrial genome and its protein-coding gene repertoire involved in the oxidative phosphorylation pathway (© 2008 da Fonseca et al; licensee BioMed Central Ltd).

Mitochondrial Toxicity in HepG2 Cells Induced by Thiazolidinediones

Figure 4. Comparison of mitochondrial toxicity induced by rosiglitazone and troglitazone/ The MILLIPLEX MAG Human OXPHOS 6-Plex Panel showed that Complex I and Complex IV is significantly reduced in troglitazone-treated cells (upper graph), whereas rosiglitazone-treated cells (lower graph) did not exhibit any reduction in Complex I. Inferring from this experiment, troglitazone is more mitochondria-toxic than rosiglitazone. Interestingly, troglitazone (aka Rezulin) has been taken off the market due to severe hepatotoxicity, whereas rosiglitazone (aka Avandia) is still on the market to treat type 2 diabetes, but with safety restrictions.

Abstract # 1896

Figure 3. Significant reduction of Complex I, III and IV of OXPHOS in antiretroviral drug induced mitochondrial toxicity. Equal amount of cell lysates were analyzed in triplicates with the MILLIPLEX MAG Human OXPHOS 6-Plex Panel (left graph) and the ELISAs (right graph) to determine the quantity of each analyte within OXPHOS. Drug induced mitochondrial toxicity was evaluated by normalizing the quantity of each analyte to mock-treated cells as a percentage. The data from the OXPHOS panel are consistent with the respective ELISAs as well as those from activity assay and the cellular oxygen consumption study (data not shown). The results indicate that antiretroviral drug ddC clearly induced mitochondrial toxicity as demonstrated by significant reduction of Complex I, III and IV while Complex II, V and NNT remained relatively stable.

ddC Causes Mitochondrial Toxicity in HepG2 Cells