Leading the Way in Life Science Technologies

GEN Exclusives

More »

Tutorials

More »
April 01, 2014 (Vol. 34, No. 7)

Screening Mitochondrial Toxicity

New Cost- and Time-Efficient Assay Kits Designed to Profile Compounds

  • Click Image To Enlarge +
    Figure 1. The mitochondrial electron transport chain and sites of inhibition. Electrons obtained from the oxidation of tricarboxylic acid (TCA) cycle intermediates enter the ETC and at complexes I and II and, through a series of redox reactions, reduce Q to QH2, which is utilized by complex III. Within the Q cycle, electrons are then passed on to cytochrome c (cytc) and terminally on to O2 via complex IV. Through this series of redox reactions, protons are translocated at complexes I, III, and IV. Inhibitors utilized in these assays, and sites at which they bind are indicated in red. Abbreviations: FMN—flavin mononucleotide (and redox couples); Q—ubiquinone; QH—ubisemiquinone; QH2—ubiquinol; Qo and Qi—the outer and inner sites of the Q-cycle, respectively; Fe-S—iron-sulfur clusters; bx—b-type hemes; Cytx—cytochromes; and Cux2+—copper centers. The mitochondrial membrane potential is indicated by ΔΨM.

    Proper mitochondrial function is critical for the maintenance of cellular homeostasis. Under aerobic conditions, the majority of ATP utilized by mammalian cells is generated by mitochondrial oxidative phosphorylation.

    Through the reduction of oxygen and oxidation of nutrients obtained through metabolic reactions, the electron transport chain (ETC) pumps protons from the mitochondrial matrix to the intermembrane space, establishing a proton gradient (Figure 1).

    This proton gradient, composed of both electrochemical (ΔψM) and pH (ΔpH) components, is harnessed by the ATP synthase (complex V) to produce ATP from ADP and inorganic phosphate. The generation of the proton gradient occurs through the oxidation of the reducing equivalents generated within the tricarboxylic acid cycle (TCA cycle).

    These reducing equivalents enter the ETC at complex I (NADH dehydrogenase), resulting in the translocation of protons (H+), and complex II (succinate dehydrogenase), respectively. Following entry, electrons are shuttled from complexes I and II via ubiquinone (Q) to complex III (ubiquinol cytochrome c oxidoreductase).

    Within complex III, through the redox cycling of ubiquinone, (Q-cycle) protons are translocated and cytochrome c is reduced. Alternatively, the Q-cycle also plays a role in cell signaling, through the generation of superoxide. The reduced form of cytochrome c is subsequently oxidized by complex IV (cytochrome c oxidase), leading to the step-wise reduction of O2 to H2O and the translocation of protons.

    Should any inhibition or inefficiencies result in poorly functioning ETC, a loss of membrane potential would occur resulting in an immediate drop in mitochondrial ATP production.

  • Drug-Induced Toxicity

    Given this unique and complex biochemistry, it is not surprising that mitochondria are susceptible to drug-induced toxicity. Over the past two decades, 80% of the drugs pulled from the market or given black box warnings were flagged due to mitochondrial toxicity-generated complications resulting in hepatotoxicity or cardiotoxicity.

    Furthermore, compounds that inhibit complex I have been linked to neurological disorders (e.g., Parkinson’s disease), while those inhibiting complex II have been linked to increased tumorigenesis.

    A number of compounds causing slight mitochondrial perturbation include certain NSAIDs, antidepressants, drugs used to treat autoimmune disorders, and diabetes drugs which, despite having the potential to disrupt the proton gradient or decrease mitochondrial efficiency, have been FDA approved.

    Historically, drugs deemed toxic to mitochondria conspicuously induce substantial, life-threatening hepatotoxicity or cardiotoxicity. Mitochondrial toxicity has only recently been appreciated for its role in adverse drug reactions, and while not yet required by the FDA, this recognition is prompting the pharmaceutical industry to implement mitochondrial toxicity screening platforms as a part of their ADME-Tox process.

    Due to the complexity of this organelle, it comes as little surprise that multiple mechanisms of mitochondrial inhibition exist, many of which have been elucidated using prototypical mitochondrial inhibitors. These well-studied inhibitors are complex specific, and include rotenone (complex I), 2-thenoyltrifluoracetone (TTFA) (complex II), Antimycin A (complex III), and potassium cyanide (KCN) (complex IV). The binding sites of these inhibitors are shown in Figure 1.

    Despite known adverse drug reactions associated with mitochondrial toxicity, few assays exist that allow for high-throughput mitochondrial toxicity screening. Furthermore, the toxicity screening assays that are available employ expensive instrumentation and consumables, or lengthy immunocapture-based reactions.

    Therefore, Cayman Chemical has generated convenient MitoCheck ETC activity assays to screen for inhibitors of the mitochondrial ETC. Isolated bovine heart mitochondria, the composition and structure of which are conserved in mammals, are provided at optimal concentrations for assaying activities of the individual complexes. These assays can be performed in a standard 96-well plate without preincubation with antibodies, making them ideal for high-throughput screening applications.

  • Methods

    Complex I activity was measured by monitoring the decrease in A340 corresponding to oxidation of NADH (A340). Complex I assays were carried out in the presence of KCN (2 mM) to inhibit complex IV. Rotenone was titrated at 12-point half-log dilutions with a maximal concentration of 10 µM.

    Complex II activity was measured as the succinate-dependent decrease in absorbance of DCPIP (A600). Complex II assays were carried out in the presence of rotenone (1 µM), antimycin A (10 µM), and KCN (2 mM) to inhibit complexes I, III, and IV, respectively. TTFA was titrated in 12-point half log dilutions with a maximal concentration of 10 mM.

  • Click Image To Enlarge +
    Figure 2. Concentration response curves for inhibition of ETC complexes I, II, III, and IV obtained using Cayman’s MitoCheck ETC Activity Assay Kits. IC50 values are reported in the Table.

    Complex II/III activity was measured as the succinate-dependent reduction of cytochrome c (A550). Complex II/III assays were carried out in the presence of rotenone (1 µM) and KCN (2 mM) to inhibit complexes I and IV, respectively. Antimycin A was titrated at 12-point half-log concentrations with a maximal concentration of 10 µM.

    Complex IV activity was measured in the absence of mitochondrial inhibitors using reduced cytochrome c as a substrate. Oxidation of cytochrome c was measured as a decrease in A550. KCN was titrated in 12-point half log dilutions with a maximal concentration of 1 mM.

  • Results and Discussion

    Click Image To Enlarge +
    Table 1. Calculated IC50 Values for Inhibition of Complexes I–IV

    The concentration response curves for inhibition of ETC complexes I, II, III and IV generated using Cayman’s MitoCheck ETC activity assays are shown in Figure 2. The calculated IC50 values for inhibition of complexes I–IV (Table 1) coincide with those previously reported using immunocapture-based methods.

    Whereas these results are comparable to previously developed assays, Cayman’s MitoCheck ETC Assay Kits offer an advantage in that mitochondrial isolation is not required. This feature means that a single assay can be completed in as little 30 minutes.

    Given the essential role of mitochondria in cell viability, mitochondrial toxicity is an important factor to consider when screening compounds. Cayman’s MitoCheck ETC Activity Kits, coupled with Cayman’s Oxygen Consumption Rate Assay Kit and Oxygen Consumption/MitoMembrane Potential Assay Kit offer a cost-effective means to thoroughly profile compounds for mitochondrial toxicity without the need for expensive equipment, spending valuable time isolating mitochondria, or lengthy incubations. 

Related content