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Mitochondrial Respiratory Complex Assay using Permeabilized Cells (Seahorse XFe96 flux analyzer)

Understand the mechanism of mitochondrial toxicity using Cyprotex’s mitochondrial respiratory complex assay using permeabilized cells (Seahorse XFe96).

Cyprotex’s mitochondrial respiratory complex assay utilising permeabilised cells on the Seahorse XFe analyzer is in Cyprotex’s portfolio of in vitro toxicology services for measuring potential mitochondrial toxicity. Cyprotex deliver consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.

Understanding the mechanism of mitochondrial toxicity in vitro using the Seahorse Flux Analyzer

  • Impairment of mitochondrial function is implicated in the etiology of drug-induced toxicity.1
  • The Seahorse XFe96 extracellular flux analyzer is used to detect, in real time, effects of compounds on oxygen consumption rate (OCR) in order to assess mitochondrial function.
  • Permeabilization of cells which leaves the mitochondrial membrane intact allows the study of mitochondrial function without the need to isolate mitochondria.
  • The use of complex specific substrates and inhibitors allows the identification of the individual complexes (complex I, complex II, complex III and complex IV) of the electron transport chain (ETC) involved in mitochondrial toxicity.
  • The mitochondrial respiratory complex assay can be used in conjunction with other mitochondrial assays (e.g., the Seahorse functional mitochondrial toxicity assay, glu/gal assay or the HCS-based mitochondrial assay) to determine the potential for mitochondrial toxicity along with an understanding of the mechanism.
Drug-induced mitochondrial toxicity is rapidly gaining recognition within the pharmaceutical industry as a contributor to compound attrition and post-market drug withdrawals.

1 Nadanaciva S and Will Y (2011) Current Pharmaceutical Design 17; 2100-2112

Protocol

Mitochondrial respiratory complex assay protocol

Cell Type HepG2 (others available on request)
Analysis Platform Seahorse XFe96 flux analyser
(Agilent Technologies)
Analysis Method Use of solid state fluorescent sensors to measure oxygen consumption rate (OCR)
Mechanism* Pyruvate respiration
Succinate respiration
Ascorbate respiration
Test Article Requirements 50 µL of a DMSO stock solution to achieve 100x Cmax (200x top concentration to maintain 0.5% DMSO) or equivalent amount in solid compound
Test Article Concentration* 7 point dose response curve with top concentration based on 100x Cmax orsolubility limit
Number of Replicates* 3 replicates per concentration
Quality Controls* Negative control: 0.5% DMSO (vehicle)
Positive control: Assay appropriate control
Data Delivery Minimum effective concentration (MEC) and AC50 values with dose response curves for each measured parameter

*Other options available on request.
Related Services
Glucose/galactose mitochondrial toxicity assay
HCS based mitochondrial toxicity assay
Functional mitochondrial toxicity assay (Seahorse XFe96)

Data

Data for mitochondrial respiratory complex assay using permeabilized cells

Known mitochondrial toxicants and non-toxicants were screened in the mitochondrial respiratory complex assay. The identified mechanisms of action were compared to those published in the literature.

 

Figure 1
Representative data assessing the effects on Complex I, Complex II/III and Complex IV mitochondrial respiration on permeabilized HepG2 cells. Compounds tested were a) rotenone, b) thenoyltrifluoroacetone (TTFA), c) antimycin A, d) sodium azide and e) betaine.

The oxygen consumption rate (OCR) of permeabilised HepG2 cells was measured in the presence of an appropriate complex I substrate (pyruvate). The test compound was injected directly onto the cells and OCR determined. Following this, a complex II/III substrate (succinate) and a complex I inhibitor were injected and further measurements taken. Finally a complex IV substrate and a complex III inhibitor were added and a final OCR was determined.

A reduction in OCR following the addition of test compound indicates inhibition of one of the complexes of the electron transport chain. If this inhibition is overcome by the addition of an alternative substrate, it indicates the potential site of inhibition.
  Pyruvate RespirationSuccinate RespirationAscorbate Respiration
CompoundMechanismMEC (µM)AC50 (µM)MEC (µM)AC50 (µM)MEC (µM)AC50 (µM)
Rotenone Complex I inhibitor 0.006 0.033 No response No response No response No response
Ketoconazole Complex I inhibitor 13.3 42.1 No response No response No response No response
Carboxine Complex II inhibitor No response No response 2.43 23.3 No response No response
Thenoyltrifluoro-acetone (TTFA) Complex II inhibitor 605 >2000 45.6 151 688 1810
Antimycin A Complex III inhibitor 0.019 0.027 0.012 0.019 No response No response
Sodium azide Complex IV inhibitor 177 654 79.1 411 23.5 156
Streptomycin No effect No response No response No response No response No response No response
Betaine No effect No response No response No response No response No response No response
Table 1
Summary of validation data

References

1 Nadanaciva S and Will Y (2011) New insights in drug-induced mitochondrial toxicity. Current Pharmaceutical Design 17; 2100-2112

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