Tools for detecting mitochondrial toxicity

3

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

Screening assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

Oxygen consumption and lactate production assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

Membrane potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7

ATP detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7

Mitochondrial biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8

Oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

Investigational assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Investigating energy impairment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

In vitro assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Ex vivo assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Investigating oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Direct ROS quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Quantification of antioxidant molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Antioxidant enzyme capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Investigating apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

4

Introduction

Inhibitionof OXPHOSenzymes

Disruptionof membrane

potential

Inductionof apoptosis

Inhibition ofmitochondrial

biogenesis

Free radical production

Figure 1. Processes involved in mitochondrial dysfunction

Every year an appreciable percentage of pharmaceutical drugs are withdrawn from the market due to adverse effects not discovered during preclinical and clinical testing . Often, these are cardio- and hepatotoxic effects caused by mitochondrial dysfunction . Mitochondrial toxicity assays are now considered to be an important part of the discovery process for all drugs1 and evaluation of mitochondrial toxicity early in the drug development process can prevent attrition in later clinical studies, saving time and money .

Our assays for assessing mitochondrial toxicity provide a complete solution for all stages of in vitro mitochondrial safety analysis and measurement of key parameters of mitochondrial function .

5

Primary mitochondrialscreen [p4]

Oxygen consumption [p6]

Lactate production [p7]

Membrane potential [p7]

ATP production [p7]

Mitochondrial biogenesis [p8]

Reactive oxygenspecies [p9]

Apoptosis [p9]

Investigationalassays [p5]

Investigating energyimpairment [p10]

Investigating oxidativestress [p12]

Investigatingapoptosis [p14]

Candidatecompounds

Figure 2. Procedure for investigating mitochondrial toxicity

Assays are available for initial screening of mitochondrial function upon candidate drug treatment . If mitochondrial toxicity is confirmed during the in vitro screening, then the mechanism of action can be further investigated with in vitro investigational assays or in animal models .

6

Screening assays

Oxygen consumption and lactate production assays

Our high-throughput microplate-based assays allow you to measure extracellular oxygen consumption and lactate production on your plate reader . Reactions are non-destructive and fully reversible, allowing measurement of time course and drug treatments .

Product Readout Sample types

Extracellular oxygen consumption assay (ab197243)

Fluorescent microplate Whole cells, isolated mitochondria, 3D cultures, isolated enzymes, bacteria and yeasts

Glycolysis assay [extracellular acidification] (ab197244)

Fluorescent microplate Whole cells, isolated mitochondria, 3D cultures, isolated enzymes, bacteria and yeasts

Product highlight

20,000

15,000

10,000

5,000

025 µM Rotenone

20 µM

Vehicle control

Lum

ine

sce

nt c

ou

nts

500

400

300

200

100

0FCCPUntreated Antimycin A

% E

ffec

t

350

300

250

200

150

100

50

0

Camptothecin (µM)

JC–1

JC–1

JC–10

JC–10

0 10

Ratio

(52

0/59

0) %

of C

on

trol

400

300

200

100

0

Vehicle

50 µM TBHP

None

Flu

ore

sce

nc

e in

ten

sity

0.3

0.2

0.1

0CPTCTRL α-Fas

O.D

. (40

5 n

m)

Cellular and energy flux

HepG2 cells (seeded at 6.5 x 104 cells/well) were treated with 1 µM antimycin A and 2.5 µM FCCP. Oxygen consumption (light grey column, ab197243), extracellular acidification rate (dark grey column, ab197244) and ATP concentration (red column, ab113849) data are shown as percentage of untreated control.

7

Membrane potential

Information on basic mitochondrial function can be gained by looking at mitochondrial membrane potential . Our wide range of assay kits enable you to measure membrane potential whatever your application .

Product Applications

JC-1 Mitochondrial Membrane Potential Assay Kit (ab113850)

Live adherent or suspension cells, microplate reader

JC-10 Mitochondrial Membrane Potential Assay Kit- flow cytometry (ab112133)- microplate reader (ab112134)

Live adherent or suspension cells microplate reader or flow cytometry

TMRE Mitochondrial Membrane Potential Assay Kit (ab113852)

Live adherent or suspension cells, flow cytometry, microplate and fluorescent microscopy

Product highlight

20,000

15,000

10,000

5,000

025 µM Rotenone

20 µM

Vehicle control

Lum

ine

sce

nt c

ou

nts

500

400

300

200

100

0FCCPUntreated Antimycin A

% E

ffec

t

350

300

250

200

150

100

50

0

Camptothecin (µM)

JC–1

JC–1

JC–10

JC–10

0 10

Ratio

(52

0/59

0) %

of C

on

trol

400

300

200

100

0

Vehicle

50 µM TBHP

None

Flu

ore

sce

nc

e in

ten

sity

0.3

0.2

0.1

0CPTCTRL α-Fas

O.D

. (40

5 n

m)

Tracking mitochondrial membrane potential

Mitochondrial membrane potential changes monitored with JC-10 (ab112134, red) and JC-1 (ab113850, grey). Jurkat cells were untreated (0 µM) or treated with camptothecin (10 µM) for 4 h. Fluorescence intensities for J-aggregates and monomeric forms of JC-1 and JC-10 were measured at Ex/Em = 490/525 nm and 490/590 nm, respectively.

ATP detection

Luminescent ATP detection kit (ab113849)

Quantitative assay to measure cellular ATP concentration . Lysis step irreversibly inactivates ATPases present in the sample, ensuring that the luminescent signal obtained truly corresponds to endogenous ATP levels .

- Results in less than 30 minutes - Detection limit of five cells/well - Wide linear dynamic range: 0.1 nM – 1 µM

8

Product highlight

20,000

15,000

10,000

5,000

025 µM Rotenone

20 µM

Vehicle control

Lum

ine

sce

nt c

ou

nts

500

400

300

200

100

0FCCPUntreated Antimycin A

% E

ffec

t

350

300

250

200

150

100

50

0

Camptothecin (µM)

JC–1

JC–1

JC–10

JC–10

0 10

Ratio

(52

0/59

0) %

of C

on

trol

400

300

200

100

0

Vehicle

50 µM TBHP

None

Flu

ore

sce

nc

e in

ten

sity

0.3

0.2

0.1

0CPTCTRL α-Fas

O.D

. (40

5 n

m)

ATP detection upon rotenone treatment

Cells were treated with either 25 µM rotenone or DMSO (vehicle control) for 4 h. After treatment, cells were lysed before measuring ATP with Luminescent ATP Detection Kit (ab113849).

Mitochondrial biogenesis

Measurement of mitochondrial biogenesis is becoming a standard component of early drug safety characterization2 and may become a regulatory requirement for antiviral and antibiotic drugs . Our MitoBiogenesisTM assays are the ideal tool to uncover chronic effects of mitochondrial DNA replication and protein synthesis .

Product Readout

MitoBiogenesis™ In-Cell ELISA - IR Dyes (ab110216) - colorimetric (ab110217) - fluorescent (ab140359)

ELISA - IRDyes®, colorimetric or fluorometric

MitoBiogenesis™ Western Blot Cocktail (ab123545)

Western blot

MitoBiogenesis™ Flow Cytometry Kit (ab168540)

Flow cytometry

Product highlight

0.0

0.5

1.0

1.5

0 10 100

Chloramphenicol [µM]

SDH–A COX–I

Rela

tive

sig

na

l

Chloramphenicol inhibition of mitochondrial biogenesis

Inhibition of mitochondrial biogenesis by chloramphenicol, assessed using MitobiogenesisTM In-Cell ELISA kit (Colorimetric) (ab110217) by monitoring the relative amounts of COX-I (mitochondrial DNA encoded) and SDH-A (nuclear DNA encoded)

“I am using this kit for high throughput screening of more than 500 compounds . The kit is highly reproducible and I did not observe any lot to lot variation . I would highly recommend this kit .”

– Dr Andaleeb Sajid

9

Oxidative stress

Increased reactive oxygen species (ROS) is indicative of oxidative stress that can arise through mitochondrial dysfunction . Our DCFDA cellular ROS assay (ab113851) can be used for initial pharmacological screening of ROS production by microplate reader or flow cytometry .

Product highlight

20,000

15,000

10,000

5,000

025 µM Rotenone

20 µM

Vehicle control

Lum

ine

sce

nt c

ou

nts

500

400

300

200

100

0FCCPUntreated Antimycin A

% E

ffec

t

350

300

250

200

150

100

50

0

Camptothecin (µM)

JC–1

JC–1

JC–10

JC–10

0 10

Ratio

(52

0/59

0) %

of C

on

trol

400

300

200

100

0

Vehicle

50 µM TBHP

None

Flu

ore

sce

nc

e in

ten

sity

0.3

0.2

0.1

0CPTCTRL α-Fas

O.D

. (40

5 n

m)

ROS quantification

Detection of reaction oxygen species with DCFDA Cellular Reactive Oxygen Species Assay Kit (ab113851). Jurkat cells were labeled with 20 µM DCFDA or unlabeled, and then cultured for 3 h in presence or absence of 50 µM tert-butyl hydrogen peroxide (TBHP).

Apoptosis

For initial screening of apoptosis, we provide an Annexin V-FITC Apoptosis Detection Kit (ab14085) for analysis by flow cytometry, and a colorimetric assay for detection of caspase 3 activation by microplate reader (ab39401) .

Product highlight

20,000

15,000

10,000

5,000

025 µM Rotenone

20 µM

Vehicle control

Lum

ine

sce

nt c

ou

nts

500

400

300

200

100

0FCCPUntreated Antimycin A

% E

ffec

t

350

300

250

200

150

100

50

0

Camptothecin (µM)

JC–1

JC–1

JC–10

JC–10

0 10

Ratio

(52

0/59

0) %

of C

on

trol

400

300

200

100

0

Vehicle

50 µM TBHP

NoneFl

uo

resc

en

ce

inte

nsi

ty

0.3

0.2

0.1

0CPTCTRL α-Fas

O.D

. (40

5 n

m)

Measuring caspase 3 activation

Quantification of caspase 3 activation in cell lysates with Caspase 3 Assay Kit (colorimetric) (ab39401) . Jurkat cells were treated for 3 h with 2 µM camptothecin (CPT) or 10 ng/mL anti-Fas antibody (α-Fas) .

For details on our complete range of apoptosis products, refer to our apoptosis guide, www .abcam .com/apoptosisebook

10

Investigational assays

Investigating energy impairment

Changes to oxygen consumption, membrane potential or ATP production may indicate energy impairment upon drug treatment . To identify the exact cause of energy impairment, activity of metabolic enzymes – including oxidative phosphorylation complexes and enzymes involved in wider mitochondrial metabolic pathways, such as the TCA cycle – should be assessed .

In vitro assaysOur in vitro MitoTox™ assays measure the effect of drug treatment on oxidative phosphorylation complex activity in a colorimetric microplate format that can be easily adapted for high-throughput assessment . The assays can be used to test the following:

- IC50 of two compounds in a dose-response format - Up to 23 compounds at a single concentration

MitoTox™ assays are available for each of the OXPHOS complexes:

Product

MitoTox™ Complete OXPHOS Activity Assay panel (5 assays) (ab110419)

MitoTox™ Complex I OXPHOS Activity Assay (ab109903)

MitoTox™ Complex II OXPHOS Activity Assay (ab109904)

MitoTox™ Complex II + III OXPHOS Activity Assay (ab109905)

MitoTox™ Complex IV OXPHOS Activity Assay (ab109906)

MitoTox™ Complex V OXPHOS Activity Assay (ab109907)

Product highlight

120110100

908070605040302010

010-2 10-1 100 101 102 103 104 105

Rotenone [µM]

IC50 = 17.3 µM

Co

mp

lex

l Ac

tivity

(%

)

120110100

908070605040302010

010-2 10-1 100 101 102 103 104 105

Oligomycin [µM]

IC50 = 8 µM

Co

mp

lex

V A

ctiv

ity (

%)

120110100

908070605040302010

010-2 10-1 100 101 102 103

TTFA [µM]

IC50 = 30 µM

Co

mp

lex

lI A

ctiv

ity (

%)

10-2 10-1 100 101 102 103

120110100

908070605040302010

0

KCN [µM]

IC50 = 3.2 µM

Co

mp

lex

IV A

ctiv

ity (

%)

120110100

908070605040302010

00 1 10 100 1000

Antimycin [µM]

IC50 = 22 µM

Co

mp

lex

lII A

ctiv

ity (

%)

Dose response curves of mitochondrial complexes

Dose response curves of mitochondrial complexes I – V after treatment with specific inhibitors. Activity was monitored with the MitoTox™ Complete OXPHOS Activity Assay panel (ab110419), using isolated bovine heart mitochondria provided in the kit.

11

Ex vivo assays

We also have the following microplate-based assays for determining quantity and activity of mitochondrial proteins in tissue and cell samples .

Product Sample type Reactivity

ALDH2 Activity Assay Kit (ab115348)

Cell extracts, tissue extracts Human, mouse, rat, cow

Carboxylesterase 1 Specific Activity Assay Kit (ab109717)

Cell extracts, tissue extracts Rat, human

Citrate Synthase Activity Assay Kit (ab119692)

Cell extracts, tissue extracts Human

Complex I Enzyme Activity Assay Kit (ab109721)

Mitochondria, cell extracts, tissue extracts

Human, mouse, rat, cow

Complex II Enzyme Activity Assay Kit (ab109908)

Mitochondria, cell extracts, tissue extracts

Human, mouse, rat, cow

Complex IV Human Enzyme Activity Assay Kit (ab109909)

Mitochondria, cell extracts, tissue extracts

Human, cow

Complex IV Rodent Enzyme Activity Assay Kit (ab109911)

Mitochondria, cell extracts, tissue extracts

Mouse, rat

Complex V (ATP Synthase) Activity Assay Kit (ab109714)

Mitochondria, cell extracts, tissue extracts

Human, rat, cow

ENO1 Activity Assay Kit (ab117994)

Cell extracts, tissue extracts Human

Fumarase Specific Activity Assay Kit (ab110043)

Cell extracts, tissue extracts Human, cow, rat

LDHB Activity Assay Kit (ab140361)

Cell extracts, tissue extracts Human, cow, goat

MDH2 Activity Assay Kit (ab119693)

Cell extracts, tissue extracts Human, mouse rat

MAOB Specific Activity Assay Kit (ab109912)

Cell extracts Human

PDH Enzyme Activity Assay Kit (ab109902)

Cell extracts, tissue extracts Human, mouse, rat, cow

Human Transketolase ELISA Kit (ab187398)

Cell culture extracts, tissue extracts

Human

12

Investigating oxidative stress

A thorough investigation of oxidative stress should be conducted where initial screens show increased ROS production to identify its exact cause . We have developed effective tools to measure ROS production by direct ROS measurement, quantification of ROS-induced protein modifications, and measurement of antioxidant capacity .

Direct ROS quantification

Product Application

DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit (ab113851)

Flow cytometry, microplate reader (96- or 384-well plate format)

Cellular Superoxide Detection Assay Kit (ab139477)

Fluorescence microscopy, flow cytometry

Cellular ROS/Superoxide Detection Assay Kit (ab139476)

Fluorescence microscopy, flow cytometry

Cellular Reactive Oxygen Species Detection Assay Kit (ab186027/ab186028/ab186029)

High-throughput screening liquid handling instruments, 96- or 384-well plate format . Multiple detection wavelengths available .

Hydrogen Peroxide Assay Kit (Cell-based) (ab138874)

High-throughput screening liquid handling instruments, 96- or 384-well plate format . For use in live cells only

Hydrogen Peroxide Assay Kit (ab138886) High-throughput screening liquid handling instruments, 96- or 384-well plate format . For use in cell supernatants and live cells

Quantification of antioxidant molecules

Product Readout Sample types

Total Antioxidant Capacity Assay Kit (ab65329)

Quantitative, colorimetric

Biological fluids, tissue extracts, cell extracts and cell culture media

Ascorbic Acid Assay Kit (Biological Samples) (ab65656)

Quantitative, colorimetric

Biological fluids, tissue extracts, cell extracts and cell culture media

NAD/NADH Assay Kit (ab65348/ ab176723)

Quantitative, colorimetric or fluorometric

Cell extracts, tissue extracts

NADP/NADPH Assay Kit (ab65349/ ab176724)

Quantitative, colorimetric or fluorometric

Cell extracts, tissue extracts

GSH/GSSG Ratio Detection Assay Kit (ab138881/ ab205811)

Quantitative, fluorometric

Urine, plasma, tissue extracts, cell extracts

Intracellular glutathione (GSH) Detection Assay Kit (ab112132)

Flow cytometry, quantitative, fluorometric

Adherent cells, suspension cells

13

Antioxidant enzyme capacity

Product Readout Sample types

GST Activity Assay Kit (ab65325/ab65326)

Quantitative, fluorometric or colorimetric

Biological fluids, tissue extracts, cell extracts

Superoxide Dismutase Activity Assay Kit (ab65354)

Quantitative, colorimetric

Biological fluids, tissue extracts, cell extracts

Glutathione Reductase Activity Assay (ab83461)

Quantitative, colorimetric

Biological fluids, tissue extracts, cell extracts

Xanthine Oxidase Activity Assay Kit (ab102522)

Quantitative, fluorometric or colorimetric

Biological fluids, tissue extracts, cell extracts

Glutathione Peroxidase Activity Assay Kit (ab102530)

Quantitative, colorimetric

Biological fluids, tissue extracts, cell extracts

Aconitase Activity Assay Kit (ab109712)

Quantitative, colorimetric

Cell extracts, tissue extracts

Catalase Specific Activity Assay Kit (ab118184)

Quantitative, luminescent (activity) and colorimetric (quantity)

Cell extracts, tissue extracts

Thioredoxin Reductase 1 (TXNRD1) Activity Assay Kit (ab190804)

Quantitative, colorimetric

Cell extracts, tissue extracts

14

Investigating apoptosis

Our range of assays for investigating apoptosis allow you to look at a range of different apoptotic processes .

For a more in-depth look at our apoptosis assays, view our apoptosis analysis guide .www .abcam .com/apoptosisebook

Parameters Detection methods Sample type Highlighted Products

Loss of membrane asymmetry/PS exposure

Flow cytometry analysis of Annexin V binding

Live cells ab14085

Caspase activation Colorimetric/fluorometric substrate-based assays in microtiter plates

Cell extractsTissue extracts

ab39383ab65607ab39700

Detection of cleavage of fluorometric substrate in flow cytometry/microscopy or by microtiter plates analysis

Live cells ab112130ab65614ab65613

Western blot analysis of pro- and active caspase

Cell extractsTissue extracts

ab32042ab138485ab32539

Caspase substrate (PARP) cleavage

Microplate spectrophotometry analysis with antibodies specific for cleaved PARP

Cells extractsTissue extractsLive cells (in cell ELISA)

ab174441ab140362

Mitochondrial transmembrane potential (ΔΨm) decrease

Flow cytometry/microscopy/microplate spectrophotometry analysis with MMP sensitive probes

Live cells ab113852ab113850ab112134

Increase of sub G1 population

Flow cytometry analysis of subG1 peak

Fixed cells ab14083ab139418

Nuclear condensation Flow cytometry analysis of chromatin condensation

Live cells ab139479ab112151ab115347

DNA fragmentation Analysis of DNA ladder in agarose gel

DNA ab66090ab65627ab66093

Analysis of DNA fragmentation by TUNEL

Live cells ab66110ab66108

References

1 . Dykens JA, Will Y . The significance of mitochondrial toxicity testing in drug development . Drug Discov Today 12, 777–85 (2007) .

2 . Nadanaciva S, Will Y . New insights in drug-induced mitochondrial toxicity . Curr Pharm Des 17, 2100–12 (2011) .

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