Tools for detecting mitochondrial toxicity

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Tools for detecting
mitochondrial toxicity
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
3
Introduction
Inhibition of
mitochondrial
biogenesis
Inhibition
of OXPHOS
enzymes
Disruption
of membrane
potential
Free radical
production
Induction
of apoptosis
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.
4
Primary mitochondrial
screen [p4]
Candidate
compounds
Investigational
assays [p5]
Oxygen consumption [p6]
Lactate production [p7]
Membrane potential [p7]
Investigating energy
impairment [p10]
ATP production [p7]
Mitochondrial biogenesis [p8]
Reactive oxygen
species [p9]
Investigating oxidative
stress [p12]
Apoptosis [p9]
Investigating
apoptosis [p14]
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.
5
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.
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
Cellular and energy flux
500
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.
% Effect
400
300
200
100
0
none
0
Product
Vehicle control
Untreated
10
Camptothecin (µM)
Vehicle
Fluorescence intensity
JC–10
Antimycin A
400
JC–10
JC–1
FCCP
50 µM TBHP
300
200
100
0
None
20 µM
6
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.
JC-1 Mitochondrial Membrane Potential
20,000
Assay
Kit (ab113850)
Live adherent or suspension cells,
500 reader
microplate
JC-10 Mitochondrial Membrane Potential
15,000
Assay Kit
- flow cytometry (ab112133)
10,000
- microplate
reader (ab112134)
Live adherent
or suspension cells
400
microplate reader or flow cytometry
TMRE5,000
Mitochondrial Membrane Potential
Assay Kit (ab113852)
Live adherent or suspension cells, flow
100
cytometry,
microplate and fluorescent
microscopy
0
% Effect
Applications
Luminescent counts
Product
300
200
0
25 µM Rotenone
Vehicle control
Untreated
FCCP
Antimycin A
Product highlight
JC–10
300
250
JC–1
200
150
JC–1
JC–10
100
50
0
0
10
Camptothecin (µM)
Tracking mitochondrial membrane
potential
400
Vehicle
Mitochondrial
membrane potential 50 µM TBHP
300
changes monitored with JC-10
(ab112134,
red) and JC-1 (ab113850,
200
grey). Jurkat cells were untreated (0 µM)
or treated with camptothecin (10 µM)
100
for 4 h. Fluorescence intensities for
J-aggregates and monomeric forms
0
of JC-1 and
JC-10 were measured at
None
20 µM
Ex/Em = 490/525 nm and 490/590
nm,
respectively.
Fluorescence intensity
Ratio (520/590) % of Control
350
ATP detection
0.3
Luminescent ATP detection kit (ab113849)
O.D. (405 nm)
0.2
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.
0.1
-- Results in less than 30 minutes
-- Detection limit of five cells/well
0
-- Wide linear
CTRLdynamic
CPT range:
α-Fas0.1 nM – 1 µM
7
Product highlight
500
ATP detection
upon rotenone treatment
400
15,000
300
Cells were
treated with either 25 µM
rotenone or DMSO (vehicle control) for 4 h.
200
After treatment, cells were lysed before
measuring
100 ATP with Luminescent ATP
Detection Kit (ab113849).
% Effect
Luminescent counts
20,000
10,000
5,000
0
0
25 µM Rotenone
Untreated
Vehicle control
FCCP
Antimycin A
Mitochondrial biogenesis
150
JC–1
Product
100
Fluorescence intensity
Ratio (520/590) % of Control
350
Measurement
of mitochondrial biogenesis
is becoming400a standard component of early
JC–10
Vehicle
2
300
drug safety
characterization and may become a regulatory requirement for antiviral
50 µM TBHP
300 ideal tool to uncover chronic
and antibiotic
drugs. Our MitoBiogenesisTM assays are the
250
JC–1
effects200
of mitochondrial DNA
replication and protein synthesis.
JC–10
Readout
200
100
ELISA - IRDyes®, colorimetric or fluorometric
MitoBiogenesis™ Western Blot Cocktail
(ab123545)
Western blot
MitoBiogenesis™ Flow Cytometry Kit
(ab168540)
0.3
Flow cytometry
O.D. (405 nm)
MitoBiogenesis™
In-Cell ELISA
50
-- IR Dyes
(ab110216)
0
0
10
-- colorimetric (ab110217)
Camptothecin (µM)
-- fluorescent (ab140359)
0
None
20 µM
0.2
Product highlight
0.1
Chloramphenicol inhibition of
mitochondrial biogenesis
0
CTRL
Relative signal
1.5
CPT
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)
α-Fas
1.0
0.5
0.0
0
10
Chloramphenicol [µM]
SDH–A
COX–I
100
“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
8
400
% Effect
300
Oxidative stress
15,000
% Effect
Vehicle control
Increased
reactive oxygen species (ROS) is indicative of oxidative stress that can arise
100
through
mitochondrial
dysfunction. Our DCFDA cellular
ROS assay (ab113851) can be
20,000
500
0 initial pharmacological screening of ROS production by microplate reader or
used for
Untreated
FCCP
Antimycin A
flow cytometry.
400
Luminescent counts
one
200
Product highlight
10,000
5,000
Fluorescence intensity
Vehicle
10
amptothecin (µM)
25 µM Rotenone
50 µM TBHP
Vehicle control
200
100
350
0
300
Vehicle
20 µM
250
JC–1
200
JC–1
Apoptosis
100
0
FCCP
Antimycin
A
Detection ofUntreated
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
400peroxide (TBHP).
JC–10
None
150
ROS quantification
Fluorescence intensity
JC–10
Ratio (520/590) % of Control
JC–1
0
200
100
400
JC–10
300
300
JC–10
300
50 µM TBHP
200
100
50
For initial screening of apoptosis, we provide an Annexin V-FITC Apoptosis Detection
0
0
Kit (ab14085)
for analysis
by flow
cytometry, and a colorimetric
assay for detection of
0
10
None
20 µM
Camptothecin
(µM)
caspase 3 activation
by microplate
reader (ab39401).
Product highlight
0.3
Measuring caspase 3 activation
α-Fas
O.D. (405 nm)
CPT
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).
0.2
0.1
0
CTRL
CPT
α-Fas
For details on our complete range of apoptosis products, refer to our apoptosis guide,
www.abcam.com/apoptosisebook
9
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 assays
Our 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)
120
110
100
90
80
70
60
50
40
30
20
10
0
Complex lI Activity (%)
Complex l Activity (%)
Product highlight
IC50 = 17.3 µM
10-2
10-1
100
101
102
103
104
120
110
100
90
80
70
60
50
40
30
20
10
0
105
IC50 = 30 µM
10-2
10-1
Complex IV Activity (%)
Complex lII Activity (%)
IC50 = 22 µM
0
1
10
100
1000
Complex V Activity (%)
Antimycin [µM]
120
110
100
90
80
70
60
50
40
30
20
10
0
101
102
103
TTFA [µM]
Rotenone [µM]
120
110
100
90
80
70
60
50
40
30
20
10
0
100
120
110
100
90
80
70
60
50
40
30
20
10
0
IC50 = 3.2 µM
10-2
10-1
100
KCN [µM]
101
102
103
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.
IC50 = 8 µM
10-2
10-1
100
101
102
103
104
105
Oligomycin [µM]
10
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
11
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 Flow cytometry, microplate reader (96- or
Detection Assay Kit (ab113851)
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 Quantitative,
Samples) (ab65656)
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/ Quantitative,
ab176724)
colorimetric or
fluorometric
Cell extracts, tissue extracts
GSH/GSSG Ratio Detection Assay Quantitative,
Kit (ab138881/ ab205811)
fluorometric
Urine, plasma, tissue extracts,
cell extracts
Intracellular glutathione (GSH)
Detection Assay Kit (ab112132)
Adherent cells, suspension
cells
Flow cytometry,
quantitative,
fluorometric
12
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) Quantitative,
Activity Assay Kit (ab190804)
colorimetric
Cell extracts, tissue extracts
13
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 extracts
Tissue extracts
ab39383
ab65607
ab39700
Detection of cleavage of
fluorometric substrate in flow
cytometry/microscopy or by
microtiter plates analysis
Live cells
ab112130
ab65614
ab65613
Western blot analysis of pro- and Cell extracts
active caspase
Tissue extracts
ab32042
ab138485
ab32539
Caspase substrate
(PARP) cleavage
Microplate spectrophotometry
analysis with antibodies specific
for cleaved PARP
Cells extracts
Tissue extracts
Live cells (in
cell ELISA)
ab174441
ab140362
Mitochondrial
transmembrane
potential (ΔΨm)
decrease
Flow cytometry/microscopy/
microplate spectrophotometry
analysis with MMP sensitive
probes
Live cells
ab113852
ab113850
ab112134
Increase of sub G1
population
Flow cytometry analysis of subG1 Fixed cells
peak
ab14083
ab139418
Nuclear condensation
Flow cytometry analysis of
chromatin condensation
Live cells
ab139479
ab112151
ab115347
DNA fragmentation
Analysis of DNA ladder in
agarose gel
DNA
ab66090
ab65627
ab66093
Analysis of DNA fragmentation
by TUNEL
Live cells
ab66110
ab66108
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).
14
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