Quantitative Analysis of Genotoxicity and

Quantitative Analysis of Genotoxicity and Cytotoxicity to DNA Damaging Agents
Using High-Content Imaging
Sangeeta Rojanala, Bhaskar Mandavilli, Robert Aggeler, Kapil Kumar, and Michael S Janes
Invitrogen Corporation - Molecular Probes® Labeling and Detection Technologies • 29851 Willow Creek Road • Eugene, OR 97402 • USA
Figure 7. Induction of Genotoxicity by DNA Damaging Agents
Figure 3. Multiplex Imaging of Genotoxicity & Cytotoxicity
We developed an assay to measure the effects of compounds or drugs
that induce DSBs in cells. This assay enables the simultaneous
quantitation of two cell health parameters in the same cell by high content
analysis: genotoxicity and cytotoxicity. DNA damage is measured as an
indicator of genotoxicity using specific antibody-based detection of
phosphorylated H2AX (Ser139) in the nucleus. Cytotoxicity is measured
using a new dead cell dye that does not stain nuclear DNA in intact live
cells due to the impermeability of the plasma membrane for this probe.
Drugs and test compounds leading to serious cell injuries, including
plasma membrane permeability, allow entry of the dye and reflect general
cytotoxicity. A549 and HeLa cells treated with a variety of compounds
known to induce DNA damage were used to validate the consistency and
robustness of this assay for cytotoxicity measurements. The data
demonstrated that this multi-parametric approach revealed cells which
underwent pre-lethal DNA damage while lethal damage was induced in
other cells within the same wells, underscoring the value of high content
imaging-based assays in cytotoxicity. In addition to pH2AX detection and
determination of plasma membrane integrity, compatibility with a new
click chemistry-based Tdt-mediated dUTP nick end labeling (TUNEL)
assay for DNA damage is shown.
A.
DMSO
30 µM
DMSO
120 µM
Average Intensity
Concentration
0.8 M
25 M
350
Cell Membrane
Permeability
DMSO
12.5 M
Concentration
50 M
180
160
Etoposide
60
concentration
300
250
Valinomycin
200
150
100
50
Figure 3. A549 cells were treated with increasing concentrations of valinomycin for 24
hours and showed genotoxic and cytotoxic effects as indicated by pH2AX antibody
labeling and the Image-iT® DEAD Green™ viability stain. 30 M valinomycin treatment
resulted in an pre-lethal increase of pH2AX but not loss of plasma membrane integrity.
0
Concentration
Figure 7. A549 cells were treated with increasing concentrations of H2O2,
camptothecin, etoposide, and valinomcin before fixation and permeabilization.
Detection of pH2AX using the HCS DNA Damage Kit revealed increased levels of
pH2AX in the nuclear regions of treated cells which was readily quantifiable.
Figure 4. Imaging of Genotoxicity & Cytotoxicity
Figure 8. Multiplex Assessment of DNA Damage: Combining
the Click-iT® TUNEL Assay with pH2AX Detection
Control
200
200
175
175
150
150
125
125
Cir cAvgIn te nCh3
225
100
75
200
100
0
-2
-1
0
1
2
3
100
50
50
0
-2
-1
0
1500
Phopsho-H2AX
Nuclear Intensity
Phopsho-H2AX
Nuclear Intensity
0
1
2
3
Log valinomycin ( M)
Log Valinomycin ( M)
EC50=61 M
300
200
100
-2
-1
0
1
2
25
50
75
100
125
150
CircAvgIntenCh2
175
200
225
250
0
25
75
-3
-2
-1
0
Log H2O2 ( M)
%CV
Z factor
Fold Change
0.47
0.05
10.2
1000
750
Cir cAvgIn te nCh3
750
500
250
500
250
0
1000
1500
2000
2500
CircAvgIntenCh2
3000
3500
4000
0
500
1000
1500
2000
2500
CircAvgIntenCh2
3000
3500
3.5
0.37
0.16
12.28
• The multi-parametric nature of the HCS DNA damage assay revealed drug
concentrations at which only pre-lethal genotoxicity was observed while higher
drug concentrations induced both genotoxicity and a lethal loss of plasma
membrane integrity.
• The DNA damage assay is robust and consistent as indicated by a coefficient
of variation that is less than 20%, Z factors >0.3, and by valinomycin-induced
increases in pH2AX and Image-iT® DEAD Green™ (I10291) signals of >3-fold
and >10-fold, respectively.
• The HCS DNA damage assay can be combined with other assays for DNA
damage such as the Click-iT® TUNEL assay (A10028, C10081, C10082), a
biomarker typically associated with apoptosis, to facilitate more mechanistic
insight in drug profiling with respect to genotoxicity.
1.1
1.4
1. Nikiforovam, M.N.; et al. Science 2001, 290, 138-141.
60 min at room temperature, protected from light
2. Vamvakas, S., Vock,; et al. Crit. Rev. Toxicol. 1997, 27, 155-174.
Figure 6. The data represents robustness and consistency achieved in measuring DNA
damage and cytotoxicity between 3 min/max plates.
4000
Figure 8. A549 cells were treated with etoposide or valinomycin followed by
assessment of DNA damage using the click chemistry-based Tdt-mediated dUTP
nick end labeling (TUNEL) assay and the HCS DNA Damage Kit. Most, but not all
cells treated with 800 M etoposide or 120 M valinomycin showed significant
increases in both TUNEL (Ch2) and pH2AX (Ch3), demonstrating that these two
assays may be combined to provide mechanistic insight in drug profiling with respect
to genotoxicity.
References
15.3
250
1000
Incubate cells with the pH2AX primary antibody
3.6
225
1250
• The HCS DNA Damage Kit (H10292) enables sensitive and simultaneous
detection of genotoxicity (pH2AX) and cytotoxicity (plasma membrane integrity)
by automated imaging and analysis.
500
Fix, permeabilize and block cells
Image-iT®
Dead Green™ Viability Stain
200
1000
3
13.5 3
175
Conclusions
Figure 6. HCS DNA Damage Assay: Robustness & Consistency
DNA Damage (valinomycin)
100
125
150
CircAvgIntenCh2
120 uM Valinomycin
Control
1250
50
EC50=0.4 mM
Figure 5. A549 cells were treated with valinomycin, etoposide and H2O2 for 24 hours
and assayed using the HCS DNA Damage Kit. EC50 values were generated for the
three test compounds.
Measured Parameter
0
Cir cAvgIn te nCh3
100
50
0
500
150
75
25
25
200
150
800 uM Etoposide
250
225
Cir cAvgIn te nCh3
Phopsho-H2AX
Nuclear Intensity
300
200
Cells/field
Cells/field
Cell membrane
permeability
Cell membrane
permeability
(mean avg
avg intensity)
intensity)
(mean
EC50=32.6 M
Incubate cells with Image-iT® DEAD Green™ viability stain
Image acquisition and analysis
80
60 M
15 M
DMSO
(Hoechst 33342)
Plate cells
Seal plate
100
0
Nuclear morphology
Log Etoposide( M)
Final washes and addition of PBS to each well
120
20
-3
Incubate cells with Alexa Fluor® 555 conjugated secondary
antibody / Hoechst 33342 nuclear counterstain
140
40
0
60 min at room temperature
100
DMSO
400
30 min under normal cell culture conditions
150
200
Figure 2. HCS DNA Damage Assay Work Flow
Test compound or drug treatment
200
0
(Image-iT® DEAD
Green™ viability stain)
400
HCS DNA Damage Kit Configuration:
• Hoechst 33342
• Image-iT® DEAD Green™ viability stain
• Mouse anti-pH2AX IgG
• Alexa Fluor® 555 goat anti-Mouse IgG
250
50
Figure 4. Quantitative representation of DNA damage, cytotoxicity, and cell loss with
increasing concentrations of valinomycin in A549 cells. The DNA damage assay with Factin and nuclear counterstaining (Alexa Fluor® 647 phalloidin – purple, Hoechst - blue)
was performed on fixed and permeabilized cells. The left-side image represents untreated
cells with intact F-actin cytoskeleton and no evidence of toxicity. The right-side image
illustrates both genotoxicity and cytotoxicIty (pH2AX detected with Alexa Fluor®
555 secondary – orange and Image-iT® DEAD Green™ viability stain - green) as well as
completely disrupted actin cytoskeletons in cells treated with 120 μM valinomycin.
Figure 1. DNA damaging agents induce DSBs that could lead to
reversible or irreversible damage in cells. Irreparable damage could
lead to genomic instability, tumorgenesis or cell death.
300
Average Intensity
Camptothecin
50
apoptosis
400
400
Figure 5. Dose Response – Valinomycin, Etoposide, H2O2
cancer
600
0
DMSO
Metabolism
Transducers
800
200
100
Sensors
1000
(pH2AX antibody/Alexa Fluor®
555 secondary)
endogenous
Damage
1200
H2O2
DNA damage
Figure 1. DNA Damage Response Pathway
cell cycle
arrest
500 M
500uM
250
exogenous
250 M
Average Intensity
A double-strand break (DSB) in genomic DNA is potentially a lethal lesion.
One of the known responses to DSB formation is phosphorylation of H2A
histones. Specifically, exogenous or endogenous agents that lead to DNA
damage induce phosphorylation of histone variant H2AX at Ser139
forming DNA foci at the site of DNA DSBs. Phosphorylated H2AX aids in
the recruitment of proteins responsible for double-strand break repair. In
mammalian cells, phosphatidylinositol 3-kinase-like protein kinases such
as ATM (ataxia-telangiectasia mutated), ATR (ATM- and Rad3-related),
and DNA-PKcs (DNA-dependent protein kinase catalytic subunit)
phosphorylate histone variant H2AX. In general, cells have intrinsic
mechanisms to adapt to very low levels of irreparable damage. However,
if a DSB inactivates an essential gene or triggers apoptosis, it can be
sufficient to kill a cell. DNA DSBs are biologically very important because
their repair is intrinsically more difficult than that of other types of DNA
damage. Thus, the intrinsic ability of cells to monitor DSB formation is
essential to the viability of the cell.
Average Intensity
Abstract
3. Richardson, C.; et al. Nature. 2000, 406, 697-700.
4. Karran, P.; et al. Curr. Opin. Genet. Dev. 2000, 10, 144-150.

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