High-Content Imaging Analysis of Cell Loss, Cellular Redox State, ROS Production and
Mitochondrial Membrane Potential Predict Drug Hepatotoxicity
Suk J. Hong,1 Brent A. Samson,1 Chandrasekaran Vasudevan,1 Jeffrey R. Haskins,1 and Richik, N. Ghosh1
Thermo Fisher Scientific • Pittsburgh, PA, USA
1
Thermo Fisher Scientific • 3747 N. Meridian Rd., PO Box 117, Rockford, Illinois U.S.A. 61105
1
10
0
100
1
10
Cmax [fold]
Cmax [fold]
1
0.0
1
10
100
Cmax [fold]
Phenylbutazone - HepG2 (24h)Trazodone HCl
+
6
4
3
Cyproheptadine HCl
1
2
1
0.0
1
10
0
100
1
10
0
100
1
10
Cmax [fold]
Cmax [fold]
Cmax [fold]
100
Novobiocin
0
1
10
100
Cmax [fold]
Troglitazone
30
0.5
10
100
1.5
1.0
0.5
0.0
1
10
6.25 Cmax
Cell Number
#1
#2
#3
1.0
0.5
Cmax [fold]
100
6
#1
#2
#3
5
4
3
2
0
100
10
Cmax [fold]
100
1
2
Fluoxetine1
Fluoxetine2
Fluoxetine3
1
0
1
10
10
100
15
Melatonin1
Melatonin2
Melatonin3
1
0
1
10
1.5
1.0
10
Cmax [fold]
100
0
1
0.5
10
Cmax [fold]
1.0
100
1
0
1
100
10
Cmax [fold]
2.5
6
5
4
3
2
1
1.0
0.5
1
10
100
Cmax [fold]
12.5 Cmax
10
100
6
Troglitazone1
Troglitazone2
Troglitazone3
5
4
3
100
1
10
Cmax [fold]
0.5
1
10
0
1
3.0
2.5
2.0
1.5
1.0
0.5
0.0
1
10
10
100 Cmax [fold]
Cmax [fold]
FCCP 1
FCCP 2
FCCP 3
100
100
100
2.0100
5.0
0.0
1.5
1
10
100
3
Dantrolene -
2
0.5
10
100
Dantrolene - HepG2 (24h)
Fluoxetine - HepG2 (24h)
0.0
100
Cmax [fold]
2
10
Cmax [fold]
1
0
1
10
100
Rosiglitazone - HepG2 (24h)
100
100
Rosiglitazone1
Rosiglitazone2
2
Rosiglitazone1
Rosiglitazone2
Rosiglitazone3
0.5
0.0
1
0.5
10
Cmax [fold]
10
Cmax [fold]
10
1
10
3
Cmax [fold]
0.5
2
1
0
1
2
1
2.0
Aspirin1
Aspirin2
Aspirin3
1.5
1.0
1
10
100
2.5
10
Cmax [fold]
100
Mitochondrial Membrane
Potential Change
Cyprohep1
Cyprohep2
Cyprohep3
1
10
1.5
1.0
0.5
0.0
1
2.0
1.5
1.0
0.5
0.0
1
10
10
Cmax [fold]
100
2
1
0
1
100
12.5
7.5
2.5
Cmax [fold]
1
10
3
2
1
0
1
10
Cmax [fold]
100
25
Cyprohep1
Cyprohep2
Cyprohep3
20
15
10
5
0
1
Cmax [fold]
10
Cmax [fold]
100
100
6
4
3
2
1
0
1
10
Dantrolene1
Dantrolene2
Dantrolene3
4
3
2
1
0
1
10
Melatonin - HepG2 (24h)
Fluoxetine - HepG2 (24h)
100
2
Fluoxetine1
Fluoxetine2
Fluoxetine3
1
0
1
10
Cmax [fold]
100
2
100
Melatonin1
Melatonin2
Melatonin3
1
0
1
10
Cmax [fold]
100
2.5
1.5
1.0
0.5
0.0
1
10
Cmax [fold]
100
6
0
1
10
GSH total
5
4
3
2
0
100
1
0.0
10
Primary rat
hepatocytes
evaluated on
the ToxInsight
HCS Reader:
Phenylb1
Phenylb2
Phenylb3
1
10
Cmax [fold]
100
2.5
Troglitazone1
Troglitazone2
Troglitazone3
2.0
1.5
2
0.0
10
Cmax [fold]
100
1
10
10
Cmax [fold]
1
0
0
-
-
+
+
Ticlopidine
+
Trazodone HCl
100
1
1
-
-
100
>2
0.5
0.0
0
1.0
-
0.0
> 2.5
1
+
-
-
-
-
+
5
Aspirin1
Aspirin2
Aspirin3
3
1
1
+
3
1
10
P
1
P
ROS total
P
N
N
1.0
N
0.5
0.0
0.5
Known
Cmax [fold]
DILI
10
P
P
P
P
P
P
P
P
P
P
3
P
P
-
+
-
-
-
1
P
P
Phenylbutazone
+
-
+
+
+
5
P
P
Cyproheptadine HCl
+
+
+
+
-
4
P
P
Novobiocin
+
+
-
+
+
4
P
P
Troglitazone
+
+
+
-
-
3
P
P
Rosiglitazone
-
-
-
-
-
0
N
N
Aspirin
-
-
-
-
0
N
N
Melatonin
2.5
2.0
-
1.5
1.0
1
10
Cmax [fold]
100
-
1.5
Mefe1
Mefe2
Mefe3
-
1.0
-
-
Gemfibrozil- HepG2 (24 h)
2
Gemfib1
Gemfib2
Gemfib3
-
-
0
N
N
0
N
N
1
False Negative = 0
False Positive = 0
0.5
< 0.5
0.0
1
>2
10
Cmax [fold]
100
>2
0
>2
1
10
Cmax [fold]
> 2.5
100
2.5
0.0
100
1
10
1.0
Drug Name
1
10
Cmax [fold]
Cyproheptadine HCl
0.5
1
10
Cmax [fold]
Ticopidin1
Ticopidin2
Ticopidin3
FCCP
0
1
10
Ticlopidine
Trazodone HCl
100
1
10
Cmax [fold]
Dantrolene1
Dantrolene2
Dantrolene3
Rosiglitazone
1.0
0.5
Threshold Value =
Sample/Control
100
Cmax [fold]
-
1.0
0.5
100
100
Cmax [fold]
Trazodo1
Trazodo2
Trazodo3
-
10
2
-
1.5
+
100
0
0.5
0.0
1
-
10
Cmax [fold]
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
+
-
+
100
-
< 0.76
1
-
> 1.42 or
< 0.62
10
-
-
2
P
P
100
Cmax [fold]
1
0.5
+
3
P
P
-
3
P
P
+
+
5
P
P
-
+
1
P
P
-
-
2
P
P
-
0
N
N
-
0
N
N
-
-
0
N
N
-
-
0
N
N
+
10
Cmax [fold]
100
Troglitazone1
Troglitazone2
Troglitazone3
1
-
10
100
Cmax [fold]
Melatonin - HepG2 (24h)
1.5
1.0
Melatonin1
Melatonin2
Melatonin3
0.5
0.0
> 2.28
Phenylb1
Phenylb2
Phenylb3
1.0
0.0
-
0.5
0.0
P
Troglitazone - HepG2 (24h)
-
Fluoxetine1
Fluoxetine2
Fluoxetine3
-
1.0
P
100
-
-
Fluoxetine - HepG2 (24h)
1.5
2
10
1.5
-
-
-
+
1
Phenylbutazone - HepG2 (24h)
+
Novobio1
Novobio2
Novobio3
+
1.0
-
ROS
1
+
+
2.0
Rosiglitazone - HepG2 (24h)
Aspirin1
Aspirin2
Aspirin3
10
+
Cmax [fold]
Known
DILI
3
1
10
> 1.83
Cmax [fold]
100
> 2.02 or
< 0.31
Aspirin - HepG2 (24h)
1.5
1
+
-
1.5
10
+
+
2.5
-
100
Melatonin
1
1
Boolean
“OR”
4
Novobiocin - HepG2 (24h)
Cyprohep1
Cyprohep2
Cyprohep3
2
0.0
0.0
Mito
Change
# of
Positive
Outputs
Tetracyc1
Tetracyc2
Tetracyc3
5
Cmax [fold]
+
0.5
GSH
6
100
1.0
+
Cyproheptadine HCl - HepG2 (24h)
3
1
10
Cmax [fold]
Trazodone HCl - HepG2 (24h)
+
100
Cmax [fold]
1
1.5
-
1
Nuclear
Dye
1
-
Ticopidine - HepG2 (24h)
2
Dantrolene Sodium
Mefenamic Acid
2
0
100
Tetracycline - HepG2 (24h)
Hycant1
Hycant2
Hycant3
Cell
Number
1.5
Dantrolene - HepG2 (24h)
Melatonin1
Melatonin2
Melatonin3
1.0
0.0
100
Cmax [fold]
1.5
Hycanthone- HepG2 (24 h)
3
Nalidixic1
Nalidixic2
Nalidixic3
2.0
1.5
100
1.0
0.5
0.0
2
Rosiglitazone1
Rosiglitazone2
Rosiglitazone3
Aspirin1
Aspirin2
Aspirin3
1
0
False Negative = 0
False Positvie = 0
Sensitivity%
75
50
25
0
0
25
50
75
100
125
GSH
Melatonin1
Melatonin2
Melatonin3
1.0
1
100
100
100
1.5
0.0
75
% False Positive (1 - Specificity)
10
+
M efenamic Acid - HepG2 (24 h)
Troglitazone1
Troglitazone2
Troglitazone3
NCmax [fold]
-
2.0
50
125
N
1
-
2.5
100
Troglitazone - HepG2 (24h)
+
-
10
Cmax [fold]
P
FCCP 1
FCCP 2
FCCP 3
Phenylb1
Phenylb2
Phenylb3
P
P
3.0
% True Positive (Sensitivity)
ROS total
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
4
3.5
4
100
25
% False Positive (1 - Specificity)
DNA
P
-
Cmax [fold]
0
100
Phenylbutazone - HepG2 (24h)
+
Cmax [fold]
10
P
Boolean
“OR” in
Assay
Aspirin - HepG2 (24h)
2
0
100
0
Cmax [fold]
False Negative = 0
False Positive = 0 Melatonin - HepG2 (24h)
# of
Positive
Cmax [fold]
Outputs
10
P
1
+
ROS
-
+
-
Rosiglitazone1
Rosiglitazone2
Rosiglitazone3
1
0
1.5
Fluoxetine1
Fluoxetine2
Fluoxetine3
0.5
2
1
N
100
1.0
+
100
0
25
P
3
N
N
Fluoxetine - HepG2 (24h)
+
10
10
Cmax [fold]
4
P
Novobio1
Novobio2
Novobio3
Tetracyc1
Tetracyc2
Tetracyc3
P
5
P
100
1.5
+
1
-
1.5
0.0
Rosiglitazone - HepG2 (24h)
1.0
0
ROS
GSH
100
Cmax [fold]
1.5
-
P
P
0
Mito
Change
10
5
Trazodo1
Trazodo2
Trazodo3
5
2.0
Dantrolene1
Dantrolene2
Dantrolene3
1
10
Cmax [fold]
2.5
0.5
0.0
1
0.5
1.0
P
Novobiocin - HepG2 (24h)
-
>2
Dantrolene - HepG2 (24h)
1.5
2
5
0.5
+
-
P
+
6
P
100
1
5
1.0
0.0
Cyprohep1
Cyprohep2
Cyprohep3
2
+
+
10
Ticopidin1
Ticopidin2
Ticopidin3
-
10
Cmax [fold]
1.5
+
-
1
P
P
Trazodone HCl - HepG2 (24h)
+
-
Cmax [fold]
-
Tetracycline
10
-
2
0
+
+
+
+
+
-
+
Cyproheptadine HCl - HepG2 (24h)
3
ROS total
-
+
Cmax [fold]
1
-
+
+
1
5
ROS total
+
100
+
2
Hycant1
Hycant2
Hycant3
-
0
Melatonin - HepG2 (24h)
0.5
Tetracyc1
Tetracyc2
Tetracyc3
1
10
Cmax [fold]
3
P
Tetracycline - HepG2 (24h)
+
Aspirin
100
Cmax [fold]
Fluoxetine1
Fluoxetine2
Fluoxetine3
1.0
1
0
+
+
+
+
Troglitazone
1.0
0.5
1
1.5
0.0
10
1
Ticopidine - HepG2 (24h)
+
+
0.0
0.5
Cmax [fold]
+
-
Nuclear
Dye
+
Fluoxetine
Rosiglitazone1
Rosiglitazone2
Rosiglitazone3
1
0.0
Cell
Number
100
Hycanthone
100
Troglitazone - HepG2 (24h)
Novobio1
Novobio2
Novobio3
Fluoxetine - HepG2 (24h)
1.0
+
+
P
50
100
125
Sensitivity%
100
75
50
25
0
0
25
50
75
100
125
% False Positive (1 - Specificity)
ROS
125
Sensitivity%
100
75
50
25
0
0
25
50
75
100
125
% False Positive (1 - Specificity)
Mitochondrial Memabrane Potential Change
125
Sensitivity%
100
75
50
25
0
0
25
50
75
100
125
% False Positive (1 - Specificity)
combined
1
0.8
Cell #
Nuc size
GSH
DNA
ROS
Mito
Boolean "OR"
0.6
0.4
0.2
0
0
0.2
0.4
0.6
0.8
1
False Positive Rate
1
10
100
Figure 4: Hepatotoxicity Assay on HepG2 cells and rat primary hepatocytes. Based on the dose-responsiveness of each target against hepatotoxic
and non-hepatotoxic chemicals, positive or negative assay targets were identified. Hepatotoxicity threshold values were calculated from the
assay target value of non-hepatotoxic drugs in each assay model. A. The ArrayScan Vti was used to obtain and analyze the hepatotoxicity data of
different drugs on HepG2 cells. B. Data acquired using the ToxInsight platform on HepG2 cells. C. Primary rat hepatocytes were used to validate
hepatotoxicity measurements using the ToxInsight platform. D. ROC Curves were generated from the ArrayScan data from (A) on HepG2 cell.
If all five assay outputs are combined with Boolean logic “OR” operation, the assay produces 100% sensitivity and the 100% specificity for this
experimental set of compounds. ToxInsight data from (B) and (C) show the same high specificity and sensitivity.
1
10
Cmax [fold]
100
Cmax [fold]
2
Aspirin1
Aspirin2
Aspirin3
1
0
1
10
100
Conclusions
Troglitazone1
Troglitazone2
Troglitazone3
5
4
3
2
1
0
1
10
100
Cmax [fold]
Figure 3: Profiling drug-induced hepatotoxicity using dose-dependent responses of assay targets in
HepG2 cells treated with hepatotoxic and non-hepatotoxic compounds for 24 hrs. Each drug shows a
distinct dose response pattern for each assay target per each drug, which demonstrates the importance
of multiple target measurements for hepatotoxicity evaluation. Among the five assay outputs, cell number
and DNA are the two outputs showing the most dramatic changes produced by hepatotoxic chemicals in
HepG2 cells. Some drug treatments result in biphasic responses of assay targets in HepG2 cells, which
indicate that hepatotoxicity measurements of many drugs should be tested in different doses and that dose
response data in HepG2 cells should be used to determine drug-induced hepatotoxicity.
References
Cmax [fold]
Figure 2: A. Assay targets and fluorescent channels for this hepatotoxicity measurement. DNA content is detected in channel 1, reduced
glutathione is being detected in channel 2, reactive oxygen species in channel 3 and mitochondria and its membrane potential are
measured in channel 4, respectively. B. Drug chemicals used in this study and its Cmax values. Chemicals in blue are control compounds
and chemicals in red are compounds known to induce hepatotoxicity. Drug dose response (up to 100 Cmax) was investigated in this
study. C. Representative hepatotoxic drug dose response in HepG2 cells acquired by high content imaging. HepG2 cells were treated with
troglitazone in triplicates in different doses for 24 hours. The changes of individual assay targets (DNA, GSH, ROS and mitochondria) are
shown in the dose response curves.
6
1.0
100
100
10
Cmax [fold]
1.5
T roglitazone - HepG2 (24h)
Rosiglitazone1
Rosiglitazone2
Rosiglitazone3
2.0
Cmax [fold]
0.5
Phenylbutazone - HepG2 (24h)
2.0
Cmax [fold]
Cmax [fold]
Rosiglitazone - HepG2 (24h)
1
2.5
100
Dantrolene - HepG2 (24h)
5
10
0.5
3
Nalidixic1
Nalidixic2
Nalidixic3
P
100
75
Aspirin - HepG2 (24h)
Novobio1
Novobio2
Novobio3
5
1
1.0
>2
Aspirin - HepG2 (24h)
Novobiocin - HepG2 (24h)
Cyproheptadine HCl - HepG2 (24h)
5.0
0.0
100
4
-
0.5
Cmax [fold]
1
Tetracycline - HepG2 (24h)
5
Cmax [fold]
Phenylb1
Phenylb2
Phenylb3
10.0
10
6
5.0
0.0
3.0
Melatonin1
Melatonin2
Melatonin3
Dantrolene1
Dantrolene2
Dantrolene3
10
-
Nalidixic Acid - HepG2 (24 h)
Tetracyc1
Tetracyc2
Tetracyc3
7.5
100
Trazodo1
Trazodo2
Trazodo3
Cmax [fold]
100
Hycant1
Hycant2
Hycant3
C.
Novobiocin - HepG2 (24h)
100
100
1
Aspirin1
Aspirin2
Aspirin3
100
8
7
10
3.5
Rosiglitazone - HepG2 (24h)
9
1
2
0
1
0
Hycanthone- HepG2 (24 h)
Nalidixic1
Nalidixic2
Nalidixic3
Phenylbutazone - HepG2 (24h)
Trazodo1
Trazodo2
Trazodo3
Cmax [fold]
0
100
2.0
3
GSH total
1
Dantrolene - HepG2 (24h)
0.0
Nalidixic Acid - HepG2 (24 h)
Gemfib1
Gemfib2
Gemfib3
Trazodone HCl - HepG2 (24h)
Ticopidin1
Ticopidin2
Ticopidin3
0.5
0.0
10
2.5
100
3
100
2
Cmax [fold]
Gemfibrozil- HepG2 (24 h)
Mefe1
Mefe2
Mefe3
10
0.5
10
-
Nalidixic Acid
T etracycline - HepG2 (24h)
10.0
1
2
1
-
Gemfibrozil
0.0
100
12.5
Hycant1
Hycant2
Hycant3
Trazodone HCl - HepG2 (24h)
Ticopidin1
Ticopidin2
Ticopidin3
1.5
10
10
Cmax [fold]
100
-
Melatonin1
Melatonin2
Melatonin3
0.5
1
-
Troglitazone1
Troglitazone2
Troglitazone3
1.0
Threshold Value =
Sample/Control
2
Cmax [fold]
Troglitazone1
Troglitazone2
Troglitazone3
1.0
Aspirin1
Aspirin2
Aspirin3
1
2
0
1
0
1
3
0
-
1.5
GSH total
GSH total
GSH total
GSH total
GSH total
1.5
0.5
10
1
100
10
100 HCl - HepG2 (24h)
Cyproheptadine
Cmax
[fold]
4
1
100
4
1
Hycanthone- HepG2 (24 h)
Cmax [fold]
Cmax [fold]
Cmax [fold]
10
Cmax [fold]
3
Cmax [fold]
Ticopidine - HepG2 (24h)
0
Cmax [fold]
10
10
1
0
1
10
Cmax [fold]
3
Melatonin1
Melatonin2 0.0
Melatonin3
100
1
100
100
1.0
2
1
0
1
100
Aspirin Rosiglitazone3
- HepG2 (24h)
1.0
0.0
10
1.5
Aspirin -0.0HepG2 (24h)
Cmax [fold]
1.0
Rosiglitazone - HepG2 (24h)
1.5
1
1.5
100
Fluoxetine1
Fluoxetine2
Fluoxetine3
1
Cmax [fold]
Melatonin1- HepG2 (24h)
10
Cmax [fold]
M elatonin - HepG2 (24h)
0.5
0.0
Cmax [fold]
0.0
Aspirin1
Aspirin2
Aspirin3
10
Cmax [fold]
-
3
10
Cmax [fold]
1
Troglitazone1
Troglitazone2
100
Troglitazone3
0.5
2
0.5
1
100
3
1
0
GSH total
GSH total
GSH total
10
Troglitazone1
Troglitazone2
Troglitazone3
1.0
Fluoxetine1
Fluoxetine2
0.0
Fluoxetine3 1
100
1.0
100
4
0
1.5
1.5
Dantrolene1
Dantrolene2
1
Dantrolene3
5 [fold]
Cmax
10
1.0
0.5
10
(24h)
1.0 [fold]
Cmax
Fluoxetine -1HepG2 (24h)
Dantrolene1
100
Cmax Dantrolene2
[fold]
Dantrolene3
1
Cmax [fold]
3
Melatonin1
Melatonin2
10
Melatonin3
100
Cmax [fold]
1.5
10
3
HepG22 (24h)
1
0
6
1
0
5
+
Aspirin1
2.5
GSH total
4
1
Novobio1
Novobio2
Novobio3
Phenylb1
Phenylb2
Phenylb3
Troglitazone - HepG2 (24h)
1.5
Troglitazone - HepG2 (24h)
0.0
100
DNA + Lipid
5
9
8
7
6
5
4
100
2
1
Nalidixic1
Nalidixic2
0
Nalidixic3
2
1
ROS total
1.0
1
Novobiocin - HepG2 (24h)
1
10
Cyprohep1
Cmax [fold]
Cyprohep2
Cyprohep3
GSH total
ROS total
2
Cmax [fold]
1.0
100
Phenylbutazone - HepG2 (24h)
Phenylb1
Phenylb2
Phenylb3
0
Novobio1
Novobio2 1
Novobio3
2.5
100
2
GSH total
Novobiocin - HepG2 (24h)
Mitochondrial Membrane
Potential Change
ROS total
Mitochondrial Membrane
Potential Change
7.5
3
4
+
FluoxetineFCCP- HepG2 (24 h)
Gemfib1
Gemfib2
Gemfib3
6
+
Aspirin2
Mefenamic
Acid
Aspirin3
ROS total
10
Cmax [fold]
1
10.0
2.5
6
5
7
GSH total
1
Cyprohep1
1
10
Cyprohep2
Cmax [fold]
Cyprohep3
100
Mefe1
Mefe2
Mefe3
6
GSH total
Trazodo1
Trazodo2
Trazodo3
10
4.5
10
4.0 [fold]
Cmax
3.5
3.0
2.5
2.0
1.5- HepG2
T roglitazone
1.0
0.5
0.0
1
7
+
FCCP
ROS total
10
Cmax [fold]
Gemfibrozil- HepG2 (24 h)
Cmax [fold]
1
1
10
1
100
10
100
M efenamic Acid - HepG2 (24 h)
GSH total
1
1
Novobio1
Novobio2
10
Novobio3
Cmax [fold]
2
Tetracyc1
Tetracyc2
Tetracyc3
1
GSH total
0.0
10
1
Nalidixic Acid - HepG2 (24 h)
0
1
Trazodo1
Trazodo2
Trazodo3
1
1
GSH total
2
0
2
DNA + Lipid
DNA + Lipid
Cmax [fold]
Cmax [fold]
Cmax [fold]
2
1
10
Cmax [fold]
Aspirin3 - HepG2 (24h)
Cmax [fold]
Rosiglitazone1
5
10
100
Rosiglitazone2
Cmax
[fold]
4
Rosiglitazone3
Phenylbutazone - HepG2 (24h)
DNA + Lipid
4
0
3
Cmax [fold]
2.0
1.0
HepG2 (24h)
2
0
ROS total
DNA + Lipid
0
0.5
0.0
1.5
4
1
- HepG2 (24h)
Trazodone HCl - HepG2 (24h)
5
100
0
FCCP 1
FCCP 2
FCCP 3
Tetracyc1
Tetracyc2
Tetracyc3
2
1
100
Mitochondrial Membrane
Potential Change
100
2.5
4
ROS total
10
1.0
3
5
5
Cmax [fold]
Cmax [fold]
100
ROS total
10
Rosiglitazone
Cmax
[fold]
10
6
Mitochondrial Membrane
Potential Change
DNA + Lipid
ROS total
DNA + Lipid
Mitochondrial Membrane
Potential Change
DNA + Lipid
10
Ticopidin1
Cmax [fold]
Ticopidin2
Ticopidin3
100
6
3
0
0.0
1
GSH
FCCPTetracycline - HepG2
(24h)HepG2 (24 h)
Cmax [fold]
Gemfib1
Gemfib2
Gemfib3
#1
#2
#3
4
0.5
1
2
50 Cmax
5
0.5
Tetracyc1
Tetracyc2
Tetracyc3
Cmax [fold]
6
GSH total
DNA + Lipid
DNA + Lipid
ROS total
Mitochondrial Membrane
Potential Change
ROS total
100
1
10
Novobiocin3 - HepG2 (24h)
1.0
2
Hycant1
Trazodone HCl Hycant2
Hycant3
1.5
ROS total
10
ROS total
DNA + Lipid
1
100
DNA + Lipid
DNA + Lipid
ROS total
Mitochondrial Membrane
Potential Change
Mitochondrial Membrane
Potential Change
GSH total
GSH total
2
Mitochondrial Membrane
Potential Change
DNA + Lipid
GSH total
Cell Number
Cell Number
Cell Number
ROS total
Mitochondrial Membrane
Potential Change
GSH total
Mitochondrial Membrane
Potential Change
Mitochondrial Membrane
Potential Change
Mitochondrial Membrane
Potential Change
GSH total
0.0
3
1
1
Mefenamic Acid - HepG2 (24 h)
Tetracyc1
Tetracyc2
Tetracyc3
4.5
4.0
3.5
Cmax [fold]
T roglitazone - HepG2 (24h)
1.5
0.0
1
1
Melatonin1
0
1
Melatonin2
Melatonin3
Aspirin1
Aspirin2
Aspirin3
Ticopidine - HepG2 (24h)
Novobio1
Novobio2
Novobio3
6
0.5
1
1
4
6
3
100
1.5
Cyprohep1
0.0
Cyprohep21
Cyprohep3
100
1
2
Tetracycline - HepG2 (24h)
1
0.5
Cyproheptadine HCl - HepG2
(24h)
3
7
Cmax [fold]
Aspirin - HepG2
(24h)
2.5
Cmax [fold]
Rosiglitazone1
Rosiglitazone2
Rosiglitazone3
2.0
100
1
Novobiocin - HepG2 (24h)
0
GSH total
1.0
0.0
100
Troglitazone2
Troglitazone3
Melatonin - HepG2 (24h)
Aspirin1
Aspirin2
Aspirin3
0.5
FCCP- HepG2 (24 h)
2
Mitochondrial Membrane
Potential Change
2
MMP Change
#1
#2
#3
1
10
Cmax [fold]
Mitochondrial Stain
1.0
0.0
3
Mitochondrial Membrane
Potential Change
Mitochondrial Membrane
Potential Change
100
1.5
2.0
4
Rosiglitazone - HepG2 (24h)
ROS
#1
#2
#3
5
ROS Staining
6.25 Cmax
188
10
Cmax [fold]
2.5
1
20
100
2
0.78 Cmax
50 Cmax
0.0
2.5
Cmax [fold]
Cmax [fold]
0.5
1
Mitochondrial Membrane
Potential Change
Mitochondrial Membrane
Potential Change
1
10
5.0
0.0
5
282
Cyprohep1
Cyprohep2
Cyprohep3
1.88
Melatonin - HepG2 (24h)
GSH
1
10
2
12.5 Cmax
DNA
DNA content
Cell Number
1.5
1
3
1
7.5
25
Cmax [fold]
ROS total
0.78 Cmax
0.0
Dantrolene1
Dantrolene2
Dantrolene3
4
GSH Staining
GSH total
Mitochondrial damage may result in the release of excessive amounts of ROS,
which damages itself and other hepatic cells.5 The redox state of the cell is critical
to maintain hepatic physiology, where oxidative stress induced by drug chemical
damages hepatic cell. One of the cellular primary defense systems against cellular
oxidation is glutathione mediated reduction of molecules. Therefore, the glutathione
level in the cell can serve as a good indicator of cellular response against damage
mediated by oxidation reaction.
5
0
100
100
Cyproheptadine HCl - HepG2 (24h)
Phenylb1
Phenylb2
Phenylb3
10.0
Fluoxetine - HepG2 (24h)
Dantrolene - HepG2 (24h)
Nuclear Staining
12.5
Cmax [fold]
Cmax [fold]
10
2.82
Phenylbutazone - HepG2 (24h)
Trazodone HCl
2.0
1
213
6
Cmax [fold]
Mitochondrial Membrane
Potential Change
1
Trazodo1
Trazodo2
Trazodo3
100
0
8
7
100
1.0
Tetracycline - HepG2 (24h)
Hycant1
Hycant2
Hycant3
931.7
1
1.0
1.0
1
Drug Name
ROS total
Rosiglitazone1
Rosiglitazone2
Rosiglitazone3
10
10
1.5
8
100
1.0
0.0
0
5
Cmax [fold]
Cyproheptadine HCl - HepG2 (24h)
0
100
Aspirin - HepG2 (24h)
1.5
0.5
Cmax [fold]
Cmax [fold]
Aspirin - HepG2 (24h)
1
10
1.5
10
0.0
Cmax [fold]
Rosiglitazone - HepG2 (24h)
Cmax [fold]
10
0
5
6
10
Ticopidin1
Ticopidin2
Ticopidin3
Troglitazone1
1.5
1 Troglitazone2
10
1.0
0.5 Cmax [fold]
Troglitazone3
Melatonin - HepG2 (24h)
0.5
Rosiglitazone1
Rosiglitazone2
Rosiglitazone3
10
1
100
10
Cmax [fold]
2
Hycant1
Hycant2
Hycant3
0.5
15
0
Melatonin1
Melatonin2
1
Melatonin3
Ticopidine - HepG2 (24h)
3.0
2.5
2.0
10
Cmax [fold]
100
7
100
4.0
3.5
Cmax
[fold]
Troglitazone1
1.5
Fluoxetine1
Fluoxetine2
100
Fluoxetine3
1
20
1
1
3
0.5
0.5
0.5
1.0
0.0
1.0
1
1.0
25
1
4.5
Cmax [fold]
1.0
0.0
0
4
2.0
Cyproheptadine HCl - HepG2 (24h)
1
8
1
100
0.0
1
Fluoxetine11
Fluoxetine2
Fluoxetine3
100
Cmax [fold]
Cmax [fold]
Mitochondrial Membrane
Potential Change
1.0
2.5
10
Cmax [fold]
2
Cmax [fold]
0.5
0.0
100
Tetracycline - HepG2 (24h)
Mitochondria and Mitochondrial Membrane Potential Change
Hycanthone- HepG2 (24 h)
9
Fluoxetine - HepG2 (24h)
10
10
Cmax [fold]
Nalidixic3
0
100
1.5
1.5
1
0.0
10
100
Rosiglitazone
- HepG2
(24h)
0.5
Cmax [fold]
1.5
1
Mitochondrial Membrane
Potential Change
1.5
0.0
1
Troglitazone
Mitochondrial Membrane
Potential Change
Mitochondrial Membrane
Potential Change
C.
0.0
9.317
2.13
0.5
Trazodone HCl - HepG2 (24h)
Ticopidin1
Ticopidin2
Ticopidin3
2.0
100
1.0
Cmax [fold]
Ticopidine - HepG2 (24h)
2.5
10
1.5
Nalidixic1
Nalidixic2
Nalidixic3
10
1.5
Rosiglitazone1
Rosiglitazone2
Rosiglitazone3
2
GSH total
0.5
1
100
Novobio1
Novobio2
Novobio3
5
GSH total
100
Cmax [fold]
0.0
10
100
Hycant1
Hycant2
Hycant3
6
2.5
10
Hycanthone- HepG2 (24 h drug treatment)
2
Cmax [fold]
0.5
1
10
Cmax [fold]
DNA + Lipid
10
1.5
Cmax [fold]
1.0
0.0
Troglitazone - HepG2
(24h)
Fluoxetine - HepG2 (24h)
Dantrolene1
Dantrolene2
Dantrolene3
100
1.0
2
0
2
ROS total
0.0
10
2.5
10
Phenylb1
2.0 Cmax [fold]
Phenylb2
1.5
Phenylb3
1
3
3
Mefe1
Mefe2
Mefe3
2
ROS total
Cmax [fold]
1.5
Mitochondrial Membrane
Potential Change
1
2.0
3
HepG2 cells
evaluated on
the ToxInsight
HCS Reader:
1
+
Threshold Value =
Melatonin - HepG2 (24h)
Sample/Control
< 0.7
ROS total
Cell Number
GSH total
Cell Number
Cell Number
Dantrolene1
Dantrolene2
Dantrolene3
100
1
1
Nalidixic1
ROS total
10
1
0.0
15000
Mitochondrial Membrane
Potential Change
100
Ticlopidine
Gemfib1
Gemfib2
Gemfib3
Mitochondrial Membrane
Potential Change
10
Cmax [fold]
0
Mitochondrial Membrane
Potential Change
1
1
2.5
Mitochondrial Membrane
Potential Change
1.0
0.5
0.0
Tetracycline
2
Nalidixic Acid - HepG2 (24 h)
Mitochondrial Membrane
Potential Change
3.0
2.5
2.0
1.5
Mefe1
Mefe2
Mefe3
Mitochondrial Membrane
Potential Change
FCCP 1
FCCP 2
FCCP 3
Mitochondrial Membrane
Potential Change
Mitochondrial Membrane
Potential Change
4.5
4.0
3.5
150
Gemfibrozil- HepG2 (24 h)
0.0
100
1
Nalidixic1
Nalidixic2
Nalidixic3
Ticopidine
Nalidixic2 - HepG2 (24h)
3.0
2.0
1.0
0.5
Dantrolene - HepG2 (24h)
1.0
Aspirin1
Aspirin2
Aspirin3
Novobio2 100
1
1
0
DNA + Lipid
Cmax [fold]
10
Cmax [fold]
1.0
1
0
100
1.5
2.5
0.5
Novobio1
0.0
3.5
ROS total
10
10
Cmax [fold]
Novobio3
100
Phenylb1
Phenylb2
Phenylb3
Nalidixic Acid - HepG2 (24 h drug treatment)
2.5
DNA + Lipid
1
1
10
Cmax [fold]
100
5.0
Troglitazone - HepG2 (24h)
GSH total
Dantrolene - HepG2 (24h)
1
1.5
0.0
2.0
Cmax [fold]
Phenylb1
Phenylb2
Phenylb3
1.0
Cell Number
0.5
Cell Number
Cyprohep1
Cyprohep2
Cyprohep3
1.0
3000
Mitochondrial Membrane
Potential Change
Phenylbutazone
Mefenamic Acid - HepG2 (24 h)
3
GSH total
Cell Number
GSH total
DNA + Lipid
Mitochondrial Membrane
Potential Change
Cell Number
GSH total
Mitochondrial Membrane
Potential Change
Cell Number
GSH total
0.5
0
2.5
0.0
1.5
Mitochondria
and Mitochondrial Membrane
Potential
Change
Novobiocin
62.5
6250
FCCP- HepG2 (24 h)
1
GSH total
1.0
Phenylbutazone - HepG2 (24h)
1.5
0.5
0.0
Nalidixic Acid
3.0
1
0.0
Melatonin
-1HepG2 (24h)
10
10
1.0
7.5
100
Fluoxetine1
Fluoxetine2
1
Fluoxetine3
0
Trazodo1
Trazodo2
Trazodo3
10.0
1.5
0.5
0.5
10 2
100
DNA content
4
FCCP 1
FCCP 2
FCCP 3
2.0
3
0
3
Cmax [fold]
100
2.5
1
4
1
1
12.5
5
0.0
Fluoxetine - HepG2
(24h)
1
10
3.5
6
100
2
Novobiocin - HepG2 (24h)
0.5
0.0
1.0
Trazodo1
Cmax [fold]
Trazodo2
Trazodo3
0.5
Novobiocin - HepG2 (24h)
2
100
Cmax [fold]
100
1.0
Cmax [fold]
0.0
650
10
10
1
10
6
0.0
0.0
8
7
0
0
1
1
10
100
10
100
HycanthoneHepG2 (24
h) (24 h drug treatment)
FCCP- HepG2 (24 h drug treatment) Nalidixic Acid - Mefenamic
HepG2 (24Acid
h) - HepG2 (24 h drug treatment)
GemfibrozilHepG2
Cmax [fold]
Cmax [fold]
1
Tetracyc1
Phenylbutazone
- HepG2 (24h)
Tetracyc2
Tetracyc3
10
Cmax [fold]
0.5
HepG2 (24 h)
1
GSH total
1
Mitochondrial Membrane
Potential Change
Cell Number
GSH total
DNA + Lipid
0
1
2
1 Hycant3
100
1.5
Cyprohep1
100
Cyprohep2
Cyprohep3
10
Cmax [fold]
Cmax [fold]
Cell Number
Cmax [fold]
1.5
100
1
Tetracycline - HepG2 (24h)
1.5
1
Nalidixic1
Nalidixic2
Nalidixic3
1.0
2
9
GSH total
Cmax [fold]
6.5
1
Cyproheptadine
HCl - HepG2 (24h)
0
10
Cmax [fold]
10
Gemfib1
Gemfib2
Gemfib3
1.5
Gemfib1
Gemfib2
Gemfib3
Melatonin
0.0
0.5
Gemfibrozil- HepG2 (24 h)
2
Mefe1
Mefe2
HycanthoneMefe3
1.0
+
+
-
1.5
Fluoxetine
Fluoxetine1
Fluoxetine2
Fluoxetine3
1.0
0.0
100
Cmax [fold]
0.0
Mitochondrial Membrane
Potential Change
10
10
Rosiglitazone - HepG2 (24h)
Mitochondrial Membrane
Potential Change
1
DNA + Lipid
DNA + Lipid
DNA + Lipid
0.0
1
100
B.
1
0
100
100
2.0
3
Mitochondrial Membrane
Potential Change
Cmax [fold]
0.5
2
Ticopidin1
Ticopidin2
Ticopidin3
1
Hycant1
0.0
0.0
Phenylbutazone
- HepG2 (24h)
2
Cmax [fold]
Trazodone HCl - HepG2Cmax
(24h)[fold]
0Cyproheptadine HCl - HepG2 (24h)
1
10
100
1.5
100
Melatonin1
Melatonin2
Melatonin3
4640
1000
Cmax [fold]
10
Cmax [fold]
Fluoxetine - HepG2 (24h)
1.5
M efenamic Acid - HepG2 (24 h)
Mitochondrial Membrane
Potential Change
Known
Hepatotoxic
Compounds
Mefenamic Acid
100
10
1.5
100
1
100
1
1
Aspirin - HepG2 (24h)
Rosiglitazone1
Rosiglitazone2
Rosiglitazone3
10
2.5
Mitochondrial Membrane
Potential Change
10
2
M elatonin - HepG2 (24h)
2
10
1
1.5
Dantrolene1
Dantrolene2
Dantrolene3
Cmax [fold]
FCCP 1
FCCP 2
DNA + Lipid
1
46.4
n/a (10)
1.0
10
1
100
0.5
Mitochondrial Membrane
Potential Change
Cmax [fold]
Hycanthone
1
Cmax [fold]
Rosiglitazone - HepG2 (24h)
1.5
0.5
0.0
100
Cmax [fold]
Aspirin1
Aspirin2
Aspirin3
1.0
0.0
100
1
100
Aspirin - HepG2 (24h)
NalidixicFCCP
Acid3- HepG2 (24 h)
0.0
Mitochondrial Membrane
Potential Change
10
10
10
1
1
1.0
100
1.0
7.5
Trazodo1
Trazodo2
10
100
Cmax [fold]0.0Trazodo3
1.5
1.5
Tetracyc1
5
Tetracyc2
4
Tetracyc3
5.0
2.0
0.5
Gemfib1
Gemfib2
Gemfib3
10.0
100
2
Dantrolene1
0
Dantrolene2
Dantrolene3
[fold]
TicopidineCmax
- HepG2
(24h)
3
0.54
1
Cmax [fold]
14
0.0
3
0
Fluoxetine1
Fluoxetine2
Fluoxetine3
1.0
0.0
10
100 - HepG2 (24h)
Dantrolene
5
3
1
100 uM
0.5
10
Cmax [fold]
2.5
Mitochondrial Membrane
Potential Change
1
1
1.0
0.5
1
0
1
3.0
(24 h)
2.0
DNA + Lipid
0.0
0
100
Cmax [fold]
124
0.5
3.5
2.5
T etracycline - HepG2 (24h)
Mitochondrial Membrane
Potential Change
0.5
10
n/a (1 uM)
1
Troglitazone1
Troglitazone2
Troglitazone3
4
0.5
1
1
2.0
100
3
10
Cmax [fold]
2.5
0
10
1.5
Cmax [fold]
1
12.5
Hycant1
Hycant2
1
Hycant3
Trazodone HCl - HepG2Hycant2
(24h)
1.0
2
Cmax [fold]
2.5
1.0
Hycanthone- HepG2 (24 h drug treatment)
0.5
1.5
Ticopidin1
Ticopidin2
Ticopidin3
Cmax [fold]
100
5
10
3
Mitochondrial Membrane
Potential Change
Cell Number
Rosiglitazone1
Rosiglitazone2
Rosiglitazone3
1.0
1
2
Gemfibrozil
1.5
Cell Number
Channel 4
(Orange)
0.0
100
Aspirin - HepG2 (24h)
Rosiglitazone - HepG2 (24h)
0.5
Novobio1
Novobio2
Novobio3
1
0.5
0
Troglitazone - HepG2 (24h)
Nalidixic1
Nalidixic2
Nalidixic3
100
10
Cmax [fold]
1
Mefe1
Mefe2
Mefe3
4
100
Gemfibrozil- HepG2 (24 h drug treatment)
Trazodone HCl - HepG2 (24h)
1
5
Mitochondrial Membrane
Potential Change
10
Cmax [fold]
1.5
1.0
10
6
Fluoxetine - HepG2 (24h)
1.5
Dantrolene1
Dantrolene2
Dantrolene3
100
Cmax [fold]
10
Cell Number
1
Cmax [fold]
Mitochondria
(mitochondrial
membrane potential
and plasma
membrane integrity)
3
Melatonin1
Melatonin2
Melatonin3
100
Ticopidine - HepG2 (24h)
0.0
100
Cmax [fold]
10
100
0
2
Ticopidin1
Ticopidin2
1
Ticopidin3 0
6
(24 h)
0
Mefe1
Mefe2
Mefe3
0.00
Cmax [fold]
10
0.0
1.02
1.5
3000
10
0.25
1
2.5
1
0.50
GSH total
0.0
100
1.24
1
Ticopidine - HepG2 (24h)
0.75
2.5
2.0
1
10
1
Cmax [fold]
3
Mitochondrial Membrane
Potential Change
10
30
100
Dantrolene - HepG2 (24h)
1.5
FCCP
0.5
10
Cmax [fold]
Melatonin - HepG2 (24h)
1.0
1
5.0
0.0
37.3
Melatonin1
Melatonin2
Melatonin3
0.0
100
10
2
Aspirin1
Aspirin2
Aspirin3
FCCP- HepG2 (24 h)
Gemfib1
Gemfib2
GemfibrozilGemfib3 HepG2
1
1.00
Mitochondrial Membrane
Potential Change
1
2
0
100
7.5
0.557
3
2
1
0
Phenylb1
Phenylb2
Phenylb3
1.25
Nalidixic3
1
1
7
Mefe1
Mefe2
HepG2
Mefe3
2
1
Hycanthone(24 h)
Mefenamic Acid - HepG2
(24 h drug HepG2
treatment)
100
FCCP 1
Nalidixic1
FCCP 2
Nalidixic2
FCCP 3
1.5
4
3
Cell Number
Cmax [fold]
Cmax [fold]
9
8
7
6
5
4
100
Cell Number
0.5
3
100
10.0
Trazodo1
Trazodo2
Trazodo3
100
Cmax [fold]
10
Cmax [fold]
1.0
0.5
0.0
0
Phenylbutazone - HepG2 (24h)
1.5
10
1.0
Gemfibrozil- HepG2 (24 h)
Mefenamic Acid -
5
0
Cell Number
Cell Number
1.0
10
Dantrolene
Fluoxetine1
Fluoxetine2
Fluoxetine3
0.373
4
1
1
1
10
6
1
1.0
Nalidixic Acid - HepG2 (24 h drug treatment)
0.5
1.5
Novobiocin - HepG2 (24h)
Cyprohep1
Cyprohep2
Cyprohep3
5
Cmax [fold]
1.5
Dantrolene1
Dantrolene2
Dantrolene3
0.0
Cmax [fold]
Fluoxetine - HepG2 (24h)
Dantrolene - HepG2 (24h)
0.5
Cyproheptadine HCl
100
0
100
7
6
1
FCCP 1
FCCP 2
FCCP 3
FCCP 1
3
FCCP 2
2
FCCP 3
3.0
2.5
2.0 1
1.5
0.5
GSH total
Cmax [fold]
10
0.015
1
0.00557
8
Troglitazone1
Troglitazone2
Troglitazone3
1.0
0.0
1
10
1
Cmax [fold]
3
2
1
1.02
Tetracyc1
Tetracyc2
Tetracyc3
0
99.5
4
10
2
Rosiglitazone1
Rosiglitazone2
Rosiglitazone3
0.0
100
7
4.5
4.0
3.5
0.0
100
5
1
1
0.0
Dantrolene - HepG2 (24h)
1.0
Cmax [fold]
Rosiglitazone - HepG2 (24h)
M efenamic Acid - HepG2 (24 h)
1
10
FCCPHepG2 (24
h drug
treatment)
Nalidixic
Acid
- HepG2
(24 h)
Tetracycline - HepG2 (24h)
n/a
6
Ticopidin1
Ticopidin2
Ticopidin3
100
2
Trazodone HCl - HepG2 (24h)
Cyproheptadine HCl - HepG2 (24h)
Rosiglitazone
0.5
1
10
Cmax [fold]
Cmax [fold]
1.5
Novobio1
Novobio2
Novobio3
1
10
Cmax [fold]
Cell Number
100
1.5
1.0
Troglitazone - HepG2 (24h)
1.0
0.0
1.5
Cell Number
Reactive Oxygen
Species (ROS)
Cell Number
0.5
100
Cmax [fold]
3.0
2.5
2.0
1
GSH total
1.0
10
100
n/a
0.995
4.0
3.5
0.5
0.0
Melatonin
1.5
Cyprohep1
Cyprohep2
Cyprohep3
10
Cmax [fold]
0.5
1
2
10
0.0
100
1.5
Cmax [fold]
FCCP- HepG2 (24 h)
2
0
Cell Number
1.5
1
100
Novobiocin - HepG2 (24h)
Cyproheptadine HCl - HepG2 (24h)
0.0
10
1.0
0.0
3
0
10
100
100x Cmax
(µg/ml)
4
0.0
Cmax [fold]
0.0
Hycant1
Hycant2
Hycant3
5
1
1
10
Cmax [fold]
6
0.5
4.5
Phenylb1
Phenylb2
Phenylb3
Fluoxetine
1
Ticopidine - HepG2 (24h)
Aspirin
0.5
1
1.0
DNA + Lipid
Cmax [fold]
Reduced Glutatione
(GSH)
Channel 3
(Green)
100
Cmax [fold]
1.5
Trazodo1
Trazodo2
Trazodo3
1.0
0.0
100
1.5
1.0
0.5
7
DNA + Lipid
Cell Number
Channel 2
(Blue)
0.5
10
2.0
1
8
Cmax (µg/ml)
2.5
1
10
0.5
1.0
Cmax [fold]
1.5
Fluoxetine1
Fluoxetine2
Fluoxetine3
Cmax [fold]
0.5
FCCP- HepG2 (24 h)
1.5
Hycanthone- HepG2 (24 h drug treatment)
Nalidixic1
Nalidixic2
Nalidixic3
3.0
Phenylbutazone - HepG2 (24h)
Control
(NonHepatotoxic)
Compounds
1.0
Cell Number
Cell Number
DNA
(lysosome in some
cases)
1
100
3.5
DMSO Control (1 %)
1.5
Ticopidin1
Ticopidin2
Ticopidin3
10
100
Trazodone HCl - HepG2 (24h)
1.5
1
10
0.0
Cmax [fold]
Ticopidine - HepG2 (24h)
0.0
1
0.5
10
Cmax [fold]
1.0
0.0
100
3
Gemfib1
Gemfib2
Gemfib3
2.0
2
DNA + Lipid
0.0
100
Cmax [fold]
Targets
Channel 1
(Red)
Drug-induced toxicity is a complex process affecting multiple pathways in general.
The ability to predict compound toxicity using in vitro assay is enhanced if early
toxicity and cell stress mechanisms are assessed, if the assays are conducted in the
proper biological context using cells, and also if several such assays are multiplexed
to simultaneously monitor and correlate multiple toxicity indicators.1-4 Cell based
assays based on High-content imaging present a viable option for simultaneously
analyzing and correlating different mechanisms involved in cytotoxicity based on
cell morphological and biochemical features. High-content imaging is based on
automated fluorescent microscopy and quantitative image analysis which enables
simultaneous measurement of different cellular targets in a 96 or 384 well format.
Critical factors contributing to the predictive nature of such assays is the ability to
measure early indicators of cell stress, toxicity and apoptosis, and to simultaneously
monitor multiple different parameters of cell stress and toxicity in the same cell.
10
0.5
Name
1.0
1
3
0
Nalidixic Acid - HepG2 (24 h drug treatment)
Tetracyc1
Tetracyc2
Tetracyc3
DNA + Lipid
Imaging Channel
(Fluorescence
Emission Color)
1
1.0
Cell Number
0.0
100
DNA + Lipid
0.5
10
Cmax [fold]
1.5
Hycant1
Hycant2
Hycant3
Cell Number
B.
1.0
1
Tetracycline - HepG2 (24h)
1.5
Nalidixic1
Nalidixic2
Nalidixic3
Cell Number
Cell Number
A.
100
Cell Number
1.5
10
Cmax [fold]
Hycanthone- HepG2 (24 h drug treatment)
Nalidixic Acid - HepG2 (24 h drug treatment)
2
0
DNA + Lipid
1
0
DNA + Lipid
0.00
100
3
1
Cell Number
10
4
2.5
DNA + Lipid
0.25
1
FCCP 1
FCCP 2
FCCP 3
5
10
Gemfibrozil- HepG2 (24 h drug treatment)
Mefe1
Mefe2
Mefe3
DNA + Lipid
0.50
1
4
DNA + Lipid
0.75
0.5
Cmax [fold]
1
0
M elatonin - HepG2 (24h)
1.0
1.5
Mitochondria
and Mitochondrial Membrane Potential
Change
GSH
ROS
Mefenamic Acid - HepG2 (24 h drug treatment)
FCCP- HepG2 (24 h drug treatment)
6
Gemfib1
Gemfib2
Gemfib3
DNA + Lipid
1.00
0.5
Gemfibrozil- HepG2 (24 h drug treatment)
Mefe1
Mefe2
Mefe3
Cell Number
FCCP 1
FCCP 2
FCCP 3
Cell Number
Cell Number
1.25
Cmax [fold]
Aspirin1
Aspirin2
Aspirin3
DNA content
Thermo Scientific Cellomics HCS Assay for Automated Compound
Hepatotoxicity Determination
Mefenamic Acid - HepG2 (24 h drug treatment)
1.0
1
0
100
Cmax [fold]
100
2.0
0.5
Aspirin - HepG2 (24h)
1.5
Rosiglitazone1
Rosiglitazone2
Rosiglitazone3
0.0
DNA + Lipid
Analysis & Visualization
(Thermo Scientific vHCS Discovery ToolBox)
1.0
Cell Number
Automated Data
Management
(Cellomics Store)
10
1
10
100
1.5
Dantrolene1
Dantrolene2
Dantrolene3
1
2.0
Troglitazone - HepG2 (24h)
2.5
Aspirin
2.5
ROS total
Rosiglitazone - HepG2 (24h)
1.5
10
Cmax [fold]
Fluoxetine - HepG2 (24h)
2
Aspirin - HepG2 (24h)
Mitochondrial Membrane
Potential Change
Decisions
(Thermo Scientific HCSExplorer)
0.5
Cmax [fold]
Cmax [fold]
1
2
Rosiglitazone
Novobio1
Novobio2
Novobio3
3.0
1
0
Dantrolene - HepG2 (24h)
3
Melatonin1
Melatonin2
Melatonin3
1
100
2
1
Cmax [fold]
1.0
0.0
100
10
3
3.5
Cyprohep1
Cyprohep2
Cyprohep3
3
ROS total
10
1
Melatonin - HepG2 (24h)
0.5
1
0
100
1.5
Fluoxetine1
Fluoxetine2
Fluoxetine3
0.0
100
10
Cmax [fold]
1.0
3
2
1
0
4
GSH total
10
2
4
GSH total
Cell Number
Cell Number
0.5
1
1
100
1.5
Dantrolene1
Dantrolene2
Dantrolene3
1.0
3
1
Fluoxetine - HepG2 (24h)
Dantrolene - HepG2 (24h)
1.5
0.0
10
Cmax [fold]
4
Troglitazone1
Troglitazone2
Troglitazone3
5
DNA + Lipid
1
5
Novobio1
Novobio2
Novobio3
Novobiocin - HepG2 (24h)
Cyproheptadine HCl - HepG2 (24h)
6
9
8
7
6
5
4
DNA + Lipid
100
0.5
6
DNA + Lipid
10
Cmax [fold]
Cyprohep1
Cyprohep2
Cyprohep3
7
DNA + Lipid
1
1.0
0.0
0.0
Cell Number
0.0
0.5
8
Troglitazone1
Troglitazone2
Troglitazone3
DNA + Lipid
0.5
1.0
Cell Number
1.0
1.5
Novobio1
Novobio2
Novobio3
Troglitazone - HepG2 (24h)
Novobiocin - HepG2 (24h)
Cyproheptadine HCl - HepG2 (24h)
DNA + Lipid
1.5
Cyprohep1
Cyprohep2
Cyprohep3
Cell Number
Cell Number
1.5
Troglitazone - HepG2 (24h)
-
2
Phenylb1
Phenylb2
Phenylb3
+
5
1.5
+
Phenylbutazone
2
-
Trazodo1
Trazodo2
Trazodo3
ROS total
GSH total
3
Ticopidin1
Ticopidin2
Ticopidin3
Tetracycline
Ticlopidine
Trazodone HCl - HepG2 (24h)
2.5
100
100
2.0
P
P
1
% True Positive (Sensitivity)
5.0
10
Cmax [fold]
2.5
5
Hycanthone- HepG2 (24 h)
0
ROS total
2
1
-
P
100
% True Positive (Sensitivity)
Cmax [fold]
3
0
100
2
Phenylb1
Phenylb2
Phenylb3
7.5
1
0.5
0.0
100
4
10
Cmax [fold]
Ticopidine - HepG2 (24h)
10.0
Trazodo1
Trazodo2
Trazodo3
5
1.5
1.0
1
-
ROS total
3.0
2.5
2.0
0
100
Phenylbutazone - HepG2 (24h)
6
Ticopidin1
Ticopidin2
Ticopidin3
4.0
3.5
10
Cmax [fold]
+
Nalidixic Acid - HepG2 (24 h)
5
Sensitivity%
100
% True Positive (Sensitivity)
Novobiocin - HepG2 (24h)
10
1
+
10
Cmax [fold]
Known
DILI
125
% True Positive (Sensitivity)
Cyproheptadine HCl - HepG2 (24h)
1
0
100
+
1
Boolean
“OR” in 1
Assay
Gemfib1
Gemfib2
Gemfib3
Cell Number
True Positive Rate
Cmax [fold]
0.5
10
Cmax [fold]
+
+0.0
100
2
Mefe1
Mefe2
Mefe3
ROS total
Cmax [fold]
1.0
0.0
100
1
Trazodone HCl - HepG2 (24h)
4.5
Phenylb1
Phenylb2
Phenylb3
+
5.0
+
+
10
Cmax [fold]
D.
Gemfibrozil- HepG2 (24 h)
ROS total
10
0
100
+
Tetracyc1
Tetracyc2
Tetracyc3
+
ROS total
0.5
10
Cmax [fold]
+
1
ROS total
1.0
1
1
+
2.5
Ticopidine - HepG2 (24h)
1.5
Trazodo1
Trazodo2
Trazodo3
0.0
100
0.0
100
+
0.0
0.5
ROS total
0.5
10
Cmax [fold]
7.5
Hycanthone
1
+
+
10.0
2
+
T etracycline - HepG2 (24h)
12.5
Hycant1
0.5
+
ROS total
Ticopidin1
Ticopidin2
Ticopidin3
1.0
10
1
Phenylbutazone - HepG2 (24h)
1.5
Cell Number
Cell Number
1.5
1
0.0
100
Trazodone HCl - HepG2 (24h)
Ticopidine - HepG2 (24h)
0.0
10
Cmax [fold]
Cmax [fold]
Mefenamic Acid
Gemfibrozil
100
ROS total
1
10
ROS total
0.0
100
1
1
Hycant2
Nalidixic
Acid
Hycant3
1
DNA + Lipid
10
Cmax [fold]
DNA + Lipid
1
Cell Number
0.0
3
Nalidixic1
Nalidixic2
Nalidixic3
0
ROS total
2
Cmax [fold]
1
FCCP
2.0
Mito
1.5
1.0
Change
ROS
ROS total
3
0.5
2
1
100
Hycanthone- HepG2 (24 h)
Nalidixic Acid - HepG2 (24 h)
Tetracyc1
Tetracyc2
Tetracyc3
10
1.0
ROS total
4
Cmax [fold]
1
GSH
1.5
ROS total
5
0
100
ROS total
6
10
2.0
ROS total
1.0
1
2.5
# of
Positive
Outputs
2.5
FCCP 1
FCCP 2
FCCP 3
3.0
Nuclear
Dye
ROS total
1.5
Cmax [fold]
2
Hycant1
Hycant2
Hycant3
7
2.0
0
100
ROS total
2.5
10
Tetracycline - HepG2 (24h)
8
Nalidixic1
Nalidixic2
Nalidixic3
3.0
0.5
1
ROS total
1.0
Cmax [fold]
1
0.0
100
Hycanthone- HepG2 (24 h drug treatment)
3.5
Tetracyc1
Tetracyc2
Tetracyc3
10
ROS total
0.5
Cmax [fold]
1
2
ROS total
1.0
0
100
Nalidixic Acid - HepG2 (24 h drug treatment)
1.5
Hycant1
Hycant2
Hycant3
10
3
Drug Name
2
3
ROS total
0.5
Cmax [fold]
1
4
ROS total
Cell Number
1.0
0
100
Tetracycline - HepG2 (24h)
1.5
Nalidixic1
Nalidixic2
Nalidixic3
10
Cell
Number
5
M efenamic Acid - HepG2 (24 h)
3.5
Gemfib1
Gemfib2
Gemfib3
6
4
P: positive
ROSN: negative
FCCP- HepG2 (24 h)
7
Mefe1
Mefe2
Mefe3
GSH total
Cmax [fold]
1
Hycanthone- HepG2 (24 h drug treatment)
1.5
Cell Number
0
100
5
HepG2 cells
evaluated on
the ArrayScan
HCS Reader:
1
GSH total
10
6
0.5
GSH total
1
1
1.0
2
Gemfibrozil- HepG2 (24 h)
GSH total
1
0.00
100
DNA + Lipid
10
Cell Number
1
2
1.5
7
FCCP 1
FCCP 2
FCCP 3
GSH total
2
2.0
GSH total
3
3
DILI : Drug-induced Liver Injury
M efenamic Acid - HepG2 (24 h)
3
Gemfib1
Gemfib2
Gemfib3
GSH total
4
DNA + Lipid
5
2.5
Mefe1
Mefe2
Mefe3
DNA + Lipid
1
4
FCCP 1
FCCP 2
FCCP 3
GSH
FCCP- HepG2 (24 h)
Gemfibrozil- HepG2 (24 h drug treatment)
DNA + Lipid
0.50
6
Gemfib1
Gemfib2
Gemfib3
DNA + Lipid
0.5
0.75
Mefenamic Acid - HepG2 (24 h drug treatment)
FCCP- HepG2 (24 h drug treatment)
DNA + Lipid
1.00
Nalidixic Acid - HepG2 (24 h drug treatment)
Cmax [fold]
We demonstrate our assay on two different high-content imaging platforms. The
Thermo Scientific Cellomics® ArrayScan® HCS Reader is a versatile, flexible solution
for high-content imaging and we first demonstrate the new hepatotoxicity assay on
it by evaluating many known hepatotoxicity compounds and control compounds.
We then applied this assay to the new Thermo Scientific Cellomics ToxInsight IVT
Platform. The ToxInsight is a recently developed complete solution for cell-based
predictive toxicology, comprising of a cell imaging instrument, software, reagents
and other consumables, and enables compound safety assessments by cellular
imaging in an optimized, simplified, efficient turn-key manner. We show that the
assay has high specificity and sensitivity, and is thus highly predictive for identifying
the drugs that cause hepatotoxicity.
Mefe1
Mefe2
Mefe3
Cmax [fold]
0.0
Using live human hepatocytes, Xu et al.1 had reported a predictive high-content
cellular-imaging assay for drug-induced liver injury. We have adapted and simplified
this assay for its use on either live or fixed HepG2 cells or live rat primary hepatocytes.
The targets in this assay are DNA (and/or lysosome), reduced glutathione, reactive
oxygen species (ROS) and mitochondria (and their membrane potential).
1.25
FCCP 1
FCCP 2
FCCP 3
0.0
FCCP- HepG2 (24 h drug treatment)
Hepatotoxicity is a major reason for drug withdrawals from the market,
nonapprovable decisions of new drug applications, and drug development failures in
pharmaceutical companies.1-4 An assay system for the early and accurate prediction
of compound-induced hepatotoxicity with high sensitivity and specificity would
benefit drug development by improving the safety profile of newly developed drugs.
Efficiency, costs, bioethics and recent trend towards replacing, refining, and reducing
animal usage have accelerated the movement using in vitro toxicity testing early in
drug discovery to maximize compound attrition. However, developing an in vitro
assay that accurately predicts the toxicology of drug candidates remains a major
challenge in drug development process.
1.0
Gemfibrozil- HepG2 (24 h drug treatment)
0.25
Cell Number
Introduction
Mefenamic Acid - HepG2 (24 h drug treatment)
FCCP- HepG2 (24 h drug treatment)
A.
DNA content
Cell Number
Real Time Quantitative
Image Analysis
DNA + Lipid
Automated Image Acquisition
Cell Number
Assay Targets Labeled with HCS
Reagents & Kits
Cell Number
Thermo Scientific Cellomics
ArrayScan VTI HCS Reader &
ToxInsight IVT Platform
Cell Number
Cellular redox homeostasis is critical for maintaining normal cell physiology against
damage induced by oxidative stress. Cellular redox processes are involved in many
cell-signaling events that can lead to cytoprotection, differentiation, cell proliferation
and cell death. Reactive oxygen species (ROS) are primarily generated through
normal metabolic processes but it is also produced by toxic insults to cells. Once
ROS production exceeds the cell’s antioxidant capacity, it can directly damage
components that maintain cell physiology, such as cellular proteins, lipids and
DNA or by affecting the signaling pathways. Because of the danger posed by ROS
and cellular oxidation in cells and tissues, measuring ROS production and cellular
redox status in combination with other cytotoxicity markers is used to predict
the hepatotoxicity of different compounds. We thus developed a quantitative and
multiplexed cell-based high-content imaging assay to monitor cell loss, cellular
redox status with glutathione levels, ROS generation and mitochondrial membrane
potential changes using specific fluorescent probes. Hepatic cell models treated
with a panel of control compounds and compounds known to induce liver injury
were labeled with the fluorescent probes, automatically imaged, and analyzed by
individual assay targets or by multiplexing all the targets. ROC (receiver-operating
characteristic) curve analysis and Boolean logic showed that multiplexed targets give
better prediction on drug hepatotoxicity with high specificity and high sensitivity.
Thus, with high-content imaging analysis, we were able to perform fast and accurate
hepatotoxicity detection of drugs in both live and fixed cells on high density
microplates.
Hepatotoxicity Assessment by Assay Target Changes
GSH total
Assay Target Dose Responses with Different Drugs
High Content Image-Based Hepatotoxicity Profiling Workflow
Mitochondrial Membrane
Potential Change
Abstract
1. Xu et al. Toxicol Sci. 105 (1): 97-105, 2008.
2. O’Brien, PJ, et al. Arch Toxicol. 80: 580-604, 2006.
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• A high-content cell-imaging based
hepatotoxcity assay was developed, which
simultaneously monitors cellular redox status
and mitochondrial and oxidative stress. This
assay was validated with selected known
hepatotoxic and non-hepaototoxic drugs.
• Different hepatotoxic drugs show distinct
changes of assay targets after 24 hrs of drug
treatment.
• This assay identifies hepatotoxic versus nonhepatotoxic drugs efficiently and accurately
with high specificity and sensitivity both in the
Cellomics ToxInsight HCS Platform as well as
in the Cellomics ArrayScan HCS Reader.
• Both live cells from a human hepatic cell
line (HepG2) and live rat primary hepatocytes
were used to measure hepatotoxicity with this
• This demonstrates the successful use of
assay. All the selected hepatotoxic drugs were
high content imaging for efficient, highly
accurately identified as hepatotoxic in both
predictive in vitro assessments for compound
cell types and all non-hepatotoxic chemicals
heptatoxocity.
were identified negative in this assay.