Evaluating Existing in vitro Endocrine Data
Jeff Pregenzer,
Director of Endocrine Studies, CeeTox
EDSP in vitro assays
EDSP in vitro Tier 1 Battery
ER binding
ERa transcriptional activation
AR binding
Steroidogenesis
Aromatase
Receptor Binding Assays
• Potential false positives
– receptor denaturation due to test chemical
– non-specific displacement at high concentrations
• Examine curve fit parameters
• Potential false negatives
– Solubility issues
• Measure precipitation in buffer
– Detection method interference (assay specific)
• Test for detection interference
Interpretation of Binding data
ER Binding
ER Binding
cyclobutyl phenyl ketone
p-n-nonylphenol
Solubility limit
100
E2 3-1-07
E2 3-2-07
CBP 3-1-07
CBP 3-2-07
90
E2 Binding (%)
80
E2 080929
E2 081007
E2 081028
NON 081028
NON 081028
70
60
50
40
30
20
10
-8
-7
-6
-5
Log Concentration (M)
binder
-4
-3
-2
L
-10 -9
CT
R
L
0
TR
100
90
80
70
60
50
40
30
20
10
0
-10
-20
C
E2 Binding (%)
Solubility limit
-10 -9
-8
-7
-6
-5
-4
Log Concentration (M)
non-binder
-3
-2
Interpretation of Binding data
Hu ERa Binding
fluorescence polarization
Compound interference
E2 Binding (%)
100
50
0
E2
E2
cmpd R
cmpd R
-10 -9
-8
-7
-6
-5
Log Concentration (M)
-4
-3
-2
Cmpd R - ER transactivation
T47D-KBluc Gene Expression
T47D-KBluc Gene Expression Antagonism
150
% 1nM E2 response
100
Cmpd R+ICI, 090522
Cmpd R+ICI, 090529
50
Cmpd R+0.1nM E2, 090522
Cmpd R+0.1nM E2, 090529
% of E2 control response
Cmpd R, 090522
Cmpd R, 090529
0
-8
-7
-6
-5
-4
100
Cmpd R+100nM E2, 090522
Cmpd R+100nM E2, 090529
50
0
-8
-3
-7
-5
-4
-3
Log Concentration (M)
Log Concentration (M)
Comments: no activity
Comments: no activity
T47D-KBluc Cytotoxicity
Solubility in T47D-KBluc Media
150
50
45
40
Cmpd R, 090522
Cmpd R, 090529
100
Cmpd R, 090522
Cmpd R, 090529
35
% Viable
Precipitation, RNU
-6
30
25
20
50
15
10
5
0
-8
-7
-6
-5
Log Concentration (M)
-4
-3
0
-8
-7
-6
-5
-4
Log Concentration (M)
-3
Transactivation Reporter Assays
• Cell-based - reporter human cell lines
• Provide functional biological response data
(agonist vs antagonist)
• Highly sensitive, High throughput
• Validated for ER agonism as of 9-2009,
(antagonism and AR to follow).
Transactivation Reporter Model Agonist Induction
Agonist
E2
ER
+ATP =
luminescence
luciferase
Transcription apparatus
ERE
LUC
ER Transactivation Agonism
Estradiol (E2) ER Induction
T47D-KBluc assay
Agonist: E2
(average of 6 assays)
10
Induction
+1µM ICI182780
Antagonist
Fold Induction
8
6
Antagonizable induction
4
2
background
ve
hi
cl
e
ve
hi
cl
e
3E
-1
4
1E
-1
3
3E
-1
3
1E
-1
2
3E
-1
2
1E
-1
1
3E
-1
1
1E
-1
0
1E
-0
9
0
Concentration (M)
Luciferase reporter gene results expressed as fold of vehicle control. Data calculations performed using
Microsoft Excel and graphed with GraphPad Prism.
Controls in Transactivation Assays
• Blank, positive, and negative controls in all plates
• use of dextran-charcoal stripped serum
• Solubility check (i.e. via nephelometry)
• Cytotoxicity Assay
• “Agonist” plates - Specific antagonist for receptor
specificity
• “Antagonist” plates - Excess agonist for non receptor
related signal interference
ER Transactivation Antagonism
T47D-KBluc Estrogen transactivation reporter model
Antagonist
“spike” with agonist
E2
antagonist
+ATP =
luminescence
ER
luciferase
Transcription apparatus
ERE
LUC
Limiting False Positives
Transactivation Reporter Model
Non-Receptor Specific Signal Inhibition
E2
chemical
+ATP =
luminescence
ER
luciferase
Transcription apparatus
ERE
LUC
Limiting False Positives
Control for Non-Estrogen Receptor
related Reduction
Test compound concentrations are co-incubated with 0.01nM E2
ICI 182780 ER Induction Antagonism
T47D-KBluc assay
4-n-pentylcyclohexanone (NCH) ER Induction Antagonism
T47D-KBluc assay
+ 0.01nM E2
80
60
40
20
% 0.01 nM E2 response
120
100
+0.01nM E2
100
80
60
40
20
0
Concentration (M)
ICI antagonizes 0.01 nM E2 response
-8
.5
-8
.0
-9
.5
-9
0.
0
-1
0.
5
-1
1.
0
-1
1.
5
-1
nt
ro
l
0
co
% 0.01 nM E2 response
120
ol
r
nt
o
c
.5
-5
.0
-5
.5
-4
.0
-4
.5
-3
.0
-3
.5
-2
-2
Concentration (M)
NCH appears to show antagonism with 0.01 nM E2.
Limiting False Positives
Transactivation Reporter Model
Antagonist –
excess agonist control
E2
E2
E2
 Non-Receptor Specific
Signal Inhibition
E2
E2
antagonist
chemical
+ATP =
luminescence
ER
luciferase
Transcription apparatus
ERE
LUC
Limiting False Positives:
Control for Non-Estrogen Receptor
related Reduction
Test compounds co-incubated with 0.01nM E2 and 100nM “excess agonist” controls.
ICI 182780 ER Induction Antagonism
T47D-KBluc assay
+ 0.01nM E2
% 0.01 nM E2 response
100
80
60
40
20
100
80
60
40
20
Concentration (M)
ICI does not affect 100 nM E2 response
ICI antagonizes 0.01 nM E2 response
-8
-2
.5
-8
-2
.5
.0
-9
-3
.0
.5
-9
-3
.5
0
0.
-1
-4
.0
5
0.
-1
-4
.5
0
1.
-5
.0
-1
-5
.5
5
1.
-1
ro
l
o
tr
on
l
0
0
c
+0.01nM E2
+100 nM E2
120
+100 nM E2
co
nt
% 0.01 nM E2 response
120
4-n-pentylcyclohexanone (NCH) ER Induction Antagonism
T47D-KBluc assay
Concentration (M)
NCH appears to show antagonism with 0.01 and 100
nM E2. Suggests apparent antagonism may really be
result of non binding site related signal inhibition.
Transactivation Assay Plate
Layout (CeeTox)
Issue
Solutions
Edge effect
Plate layout, outlier rejection
1
A
B
C
D
E
F
G
H
2
1nM E2
3
medium
controls
4
5
0.1nM E2
control 2: conc. 1
same as above, with high E2 (100nM)
same as above, with high E2 (100nM)
same as above, with high E2 (100nM)
same as above, with high E2 (100nM)
6
conc. 2
7
conc. 3
8
conc. 4
9
conc. 5
10
11
12
conc. 6
conc. 7
conc. 8
in vitro Metabolism check
(possible future assay)
Metabolism testing -/+ S9 microsomes.
Phase I and Phase II enzymes both in the liver and in hormonally active tissues
could lead to:
false-positive data (due to lack of detoxification) or
false-negative data (lack of activation)
in vitro metabolism testing could test potential for metabolism.
• End
Steroidogenesis
inhibition example
M. Hecker et al. / Toxicology and Applied Pharmacology 217 (2006) 114–124
Steroidogenesis
• 5.2.9 Known False Negatives and False Positives
• The assay will almost certainly produce false negative results. As for
false negatives, this is most likely to occur for those test substances
that require metabolic activation, since the testes do not include
pathways for metabolism. Other examples of false negatives involve
those instances when a substance evokes an indirect effect on
steroidogenesis, e.g., site of action is at the hypothalamus or
pituitary gland.
• Finally, if the effect of the toxicant is delayed for a time greater than
the duration of the incubation period, then a false negative result will
occur. An example of a delayed effect was observed when lead was
tested for its effect on steroidogenesis, which inhibited steroid
hormone production 4 hours after initiation of the incubation
(Thoreux-Manlay et al., 1995).
• There are no known false positive instances to report at this time.
Transactivation
• Potential reasons for false positives
– non-specific interaction (agonism)
• Solution – specific inhibitor control
– Assay signal inhibition (antagonism)
• Solution – controls: constitutive luciferase or excess agonist
to out compete specific antagonism
• Potential reasons for false negatives
– Solubility issues in assay medium
• Edge effect
• Plate layout
• outlier rejection
Binding data and Transactivation data corroborate?
AR Transactivation Antgonism
MDA-kb2 Gene Expression Antagonism
150
% DHT control response
Nilutamide+1nM DHT, 090429
Nilutamide+1nM DHT, 090429b
100
Nilutamide+1µM DHT, 090429
Nilutamide+1µM DHT, 090429b
50
0
-8
-7
-6
-5
-4
-3
Log Concentration (M)
In the example graph above luciferase reporter gene results are expressed as fold of vehicle
control. Data calculations are performed using Microsoft Excel and graphed with GraphPad Prism.
Limiting False Positives – Assay inhibition
Bisphenol A (BPA)
ER
Aldrich 239658 lot 06326PO CAS 80-05-7
key monomer in production of polycarbonate plastic and epoxy resins.
T47D-KBluc Gene Expression Antagonism
T47D-KBluc Gene Expression
150
% 1nM E2 response
100
50
% of E2 control response
BPA+0.1nM E2, 080228
BPA, 080228
BPA+ICI, 080228
0
-9
-8
-7
-6
-5
-4
100
BPA+100nM E2, 080228
50
0
-9
-3
-8
-7
-6
-5
-4
Log Concentration (M)
Log Concentration (M)
Solubility in T47D-KBluc Media
T47D-KBluc Cytotoxicity
150
50
45
BPA, 080228
-3
100
BPA, 080228
35
% Control
Solubility RNU
40
30
25
50
20
15
10
5
0
-9
-8
-7
-6
-5
Log Concentration (M)
-4
-3
0
-9
-8
-7
-6
-5
Log Concentration (M)
-4
-3