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
ER 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
binder non-binder
-20
-10
0
10
20
30
40
50
60
70
80
90
100
-10 -9 -8 -7 -6 -5 -4 -3 -2
ER Bindingp-n-nonylphenol
CTRL
E2 080929
NON 081028
E2 081007
NON 081028
Solubility limit
E2 081028
Log Concentration (M)
E2
Bin
din
g (
%)
0
10
20
30
40
50
60
70
80
90
100
-10 -9 -8 -7 -6 -5 -4 -3 -2
ER Bindingcyclobutyl phenyl ketone
CTRL
Solubility limit
E2 3-1-07
CBP 3-1-07
E2 3-2-07
CBP 3-2-07
Log Concentration (M)E
2 B
ind
ing
(%)
Interpretation of Binding data
0
50
100
-10 -9 -8 -7 -6 -5 -4 -3 -2
Hu ER Bindingfluorescence polarization
E2
cmpd R
E2
cmpd R
Compound interference
Log Concentration (M)
E2
Bin
din
g (
%)
T47D-KBluc Gene Expression
-8 -7 -6 -5 -4 -3
0
50
100Cmpd R, 090522
Cmpd R, 090529
Cmpd R+ICI, 090529Cmpd R+ICI, 090522
Log Concentration (M)
% 1
nM
E2
resp
on
se
Solubility in T47D-KBluc Media
-8 -7 -6 -5 -4 -30
5
10
15
20
25
30
35
40
4550
150
Cmpd R, 090522
Cmpd R, 090529
Log Concentration (M)
Pre
cip
itatio
n, R
NU
T47D-KBluc Gene Expression Antagonism
-8 -7 -6 -5 -4 -30
50
100
150
Cmpd R+0.1nM E2, 090529
Cmpd R+100nM E2, 090529
Cmpd R+100nM E2, 090522
Cmpd R+0.1nM E2, 090522
Log Concentration (M)
% o
f E
2 co
ntr
ol r
esp
on
se
T47D-KBluc Cytotoxicity
-8 -7 -6 -5 -4 -30
50
100 Cmpd R, 090522
Cmpd R, 090529
Log Concentration (M)
% V
iab
le
Cmpd R - ER transactivation
Comments: no activity Comments: no activity
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).
Agonist
ERER
ERE LUC
+ATP = luminescence
Cofactor
E2E2
Transcription apparatusTranscription apparatus
luciferaseluciferase
Transactivation Reporter Model Agonist Induction
Estradiol (E2) ER InductionT47D-KBluc assay(average of 6 assays)
vehic
le
vehic
le
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
90
2
4
6
8
10 Induction+1µM ICI182780
Concentration (M)
Fo
ld In
du
ctio
n
ER Transactivation Agonism
Luciferase reporter gene results expressed as fold of vehicle control. Data calculations performed using Microsoft Excel and graphed with GraphPad Prism.
Agonist: E2Agonist: E2
AntagonistAntagonist
background
Antagonizable inductionAntagonizable induction
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
Antagonist
ERER
ERE LUC
+ATP = luminescenceCofactor
E2E2
Transcription apparatusTranscription apparatus
antagonist
luciferaseluciferase
ER Transactivation AntagonismT47D-KBluc Estrogen transactivation reporter model
“spike” with agonist
Limiting False Positives Transactivation Reporter Model
Non-Receptor Specific Signal Inhibition
ERER
ERE LUC
+ATP = luminescenceCofactor
E2E2
Transcription apparatusTranscription apparatus
chemicalchemical
luciferaseluciferase
Limiting False PositivesControl for Non-Estrogen Receptor
related Reduction
ICI 182780 ER Induction AntagonismT47D-KBluc assay
contro
l
-11.
5-1
1.0
-10.
5-1
0.0
-9.5
-9.0
-8.5 -8
0
20
40
60
80
100
120
+ 0.01nM E2
Concentration (M)
% 0
.01
nM
E2
resp
onse
NCH appears to show antagonism with 0.01 nM E2.
Test compound concentrations are co-incubated with 0.01nM E2
4-n-pentylcyclohexanone (NCH) ER Induction AntagonismT47D-KBluc assay
contro
l-5
.5-5
.0-4
.5-4
.0-3
.5-3
.0-2
.5 -20
20
40
60
80
100
120
+0.01nM E2
Concentration (M)
% 0
.01
nM
E2
resp
onse
ICI antagonizes 0.01 nM E2 response
Antagonist – excess agonist control
ERER
ERE LUC
+ATP = luminescenceCofactor
E2E2
Transcription apparatusTranscription apparatus
antagonist
luciferaseluciferase
Limiting False Positives Transactivation Reporter Model
E2E2E2E2
E2E2
E2E2chemicalchemical
Non-Receptor Specific Signal Inhibition
Limiting False Positives:Control for Non-Estrogen Receptor
related Reduction
ICI 182780 ER Induction AntagonismT47D-KBluc assay
contro
l
-11.
5-1
1.0
-10.
5-1
0.0
-9.5
-9.0
-8.5 -8
0
20
40
60
80
100
120+ 0.01nM E2
+100 nM E2
Concentration (M)
% 0
.01
nM
E2
resp
onse
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.
Test compounds co-incubated with 0.01nM E2 and 100nM “excess agonist” controls.
4-n-pentylcyclohexanone (NCH) ER Induction AntagonismT47D-KBluc assay
contro
l-5
.5-5
.0-4
.5-4
.0-3
.5-3
.0-2
.5 -20
20
40
60
80
100
120+0.01nM E2+100 nM E2
Concentration (M)
% 0
.01
nM
E2
resp
onse
ICI does not affect 100 nM E2 response
ICI antagonizes 0.01 nM E2 response
Transactivation Assay Plate Layout (CeeTox)
1 2 3 4 5 6 7 8 9 10 11 12
A 1nM E2medium controls
0.1nM E2 control 2: conc. 1 conc. 2 conc. 3 conc. 4 conc. 5 conc. 6 conc. 7 conc. 8
BCDE same as above, with high E2 (100nM)F same as above, with high E2 (100nM)G same as above, with high E2 (100nM)H same as above, with high E2 (100nM)
Issue Solutions
Edge effect Plate layout, outlier rejection
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
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.
MDA-kb2 Gene Expression Antagonism
-8 -7 -6 -5 -4 -30
50
100
150Nilutamide+1nM DHT, 090429Nilutamide+1nM DHT, 090429b
Nilutamide+1µM DHT, 090429Nilutamide+1µM DHT, 090429b
Log Concentration (M)
% D
HT
co
ntr
ol r
esp
on
se
T47D-KBluc Gene Expression
-9 -8 -7 -6 -5 -4 -3
0
50
100 BPA, 080228BPA+ICI, 080228
Log Concentration (M)
% 1
nM
E2
resp
on
se
Solubility in T47D-KBluc Media
-9 -8 -7 -6 -5 -4 -30
5
10
15
20
25
30
35
40
4550
150
BPA, 080228
Log Concentration (M)
So
lub
ility
RN
U
T47D-KBluc Gene Expression Antagonism
-9 -8 -7 -6 -5 -4 -30
50
100
150BPA+0.1nM E2, 080228
BPA+100nM E2, 080228
Log Concentration (M)
% o
f E
2 co
ntr
ol r
esp
on
se
T47D-KBluc Cytotoxicity
-9 -8 -7 -6 -5 -4 -30
50
100 BPA, 080228
Log Concentration (M)
% C
on
tro
l
ER Bisphenol A (BPA)Aldrich 239658 lot 06326PO CAS 80-05-7
key monomer in production of polycarbonate plastic and epoxy resins.
Limiting False Positives – Assay inhibition