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