Applying Adverse Outcome Pathway Concepts to ER-mediated Effects Pat Schmieder US Environmental Protection Agency Office of Research and Development National

Applying Adverse Outcome Pathway Concepts to ER-mediated Effects

Pat SchmiederUS Environmental Protection AgencyOffice of Research and Development

National Health and Environmental Effects Research LaboratoryMid-Continent Ecology Division

Duluth, MN

Toxicity Pathway: NAS report – ToxicityTesting in the 21st Century:

A Vision and A Strategy

“Toxicity Pathway: A cellular response pathway that, when sufficiently perturbed, is expected to result in an adverse health effect.”

BiologicInputs

NormalBiologicFunction

Morbidityand

Mortality

Cell Injury

Adaptive StressResponses

Early CellularChanges

Exposure

Tissue Dose

Biologic Interaction

Perturbation

Low DoseHigher Dose

Higher yet

A New Paradigm: Activation of Toxicity PathwaysA New Paradigm: Activation of Toxicity Pathways

NAS Report: Toxicity Testing in the 21st Century: A Vision and A Strategy

Adverse Outcome Pathway:

Sequence of events from Initiating Point,

through Toxicity Pathway, to Adverse Outcome,

BiologicInputs

NormalBiologicFunction

Morbidityand

Mortality

Cell Injury

Adaptive StressResponses

Early CellularChanges

Exposure

Tissue Dose

Biologic Interaction

Perturbation

Low DoseHigher Dose

Higher yet

A New Paradigm: Activation of Toxicity PathwaysA New Paradigm: Activation of Toxicity Pathways

NAS Report: Toxicity Testing in the 21st Century: A Vision and A Strategy

MolecularInitiating Event(chemical-biological interaction)

Toxicity Pathway

Adverse Outcome

Adverse Outcome Pathwayand Chemical Extrapolation

CellularResponse

Tissue/Organ

Individual

AlteredCell Structure,Cell Function

Altered Tissue Structure

(pathology),Function

Altered Function,Behavior;Pathology

MolecularTargets

Toxicity Pathway

Adverse Outcome Pathway

Receptors,Enzymes,

Ion channels,Membrane proteins,

etc

Parent Chemical

(Metabolites/Speciation)

Test Multiple Chemicals to Extrapolate within Agency inventories(QSAR;Categories;Analogues)

Example:ER-mediated Reproductive Impairment

ReproductiveImpairment

Adverse Outcome PathwayER-mediated Reproductive ImpairmentMeasurements across levels of biological organization

In vivo

INDIVIDUAL

Sex reversal;

Altered behavior;

Repro.


In vivo

INDIVIDUAL

Skewed Sex

Ratios;

Yr Class

POPULATION

Sex reversal;

Altered behavior;

Repro.


In vivo

INDIVIDUAL

Skewed Sex

Ratios;

Yr Class

POPULATION

GonadOva-testis;

Sex-reversed Gonad;

Fecundity

TISSUE/ORGAN

Liver Altered gene products

(timing, amt)

Gonad Ova-testis; Sex-reversed; Fecundity

Sex reversal;

Altered behavior;

Repro.


In vivo

TISSUE/ORGAN INDIVIDUAL

Skewed Sex

Ratios;

Yr Class

POPULATION

Liver CellsAltered Protein

Expression(marker)(effect)

Vitellogenin


(timing, amt)


Sex reversal;

Altered behavior;

Repro.


In vivo

CELLULARResponse


Skewed Sex

Ratios;

Yr Class

POPULATION

ReceptorBinding

ER-Chemical Binding

Liver CellsAltered Protein

Expression(marker)(effect)

Vitellogenin


(timing, amt)


Sex reversal;

Altered behavior;

Repro.


In vivo

MOLECULAR Target

CELLULARResponse


Skewed Sex

Ratios;

Yr Class

POPULATION

QSAR focus area

Chemicals

ReceptorBinding

ER Binding

Liver CellsAlteredProtein

Expression

Vitellogenin

LiverAltered proteins

GonadOva-testis;

Sex-reversed;Fecundity

Sex reversal;

Altered behavior;

Repro.

Adverse Outcome PathwayER-mediated Reproductive Impairment

Measurements made across levels of biological organization

In vivo

MOLECULAR Target

CELLULARResponse


Skewed Sex

Ratios;

Yr Class

POPULATION

In vitro Assayfocus area

Toxicity PathwayAdverse Outcome Pathway

QSAR focus area

Chemicals

ReceptorBinding

ER Binding

Liver CellsAlteredProtein

Expression

Vitellogenin

LiverAltered proteins

GonadOva-testis;

Sex-reversed;Fecundity

Sex reversal;

Altered behavior;

Repro.



In vivo

MOLECULAR Target

CELLULARResponse


Skewed Sex

Ratios;

Yr Class

POPULATION


Risk Assessment RelevanceToxicological Understanding

Uses of Adverse Outcome Pathway:

•Organizing tool:

What do we know (what boxes can we fill)?

What don’t we know (empty boxes)?

What do we need to know?

Uses of Adverse Outcome Pathway:

•Basis for:

Chemical Extrapolation

Species Extrapolation

Levels of Biological Organization

Uses of Adverse Outcome Pathway

Defining the RISK CONTEXT guides what you need to know:

-Prioritizing chemicals for further testing

-High Tier Risk Assessment (e.g., to set a reference dose, criterion concentration)

Type of information needed for each is very different, acceptable uncertainty bounds for each are different, etc.

Risk Context - Regulatory NeedQSAR for Prioritization

• Food Quality Protection Act For lists of hundreds of chemicals where ‘endocrine data’ is lacking, is there a way to prioritize in vivo testing options so that chemicals most likely to have the effect are tested first

• Inert ingredients used in formulations of pesticides used on crops • Antimicrobial active ingredient pesticides

• Prioritization approach• Determine potential of a chemical to initiate ER-mediated Reproductive Impairment Pathway

(strategic testing -- in vitro assays)• Use QSAR-based approach to prioritize ‘estrogenic potential’ of untested inert ingredients (FI) and antimicrobial (AM) pesticides

QSAR focus area

Inert; Antimicrobial Chemicals

ReceptorBinding

ER Binding

Liver CellProtein

Expression(marker)

Vitellogenin (egg protein induced in male fish)

LiverAltered

proteins, hormones;

GonadOva-testis

Sex- reversed

Sex reversal;

Altered behavior;

Repro.



In vivo

MOLECULAR Target

CELLULARResponse


Skewed Sex

Ratios;

Yr Class

POPULATION


Toxicity PathwayAdverse Outcome Pathway

A QSAR is only be as good as the underlying toxicological understanding and associated data

• Pathway provides plausible connection of initiating event to adverse outcome

• In vitro assays used had to be optimized for the chemicals of regulatory concern (FI; AM)– low affinity ER-binders– chemical dosimetry

• Similar toxicological activity is used to group “similar” chemicals (e.g., binding types)

R 394

E 353 H 524

CC

T 347

HOOH

CH3 H

H H

H

AA BB

A_B Interaction A_B Interaction

Distance = 10.8 for 17-EstradiolJ. Katzenellenbogen

R 394

E 353 H 524

CC

T 347

HOAA BB

““A_site only” Interaction A_site only” Interaction

CH3

J. Katzenellenbogen

R 394

E 353 H 524

CC

T 347

AA BB

““B_site only” Interaction B_site only” Interaction

H3C

NH2

J. Katzenellenbogen

A QSAR can only be as good as the underlying toxicological understanding and associated data

• well-defined endpoint = ER-mediated Adverse Outcome PathwayER binding confirmed by gene activation (VTG induction);

- binding-additional assays: Ki; rec rtER; -chemical conc in assay buffer over time;-chemical solubility in assay buffer with proteins

- liver slice Vtg mRNA induction – agonism; antagonism-chemical/metabolites in assay media & slices-chemical solubility in assay buffer with proteins

• well-defined assays = chemical dosimetry measured total and free (bioavailable) chem conc in:

binding assayslice assay

• Seek mechanistic interpretation = Similar toxicological activity used to group “similar” chemicals –

ER binding types

Problem Definition (Risk Context)

• Risk assessment target = QSAR-based approach for priority setting for testing

• Chemicals = defined by regulators (EPA Office of Pesticide Programs):

Antimicrobials (AM) & Food Use Inerts (FI)Expand ‘structure space’ of previously known data to adequately cover chemicals of regulatory concern (APPLICABILITY DOMAIN)– Get lists from regulators (FI and AM)– Characterize structures on lists– Assess coverage of existing data and use the data that applies– Select chemicals for testing to strategically expand structure space

(i.e., maximize information gained from every structure tested)

In-lab Testing to expand Training Set The Issue:Assay protocols optimized to increase confidence in quantifying activity of

low potency compounds

Compare EPA Office of Pesticide Programs chemical structures to pre-existing ER data

The Assays:

1) ER binding –

• standard competitive binding assay optimized for low affinity binders

• rainbow trout liver cytosol estrogen receptors (rtER)

2) Gene Activation

• trout liver slice - vitellogenin mRNA

• endogenous metabolism

Data Example - Primary Assay: In vitro Estrogen Receptor Binding Displacement Assay

rtER Binding

0

10

20

30

40

50

60

70

80

90

100

110

120

-10 -9 -8 -7 -6 -5 -4 -3 -2Log Concentration (M)

E2

VN

HCP

CRTL

HOB

OHP

[3H

]-E

2 B

indi

ng (

%)

rtER

Positive Control:Estradiol

Test Chemicals: Negative response

CTRL

0

10

20

30

40

50

60

70

80

90

100

110

120

-10 -9 -8 -7 -6 -5 -4 -3 -2 -1

E2

P

PNEP

PNMP

PNBP

PNPrPPIPrP

Log Concentration (M)

[3H

]-E

2 B

ind

ing

(%)


Test Chemicals: Positive response

CTRL0.00001

0.0001

0.001

0.01

0.1

1

10

-10 -9 -8 -7 -6 -5 -4 -3 -2

PTOP

PTAP E2 control


Vtg

mR

NA

(F

ract

ion

of

max

imu

m E

2ef

fica

cy)

0.0001

0.001

0.01

0.1

1

10

-10 -9 -8 -7 -6 -5 -4 -3 -2

IAB E2 control

CTRL


Vtg

mR

NA

(Fra

ctio

n o

f max

imu

m E

2ef

fica

cy)



Test Chemical: Negative response


Data Example – Confirmatory Assay: In vitro Gene Activation Assay

Optimize assay for your question and your chemicals.

rtER Binding

0

10

20

30

40

50

60

70

80

90

100

110

120

-10 -9 -8 -7 -6 -5 -4 -3 -2Log Concentration (M)

E2

VN

HCP

CRTL

HOB

OHP

[3H

]-E

2 B

indi

ng (

%)

rtER


Test Chemicals: Negative response

CTRL

0

10

20

30

40

50

60

70

80

90

100

110

120

-10 -9 -8 -7 -6 -5 -4 -3 -2 -1

E2

P

PNEP

PNMP

PNBP

PNPrPPIPrP


[3H

]-E

2 B

ind

ing

(%)



Total vs. free concentration of octylphenol in an estrogenicity reporter gene assay

using SPME to measure “free” chemical

-9 -8 -7 -6 -5 -40

100000

200000

300000

4000005% serum20% serum50% serum

log nominal conc. (M)

resp

on

se

-9 -8 -7 -6 -5 -40

100000

200000

300000

4000005% serum20% serum50% serum

log free conc. (M)

resp

on

se

Heringa M.B. et al. Environ. Sci. Technol. 2004, 38, 6263-6270Picture from J. Hermens, Utrecht University

Nominal / total concentration

Free aqueous concentration

Dose

Relative Binding AffinityChemical Log Kow cyto rtER rec rtER rec hERAAN 3.39 0.0011 0.0038 0.0089

pnBP 3.65 0.0035 0.065 0.024

pnOP 5.68 0.009 0.32 0.17

ptOP 5.16 0.009 0.56 0.25

pDoP 7.80 0.012 0.46 0.50

Assay differences vs Species differencesFor the same chemical,

RBAs determined using trout and human recombinant ER assaysare similar (same order of magnitude), and both are

10x > RBAs determined using trout ER isolated from cytosol

cyto rtER rec rtER rec hERProtein conc = 7 mg/ml 0.01 mg/ml ~0.01?Chemical free fraction = 27% 66% --

-11 -10 -9 -8 -7 -6 -5 -4 -3 -20

20

40

60

80

100

120E2-CRUDEE2-ENGOA-CRUDEOA-ENG

Fig 3. Octylaniline tested in both engineered and crude preps, n=3.

0.0042

0.018

Competitor (M)

Sp

ecif

ic B

ind

ing

(%

)

RBA

E2 cyto rtER

E2 rec rtER

OA cyto rtER

OA rec rtER

Assay differences vs Species differencesTake care not to misinterpret differences in assay conditions

which affect dosimetry (chemical availability) as species differences.

Pathway used to compare in vitro and in vivo:

ER Toxicity Pathway

In vitroAssayEndpts

Troutcyto rtERBinding

MaleTrout Liver Slice Vtg Induction

ER Binding

AlteredProtein

Expression

Chg 2ndry Sex Char

AlteredRepro.

Skewed Sex

Ratios,AlteredRepro.

MOLECULARInitiating Event

CELLULARRespopse

TISSUE/ORGANResponse

INDIVIDUALResponse

POPULATIONResponse

Altered proteins (VTG),

Ova-testis

In vivoAssay Endpts

Male MedakaLiver Vtg Induction

Female MedakaLiver Vtg decr.

Male MedakaChg in 2ndary sex characteristic; behavior?Papillary Processes

Male MedakaGonadComplete conversion to ovary

Female MedakaFecundityHatch

Sex ReversalGenetic Males to Phenotypic Females

Vitellogenin Induction

In vitro – Liver slice media conc where Vtg induction was observed Media = -4.0 LogM

In vivo – Lowest conc where Vtg Induction was observed Water = -6.16 LogM

Medaka VTG induction in Males, [Water Conc]= -6.16 Liver Conc as 160 times Water Conc = -3.95

Trout Liver Slice Media Conc for Vtg induction = -4.0

Results of Prioritization Research:

0.000001

0.00001

0.0001

0.001

0.01

0.1

0 1 2 3 4 5 6 7 8LogKow

Lo

gR

BA

46 diverse chemicals tested of LogKOW <1.3 were all non-binders

Many chemical sub-classes were found to be ER “Inactive”

Relationships determined: lipophilicity; LogP cutoffs

High Potency Chemicals Estradiol

Ethinyl Estradiol

ER Binders

0.000001

0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

0 1 2 3 4 5 6 7 8

Log(Kow)

Lo

g(R

BA

)

EstradiolEthinyl Estradiol

InertsAntimicrobials

Diverse classes of Low affinity binders were found

Food Use Inerts Antimicrobials

393 Total Chemicals 211

379 (96%) Lower Priority 196 (93%)

14 ( 4%) Higher Priority 15 ( 7%)

Small percentage are high priority for likelihood to elicit adverse effect through this pathway

Chemical Liste.g., Food Use Inerts

AntimicrobialsContains a Cycle

Yes Contains 2 OH, or OH and =0, at

Spec. Dist

Possible High Affinity,“A-B”; “A-C”; or

“A-B-C” type binder

Contains some attenuating feature

steric?; other?

High Binding Affinity “A-B”;“A-C” or “A-B-C” type

No

Non-binder Ex: Progesterone

Corticosterone(RBA<0.00001)

Log KOW <1.3

Low Affinity Binder“A-B”,“A-C” or “A-B-C” type

Assess strength of attenuation steric?;

other?

Some

Complete

Meets a “Special Class” Rule

Type “A”Contains Phenol

Fragment

• Alkylanilines• Alkyloxy anilines• Phthalates• Phenones – (branched)• Cyclohexanols• Cyclohexanones• Benzoates – (o-CH3 ring subst) • Benzoates – (tri-CH3 ring subst)

VI

Possible Low AffinityA-Type, B-Type

Type “B”Contains “Specified”

Fragment

• Alkylphenols • Alkoxy phenols• Parabens• Salicylates

Belongs to known Active

Sub-class Potency Rules / Equations

Yes

No

IV

Needs testing

Yes

No

No

Yes

Yes

Belongs to sub-classes under investigation

with possible Type B low affinity

Belongs to untested or mixed Sub-classes with possible

Type A low affinity

• Thiophosphate Esters• Benzoates – (p-CH3 ring subst)• Mixed Organics• Cyclic Alcohols (not C6)• Cyclic Ketones (not C6)

No

No

Yes

Belongs to known Active

Sub-class

Belong to untested classNeeds testing

Needs testing

• Mixed Phenols

No

V

No

Belongs to known Inactive

Sub-class

• Alkylaromatic Sulfonic Acids• Sulfonic Acid Dyes• Alkylfluorobenzenes(p-n chain)• Bis–anilines•Imidazolidines• Isothiazolines• Oxazoles• Benzamides

Yes

• Hydrofurans (ketone & alcohol)• Sorbitans• Triazines• Alkylbenzthiols• Pyrrolidiones• Alkylphenols (fully-hindered)• Phenones (n-alkyl)• Benzoates (non ring-subst)• Mono-Cyclichydrocarbons

• DDT-Like • Tamoxifen-Like• Multi-Cyclichydrocarbons• Alkylchlorobenzenes

Yes

No

No

Yes

Potency Rules / Equations

Non-binder(RBA<0.00001)

Low - Moderate Affinity

I

Non-binder(RBA<0.00001)

Yes

No

III

II

p-n chain

The small % of chemicals having potential to bind should be prioritized for testing above others;

Pathway gives context for why to test, what to test, context for formulating hypotheses, etc

Pathway helps guide in vivo research: Q - How does binding affinity (with proven ER-mediated gene

activation) translate to in vivo effects?

Q - How is in vitro dosimetry best translated to in vivo?

Q – Do chemicals yielding half-maximal efficacy have in vivo effects through the ER-mediated pathway?

0.000001

0.00001

0.0001

0.001

0.01

0.1

0 1 2 3 4 5 6 7 8LogKow

Lo

gR

BA

Chemicals selected for in vivo testing

SUMMARY:

Adverse Outcome Pathways:Organizing toolChemical ExtrapolationSpecies ExtrapolationCompare across Levels of Biological OrganizationCompare in vitro to in vivo

effectsdosimetry

Regulatory Purpose-Priority Setting

Defined Endpoint-ER-mediated Reproductive Impairment

AcknowledgementsEPA ORD/NHEERL/MED-Duluth:

R. Kolanczyk, M. Tapper, J. Denny, B. Sheedy, M. Hornung

Univ. Minnesota-Duluth:H. Aladjov

EPA Student Services Contractors:C. Peck, B. Nelson, T. Wehinger, B. Johnson, L. Toonen, R.

Maciewski, W. Backe, M. Dybvig, M. Mereness

International QSAR Foundation:G. Veith

EPA Office of Pesticide Programs:S. Bradbury, J. Chen, N. Shamim, D. Smegal, J. Holmes, B.

Shackleford, K. Leifer, P. Wagner, P.V. Shah

OASIS software (Laboratory of Mathematical Chemistry, Bourgas, Bg): O. Mekenyan

Effectopedia

Cause Link Effect

ER-mediated Adverse-outcome PathwayOctylphenol

Molecular Cellular Organ Individual Population• Octylphenol

binding to ER• Liver slice

Vtg (mRNA)

•Altered reproduction

•Altered developmentDecreased numbers of animals

In-vitro pathwaySchmieder et.al.

• ER transcription factor

In-vivo pathwayMultigen assay

-dose: sex reversal (altered gamete ratios)

-dose: reduced fecundity

Population reductionOctylphenol-ER binding

ER transcriptionfactors

♂ Liver Vtg (mRNA)

Anal fin papillae?

Gonadal morphology?

?

-dose: mixed-sex gonad

Molecular PopulationCellular IndividualOrgan

Altered sex-ratios?

?

ER-mediated Adverse-outcome PathwayAmylaniline (AAN)

Molecular Cellular Organ Individual Population• AAN binding

to ER• Liver slice

Vtg (mRNA)

• Liver slice toxicity

•Altered reproduction

•Altered developmentDecreased numbers of animals

In-vitro pathwaySchmieder et.al.

• ER transcription factor


dose: Sex reversal (altered gamete ratios)

Population reduction

AAN bindingto ER

ER transcriptionfactors

♂ Liver Vtg (mRNA)

Anal fin papillae?

Gonadal morphology??

dose: Mixed-sex gonad

Molecular PopulationCellular IndividualOrgan

Altered sex-ratios?

AAN bindingto Hbg ?

Splenic/head-kidney pathology

?

dose: Reduced fecundity

dose: Reduced growth ?

?

ER-mediated Adverse-Outcome PathwayOctylphenol

Octylphenol-ER binding

ER gene product Anal fin papillae?

-dose: mixed-sex gonad


MolecularInitiating Event

PopulationCellular IndividualOrgan

♂ Liver Vtg (mRNA)Octylphenol-ER binding ER gene product

-dose: sex reversal (altered gamete ratios)

Population reduction?

Altered sex-ratios?

Gonadal morphology?Octylphenol-ER binding

ER gene product

Medaka (M), __day old, exposed at __ conc for __ days


-dose: reduced fecundity Octylphenol-ER binding ER gene product

Medaka (F), __day old, exposed at __ conc for __ days

Population reduction?

Altered sex-ratios?

Gonadal morphology?Octylphenol-ER binding

ER gene product Population reduction?

Altered sex-ratios?



Dose-Response and Critical WindowDioxins, Furans and PCBs - Hearing Loss

Exposure Hepatic Phase II Enzymes

Hepatic Parent or Metabolite

SerumT4 & T3

TissueT3

Alter TR Mediated Proteins

Loss of cochlear hair cells

HearingLoss

Binding to PXR

Binding to AhR

Dose-Response Perchlorate, Propylthiouracil and Hippocampal

Physiology

Exposure SerumT4 & T3Thyroid

Perchlorate

HippocampalT3

Alter TR Mediated Proteins

Synaptic Malformation

LearningImpariment

Binding to TPO

Inhibition Of TPO

ThyroidPTU

Altered SynapticFunction

Adverse Outcome Pathways Adverse Outcome Pathways for Thyroid Disruptionfor Thyroid Disruption

Perchlorate

Methimazole

NIS Inhibition

TPO Enzyme Inhibition

ThyroidFollicular Cells T4 Synthesis

Serum T4 Systemic T4 Insufficiency

ArrestedAmphibian

Metamorphosis

ChemicalChemicalSubcellularSubcellular

TargetTargetCells Cells

EffectedEffectedAdverse Adverse OutcomeOutcomeTissue/OrganTissue/Organ

ThyroidFollicular Cells T4 Synthesis

Serum T4 Systemic T4 Insufficiency

ArrestedAmphibian

Metamorphosis

Toxicity PathwayToxicity Pathway

Adverse Outcomes PathwayAdverse Outcomes Pathway

Amphibian Metamorphosis Assay

Documents

Applying Adverse Outcome Pathway Concepts to ER-mediated Effects Pat Schmieder US Environmental Protection Agency Office of Research and Development National