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Transcript of Applying Adverse Outcome Pathway Concepts to ER-mediated Effects Pat Schmieder US Environmental...
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.
Adverse Outcome PathwayER-mediated Reproductive ImpairmentMeasurements across levels of biological organization
In vivo
INDIVIDUAL
Skewed Sex
Ratios;
Yr Class
POPULATION
Sex reversal;
Altered behavior;
Repro.
Adverse Outcome PathwayER-mediated Reproductive ImpairmentMeasurements across levels of biological organization
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.
Adverse Outcome PathwayER-mediated Reproductive ImpairmentMeasurements across levels of biological organization
In vivo
TISSUE/ORGAN INDIVIDUAL
Skewed Sex
Ratios;
Yr Class
POPULATION
Liver CellsAltered Protein
Expression(marker)(effect)
Vitellogenin
Liver Altered gene products
(timing, amt)
Gonad Ova-testis; Sex-reversed; Fecundity
Sex reversal;
Altered behavior;
Repro.
Adverse Outcome PathwayER-mediated Reproductive ImpairmentMeasurements across levels of biological organization
In vivo
CELLULARResponse
TISSUE/ORGAN INDIVIDUAL
Skewed Sex
Ratios;
Yr Class
POPULATION
ReceptorBinding
ER-Chemical Binding
Liver CellsAltered Protein
Expression(marker)(effect)
Vitellogenin
Liver Altered gene products
(timing, amt)
Gonad Ova-testis; Sex-reversed; Fecundity
Sex reversal;
Altered behavior;
Repro.
Adverse Outcome PathwayER-mediated Reproductive ImpairmentMeasurements across levels of biological organization
In vivo
MOLECULAR Target
CELLULARResponse
TISSUE/ORGAN INDIVIDUAL
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
TISSUE/ORGAN INDIVIDUAL
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.
Adverse Outcome PathwayER-mediated Reproductive Impairment
Measurements made across levels of biological organization
In vivo
MOLECULAR Target
CELLULARResponse
TISSUE/ORGAN INDIVIDUAL
Skewed Sex
Ratios;
Yr Class
POPULATION
In vitro Assayfocus area
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.
Adverse Outcome PathwayER-mediated Reproductive Impairment
Measurements made across levels of biological organization
In vivo
MOLECULAR Target
CELLULARResponse
TISSUE/ORGAN INDIVIDUAL
Skewed Sex
Ratios;
Yr Class
POPULATION
In vitro Assayfocus area
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
(%)
Positive Control:Estradiol
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
Log Concentration (M)
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
Log Concentration (M)
Vtg
mR
NA
(Fra
ctio
n o
f max
imu
m E
2ef
fica
cy)
Positive Control:Estradiol
Positive Control:Estradiol
Test Chemical: Negative response
Test Chemicals: Positive 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
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
(%)
Positive Control:Estradiol
Test Chemicals: Positive response
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
In-vivo pathwayMultigen assay
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
In-vivo pathwayMultigen assay
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
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?
Medaka (M), __day old, exposed at __ conc for __ days
Medaka (M), __day old, exposed at __ conc for __ days
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