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Adverse Outcome Pathways in Adverse Outcome Pathways in Ecotoxicology ResearchEcotoxicology Research
US Environmental Protection Agency, Mid-Continent Ecology Division,
Duluth, MN
Meeting of the Northland Chapter of SOTOctober 7, 2010
St. Paul, MN
The views expressed in this presentation are those of the author and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency.
Michael W. Hornung
2
Background
3
Background
• Historically, regulatory toxicity testing has relied on the direct observation of adverse effects in whole animal toxicity tests.
� Expensive� Time-consuming� Intensive animal use� Few chemicals comprehensively tested
• Often limited mechanistic information� Limits extrapolation across chemicals,
species, mixtures…� Relevance to emerging chemicals of
concern; acute toxicity vs low dose effects
4
Four competing objectives
• Depth – providing the most accurate, detailed, characterization possible.
• Breadth – providing data on the broadest universe of chemicals, endpoints, species, life-stages, etc.
• Animal welfare – using the fewest animals possible and minimizing suffering.
• Conservation – minimizing expenditure of money and time on testing and review.
Background
5
Ecotox is faced with the same competing objectives
� Depth, Breadth vs. Animal welfare, Conservation
Some particular challenges to implementation in Eco tox
� Less willingness to apply precautionary principle in weighing risk-benefit; higher bar for “proof of adversity”
� Unit of concern is sustainable populations and ecosystem functions , not individual health
� Much broader species extrapolation challengevertebrates, invertebrates, microorganisms, plants
Background
6
Background
How do the topics and suggestions in this report parallel new emerging research trends and thinking in ecotoxicology research ?
How do we translate information at the level of the toxicity pathway to the needs of risk assessment?
7
Adverse Outcome Pathway
Definition:
An Adverse Outcome Pathway (AOP) is a conceptual
framework that portrays existing knowledge concerni ng the
linkage between a direct molecular initiating event and an
adverse outcome , at a level of biological organization relevant
to risk assessment.
Designed for the translation of mechanistic informa tion into
endpoints meaningful to ecological risk
8
What is an Adverse Outcome Pathway ?
� Provides consistent structure and terminology for organizing ecotoxicological understanding across le vels of biological organization
9
What is an Adverse Outcome Pathway ?
ChemicalProperties
Receptor/LigandInteraction
DNA Binding
Protein Oxidation
ToxicantMacro-Molecular
Interactions
Anchored by a Molecular Initiating Event in which a chemical interacts with a specific biomolecule...
Anchor 1Molecular Initiating
Event
10
What is an Adverse Outcome Pathway ?
Lethality
Impaired Development
Impaired Reproduction
Cancer
OrganismResponses
Structure
Recruitment
Extinction
PopulationResponses
Anchor 2Adverse Outcome
... and anchored at an Adverse Outcome at the organism- or population-level that is relevant to risk assessment
11
What is an Adverse Outcome Pathway ?
ChemicalProperties
Receptor/LigandInteraction
DNA Binding
Protein Oxidation
Lethality
Impaired Development
Impaired Reproduction
Cancer
Toxicant
Macro-Molecular
InteractionsOrganism
Responses
Structure
Recruitment
Extinction
PopulationResponses
Anchor 1Molecular Initiating
EventAnchor 2
Adverse Outcome
The cellular and organ responses through which the molecular initiating event produces an adverse outcomecompletes the Adverse Outcome Pathway
Gene Activation
Protein Production
Altered Signaling
Protein Depletion
OrganResponses
Cellular Responses
Altered Physiology
Disrupted Homeostasis
Altered Tissue Developmentor Function
12
Linked Levels of Biological Organization
ChemicalProperties
Receptor/LigandInteraction
DNA Binding
Protein Oxidation
Gene Activation
Protein Production
Altered Signaling
Protein Depletion
Altered Physiology
Disrupted Homeostasis
Altered Tissue Developmentor Function
Lethality
Impaired Development
Impaired Reproduction
Cancer
Toxicant
Macro-Molecular
InteractionsCellular
ResponsesOrgan
ResponsesOrganism
Responses
Structure
Recruitment
Extinction
PopulationResponses
Linkages between levels of organization may be caus al, mechanistic, inferential, or correlation based
Establishes scientifically defensible connection be tween initiating event and adverse outcome.
13
What is an Adverse Outcome Pathway ?
The Adverse Outcome Pathway
� has origins in previous toxicological terms / conce pts
� developed, in part, due to ambiguities in usage of terms
14
Adverse Outcome Pathway vs Toxicity Pathway
ChemicalProperties
Receptor/LigandInteraction
DNA Binding
Protein Oxidation
Gene Activation
Protein Production
Altered Signaling
Protein Depletion
Altered Physiology
Disrupted Homeostasis
Altered Tissue Developmentor Function
Lethality
Impaired Development
Impaired Reproduction
Cancer
Toxicant
Macro-Molecular
InteractionsCellular
ResponsesOrgan
ResponsesOrganism
Responses
Structure
Recruitment
Extinction
PopulationResponses
Toxicity PathwayToxicity Pathway
Adverse Outcome Pathway
“Cellular response pathways that when sufficiently perturbed are expected to result in adverse health effects”Toxicity Testing in 21st Century, NRC 2007.
15
Adverse Outcome Pathway vs Mechanism of Action
ChemicalProperties
Receptor/LigandInteraction
DNA Binding
Protein Oxidation
Gene Activation
Protein Production
Altered Signaling
Protein Depletion
Altered Physiology
Disrupted Homeostasis
Altered Tissue Developmentor Function
Lethality
Impaired Development
Impaired Reproduction
Cancer
Toxicant
Macro-Molecular
InteractionsCellular
ResponsesOrgan
ResponsesOrganism
Responses
Structure
Recruitment
Extinction
PopulationResponses
Mechanism of ActionMechanism of Action : a complete and detailed understanding of each and every step in the sequence of events that leads to a toxic outcome
But often only refers to Toxicity Pathway
16
Adverse Outcome Pathway vs Mode of Action
ChemicalProperties
Receptor/LigandInteraction
DNA Binding
Protein Oxidation
Gene Activation
Protein Production
Altered Signaling
Protein Depletion
Altered Physiology
Disrupted Homeostasis
Altered Tissue Developmentor Function
Lethality
Impaired Development
Impaired Reproduction
Cancer
Toxicant
Macro-Molecular
InteractionsCellular
ResponsesOrgan
ResponsesOrganism
Responses
Structure
Recruitment
Extinction
PopulationResponses
Mode of ActionMode of Action: a common set of biochemical, physiological, or behavioral responses that characterize an advers e biological response where major, but not necessaril y all, linkages between a direct initiating event and an a dverse outcome are understood
17
What is an Adverse Outcome Pathway ? AOPs are a sequential series of eventsBut they are not isolated from other biological pro cesses
Exposure
Outcome
Cell Tissues&
Organ Systems
Organism&
Population
18
Examples of Adverse Outcome Pathways
� Narcosis
� Photo-Activated Toxicity
� AhR Mediated Toxicity
� Estrogen Receptor Activation
� Impaired Vitellogenesis
19
AOPs in Ecotoxicology
20
AOP Example 1:
Narcosis
21
AOP: Narcosis – Baseline Toxicity
Narcosis is “non-specific toxicity resulting from w eak and reversible hydrophobic interactions” (Overton, 1901)
Narcosis is theorized to result from hydrophobic in teractions between chemicals and cellular membranes.
Baseline toxicity: if a chemical does not produce toxicity by some more specific mechanism it will act by narcosi s, providing it is sufficiently soluble in water at hi gh enough concentrations to achieve required chemical activit y
22
AOP: Narcosis – Baseline Toxicity
CellularMembranes
Changes in fluidity
/ transport
Non-polarNarcotics
NumerousChemicals
Neurons(Multiple)
? ���� Respiration,���� Metabolic rate
CNS/ MultipleOrgan types
Equilibriumloss,
Mortality
Organism
Decliningtrajectory
Population? ?
Not all linkages are known with absolute certainty in this AOP,
… but the relationship between chemical property and adverse outcome is well established
23
AOP : Narcosis – Baseline Toxicity
Data plotted from Russom et al. 1997. ET&C, 16, 948-967
-1
0
1
2
3
4
5
6
-2 -1 0 1 2 3 4 5 6 7
Log Kow
96-h
Log
LC
50 (
mg/
L)
Fathead Minnow 96-h Toxicity
24
AOP : Narcosis – Baseline Toxicity
Predictive QSAR based upon log Kow
Log Kow 96 h LC50
CellularMembranes
Changes in fluidity
/ transport
Non-polarNarcotics
NumerousChemicals
Neurons(Multiple)
?���� Respiration,���� Metabolic rate
CNS/ MultipleOrgan types
Equilibriumloss,
Mortality
Organism
Decliningtrajectory
Population
? ?
25
AOP : Narcosis – Baseline Toxicity
• AOP defined in the context of well-established toxi city endpoints
• Do we need to know all of these linkages? NO.
• Strong weight-of-evidence shows that organismal eff ect is related to chemical property (Kow)
• Supports use of a predictive model based upon chemi cal propert
CellularMembranes
Changes in fluidity
/ transport
Non-polarNarcotics
NumerousChemicals
Neurons(Multiple)
?���� Respiration,���� Metabolic rate
CNS/ MultipleOrgan types
Equilibriumloss,
Mortality
Organism
Decliningtrajectory
Population? ?
26
AOP Example 2:
AOPs Converging at Impaired Vitellogenesis
27
Vitellogenin Production in Fish
Agonism
EstrogenReceptor
Vtgproduction
Hepatocyte
Oocytedevelopment
Ovary
Ovulation& spawning
Female
Stable orincreasing trajectory
Population
Estradiol
ERAgonist
Well-defined linkages from a MIE to normal biologic al function
OH
OH
ERE-Vtg
OH
OH
28
Three AOPs of Impaired Vitellogenesis
Tamoxifen
ERAntagonist1)
Reduced Vtgproduction
Hepatocyte
Oocytedevelopment
Ovary
Ovulation& spawning
Female
Antagonism
EstrogenReceptor
Three distinct molecular initiating events
29
Three AOPs of Impaired Vitellogenesis
Tamoxifen
ERAntagonist1)
Reduced Vtgproduction
Hepatocyte
Oocytedevelopment
Ovary
Ovulation& spawning
Female
Antagonism
EstrogenReceptor
Fadrozole
AromataseInhibitor2)
Reduced Vtgproduction
Hepatocyte
Oocytedevelopment
Ovary
Ovulation& spawning
Female
Inhibition
AromataseEnzyme
Three distinct molecular initiating events
30
Three AOPs of Impaired Vitellogenesis
Tamoxifen
ERAntagonist1)
Reduced Vtgproduction
Hepatocyte
Oocytedevelopment
Ovary
Ovulation& spawning
Female
Antagonism
EstrogenReceptor
Fadrozole
AromataseInhibitor2)
Reduced Vtgproduction
Hepatocyte
Oocytedevelopment
Ovary
Ovulation& spawning
Female
Inhibition
AromataseEnzyme
Three distinct molecular initiating events
17ß-Trenbolone
AR Agonist
3)Reduced Vtgproduction
Hepatocyte
Oocytedevelopment
Ovary
Ovulation& spawning
Female
Agonism
AndrogenReceptor
31
AOPs Converging at Impaired Vitellogenesis
Antagonism
���� Agonism
EstrogenReceptor
Reduced Vtgproduction
Hepatocyte
Inhibition
���� Substrate
AromataseEnzyme
Agonism
AndrogenReceptor
Reduced LH/FSHsynthesis/release
GnRH Neurons/ Gonadotrophs
(���� E2)ReducedE2 synthesis
GranulosaCell
ReducedT synthesis
Tamoxifen
ERAntagonist
(���� T)
Fadrozole
AromataseInhibitor
17ß-Trenbolone
AR Agonist
ER Antagonism
Decreased ER Agonism
1)
3)
2)
CH3
ON
CH3
CH3
32
AOPs Converging at Impaired Vitellogenesis
Antagonism
���� Agonism
EstrogenReceptor
Reduced Vtgproduction
Hepatocyte
Inhibition
���� Substrate
AromataseEnzyme
Agonism
AndrogenReceptor
Reduced LH/FSHsynthesis/release
GnRH Neurons/ Gonadotrophs
(���� E2)
ReducedE2 synthesis
Granulosa Cell
ReducedT synthesis
Tamoxifen
ERAntagonist
(���� T)
Fadrozole
AromataseInhibitor
17ß-Trenbolone
AR Agonist
ER Antagonism
Decreased ER Agonism
1)
3)
2)
N
N
N
33
AOPs Converging at Impaired Vitellogenesis
Antagonism
���� Agonism
EstrogenReceptor
Reduced Vtgproduction
Hepatocyte
Inhibition
���� Substrate
AromataseEnzyme
Agonism
AndrogenReceptor
Reduced LH/FSHsynthesis/release
GnRH Neurons/ Gonadotrophs
(���� E2)ReducedE2 synthesis
Granulosa Cell
ReducedT synthesis
Tamoxifen
ERAntagonist
(���� T)
Fadrozole
AromataseInhibitor
17ß-Trenbolone
AR Agonist
ER Antagonism1)
3)
2)
Decreased ER Agonism
O
OH
34
AOPs Help Focus Research
Reduced Vtgproduction
Hepatocyte
Tamoxifen
ERAntagonist
Fadrozole
AromataseInhibitor
17ß-Trenbolone
AR Agonist
1)
3)
2)Oocyte
development
Ovary
Ovulation& spawning
Female
Decreasing trajectory
Population
To evaluate how chemicals can affect Vtg production, focus research on the three MIEs for hazard evaluation of large chemical inventories & predictive models
35
AOPs Help Focus Research
Reduced Vtgproduction
Hepatocyte
Tamoxifen
ERAntagonist
Fadrozole
AromataseInhibitor
17ß-Trenbolone
AR Agonist
1)
3)
2)Oocyte
development
Ovary
Ovulation& spawning
Female
Decreasing trajectory
Population
Three distinct MIEs = three distinct QSARs
36
Predictive Models within AOPs
Reduced Vtgproduction
Hepatocyte
Tamoxifen
ERAntagonist
Fadrozole
AromataseInhibitor
17ß-Trenbolone
AR Agonist
1)
3)
2)Oocyte
development
Ovary
Ovulation& spawning
Female
Decreasing trajectory
Population
Predictive model from effect to Adverse Outcome
0
0.2
0.4
0.6
0.8
1
0 5 10 15 20
Time (Years)
Ave
rage
Pop
ulat
ion
Siz
e(P
ropo
rtion
of C
arry
ing
Cap
acity
) A
B
C
DE
Forecast Population Trajectories
0
0.2
0.4
0.6
0.8
1
0 5 10 15 20
Time (Years)
Ave
rage
Pop
ulat
ion
Siz
e(P
ropo
rtio
n of
Car
ryin
g C
apac
ity)
A
B
C
DE
0%
25%
50%
75%>95%0
0.2
0.4
0.6
0.8
1
0 5 10 15 20
Time (Years)
Ave
rage
Pop
ulat
ion
Siz
e(P
ropo
rtion
of C
arry
ing
Cap
acity
)
0
0.2
0.4
0.6
0.8
1
0 5 10 15 20
Time (Years)
Ave
rage
Pop
ulat
ion
Siz
e(P
ropo
rtion
of C
arry
ing
Cap
acity
) A
B
C
DE
Forecast Population Trajectories
0
0.2
0.4
0.6
0.8
1
0 5 10 15 20
Time (Years)
Ave
rage
Pop
ulat
ion
Siz
e(P
ropo
rtio
n of
Car
ryin
g C
apac
ity)
A
B
C
DE
0%
25%
50%
75%>95%0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Relative Vitellogenin
Rel
ativ
e F
ecun
dity
Fathead Minnow Fecundity vs Vtg
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Relative Vitellogenin
Rel
ativ
e F
ecun
dity
Fathead Minnow Fecundity vs Vtg
Fecundity = -0.042 + 0.95 * Vtg (R 2 = 0.88)
Ankley et al. 2008 Aquat. Toxicol. 88:69-74
37
AOPs and Biomarkers
Antagonism
���� Agonism
EstrogenReceptor
Reduced Vtgproduction
Hepatocyte
Inhibition
���� Substrate
AromataseEnzyme
Agonism
AndrogenReceptor
Reduced LH/FSHsynthesis/release
GnRH Neurons/ Gonadotrophs
(���� E2)
E2 synthesis
GranulosaCell
Tsynthesis
Tamoxifen
ERAntagonist
(���� T)
Fadrozole
AromataseInhibitor
17ß-Trenbolone
AR Agonist
ER Antagonism
Decreased ER Agonism
1
3
2
Where can ‘omics and systems biology
information contribute?
Identification of Potential Biomarkers
diagnostic of exposure & adverse effect
38
� Help establish relevance of MIEs and intermediate s teps
� Understand relationship of biomarkers – genes, prote ins, protein function – in relation to adverse outcomes
� Develop and improve predictive models and approache s to advance regulatory ecotoxicology
� Need to understand AOPs in the context of dosimetry
Exposure ≠ Adverse Outcome
Why use Adverse Outcome Pathway Framework ?
Dose
Effe
ct
Compensation
AdverseOutcome
39
Provides a conceptual framework in which data and k nowledge
collected at multiple levels of biological organiza tion can be
synthesized in a useful way to risk assessors and e cotoxicologists.
� Existing knowledge can be organized
� Key uncertainties and research priorities identifie d
� Promotes effective communication between research scientists, risk assessors, risk managers, others
Why use Adverse Outcome Pathway Framework ?
40
Acknowledgements
Gerald T. Ankley, Richard S. Bennett, Russell J. Erickson, Dale J. Hoff, Rodney D. Johnson, David R.
Mount, John W. Nichols, Christine L. Russom, Patricia K. Schmieder, Jose A. Serrano, Joseph E. Tietge, Daniel L. Villeneuve