Adverse Outcome Pathways in Ecotoxicology Research · 2015. 5. 15. · Michael W. Hornung. 2 Background. 3 Background • Historically, regulatory toxicity testing has relied on the

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


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


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


ChemicalProperties


DNA Binding

Protein Oxidation

Lethality



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


DNA Binding

Protein Oxidation

Gene Activation

Protein Production

Altered Signaling

Protein Depletion

Altered Physiology



Lethality



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


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


DNA Binding

Protein Oxidation

Gene Activation

Protein Production

Altered Signaling

Protein Depletion

Altered Physiology



Lethality



Cancer

Toxicant

Macro-Molecular


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


DNA Binding

Protein Oxidation

Gene Activation

Protein Production

Altered Signaling

Protein Depletion

Altered Physiology



Lethality



Cancer

Toxicant

Macro-Molecular


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


DNA Binding

Protein Oxidation

Gene Activation

Protein Production

Altered Signaling

Protein Depletion

Altered Physiology



Lethality



Cancer

Toxicant

Macro-Molecular


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


Predictive QSAR based upon log Kow

Log Kow 96 h LC50

CellularMembranes

Changes in fluidity

/ transport

Non-polarNarcotics

NumerousChemicals

Neurons(Multiple)

?�� Respiration,�� Metabolic rate


Equilibriumloss,

Mortality

Organism

Decliningtrajectory

Population

? ?

25


• 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


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


Tamoxifen

ERAntagonist1)


Hepatocyte

Oocytedevelopment

Ovary

Ovulation& spawning

Female

Antagonism

EstrogenReceptor

Fadrozole

AromataseInhibitor2)


Hepatocyte

Oocytedevelopment

Ovary

Ovulation& spawning

Female

Inhibition

AromataseEnzyme


30


Tamoxifen

ERAntagonist1)


Hepatocyte

Oocytedevelopment

Ovary

Ovulation& spawning

Female

Antagonism

EstrogenReceptor

Fadrozole

AromataseInhibitor2)


Hepatocyte

Oocytedevelopment

Ovary

Ovulation& spawning

Female

Inhibition

AromataseEnzyme


17ß-Trenbolone

AR Agonist

3)Reduced Vtgproduction

Hepatocyte

Oocytedevelopment

Ovary

Ovulation& spawning

Female

Agonism

AndrogenReceptor

31


Antagonism

�� Agonism

EstrogenReceptor


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


Antagonism

�� Agonism

EstrogenReceptor


Hepatocyte

Inhibition

�� Substrate

AromataseEnzyme

Agonism

AndrogenReceptor



(�� E2)

ReducedE2 synthesis

Granulosa Cell

ReducedT synthesis

Tamoxifen

ERAntagonist

(�� T)

Fadrozole

AromataseInhibitor

17ß-Trenbolone

AR Agonist

ER Antagonism


1)

3)

2)

N

N

N

33


Antagonism

�� Agonism

EstrogenReceptor


Hepatocyte

Inhibition

�� Substrate

AromataseEnzyme

Agonism

AndrogenReceptor



(�� E2)ReducedE2 synthesis

Granulosa Cell

ReducedT synthesis

Tamoxifen

ERAntagonist

(�� T)

Fadrozole

AromataseInhibitor

17ß-Trenbolone

AR Agonist

ER Antagonism1)

3)

2)


O

OH

34

AOPs Help Focus Research


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


Hepatocyte

Tamoxifen

ERAntagonist

Fadrozole

AromataseInhibitor

17ß-Trenbolone

AR Agonist

1)

3)

2)Oocyte

development

Ovary

Ovulation& spawning

Female


Population

Three distinct MIEs = three distinct QSARs

36

Predictive Models within AOPs


Hepatocyte

Tamoxifen

ERAntagonist

Fadrozole

AromataseInhibitor

17ß-Trenbolone

AR Agonist

1)

3)

2)Oocyte

development

Ovary

Ovulation& spawning

Female


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


Hepatocyte

Inhibition

�� Substrate

AromataseEnzyme

Agonism

AndrogenReceptor



(�� E2)

E2 synthesis

GranulosaCell

Tsynthesis

Tamoxifen

ERAntagonist

(�� T)

Fadrozole

AromataseInhibitor

17ß-Trenbolone

AR Agonist

ER Antagonism


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

Adverse Outcome Pathways in Ecotoxicology Research · 2015. 5. 15. · Michael W. Hornung. 2 Background. 3 Background • Historically, regulatory toxicity testing has relied on the

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