# Building Libraries of Adverse

Outcome Pathways
Daniel L. Villeneuve, Ph.D., Aquatic Toxicologist Toxic Effects Characterization Research Branch, MED, NHEERL, ORD, EPA

5/26/2011 1 U.S. Environmental Protection Agency

### 5/26/2011 U.S. Environmental Protection Agency 2 

# I. Paradigm Shift in Toxicology – Predicting Adversity II. Adverse Outcome Pathways – definition and example III. AOPs and alternative testing strategies 

# IV. Strategies for AOP discovery and development V. Applying AOPs VI. Conclusions 

# Outline

### 3 

## Mode of action – based Focused/targeted in vivo testing

• QSARs • Chemical Categories • High throughput screening • In vitro 

## • Biomarkers • Genomics 

# I. Paradigm Shift in Toxicology

# • QSARs • Chemical Categories 

# • High throughput screening • In vitro • Biomarkers 

# • Genomics Use in hazard/risk assessment has been limited by lack of well defined (predictive) linkages between these alternative types of data and adverse outcomes traditionally

considered relevant to ecological risk assessment. Molecular Cellular Tissue Organ Organ System Individual Population Ecosystem



# • Survival 

# • Growth/development • Reproduction 

# I. Paradigm Shift in Toxicology

### An Adverse Outcome Pathway (AOP) is a conceptual framework that portrays existing knowledge concerning the linkage between a direct molecular initiating event and an adverse outcome , at a level of biological organization relevant to risk assessment. (Ankley et al. 2010, Environ. Toxicol . Chem., 29(3): 730 - 741.)

Chemical Properties Receptor/Ligand

Interaction 

### DNA Binding Protein Oxidation Gene activation Protein 

### production Altered signaling Altered

physiology 

### Disrupted homeostasis Altered tissue development/ function Lethality

Impaired 

### Development Impaired Reproduction Toxicant Cellular Responses Organ

Responses Organism

Responses Structure

Recruitment Extinction Population Responses MacroMolecular 

### Interactions

Molecular Cellular Tissue Organ Organ System Individual Population Ecosystem 

## II. Adverse Outcome Pathways – definition and example

### 6 

## Biologic inputs “Normal” Biological Function

Adverse

Outcomes (e.g., mortality, Reproductive Impairment) Cell inury, Inability to regulate 

## Adaptive Responses Early cellular

changes 

### Exposure Uptake - Delivery to Target Tissues

Perturbation 

## Cellular response pathway

Molecular initiating event Perturbed cellular



### response pathway Adverse outcome relevant to risk assessment 

# Toxicity Pathway Adverse Outcome Pathway II. Adverse Outcome Pathways – definition and example

## Ovary Brain 

# Aromatase Inhibition

Villeneuve et al. 2006. Aquat. Toxicol. 76:353 -368 In vitro assay with small fish tissues 

## II. Adverse Outcome Pathways – definition and example

## Sanderson et al. 2001, EHP, 109: 1027 - 1031



## High throughput assay with human cell line II. Adverse Outcome Pathways – definition and exampleSAR and QSAR in Environmental Research, 20: 657 - 678. 

# QSAR, chemical categories II. Adverse Outcome Pathways – definition and example

### 10 Pregnenolone

Progesterone 17a- OH - Pregnenolone
17a- OH - Progesterone CYP17 (hydroxylase) CYP11A
3 b - HSD 3 b -HSD DHEA
Androstenedione Testosterone CYP17 (lyase) 3

b -HSD
17 b - HSD CYP19 17 b -estradiol Cholesterol

20 b -HSD 17 α20 β-dihydroxy-4- pregnen -3 -one CYP17 (hydroxylase) CYP17 (lyase) estrone CYP19 17 b - HSD
11 β- OH Testosterone CYP11B1

11 - Ketotestosterone 11β HSD 11 -deoxycorticosterone CYP21
corticosterone CYP11B1
aldosterone CYP11B2 11 -deoxycortisol CYP21

cortisol CYP11B2 

## II. Adverse Outcome Pathways – definition and example

# A bit of fish reproductive biology

11 

### Agonism Estrogen 

### Receptor Vtg

production Hepatocyte Oocyte

development Ovary Ovulation

& spawning 

### Female Stable or



### increasing trajectory Population Estradiol ER Agonist



## ERE -Vtg 

# Cellular response pathway Biologic inputs “Normal” 

### Biological Function 

## II. Adverse Outcome Pathways – definition and example

### 12 

## Fadrozole

Biochemical Reproduction (Key Events)

Ankley et al., 2002, Toxicol. Sci. 67:121 - 130



# Perturbed fish reproductive biology II. Adverse Outcome Pathways – definition and example

### 5/26/2011 13 Chemical Property Profile Receptor/Ligand

Interaction DNA Binding Protein Oxidation Gene Activation Protein Production Altered Signaling Protein Depletion Altered Physiology Disrupted Homeostasis Altered Tissue Development or Function Lethality Impaired Development Impaired Reproduction Cancer Toxicant Macro- Molecular

Interactions Cellular Responses Organ Responses Individual Responses Structure

Recruitment Extinction Population Responses Adverse Outcome Pathway



### Aromatase inhibition Reduced E2,

Vtg synthesis Impaired

vitellogenesis Reduced fecundity 

## II. Adverse Outcome Pathways – definition and example

Molecular initiating event Key events or predictive relationships spanning levels of biological organization Adverse outcome relevant to risk assessment

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## Adverse outcome pathway : a conceptual framework that portrays existing knowledge concerning the linkage between a direct molecular initiating event and an adverse outcome, at a level of biological organization relevant to risk assessment . – (eco; demographic relevance) Mode of action: the sequence of key events, starting with the interaction of an 

## agent with the target cell , through molecular, biochemical, cellular, functional and anatomical changes, resulting in cancer or other adverse health effects. Key event: empirically observable, necessary steps in the disease (cancer) or adverse outcome process. Molecular initiating event: the initial point of chemical - biological intersection within an organism that starts an AOP. First key event in a mode of action. 

## Toxicity pathway: cellular response pathways that, when sufficiently perturbed, are expected to result in adverse health effects. 

## II. Adverse Outcome Pathways – definition and example

### 5/26/2011 15 

## II. Adverse Outcome Pathways – definition and example

### 5/26/2011 U.S. Environmental Protection Agency 16 

## III. AOPs and alternative testing strategies 

# How do AOPs support the development of alternative testing strategies?

## OECD 210, OCSPP 850.1400: Fish, Early- life Stage Toxicity Test

• Most frequently used bioassay for predicting fish chronic toxicity
• Supports ERA for pesticides, biocides, pharmaceuticals, industrial chemicals.

• Requires ≥ 360 fish per test • 30 - 80 days depending on species • Cost: 50 - 80K USD/test



### • Endpoints • Hatching • Mortality • Morphological/behavioral abnormalities • Growth (weight, length)


### • Product: LOEC, NOEC, and/or EC10 • Most sensitive endpoint as hazard threshold 

## III. AOPs and alternative testing strategies

### • >50 scientists • Academia, gov’t, industry, NGOs • Europe, North America, Asia 

## III. AOPs and alternative testing strategiesAhR-mediated cardiotoxicity

AChE inhibition

Gill narcosis 

### Volz et al. in preparation 

## III. AOPs and alternative testing strategiesOther FELS toxicity pathway assays Thousands

Tens Hundreds



### Volz et al. in preparation 

## III. AOPs and alternative testing strategies

### 5/26/2011 U.S. Environmental Protection Agency 21 

## IV. Strategies for AOP discovery/development 

# How many AOPs are there? How can we systematically discover 

# and characterize them?• USEPA, NERL – Cincinnati, OH • D. Bencic, M. Kostich, A. Biales, D. Lattier, J. Lazorchak, G. Toth, R. Wang, • USEPA, NHEERL – Duluth, MN, and Grosse Isle, MI • G. Ankley, E Durhan, K Jensen, M Kahl, C. LaLone, E Makynen, D. Miller, D. Villeneuve • USEPA, NERL – Athens, GA • T. Collette, D. Ekman, M. Henderson, K. Ralston - Hooper, Q. Teng • USEPA, NHEERL, - RTP, NC • M. Breen, R. Conolly, S. Edwards, L. Burgoon • USEPA, NCER, STAR Program • N. Denslow (Univ. of Florida), E. Orlando, (Univ. Maryland), K. Watanabe (Oregon Health Sciences Univ.), M. Sepulveda (Purdue Univ.) • USACE, ERDC – Vicksburg, MS • E. Perkins, T. Habib, M. Mayo • Other partners
• Jackson St. University, N. Garcia - Reyero, • University of St. Thomas, D. Martinovic • Birmingham University, Birmingham, UK, F. Falciani, N. Turan • UC Berkeley, C. Vulpe, A. Loguinov • UC Santa Barbara, J. Shoemaker, F. Doyle III • Joint Genome Institute, DOE (Walnut Creek, CA) • Sandia, DOE (Albuquerque, NM) • Pacific Northwest National Laboratory (Richland, WA) • Environment Canada, M. McMaster (Burlington, ON) 

# SMALL FISH Linking exposures and effects using transcriptomics, proteomics, and metabolomics with small fish models

### 5/26/2011 23 

## IV. Strategies for AOP discovery/development

Adapted from Greely 2002

### 5/26/2011 24 11 βHSD P45011 β . GnRH Neuronal System GABA Dopamine Brain

Gonadotroph Pituitary D1 R

D2 R ?

? Y 2 R NPY

Y 1 R GnRH R ?

Activin Inhibin GABAA R GABAB R Y 2 R

D2 R GnRH

Circulating Sex Steroids / Steroid Hormone Binding Globlulin PACAP Follistatin Activin PAC 1 R Activin R FSH b LH b GPa Circulating LH, FSH

FSH R LH R Circulating LDL, HDL

LDL R HDL R Outer mitochondrial membrane Inner mitochondrial membrane StAR

P450scc pregnenolone 3 b HSD P450c17 androstenedione
testosterone Cholesterol

estradiol Gonad (Generalized, gonadal, steroidogenic cell)

Androgen / Estrogen Responsive Tissues (e.g. liver, fatpad, gonads) AR ER 3 4
5 6 Blood

Blood 

### Compartment

17 βHSD P450arom
11 -ketotestosterone progesterone 17 α-hydroxyprogesterone

17 α,20 β-P (MIS) 20 βHSD 

## Fipronil ( - ) Muscimol (+) Apomorphine (+) Haloperidol ( - ) Trilostane ( - ) Ketoconazole ( - ) Fadrozole ( - ) Prochloraz ( - , - ) Vinclozolin ( - ) Flutamide ( - ) 

## 17 β -Trenbolone (+) 17 α 

## - Ethynylestradiol (+) 7 8
9 10 11 12 2 1 

# Chemical ProbesFathead minnow 21 d, reproduction • Fecundity (NOEC / LOEC)

• Plasma steroids and vtg

• Targeted qpcr analyses Zebrafish 24, 48, 96 h time course •Targeted qpcr analyses

• Microarray
Fathead minnow 1, 2, 4, 8 d exposed, 1, 2, 4, 8 d dep. • Plasma steroids and vtg • Ex vivo steroid production • Targeted qpcr analyses • Microarray • Metabolomics 

### Mode of action Aromatase inhibitor Anti - androgen (metabolites) Dopamine 2 receptor antagonist Steroidogenesis inhibitor Steroidogenesis inhibitor Androgen receptor agonist Steroidogenesis inhibitor Anti - androgen (metabolites) 

### Test chemical Fadrozole (FAD) Flutamide (FLU) Haloperidol (HAL) Ketoconazole ( KTC) Prochloraz (PRO) 17β trenbolone (TRB) Trilostane (TRI) Vinclozolin (VIN) 

## IV. Strategies for AOP discovery/developmentTest chemical Mode of action 8 d time course Conc (μg/L) 21 d fecundity LOEC & NOEC (μg/L) 

### Fadrozole (FAD) Aromatase inhibitor L: 4.1 ±0.13 H: 35 ± 1.1 NOEC: ND LOEC: 2.0 

### Flutamide (FLU) Anti - androgen (metabolites) L: 56 ±3.0 H: 557 ±37 NOEC: 50 LOEC: 500 

### Haloperidol 

### (HAL) Dopamine 2 receptor antagonist 57.3 ± 4.7 NOEC: 20 LOEC: ND 

### Ketoconazole (KTC) Steroidogenesis inhibitor L: 32.7 ±3.4 H: 311 ±36 NOEC: 6.0 d LOEC: 25 d 

### Prochloraz (PRO) Steroidogenesis inhibitor L: 23.6 ± 1.4 H: 288 ±24 NOEC: 30 LOEC: 100 

### 17β trenbolone (TRB) Androgen receptor agonist L: 0.032 ±0.007 H: 0.493 ±0.055 NOEC: 0.005 LOEC: 0.050 

### Trilostane (TRI) Steroidogenesis inhibitor L: 282 ±38 H: 1620 ± 190 NOEC: 300 LOEC: 1500 

### Vinclozolin (VIN) Anti - androgen (metabolites) L: 177 ±30 H: 502 ±43 NOEC: ND LOEC: 60 

## EDC Exposures (8 chemicals x 2 fhm assays) IV. Strategies for AOP discovery/development17 β trenbolone (trb - acetate) AR binding AR - dependent somatic cell proliferation Tubercle and fatpad formation in females

Ankley et al. 2003 ET&C 22:1350- 1360 

## IV. Strategies for AOP discovery/developmentMolecular Cellular Tissue Organ Organ System Individual Population Ecosystem 17 β trenbolone (trb - acetate) AR binding Negative feedback, hypothalamic neurons Reduced steroidogenesis , vitellogenesis Reduced fecundity

Ankley et al. 2003 ET&C 22:1350- 1360 

## IV. Strategies for AOP discovery/development17 β trenbolone (trb - acetate) AR binding Negative feedback, hypothalamic neurons AR - dependent somatic cell proliferation Tubercle and fatpad formation in females Reduced steroidogenesis , vitellogenesis Reduced fecundity 

## Adverse Outcome Pathway: Human

17 β trenbolone (trb - acetate) AR binding Negative feedback, hypothalamic neurons AR - dependent somatic cell proliferation Increased body hair in females Testicular atrophy, impaired sperm production Infertility 

## Adverse Outcome Pathway: Small Fish Molecular Cellular Tissue Organ Organ System Individual Population Ecosystem 

## Cross-species comparisons

### 5/26/2011 U.S. Environmental Protection Agency 30 

# •As AOPs are developed, AOP information needs to be assembled into a properly curated, accessible, searchable, 

# and adaptable format.
• Important step toward contextualizing AOP networks IV. Strategies for AOP discovery/development

# AOPs depicted as linear 

# AOPs function in networks

• intersections • shared nodes • can influence on another AOP networks function in a broader systems biology context IV. Strategies for AOP discovery/development

# Role for Toxicogenomics

• Toxicogenomics - can survey a broader range of “biological space” • Discovery - adverse effects mediated through unknown, or poorly characterized, mechanisms of action. • Unraveling interactions between biological pathways. • Pathway perturbation motifs - Identifying those features that respond to a given stressor/types of stress.

Chemical exposure Gene expression

response Reverse engineering of

interaction network Refinement of network with binding interactions Computational model of network architecture 

### Defining interaction 

### network Relate network to known pathways and outcomes 

## IV. Strategies for AOP discovery/developmentFathead minnow 21 d, reproduction • Fecundity (NOEC / LOEC)

• Plasma steroids and vtg

• Targeted qpcr analyses Zebrafish 24, 48, 96 h time course •Targeted qpcr analyses

• Microarray
Fathead minnow 1, 2, 4, 8 d exposed, 1, 2, 4, 8 d dep. • Plasma steroids and vtg • Ex vivo steroid production • Targeted qpcr analyses • Microarray • Metabolomics 

### Mode of action Aromatase inhibitor Anti - androgen (metabolites) Dopamine 2 receptor antagonist Steroidogenesis inhibitor Steroidogenesis inhibitor Androgen receptor agonist Steroidogenesis inhibitor Anti - androgen (metabolites) 

### Test chemical Fadrozole (FAD) Flutamide (FLU) Haloperidol (HAL) Ketoconazole ( KTC) Prochloraz (PRO) 17β trenbolone (TRB) Trilostane (TRI) Vinclozolin (VIN) 

## IV. Strategies for AOP discovery/development

### 5/26/2011 U.S. Environmental Protection Agency 34 

# Mutual information a measure of how much one random variables tells us about another - can be thought of as the reduction in uncertainty about one random variable given knowledge of another Expression effects

Expression effects

across many experimental conditions 

## IV. Strategies for AOP discovery/development

### Physiological measurements Receptor hubs Targets 

# Mutual information network IV. Strategies for AOP discovery/development

### mRNA processing

Cell cycle Protein secretion DNA damage response

none none Metabolic processes Inflammatory response Regulation of biol. processes none 

# Mapping Functional Modules onto Network

system development organ development developmental process anatomical structure development intrinsic to plasma membrane 

## IV. Strategies for AOP discovery/development

# Mutual information network

Clusters of genes with statistically- coordinated expression profiles

• Identify characterize novel biological pathways/functions mediating adverse outcomes. • Anchor to features in graphical systems models • Accelerate the discovery of new AOPs and improve understanding of AOPs in a network/systems context 

## IV. Strategies for AOP discovery/developmentfipronil GABA receptor antagonist

cyproterone acetate anti -androgen 17 b -trenbolone androgen vinclozolin anti -androgen 17a-ethynylestradiol (EE2) estrogen

bisphenol A (BPA) estrogen and anti -androgen ? 

# Metabolomics

average “exposed” spectrum –minus– average “control” spectrum (using urine from male FHM) 1.0 2.0 3.0 4.0 Chemical Shift (ppm) 

## IV. Strategies for AOP discovery/development

### 5/26/2011 U.S. Environmental Protection Agency 39 

# Once AOPs are developed, how can they be applied? 1. Linkage to adversity - relevance of alternative data 2. Support focused testing strategies – associate molecular perturbations w/ probable outcomes 

# 3. Identification of uncertainties & research needs/priorities V. Applying AOPs

### 5/26/2011 U.S. Environmental Protection Agency 40 

# 4. Support development of useful QSARs and biomarkers V. Applying AOPs


# OECD WORKSHOP ON USING MECHANISTIC INFORMATION IN FORMING CHEMCIAL 

# CATEGORIES TO FILL DATA GAPS FOR REGULATORY PURPOSES US -EPA One Potomac Yard 2777 S. Crystal Drive Arlington, VA 22202, USA 8 - 10 December 2010

### 5/26/2011 U.S. Environmental Protection Agency 41 

# 5. Species extrapolation

• The better we understand the biology, the easier it is to understand how effects translate across species. V. Applying AOPs

#  Identify chemicals positive for a defined initiating event – Toxcast
 Use AOP to formulate predictions of expected adverse outcomes 

# in non - human organisms
 Test hypotheses against existing data or in new experiments

• EPA, ORD • US Army, ERDC • Environment Canada • Norwegian Institute for Water Research (NIVA) • Okazaki National Research Institute, Japan 

# V. Applying AOPs



# • Where key features are conserved, foundation for applying HH HTS data for wildlife (or v.v.) 5. Species extrapolationSchmieder et al. – case study; OECD Workshop on using mechanistic information in forming chemical categories.... 

## Test Organism ER Conserved Salmonid Yes Another fish (different order) Yes Another vertebrate (different order) Yes Planktonic crustacean ( daphnid ) No Benthic crustacean 

## (amphipod, crayfish) No Insect No Another non -vertebrate, non - arthopod (worm, mollusk, rotifer) No 

# ACR > 100,000 V. Applying AOPs 

# 5. Species extrapolation Basis for predicting which species and/or life - stages may be 

# vulnerable/susceptible (or not) to effects.

### 44 

# Phylogenetic Sensitivity to EE2 Genus or Species Chronic Value(s) ( ng/L) Notes Zebrafish 0.6, 1.5, <1.1 Life - cycle tests Fathead Minnow <0.32, 1.45 Life - cycle tests Medaka 3.2 F0 from 1 d through spawning Oncorhynchus <16 Adult exposure, fertilization success Potamopyrgus 50 Adult exposure; embryo production 

### Gammarus >7600 100 d test, population size Daphnia 45,000 Life - cycle test Tisbe >100,000 Saltwater copepod Chironomus 320,000 Larval growth and molting schedule Brachionus 800,000 72 h test, intrinsic rate of increase U.S. Environmental Protection Agency. 2008. Aquatic life criteria for contaminants of emerging concern. Part 2: Illustration of recommendations using data for 17a - ethinylestradiol (EE2). Office of Water, Washington, DC. 

# V. Applying AOPs 

# 5. Species extrapolation

### 45 

# AOPs assume sufficient perturbation to cause the adverse outcome. • Hazard assessment – capability to cause an adverse outcome • Screening and prioritization •Targeted testing Risk assessment – probability of an adverse outcome under defined circumstances.
• Requires an understanding of adaptive/homeostatic capacity of biological systems and their limits, relative to concentration and duration of exposure. 

# V. Applying AOPs 

# 5. Quantitative RA

### 46 

## Biologic 

## inputs “Normal” Biological Function

Adverse

Outcomes (e.g., mortality, Reproductive Impairment) Cell inury, Inability to regulate 

## Adaptive Responses Early cellular

changes 

### Exposure Uptake - Delivery to Target Tissues

Perturbation 

## Cellular response pathway 

# V. Applying AOPs 

# 5. Quantitative RAFathead minnow 21 d, reproduction • Fecundity (NOEC / LOEC)

• Plasma steroids and vtg

• Targeted qpcr analyses Zebrafish 24, 48, 96 h time course •Targeted qpcr analyses

• Microarray
Fathead minnow 1, 2, 4, 8 d exposed, 1, 2, 4, 8 d dep. • Plasma steroids and vtg • Ex vivo steroid production • Targeted qpcr analyses • Microarray • Metabolomics 

### Mode of action Aromatase inhibitor Anti - androgen (metabolites) Dopamine 2 receptor antagonist Steroidogenesis inhibitor Steroidogenesis inhibitor Androgen receptor agonist Steroidogenesis inhibitor Anti - androgen (metabolites) 

### Test chemical Fadrozole (FAD) Flutamide (FLU) Haloperidol (HAL) Ketoconazole ( KTC) Prochloraz (PRO) 17β trenbolone (TRB) Trilostane (TRI) Vinclozolin (VIN) 

# V. Applying AOPs

### 5/26/2011 48 Control Low 

### High Exposure Start (d. 0) 

### 24 h 48 h

Exposure phase (d. 1 -8) Depuration/recovery phase (d. 9 - 16)



### 96 h 8 d Exposure

Ends (d. 8) 

### + 24 h + 48 h + 96 h + 8 d25 oC, 12 h RIA 

### pituitary FSH
Ovary cyp19a1a E2 

### vitellogenesis
Villeneuve et al. 2009 Environ. Health Perspect. 117:624-631Theca

Granulosa 

## cyp11a 

# cyp19a1a T sensor E2 sensor 

### TRB



# T E2 E2 T



## Ekman et al. 2011, ET&C 30:319-329

# Biologically- based modeling

Overshoot
Compensation during exposure V. Applying AOPs 

# 5. Quantitative RA

### 5/26/2011 U.S. Environmental Protection Agency 52 Reif et al. Environ Health Perspect 118:1714 – 1720 (2010). doi:10.1289/ehp.1002180 [Online 8 September 2010] 

# Mixtures and mixed mode of action V. Applying AOPs 

# 5. Quantitative RA

## Systems biology – The scientific study of the dynamic interactions among the integrated networks of genes, proteins and biochemical reactions which give rise to emergent functions that support life. 

# V. Applying AOPs 

# 5. Quantitative RA

# • Understanding key components of system that must be included in an 

# assay or effectively modeled to provide predictive power.
• Critical for understanding and predicting complex duration concentration - response relationships.
• Ultimately needed to understand and predict effects of individual chemicals with mixed MOA or complex mixtures. V. Applying AOPs 

# 5. Quantitative RA

### 5/26/2011 U.S. Environmental Protection Agency 55 

# VI. Conclusions



#  Significant advances in systems biology will be needed to 

# support a transition from qualitative use of AOPs for hazard assessment, screening and prioritization to quantitative risk assessment.
 AOPs can support development of alternative methods and informed extrapolation across taxa.


#  Hypothesis - driven research is critical to establish the linkages depicted in AOPs
 Effective use of „omics‟ and HTS approaches can help accelerate AOP discovery, development, and evaluation.

#  AOPs provide a systematic framework to support the integration of diverse types of data in hazard/risk assessment.
 AOPs provide a basis to use rapid, low cost, predictive data to focus more costly whole animal 

# testing. VI. Conclusions

### 5/26/2011 U.S. Environmental Protection Agency 57 

# VII. Acknowledgements

### 5/26/2011 U.S. Environmental Protection Agency 58