Adverse Outcome Pathways in Ecotoxicology Research Michael W. Hornung US Environmental Protection Agency, Mid-Continent Ecology Division, Duluth, MN Meeting of the Northland Chapter of SOT October 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.
Background 2
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 3
Background 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. 4
Background Ecotox is faced with the same competing objectives Depth, Breadth vs. Animal welfare, Conservation Some particular challenges to implementation in Ecotox 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 challenge vertebrates, invertebrates, microorganisms, plants 5
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? 6
Adverse Outcome Pathway Definition: 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. Designed for the translation of mechanistic information into endpoints meaningful to ecological risk 7
What is an Adverse Outcome Pathway? Provides consistent structure and terminology for organizing ecotoxicological understanding across levels of biological organization 8
What is an Adverse Outcome Pathway? Anchored by a Molecular Initiating Event in which a chemical interacts with a specific biomolecule... Toxicant Chemical Properties Macro-Molecular Interactions Receptor/Ligand Interaction DNA Binding Protein Oxidation Anchor 1 Molecular Initiating Event 9
What is an Adverse Outcome Pathway?... and anchored at an Adverse Outcome at the organism- or populationlevel that is relevant to risk assessment Organism Lethality Impaired Development Impaired Reproduction Population Structure Recruitment Extinction Cancer Anchor 2 Adverse Outcome 10
What is an Adverse Outcome Pathway? The cellular and organ responses through which the molecular initiating event produces an adverse outcome completes the Adverse Outcome Pathway Toxicant Chemical Properties Anchor 1 Molecular Initiating Event Macro- Molecular Interactions Receptor/Ligand Interaction DNA Binding Protein Oxidation Cellular Gene Activation Protein Production Altered Signaling Protein Depletion Organ Altered Physiology Disrupted Homeostasis Altered Tissue Development or Function Organism Lethality Impaired Development Impaired Reproduction Cancer Population Structure Recruitment Extinction Anchor 2 Adverse Outcome 11
Linked Levels of Biological Organization Toxicant Macro- Molecular Interactions Cellular Organ Organism Population Chemical Properties 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 Structure Recruitment Extinction Linkages between levels of organization may be causal, mechanistic, inferential, or correlation based Establishes scientifically defensible connection between initiating event and adverse outcome. 12
What is an Adverse Outcome Pathway? The Adverse Outcome Pathway has origins in previous toxicological terms / concepts developed, in part, due to ambiguities in usage of terms 13
Adverse Outcome Pathway vs Toxicity Pathway Adverse Outcome Pathway Toxicant Macro- Molecular Interactions Cellular Organ Organism Population Chemical Properties 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 Structure Recruitment Extinction Toxicity Pathway Cellular response pathways that when sufficiently perturbed are expected to result in adverse health effects Toxicity Testing in 21st Century, NRC 2007. 14
Adverse Outcome Pathway vs Mechanism of Action Mechanism 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 Toxicant Macro- Molecular Interactions Cellular Organ Organism Population Chemical Properties 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 Structure Recruitment Extinction 15
Adverse Outcome Pathway vs Mode of Action Mode of Action Mode of Action: a common set of biochemical, physiological, or behavioral responses that characterize an adverse biological response where major, but not necessarily all, linkages between a direct initiating event and an adverse outcome are understood Toxicant Macro- Molecular Interactions Cellular Organ Organism Population Chemical Properties 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 Structure Recruitment Extinction 16
What is an Adverse Outcome Pathway? AOPs are a sequential series of events But they are not isolated from other biological processes Outcome Exposure Cell Tissues & Organ Systems Organism & Population 17
Examples of Adverse Outcome Pathways Narcosis Photo-Activated Toxicity AhR Mediated Toxicity Estrogen Receptor Activation Impaired Vitellogenesis 18
AOPs in Ecotoxicology 19
AOP Example 1: Narcosis 20
AOP: Narcosis Baseline Toxicity Narcosis is non-specific toxicity resulting from weak and reversible hydrophobic interactions (Overton, 1901) Narcosis is theorized to result from hydrophobic interactions between chemicals and cellular membranes. Baseline toxicity: if a chemical does not produce toxicity by some more specific mechanism it will act by narcosis, providing it is sufficiently soluble in water at high enough concentrations to achieve required chemical activity 21
AOP: Narcosis Baseline Toxicity Not all linkages are known with absolute certainty in this AOP, Non-polar Narcotics Cellular Membranes Neurons? (Multiple)? CNS/ Multiple Organ types Organism Population Numerous Chemicals Changes in fluidity / transport? Respiration, Metabolic rate Equilibrium loss, Mortality Declining trajectory but the relationship between chemical property and adverse outcome is well established 22
AOP : Narcosis Baseline Toxicity 6 Fathead Minnow 96-h Toxicity 5 96-h Log LC50 (mg/l) 4 3 2 1 0-1 -2-1 0 1 2 3 4 5 6 7 Log Kow Data plotted from Russom et al. 1997. ET&C, 16, 948-967 23
AOP : Narcosis Baseline Toxicity Non-polar Narcotics Numerous Chemicals Cellular Membranes Changes in fluidity / transport Neurons (Multiple)??? CNS/ Multiple Organ types Respiration, Metabolic rate Organism Equilibrium loss, Mortality Population Declining trajectory Log Kow 96 h LC50 Predictive QSAR based upon log Kow 24
AOP : Narcosis Baseline Toxicity Non-polar Narcotics Numerous Chemicals Cellular Membranes Changes in fluidity / transport Neurons (Multiple)??? CNS/ Multiple Organ types Respiration, Metabolic rate Organism Equilibrium loss, Mortality Population Declining trajectory AOP defined in the context of well-established toxicity endpoints Do we need to know all of these linkages? NO. Strong weight-of-evidence shows that organismal effect is related to chemical property (Kow) Supports use of a predictive model based upon chemical propert 25
AOP Example 2: AOPs Converging at Impaired Vitellogenesis 26
Vitellogenin Production in Fish Well-defined linkages from a MIE to normal biological function ER Agonist Estradiol Estrogen Receptor Agonism Hepatocyte Vtg production Ovary Oocyte development Female Ovulation & spawning Population Stable or increasing trajectory OH OH HO HO ERE-Vtg 27
Three AOPs of Impaired Vitellogenesis Three distinct molecular initiating events 1) ER Antagonist Tamoxifen Estrogen Receptor Antagonism Hepatocyte Reduced Vtg production Ovary Oocyte development Female Ovulation & spawning 28
Three AOPs of Impaired Vitellogenesis Three distinct molecular initiating events 1) ER Antagonist Tamoxifen Estrogen Receptor Antagonism Hepatocyte Reduced Vtg production Ovary Oocyte development Female Ovulation & spawning 2) Aromatase Inhibitor Fadrozole Aromatase Enzyme Inhibition Hepatocyte Reduced Vtg production Ovary Oocyte development Female Ovulation & spawning 29
Three AOPs of Impaired Vitellogenesis Three distinct molecular initiating events 1) ER Antagonist Tamoxifen Estrogen Receptor Antagonism Hepatocyte Reduced Vtg production Ovary Oocyte development Female Ovulation & spawning 2) Aromatase Inhibitor Fadrozole Aromatase Enzyme Inhibition Hepatocyte Reduced Vtg production Ovary Oocyte development Female Ovulation & spawning 3) AR Agonist 17ß-Trenbolone Androgen Receptor Agonism Hepatocyte Reduced Vtg production Ovary Oocyte development Female Ovulation & spawning 30
AOPs Converging at Impaired Vitellogenesis 1) ER Antagonist Tamoxifen ER Antagonism CH 3 O CH 3 N CH3 Estrogen Receptor Hepatocyte 2) Aromatase Inhibitor Fadrozole Aromatase Enzyme Inhibition Granulosa Cell Reduced E2 synthesis ( E2) Decreased ER Agonism Antagonism Agonism Reduced Vtg production Substrate Reduced T synthesis ( T) 3) AR Agonist 17ß-Trenbolone Androgen Receptor Agonism GnRH Neurons/ Gonadotrophs Reduced LH/FSH synthesis/release 31
AOPs Converging at Impaired Vitellogenesis 1) ER Antagonist Tamoxifen ER Antagonism 2) Aromatase Inhibitor Fadrozole N Aromatase Enzyme Decreased ER Agonism Granulosa Cell ( E2) Estrogen Receptor Antagonism Agonism Hepatocyte Reduced Vtg production Inhibition Substrate Reduced E2 synthesis N ( T) Reduced T synthesis N 3) AR Agonist 17ß-Trenbolone Androgen Receptor Agonism GnRH Neurons/ Gonadotrophs Reduced LH/FSH synthesis/release 32
AOPs Converging at Impaired Vitellogenesis 1) 2) ER Antagonist Tamoxifen Aromatase Inhibitor Fadrozole Aromatase Enzyme Inhibition Substrate ( T) Granulosa Cell Reduced E2 synthesis Reduced T synthesis ER Antagonism Decreased ER Agonism ( E2) Estrogen Receptor Antagonism Agonism Hepatocyte Reduced Vtg production 3) AR Agonist 17ß-Trenbolone Androgen Receptor Agonism GnRH Neurons/ Gonadotrophs Reduced LH/FSH synthesis/release OH O 33
AOPs Help Focus Research 1) ER Antagonist Tamoxifen To evaluate how chemicals can affect Vtg production, focus research on the three MIEs for hazard evaluation of large chemical inventories & predictive models 2) Aromatase Inhibitor Fadrozole Hepatocyte Reduced Vtg production Ovary Oocyte development Female Ovulation & spawning Population Decreasing trajectory 3) AR Agonist 17ß-Trenbolone 34
AOPs Help Focus Research 1) ER Antagonist Tamoxifen Three distinct MIEs = three distinct QSARs 2) Aromatase Inhibitor Fadrozole Hepatocyte Reduced Vtg production Ovary Oocyte development Female Ovulation & spawning Population Decreasing trajectory 3) AR Agonist 17ß-Trenbolone 35
Predictive Models within AOPs 1) ER Antagonist Tamoxifen Predictive model from effect to Adverse Outcome 2) Aromatase Inhibitor Fadrozole Hepatocyte Reduced Vtg production Ovary Oocyte development Female Ovulation & spawning Population Decreasing trajectory Fecundity = -0.042 + 0.95 * Vtg (R 2 = 0.88) 3) AR Agonist 17ß-Trenbolone Relative Fecundity Fathead Minnow Fecundity vs Vtg 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Relative Vitellogenin Average Average Population Population Size Size (Proportion (Proportion of Carrying of Carrying Capacity) Capacity) Forecast Population Trajectories 1 1 A A 0% 0.8 0.8 0.6 0.6 0.4 0.4 B B 25% 0.2 0.2 C 50% C E D 0 >95% E 75% D 0 0 5 10 15 20 0 5 10 15 20 Time (Years) Time (Years) Ankley et al. 2008 Aquat. Toxicol. 88:69-74 36
AOPs and Biomarkers 1 ER Antagonist Tamoxifen ER Antagonism Where can omics and systems biology information contribute? 2 Aromatase Inhibitor Fadrozole Aromatase Enzyme Inhibition Substrate ( T) Granulosa Cell E2 synthesis T synthesis Decreased ER Agonism ( E2) Estrogen Receptor Antagonism Agonism Hepatocyte Reduced Vtg production Identification of Potential Biomarkers 3 AR Agonist 17ß-Trenbolone Androgen Receptor Agonism GnRH Neurons/ Gonadotrophs Reduced LH/FSH synthesis/release diagnostic of exposure & adverse effect 37
Why use Adverse Outcome Pathway Framework? Help establish relevance of MIEs and intermediate steps Understand relationship of biomarkers genes, proteins, protein function in relation to adverse outcomes Develop and improve predictive models and approaches to advance regulatory ecotoxicology Need to understand AOPs in the context of dosimetry Exposure Adverse Outcome Adverse Outcome Effect Compensation Dose 38
Why use Adverse Outcome Pathway Framework? Provides a conceptual framework in which data and knowledge collected at multiple levels of biological organization can be synthesized in a useful way to risk assessors and ecotoxicologists. Existing knowledge can be organized Key uncertainties and research priorities identified Promotes effective communication between research scientists, risk assessors, risk managers, others 39
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 40