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Crofton PPDC Workshop AOP Framework 2013
- 1. Office of Research and Development National Center for Computational Toxicology PPDC 21st Century Workshop 2013 Kevin M. Crofton National Center for Computational Toxicology Adverse Outcome Pathways and Their Unifying Role in Developmental Toxicology Source to Outcome Pathways Source Environmental Contaminant Exposure Molecular Initiating Event Cellular Effects Individual Population Community Source to Outcome EPA Office of Pesticide Programsโ Stakeholder workshop http://www2.epa.gov/sites/production/files/2014-06/documents/session1-outcome.pdf
- 2. Office of Research and Development National Center for Computational Toxicology 1 Outline ๏ Introduction to โpathwaysโ โข Source-to-Outcome Pathway, 21st Century Toxicity Pathway, Mode of action, Adverse Outcome Pathways โข Example โ Perchlorate ๏ Thyroid Biology 101 ๏ Using AOP to Focus Research on Critical Data Gaps in Thyroid Disruption Induced Adverse Outcomes โข Life-Stage Specificity and Species extrapolation โข Provides structure for qualitative & quantitative predictive models โข Helps indentify data gaps in hazard assessments that, if filled, with reduce uncertainties in decisions ๏ Take home message ๏ AOPs can reduced uncertainty = better predictions of human toxicity
- 3. Office of Research and Development National Center for Computational Toxicology Exposure SOP, AOP, MOA, ToxPathway โข Definition: The continuum or cascade of measurable events starting from release into the environment and ending at an adverse outcome (USEPA 2003). โข Example: Perchlorate 2 Rocket Propellant & Fertilizer Production & Release Target Dose Molecular Initiating Event Cellular Response Organ Response Individual Response Population Community Exposure Food & Water Thyroid Gland IQ Loss in Child Inhibition of NIS Decreased TH Synthesis Altered Development of Nervous System Decreased Productivity, Increased Health Care Costs, Family and Social Issues Shift in Population IQ Uses: โข Qualitatively links actual use of chemicals to adverse outcomes โข Can be used to link exposure, toxicokinetic and toxicodynamic models to qualitatively predict outcomes โข Basis for causative & quantitative models Toxicity PathwayMode-of-ActionAdverse Outcome Pathway
- 4. Office of Research and Development National Center for Computational Toxicology Toxicity Pathway โข Definition: Cellular response pathways that, when sufficiently perturbed, are expected to result in adverse health effects are termed toxicity pathways (NRC 2007). โข Example โ Normal: Kainate receptors activated by glutamate open ion channels in neurons and regulate ion flux important for neuronal firing. 3 Biologic Inputs Normal Biologic Function Morbidity and Mortality Cell Injury Adaptive Stress Responses Early Cellular Changes Exposure Tissue Dose Biologic Interaction Perturbation NRC, 2007
- 5. Office of Research and Development National Center for Computational Toxicology Toxicity Pathway โข Example - Abnormal: Domoic acid causes glutamate-induced hyperstimulation of neurons, neurons accumulate excess Ca+, and at high enough levels this leads to cell death. 4 Use: Computational modeling will allow quantification of the significance of the perturbation. i.e., how hard does the system need to be hit to overcome compensation and result in cell injury. Watanabe et al 2011
- 6. Office of Research and Development National Center for Computational Toxicology Adverse Outcome Pathway โข Definition: An adverse outcome pathway (AOP) represents existing knowledge concerning the linkage between a molecular initiating event and an adverse outcome at the individual or population levels (Ankley et al. 2009). โข Examples: Chemical antagonism of the interaction of estrogen with the estrogen receptor, depress hepatic production of VTG, thereby decreasing plasma concentrations of the protein, its deposition to developing oocytes in the ovary, and ultimately decreased egg production and repercussions relative to population-level effects โข Use: โ Species concordance (OECD MOA Framework) โ Identified key events can be qualitatively and quantitatively linked to an adverse outcome and these events can be used in developing chemical categories. Note: AOPs are not singular โ complex interactions can occur 5
- 7. Office of Research and Development National Center for Computational Toxicology Mode of Action โข A mode-of action describes the key, rate limiting, and quantifiable events that lead to adverse outcomes (IPCS MOA Framework, see Sonich-Mullen et al., 2001) โDetermine whether animal MOA is plausible and relevant to humans โKey events must be measureable and causal โProvide guidance on establishing causal relationships between key events (modified Bradford Hill criteria) โMust predict adverse outcome from initiating key event (at least qualitatively) โCan be species specific 6
- 8. Office of Research and Development National Center for Computational Toxicology Mode of Action โข A mode-of action describes the key, rate limiting, and quantifiable events that lead to adverse outcomes (IPCS MOA Framework, see Sonich-Mullen et al., 2001) โDetermine whether animal MOA is plausible and relevant to humans โKey events must be measureable and causal โProvide guidance on establishing causal relationships between key events (modified Bradford Hill criteria) โMust predict adverse outcome from initiating key event (at least qualitatively) โCan be species specific 7 MOA is a regulatory assessment framework based on the concept of pathways AOP is a conceptual and practical tool to capture descriptions of toxicological processes.
- 9. Office of Research and Development National Center for Computational Toxicology 8 Catabolic Enzymes Hypothalamus Pit TRH Thyroid TSH Blood T3/T4 Target Tissues Liver Elimination from the body TH binding proteins 5โ-deiodinases T4 T3 Acts as a ligand for nuclear thyroid hormone receptors (TRs) Thyroid Biology 101 Synthesis, Regulation, Action and Catabolism
- 10. Office of Research and Development National Center for Computational Toxicology Thyroperoxidase NIS Hepatic Nuclear Xenoreceptors Deiodinases Thyroid Receptors Free T3 & T4 ๏จ TSH Tissue THChanges Thyroid Hyperplasia Thyroid Tumors Neurological Development Adverse Outcomes T4 & T3 Synthesis Phase 2 Catabolism Cellular Transporters Cellular T4 ๏กT3 Conversion Biliary Elimination Amphibian Metamorphosis 4 9 35 6 7 8 Transport Proteins 10 Thyroid Gland Pituitary TSH R TRH R Hypothalamus TR Blood CellularTR Signaling Altered Development 3 2 1 Hypothalamic Pituitary Feedback Bound-TH 1 2
- 11. Office of Research and Development National Center for Computational Toxicology TDCs - Two Major Adverse Outcomes of Regulatory Concern โข Pathway #1 โ Upregulation of the HPT feedback system โ Yields thyroid hypertrophy, hyperplasia and thyroid follicular tumors in rats โ This AOP is not relevant to humans due to substantial species differences in key events (Hill et al., Env Hlth Perspect 1998) โข Pathway #2 โ Decreased serum TH, decreased tissue TH, decreased tissue mRNA and protein synthesis, during development leads to: โข Amphibians โ altered metamorphosis โข Humans - decreased IQ (MOA relevant to humans) โ This AOP has be shown to be relevant to rodents and humans (Crofton & Zoeller, Crit Rev. Toxicol. 2005) 10 Weโre worried about IQ in kids, not thyroid tumors Itโs serum TH not TSH that matters!
- 12. Office of Research and Development National Center for Computational Toxicology AOPs for Thyroid Disruption Multiple MIEs and Species Specific Outcomes NIS Thyroperoxidase Hepatic Nuclear Xenoreceptors Deiodinases Thyroid Receptors โT4โSerumTransport Protein Binding ๏ฉSerum T3 & T4 ๏จ TSH Tissue THChanges Altered Development Thyroid Hyperplasia Thyroid Tumors IQ Loss Adverse Outcomes MIEs ๏ T4 & T3 Synthesis ๏ Phase 2 Catabolism ๏ Cellular Transporters ๏ฉ Tissue T4 ๏กT3 Conversion ๏ Biliary Elimination 1 2 X X X
- 13. Office of Research and Development National Center for Computational Toxicology AOPs โ What are they good for? 1. Improved predictions of toxicity via decreased uncertainty โ Increases level of confidence in the relationship between measured data and adverse outcomes that is critical for risk assessments 2. Can be Life-Stage specific 3. Enhance species to species extrapolation 4. Identification of Data Gaps โ Construction of an AOP should identify data gaps i.e., critical needs to build a useful model 5. Provide molecular targets for development of in vitro screening assays 6. Holy Grail is development of predictive computational models โ If the MIE predicts the Adverse Outcome โ then you donโt need to measure the outcome 12
- 14. Office of Research and Development National Center for Computational Toxicology Example: Linking Liver to thyroid hormones to developmental neurotoxicity Life Stage Specificity 13
- 15. Office of Research and Development National Center for Computational Toxicology 14 Description of Postulated AOP โข MIE โ Activation of hepatic nuclear receptors (i.e., CAR/PXR) โข Key Events โ Upregulation Phase II and III metabolism โ Increased hepatic clearance of TH โ Decreased Serum T3, T4 โ Decreased Tissue TH concentrations during critical developmental periods โ Altered expression of TH-responsive genes in developing brain โข Adverse Outcome โ Altered neurological development โ case study = hearing loss Note: TSH not involved
- 16. Office of Research and Development National Center for Computational Toxicology Thyroperoxidase NIS Hepatic Nuclear Xenoreceptors Deiodinases Thyroid Receptors Free T3 & T4 ๏จ TSH Tissue THChanges Thyroid Hyperplasia Thyroid Tumors Life Stage Specific Hearing Loss Adverse Outcomes T4 & T3 Synthesis Phase 2 Catabolism Cellular Transporters Cellular T4 ๏กT3 Conversion Biliary Elimination 54 2 Transport Proteins ? Thyroid Gland Pituitary TSH R TRH R Hypothalamus TR Blood CellularTR Signaling Altered Development Altered Metabolism Hypothalamic Pituitary Feedback Bound-TH 1 3 6 7
- 17. Office of Research and Development National Center for Computational Toxicology 16 Experimental Support for Linking MIE, Key Events, and Adverse Outcome Table 1: Key Events in the Animal AOP โ PHAH Example Key Event Experimental Evidence References 1. Activationof Hepatic Ah, CAR and PXR pathways YES: A lot of data demonstrating (In vivo and in vitro) evidence for activationby PHAHs Knockout models prevent subsequent key event (Safe, 1994; Schuetz et al., 1998; Honkakoski et al., 2003) 2. Induction of Phase II Catabolism YES: Hepatic NR agonists induce hepatic UGTs (Ahotupa et al., 1978; Ganem etal., 1999; Oppenheimer et al., 1968; McClainet al.,1989; Hood and Klaassen, 2000) 3. BiliaryElimination YES: Many PHAHs increase biliary eliminationof conjugated T4. (Batomsky, 1974; Beestra et al., 1991; Vansell and Klaassen, 2001; 2002) 4. Developmental Hypothyroxinemia YES:In vivo decreases in thyroxine during early critical postnatal period; thyroxine replacement ameliorates functional loss โ many chemicals and many MIEs do this T4 Therapy reverses this effect (Morse et al., 1993; Morse et al., 1996; Goldey et al., 1995a; Goldey& Crofton, 1998;Crofton et al., 2000a.) 5. Decreased Target Tissue TH Concentrations No Data: No data available for effects of PHAHss on cochlear T3. Limited data suggest induction of Type II deiodinases prevents decreases in fetal cortical T3 levels PCB exposure. (lots of evidencefromother THD Chemicals) (Morse et al., 1996) 6. Decreases in TR Regulated Cochlear Proteins No Data: No evidence in cochlea. Data gap for PHAHs 7. Structural Damage in the Cochlea YES: In vivo evidence of missing hair cells in apical turns of cochlea (also evidence fromother TH Chemicals) (Crofton et al., 2000b) 8. Loss of cochlear function YES: Loss of low-frequency hearing using behavioral audiometry, brain stem auditory evoked potentials and otoacoustic emissions. (lots of evidencefromother THD Chemicals) (Goldey et al., 1995; Crofton et al., 2000b; Herr et al., 1996; Laskey et al., 2002)
- 18. Office of Research and Development National Center for Computational Toxicology Example of Temporal relationship (lifestage) Critical-Period Model for Highly Lipophilic Chemicals Exposure and Key Event Align Temporally with Critical Window โข Exposure occurs mainly postnatally due to lactational transfer โข Impact on thyroid hormones in mostly postnatal โข Therefore, impact on developing cochlea occurs during low-frequency development BirthConception Weaning ThyroidHormone Fetal/PostnatalExposure CochlearDevelopment High - Frequency - Low Low-frequency Hearing loss
- 19. Office of Research and Development National Center for Computational Toxicology 18 Example of Correlative relationship between Key Events Serum Thyroxine on PND14 Predicts Low-Frequency Hearing Loss in Adults Data from multiple developmental studies over a ten year with various Chemicals - Propylthiouracil - PCBs - Dioxins - Brominated flame retardants
- 20. Office of Research and Development National Center for Computational Toxicology Identification of Data Gaps & Cross Species Extrapolation โข Construction of an AOP should identify data gaps i.e., critical needs to build a useful model โข Identification of missing empirical data for some key events โข Lack of identification of MIEs โ some AOPs start with key events โข Filling gaps should reduce uncertainties in decisions โข Enhance species to species extrapolation โข Compare chemical effects on species specific MIEs or key events Triclosan as an example 19
- 21. Office of Research and Development National Center for Computational Toxicology Molecular Initiating Event - Cross Species Triclosan โข Regulatory Driver: In vivo rodent studies demonstrate that triclosan decreases thyroid hormones and increases hepatic enzyme important for T4 regulation in rats โ Uncertainty: Does it do so in humans? No data = assumption yes โข AOP is well accepted for many drugs and environmental chemicals โ Evidence that MIE in humans is activation of PXR ( Jacobs et al., 2005) โข Hypothesis: MIE is activation of hepatic PXR 20 Activate Hepatic PXR ๏ Hepatic Phase II/III ๏ T4 Clearance Decreased Serum T4 Adverse Neurodevelopment Outcomes (IQ) MIE Cellular Response Organ Response Individual Response Tissue Response Activate Hepatic PXR ๏ Hepatic Phase I, II, III ๏ T4 Clearance Decreased Serum T4 Adverse Neurodevelopment Outcomes (IQ) Rat Human ๏ผ ๏ผ* *? ๏ผ ? * Empirical evidence Inferred from other chemicals or stressors Data gap ? *
- 22. Office of Research and Development National Center for Computational Toxicology Molecular Initiating Event - Cross Species Triclosan โข Hypothesis: MIE is activation of hepatic PXR 21 Activate Hepatic PXR ๏ Hepatic Phase I, II, III ๏ T4 Clearance Decreased Serum T4 Adverse Neurodevelopment Outcomes (IQ) MIE Cellular Response Organ Response Individual Response Tissue Response In vitro: TCS activates human PXR but not rat PXR Treatment concentration (ยตM) 0.1 1 10 100 %MaximalDEXInductionยฑSE 0 20 40 60 80 100 120 Dexamethasone (DEX) Triclosan (TCS) Treatment concentration (ยตM) 0.01 0.1 1 10 100 %MaximalRIFInductionยฑSE 0 20 40 60 80 100 120 Rifampicin (RIF) Triclosan (TCS) hPXR rPXR Paul et al., submitted
- 23. Office of Research and Development National Center for Computational Toxicology Triclosan Species Extrapolation - Remaining Uncertainty 22 โข Differential effects of triclosan on hepatic MIEs โข Human โ both hCAR and HPXR activated in vitro โข Rat โ only rCAR activated in vitro โข Are humans more sensitive? Unknownโฆ. Use in vitro cell cultures to check rCAR โserum T4 โ biliary elimination of THs Weak hCAR1&3 + hPXR โserum T4 โ hepatic Phase I & II mRNA & protein โ biliary elimination of THs Rat Human โ ? โ โโ hepatic Phase I & II mRNA & protein โ ? Paul et al., 2013
- 24. Office of Research and Development National Center for Computational Toxicology AOP and High-Throughput Screening Whatโs the Promise? Premise: The AOP is well developed and based on empirically established links between the MIE and the Adverse Health and Community Outcomes Promise: Upstream MIEs and Key Events can be used in lieu of costly in vivo testing โข Health predictions can be done based on upstream events โ e.g., serum T4 during fetal development is a predictor of IQ in children, and TPO inhibition is correlated with serum hormones โ Already being done in regulatory arena โข Can be used to prioritize follow up testing needs โ Data from high-throughput methods can represent a true โfirst-tierโ screen for the thousands of chemicals currently lacking data 23
- 25. Office of Research and Development National Center for Computational Toxicology Thyroperoxidase NIS Hepatic Nuclear Xenoreceptors Deiodinases Thyroid Receptors Free T3 & T4 ๏จ TSH Tissue THChanges Thyroid Hyperplasia Thyroid Tumors Adverse Outcomes T4 & T3 Synthesis Phase 2 Catabolism Cellular Transporters Cellular T4 ๏กT3 Conversion Biliary Elimination 4 9 35 6 7 8 Transport Proteins 10 Thyroid Gland Pituitary TSH R TRH R Hypothalamus TR Blood CellularTR Signaling Altered Development Altered Metabolism 3 2 1 Hypothalamic Pituitary Feedback Bound-TH MIEs as Molecular Screening Targets Developmental Neurotoxicty
- 26. Office of Research and Development National Center for Computational Toxicology 25 Screening Technology Target Existing Adaptable R & D Comments TSH Receptor Signaling X TRH Receptor Signally X NIS Mediated Iodine uptake X No commercial source TPO inhibition X ORD method under development Secretion from Thyroid Gland X Transport proteins X Two methods, no commercial source Deiodination X Sulfation and Glucuronidation X Low-med throughput Nuclear Receptors X Already done for >1000 chemicals TH Membrane Transporters X TR binding and transcription X Already done for >>8500 chemicals Screening for THDs โ State of the Science* *adapted from Murk et al., 2013
- 27. Office of Research and Development National Center for Computational Toxicology Percentinhibition โข 21-chemical training set โข Automated 384-wp testing format โข Automated data work flow for analyses (R.version 2.15.1) Paul et al., 2013
- 28. Office of Research and Development National Center for Computational Toxicology Challenges for Development and Use of AOPs โข Thyroid system disruptions are not the adverse outcome pathways โข For Example: What are all the MIEs and Pathways for environmental contaminants that cause developmental neurotoxicity? โ Linking MIEs to adverse outcomes can be very difficult for some DNT outcomes โข e.g., sodium channel disruption induced behavioral effects (pyrethroids) โ Lack of known pathobiology of outcomes can makes this very difficult โข where is the nucleus in the brain that is responsible for autismm or IQ loss, or ADHD (what are the MIEs that lead to IQ loss?) โข Development of quantitative predictive models can require a LOT of data โ How much of a change in the MIE is needed to overcome compensatory processes? (%change in serum T4 and changes in brain tissue T3? โข How can we make use of โpartialโ AOPs to reduce uncertainty in risk decisions? โข AOPs are not singular linear pathways, they are complex sets of interacting processes โ it only gets more complicated โ need to be prudent in deciding how much of a pathway is needed for a purpose 27
- 29. Office of Research and Development National Center for Computational Toxicology AOP Continuum Domains of Application Examples โข Simple correlative โข Links between inherent chemical properties and outcomes โข Qualitative โข Known links between key events and outcomes โข No complete set of key events โ contains correlative โleapsโ โข Causative โข Quantitative โข Predictive quantitative models ReadAcross Prioritizationand/or TargetedTesting LogP and fish mortality Neuronal-glia differentiation and developmental neurotoxicity QuantitativeRisk Assessments Quantitative models from MIE to health and community impacts Qualitative Thyroid/DNT AOP ToolboxCategories ModelDataNeeds ModelUncertainty JRC/EPA AOP Workgroup Meeting RTP 03/01/12
- 30. Office of Research and Development National Center for Computational Toxicology Announcement โ AOPWiki An AOPWiki is being developed and a beta version is being tested โ public release later this year โข Developed as a joint project between: โ OECD, EU Joint Research Center, Italy, US EPA, US Army Engineering Research and Development Center, Vicksburg MS โข Provides a โuser-friendlyโ interface for โcrowd sourcing the development of AOPs Link - www.aopwiki.org Contacts: Edwards.Stephen@epa.gov Clemens.Wittwehr@ec.europa.eu 29
- 31. Office of Research and Development National Center for Computational Toxicology Thanks for Listening 30