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2009 Crofton DNT Thyroid Disruptors NBTS Meeting
- 1. Developmental Neurotoxicity and Thyroid Hormone Disruptors: Past, Present and Future Kevin M. Crofton, PhD Systems Biology Branch Integrated Systems Toxicology Division National Health and Environmental Effects Research Laboratory Office of Research and Development Research Triangle Park, NC June 28, 2009 Neurobehavioral Teratology Society Roi Grande PR The views expressed in this presentation are those of the presenter and do not necessarily reflect the views or policies of the U.S. EPA
- 2. Developmental Neurotoxicity and Thyroid Hormone Disruptors: Past, Present and Future Kevin M. Crofton, PhD Systems Biology Branch Integrated Systems Toxicology Division National Health and Environmental Effects Research Laboratory Office of Research and Development Research Triangle Park, NC Wadsworth Center, Albany NY May 14, 2009 Mode-of-action, Source to Outcome, Toxicity Pathway: No Matter What You Call It Behavioral Effects Are Critical and Behavioral Toxicologists are Needed
- 3. Outline • Mode-of-Action: What is it and what is it good for • MOA Examples: Thyroid hormone disruption, thyroid cancer and developmental neurotoxicity Thyroid 101: Hormones, homeostasis and signaling Sites-of-action for TDCs • Relevancy of animal data to humans • How can MOA help identify research needs Developmental neurotoxicity of TDCs Acute neurotoxicity of pyrethroids • How can thinking of MOA help with screening for toxicity
- 4. What is a Mode-of-Action? • Mode of action is defined as the sequence of key cellular and biochemical events (measurable parameters) that result in a adverse health outcome. • Critical to MOAs are causative relationships, not just correlations (Bradford Hill’s criteria for etiologic significance in epidemiologic investigations of causality)
- 5. Mode of ActionMode of Action Exposure-Dose-Response, Toxicity Pathway,Exposure-Dose-Response, Toxicity Pathway, Adverse Outcome PathwayAdverse Outcome Pathway Mode-of-action Framework by: Schulte, Environ Res. 48:129-44 1989 Kinetics How does it get to where you don’t want it to be? • uptake • distribution • metabolism • elimination Dynamics What does it do when it gets there? • initial key target • consequent effects at molecular, cellular, organismal levels • adverse outcomes
- 7. TDCs - Multiple Targets T4 -Gluc T4 TTR/TBG Hypothalamus TRH TSH + T3 & T4 T3 & T4 T4 > T3 Ah-Receptor T4 UDPGTs CAR/PXR T4 T3 Thyroperoxidase I + tyrosine T3 & T4 _ _ Liver T4 -Gluc Biliary Excretion Blood Free- TH Bound- TH- Hypothalamus Pituitary Thyroid + + Ah-Receptor T4 CAR/PXR Iodine Perchlorate Thiocyanate T3 I + tyrosine T3 & T4 PTU Mancozeb Pronamide PTU Octyl-methoxycinnamate HO-PCBs EMD 49209 Dioxins PBDEs PCBs Peripheral Tissues _ _ Deiodinases TH TTR/TBG UDPGTs Transporters NISNISNISNIS Thyroperoxidase Transporters PBDEs
- 8. CoR TH TH CoA AAAAA TRE TRTR TDCs – Multiple Targets Thyroid Hormone Receptors TRE TR TR CoR X BPA Ho-PCB CoA
- 9. Thyroid Hyperplasia Rat Thyroid Tumors Health Risk Serum T4 & T3 Modes of Action - ExamplesModes of Action - Examples Cancer and Neurodevelopment in RatsCancer and Neurodevelopment in Rats Altered Neuroanatomy Developmental Defects Health Risk Thyroperoxidase Iodine Symporter Exposure Hepatic UDPGTs Thyroidal Extra-Thyroidal Deiodinases Thyroid Receptors ↓ T4–TTR Binding TSH Mode-of-action Framework by: Schulte, Environ Res. 48:129-44 1989
- 10. MOA or Pathway “Thinking” What is it good for? • Transparent process to characterize the weight of evidence to support an animal MOA • Allows a determination of whether there is any evidence to reasonably exclude the same MOA in humans e.g., humans lack an enzyme that generates a toxic metabolite • Importantly, the process of working through this analysis allows for identification of data needs = focused research • Knowledge of key events allows identification of target that can be used in screening chemicals Adapted from Cohen 2009
- 11. MOA Framework (IPCS, EPA, Health Canada) Boobis et al (2008) Do you have an MOA in animals and is it relevant to humans? YES NO NO YESMOA not Relevant YESMOA not Relevant NO Proceed with risk assessment Proceed with risk assessment Is the weight of evidence sufficient to establish a mode of action (MOA) in animals? Can human relevance be reasonably excluded on the basis of qualitative differences between animals and humans? Can human relevance be reasonably excluded on the basis of quantitative differences between animals and humans?
- 12. 1212 Thyroid Hyperplasia Rat Thyroid Tumors Health Risk Serum T4 & T3 Modes of Action for TDCs - RatModes of Action for TDCs - Rat Altered Neuro- Development Developmental Defects Health Risk Thyroperoxidase Iodine Symporter Exposure Hepatic UDPGTs Thyroidal Extra-Thyroidal Deiodinases Thyroid Receptors ↓ T4–TTR Binding TSH Mode-of-action Framework by: Schulte, Environ Res. 48:129-44 1989 Do these pathways apply to humans?
- 13. 1313 Thyroid Hyperplasia Rat Thyroid Tumors Health Risk Serum T4 & T3 Modes of Action for TDCs - RatModes of Action for TDCs - Rat Altered Neuro- Development Developmental Defects Health Risk Thyroperoxidase Iodine Symporter Exposure Hepatic UDPGTs Thyroidal Extra-Thyroidal Deiodinases Thyroid Receptors ↓ T4–TTR Binding TSH Mode-of-action Framework by: Schulte, Environ Res. 48:129-44 1989 Do these pathways apply to humans?
- 14. 1414 Thyroid Hyperplasia Rat Thyroid Tumors Health Risk Serum T4 & T3 Modes of Action for TDCs – HumanModes of Action for TDCs – Human Example CarbamazepineExample Carbamazepine Altered Neuro- Development Developmental Defects Health Risk Thyroperoxidase Iodine Symporter Exposure Hepatic UDPGTs Thyroidal Extra-Thyroidal Deiodinases Thyroid Receptors ↓ T4–TTR Binding TSH Mode-of-action Framework by: Schulte, Environ Res. 48:129-44 1989 = decrease; = no effect Effects of carbamazepine on thyroid function in humans = decrease; = no effect Effects of carbamazepine on thyroid function in humans = decrease; = no effect Effects of carbamazepine on thyroid function in humans
- 15. 1515 No Thyroid Hyperplasia No Thyroid Tumors No Health Risk Serum T4 & T3 MOA Thyroid Tumors – HumanMOA Thyroid Tumors – Human Example - CarbamazepineExample - Carbamazepine Altered Neuro- Development Developmental Defects Health Risk Thyroperoxidase Iodine Symporter Exposure Hepatic UDPGTs Thyroidal Extra-Thyroidal Deiodinases Thyroid Receptors ↓ T4–TTR Binding No TSH Mode-of-action Framework by: Schulte, Environ Res. 48:129-44 1989
- 16. Is the Developmental MOA in Rats Relevant to Humans? YES! • Normal thyroid hormone levels and receptor function are CRITICAL for normal development of the structure and function of the brain in both rats and humans • Humans: • Over 100 years of data for iodine deficiency • Congenital hypothyroidism • Animals: • Lots of analogous animal models
- 17. 1717 Serum T4 & T3 MOA Thyroid Tumors – HumanMOA Thyroid Tumors – Human Example – Iodine DeficiencyExample – Iodine Deficiency Altered Neuro- Development Developmental Defects Health Risk Thyroperoxidase Iodine Symporter Dietary Iodine Hepatic UDPGTs Thyroidal Extra-Thyroidal Deiodinases Thyroid Receptors ↓ T4–TTR Binding Mode-of-action Framework by: Schulte, Environ Res. 48:129-44 1989 “High dose” = Cretinism “Low dose” = IQ losss
- 18. Research Needs How MOA ‘thinking” can focus research 1. One if MOA in animals is judged to be incomplete where are the data gaps Example pyrethroid acute neurotoxicity 1. In Human relevancy analysis – where are the data gaps • Lack of data translates to assumed relevance. • Example interspecies sensitivity to TDCs 1. Screening for prioritization If you know the molecular target – then develop low-cost and high-throughput methods for that target
- 19. MOA Ex #1 - Pyrethroids Exposure Absorbed Dose Target Tissue Dose Binding Molecular Target In Vitro Human Neurons Organ Level Effect Adverse Clinical Outcome Exposure Absorbed Dose Target Tissue Dose Molecular Target In Vitro Rat Neuron Firing Rate Organ Level Effect Adverse Clinical Outcome Human Exposure & Kinetics Modeling Effects Modeling Too many unknowns for to call this an established MOA
- 20. Binding Molecular Target In Vitro Human Neurons Organ Level Effect Adverse Clinical Outcome Molecular Target In Vitro Rat Neuron Firing Rate Organ Level Effect Adverse Clinical Outcome Human Na Channel work almost all in vitro Need a good in vivo biomarker Mostly in vitro firing rates Need in vivo data Rat MOA for Acute Pyrethroid Neurotoxicity? If key initiating event is changes in VGSC kinetics, then how does this translate into changes in behavior?? Available biochem and physiology data not correlative Behavior data from high dose studies Use in vitro models to compare rat and human cell lines
- 21. 100 25 0 0 25 50 75 100 125 0 25 50 75 100 125 66 50 TH Concentration Dysfunction (%Control) 100 25 0 0 25 50 75 100 125 0 25 50 75 100 125 66 50 TH Concentration Dysfunction (%Control) Rat Human MOA Ex. #2 – Interspecies Sensitivity 100 25 0 0 25 50 75 100 125 0 25 50 75 100 125 66 50 TH Concentrations Dysfunction (%Control) Most experimental “sledgehammer” models Likely human environmental exposures Data Need: Comparative dose-response data for rats and humans
- 22. 2222 Humans dose response data is very limited Haddow et al. (1999) Even low level maternal hypothyroxinemia (second trimester) leads to adverse outcomes (~25% T4 leads to IQ loss) Animal dose response data: more, but limited Comparative Sensitivity – Rat and Human Crofton (2004) Gilbert, unpublished Synaptic plasticity vs T4Hearing vs T4 – 50% loss What amount of change is adverse? 50% during critical window ??
- 23. MOA Ex #3 - Screening • Chemicals with info on thyroid endpoint is less than 200 • Chemicals with potential for exposure and lacking any assessments has been estimated to be around ~10,000 • Regardless of all the other uncertainties, this is a high priority We cannot test our way out of this problem starting with rodent studies, especially not using developmental exposures
- 24. Developing a New Way of Doing Business + If you know the key initiating event (or events) then develop efficient screens for this target + Use these screen to prioritize for further testing not for regulation + Start with 500 or 600 chemicals – identify the ‘worst actors” – and then test them in a tiered testing strategy + e.g., in vitro TPO inhibition assay, then test in 96 well Xenopus assay, then in mammals, higher to medium to lower throughput
- 25. Ongoing Methods Development If you know the key initiating event (or events) – then develop efficient screens 1) Receptor based screening at EPA’s NCCT e.g. PXR, AhR, PPAR, TRbeta recently completed screening of 320 pesticidal chemicals 2) TPO screening 96 well medium throughput assay for inhibition of porcine TPO (Mike Hornung at ORD’s Duluth Lab) 3) TH signaling in transgenic X. laevis 96-well format for detecting alterations in TR signaling (Barbara Demeneix - Environ. Sci. Technol., 2007, 41:5908–5914) 4) OECD/IPCS Advisory Group – Collating & summarizing available assays A NEGATIVE IN THESE SCREENS DOES NOT TRANSLATE TO NO HAZARD!
- 26. Screening for Thyroid Disruptors Current Approaches Standard approach • Guidelines • ‘Triggered’ testing • Low throughput New Tiered Approach Screening for Prioritization HTP in vitro/QSAR L-MTP - alt species? Targeted mammalian testing
- 27. Paradigm Shift for Tox Testing New Tiered Approach Screening for Prioritization HTP in vitro L- MTP alt species Targeted mammalian testing 1,000s 100s 10s Screening for Prioritization Testing for Hazard Assessments
- 28. Summary • MOA Approach helps to focus research • Development of MOAs requires adverse outcome data – without it the MOA is neutered. • A number of thyroid pathways are known that if disturbed during developmental result in neurological deficiencys Multiple targets involved In vitro models are available for many of the targets • Need to develop testing methods that can be used for Screening for Prioritization Also needed --- tiered testing strategies • For this strategy to work the input and research from behavioral folks is critical!
- 29. Thank you for listening
Editor's Notes
- So how can PCBs alter transcription if they do not “bind” to the TR? The first posibility isthat PCBs may inhibit the corepressor/thyroid hormone receptor interaction which would lead to derepression without activation. PCBs may enhance the coactivator/ thyroid hormone receptor interaction which would increase transcriptional activation PCBs may interfere with the thyriod hormone response element/thyroid hormone receptor interaction which would produce a result similar to derepression without activation