Long Term Goal 1: Neurodevelopment and Thyroid Homeostasis  Reproductive Function and EDCs Mary E. Gilbert Neurotoxicology...
Linkage and Timeline for APGs to Meet LTG 1: 2004 FY03 FY04 FY05 FY06 FY07 Characterize the  effects of exposure to  multi...
2013 -  Provide OPPTS, OW, the Regions and other organizations with  new exposure assessment and risk management tools to ...
<ul><li>Low Dose Effects, Developing Appropriate Animal Models </li></ul><ul><li>Evaluation of Mixtures of EDCs </li></ul>...
Brain Malformation Induced by Prenatal  Thyroid Hormone Insufficiency Goodman and Gilbert, Endocrinology, 2007 Only seen i...
Excitatory Synaptic Transmission  is Impaired in Offspring of PTU  and Perchlorate-Treated Dams Gilbert and Sui,  2006 Gil...
Cognitive Deficits  Assessed To Mirror  Health Outcomes of Concern in Humans  <ul><li>Subtle changes in IQ function are th...
Low Levels of TH Disruption  Impact Myelination CC Oligodendrocytes CC mRNA MAG ( Zoeller  et al, under review) Cell type ...
Dose-Dependent Reductions in Myelin Associated Genes at Low Doses of PTU Consistent with  in situ  hybridization, microarr...
Persistent Long-Term Functional  Consequences of Early Exposure to EDCs <ul><li>Permanent cognitive impairments and altere...
Effects of  in utero  Exposure to EDCs Transcend Generations <ul><li>Low sperm counts, reduced sperm motility, testes dysm...
Developing a “Toxicological  Science” of EDC Mixtures <ul><li>Dose Additivity – Default Risk Assessment Model for mixture ...
Binary Mixtures of Phthalates  Induce Dose-Additive Effects Howdeshell et al., Toxicological Sciences, 2007 Both phthalate...
<ul><li>Androgen Antagonists –  vinclozolin ,    procymidone,   phthalates   </li></ul><ul><li>Estrogens –  methoxychlor, ...
Thyroid Disrupting Chemicals: Structurally  Diverse, Multiple Sites, Multiple Mechanisms T4 - Gluc Biliary Excretion Free ...
Mixtures Of Thyroid Hormone Disruptors: Evolving Statistical Models to Predict Outcome Default Dose Additivity Model predi...
<ul><li>Mixtures of chemicals that alter gonadal or thyroid hormones via a  common MOA behave in a Dose-Additive manner   ...
Studies to Examine Extrapolation Across Species and Inform Screening Efforts <ul><li>Estrogen and Androgen Receptors Acros...
 
Aromatase Activity – MOA for EDCs in Rats and Fish?   A Case Study with Atrazine Goal:   Improve species extrapolation by ...
Biomarkers, Toxicogenomics,  and Screening Tools <ul><li>Biomarkers of Reproductive & Thyroid Dysfunction </li></ul><ul><u...
Using Toxicogenomics in Risk Assessment:  A Case Study with Dibutyl Phthalate   <ul><li>Increasing use of ‘omics technolog...
Future Work on Reproductive & Thyroid  EDCs <ul><li>Low Dose   </li></ul><ul><ul><li>Biomarkers of TH disruption and neuro...
The NHEERL Players … <ul><li>RTD Scientists </li></ul><ul><ul><li>Ralph Cooper </li></ul></ul><ul><ul><li>Earl Gray </li><...
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Long Term Goal 1: Neurodevelopment and Thyroid Homeostasis ...

  1. 1. Long Term Goal 1: Neurodevelopment and Thyroid Homeostasis Reproductive Function and EDCs Mary E. Gilbert Neurotoxicology Division National Health and Environmental Effects Research Laboratory Office of Research and Development U.S. Environmental Protection Agency September 14, 2007
  2. 2. Linkage and Timeline for APGs to Meet LTG 1: 2004 FY03 FY04 FY05 FY06 FY07 Characterize the effects of exposure to multiple EDCs in various combinations including those with similar & different mechanisms of action Evaluate at least 3 existing risk management tools to reduce exposure to EDCs Determine the shape of the dose - response curve in a variety of species exposed to ambient levels of EDCs Determine degree to which EDCs with defined mechanisms/modes of action can be extrapolated across classes of vertebrates LTG 1: Provide a Better Understanding of Science Underlying the Effects, Exposure, Assessment, and Management of Endocrine Disruptors Determine critical biological factors during development resulting in toxicities later in life FY08 FY09 FY10 FY11 FY12 Identify key risk assessment issues and develop guidance for assessing endocrine disruptors Develop at least 2 new risk manage- ment tools Identify risk management EDCs research Evaluate exposure methods, measurement protocols, and models for the assessment of risk management efficacy on EDCs Provide at least 1 computational model for assessing endocrine disruptor compounds Develop systems models to test & predict vulnerabilities of neuroendocrine system to contaminant- induced effects
  3. 3. 2013 - Provide OPPTS, OW, the Regions and other organizations with new exposure assessment and risk management tools to characterize and reduce exposure to EDCs 2013 - Provide OPPTS, OW, the Regions and other organizations with systems and models to test and predict vulnerability of the neuroendocrine system to contaminant-induced effects 2013 - Provide OPPTS, OW, the Regions and other organizations with data from the development and application of high-through put and molecular approaches, including ‘omics , and computational tools for defining mechanisms of action, extrapolation across species and improving assessments for EDCs 2010 - Provide OPPTS, OW, the Regions and other organizations with improved data on the shape of the dose-response curve as a result of exposure to environmentally relevant levels of endocrine disruptors 2009 2010 2011 2012 2013 2015 2014 LTG 1: Reduction in uncertainty regarding effects, exposure, assessment, and management of EDCs so that EPA has a sound scientific foundation for environmental decision making. Linkage & Timeline for APGs to Meet LTG 1: Redefined
  4. 4. <ul><li>Low Dose Effects, Developing Appropriate Animal Models </li></ul><ul><li>Evaluation of Mixtures of EDCs </li></ul><ul><li>Species Extrapolation </li></ul><ul><li>Toxicogenomics in Risk Assessment </li></ul><ul><li>Biomarkers and Screening Tools </li></ul>Examples of Research Addressing LTG1
  5. 5. Brain Malformation Induced by Prenatal Thyroid Hormone Insufficiency Goodman and Gilbert, Endocrinology, 2007 Only seen in TH- deficient offspring Cells are born late gestation ~ mid- pregnancy in humans Incidence and size are dose - dependent Cells are neuronal phenotype, not injury response - CON PTU <ul><li>Low Dose Effects/Animal Models </li></ul><ul><li>Mixtures of EDCs </li></ul><ul><li>Species Extrapolation </li></ul><ul><li>Toxicogenomics in RA </li></ul><ul><li>Biomarkers and Screening </li></ul>NEUN GFAP GD13-16 GD17-19
  6. 6. Excitatory Synaptic Transmission is Impaired in Offspring of PTU and Perchlorate-Treated Dams Gilbert and Sui, 2006 Gilbert and Sui, under review Dose-dependent reductions in T4 induced by PTU or perchlorate impair excitatory synaptic function in hippocampus of adult offspring – an area critical for learning. Correlations between level of hormone reduction and degree of impairment. <ul><li>Low Dose Effects/Animal Models </li></ul><ul><li>Mixtures of EDCs </li></ul><ul><li>Species Extrapolation </li></ul><ul><li>Toxicogenomics in RA </li></ul><ul><li>Biomarkers and Screening </li></ul>
  7. 7. Cognitive Deficits Assessed To Mirror Health Outcomes of Concern in Humans <ul><li>Subtle changes in IQ function are the most commonly reported impairments observed in children born to women with thyroid hormone insufficiencies. Extrapolation to humans from animal models is facilitated if common endpoints are assessed in both species. </li></ul><ul><li>Dose-dependent impairments detected in behavioral and electrophysiological indicators of “learning” in response to graded degrees of thyroid dysfunction </li></ul><ul><li>Low Dose Effects/Animal Models </li></ul><ul><li>Mixtures of EDCs </li></ul><ul><li>Species Extrapolation </li></ul><ul><li>Toxicogenomics in RA </li></ul><ul><li>Biomarkers and Screening </li></ul>
  8. 8. Low Levels of TH Disruption Impact Myelination CC Oligodendrocytes CC mRNA MAG ( Zoeller et al, under review) Cell type critical for myelination is reduced by low doses of PTU <ul><li>Low Dose Effects/Animal Models </li></ul><ul><li>Mixtures of EDCs </li></ul><ul><li>Species Extrapolation </li></ul><ul><li>Toxicogenomics in RA </li></ul><ul><li>Biomarkers and Screening </li></ul>(% of Control) *** ** 0ppm 1ppm 2ppm 3ppm 0 20 40 60 80 100 120 MAG mRNA Oligodendrocyte Density (Cells/mm 2 ) *** *** 0ppm 1ppm 2ppm 3ppm 0 200 400 600 Oligodendrocyte Number
  9. 9. Dose-Dependent Reductions in Myelin Associated Genes at Low Doses of PTU Consistent with in situ hybridization, microarrays show changes in expression of myelin genes in hippocampus of PN15 offspring are associated with low level TH insufficiencies. This approach may provide sensitive biomarkers of effect. <ul><li>Low Dose Effects/Animal Models </li></ul><ul><li>Mixtures of EDCs </li></ul><ul><li>Species Extrapolation </li></ul><ul><li>Toxicogenomics in RA </li></ul><ul><li>Biomarkers and Screening </li></ul>( Royland and Gilbert, in preparation)
  10. 10. Persistent Long-Term Functional Consequences of Early Exposure to EDCs <ul><li>Permanent cognitive impairments and altered synaptic function result from transient thyroid hormone insufficiencies </li></ul><ul><li>Morphological abnormalities resulting from transient exposure to AR antagonists delay puberty and reduce fertility </li></ul><ul><li>Transgenerational effects of androgen receptor antagonists </li></ul><ul><li>Low Dose Effects/Animal Models </li></ul><ul><li>Mixtures of EDCs </li></ul><ul><li>Species Extrapolation </li></ul><ul><li>Toxicogenomics in RA </li></ul><ul><li>Biomarkers and Screening </li></ul>
  11. 11. Effects of in utero Exposure to EDCs Transcend Generations <ul><li>Low sperm counts, reduced sperm motility, testes dysmorphology in F3 generation males to dosing of F0 females to low doses of vinclozilin or methoxychlor </li></ul><ul><li>Incidence and persistence across generations suggests epigenetic reprogramming of germ line </li></ul><ul><li>CAVEAT: Failure to replicate from two independent labs; number of animals this study small; efforts in RTD are ongoing with negative findings to date </li></ul>EPA STAR Grantee Anway et al., Science, 2005 <ul><li>Low Dose Effects/Animal Models </li></ul><ul><li>Mixtures of EDCs </li></ul><ul><li>Species Extrapolation </li></ul><ul><li>Toxicogenomics in RA </li></ul><ul><li>Biomarkers and Screening </li></ul>spermatazoa spermatocyte CON VIN
  12. 12. Developing a “Toxicological Science” of EDC Mixtures <ul><li>Dose Additivity – Default Risk Assessment Model for mixture of chemicals with common MOA. </li></ul><ul><li>Response Additivity – Default Risk Assessment Model for mixtures of chemicals with different MOA </li></ul><ul><li>Thyroid & Reproductive Mixture Studies Demonstrate: 1. Response Additivity underestimates risk 2. Dose Additivity is predictive for chemicals with same and different MOA </li></ul><ul><li>3. Next generation models encompass both? </li></ul><ul><li>Low Dose Effects/Animal Models </li></ul><ul><li>Mixtures of EDCs </li></ul><ul><li>Species Extrapolation </li></ul><ul><li>Toxicogenomics in RA </li></ul><ul><li>Biomarkers and Screening </li></ul>
  13. 13. Binary Mixtures of Phthalates Induce Dose-Additive Effects Howdeshell et al., Toxicological Sciences, 2007 Both phthalates reduce testicular hormone production and expression of genes critical for steroidogenesis. <ul><li>Low Dose Effects/Animal Models </li></ul><ul><li>Mixtures of EDCs </li></ul><ul><li>Species Extrapolation </li></ul><ul><li>Toxicogenomics in RA </li></ul><ul><li>Biomarkers and Screening </li></ul>Several androgen-dependent endpoints of male reproductive tract development interact in dose-additive manner as predicted by common mechanism of toxicity during sexual differentiation. Ano-genital Distance is Reduced in Dose-Additive Manner Con DBP DEHP DBP+DEHP Fetal Testicular Insl3 mRNA Con DBP DEHP DBP+DEHP Fetal Testicular Testosterone
  14. 14. <ul><li>Androgen Antagonists – vinclozolin , procymidone, phthalates </li></ul><ul><li>Estrogens – methoxychlor, </li></ul><ul><li> bisphenol A </li></ul><ul><li>Androgens- trenbolone </li></ul><ul><li>Fetal androgen synthesis inhibitors - linuron </li></ul><ul><li>Fetal Germ Cell Toxicants- busulfan </li></ul><ul><li>Steroidogenesis Inhibitors - prochloraz </li></ul>Reproductive Toxicants: Multiple Mechanisms, Different Fetal Targets CONCLUDE : Extensive dose-response information, especially low dose levels, is needed to appropriately design and interpret Mixture Studies <ul><li>Low Dose Effects </li></ul><ul><li>Mixtures of EDCs </li></ul><ul><li>Species Extrapolation </li></ul><ul><li>Toxicogenomics in RA </li></ul><ul><li>Biomarkers and Screening </li></ul>Default: Response-Additive Model Observed: Dose-Additive Responses
  15. 15. Thyroid Disrupting Chemicals: Structurally Diverse, Multiple Sites, Multiple Mechanisms T4 - Gluc Biliary Excretion Free - TH Bound - TH T4 TTR/TBG Hypothalamus Pituitary Thyroid Gland TRH TSH + + T3 & T4 T3 & T4 Deiodinase T4 > T3 Ah - Receptor T4 UDPGTs CAR/PXR Iodine Perchlorate Thiocyanate T4 T3 Thyroperoxidase I + tyrosine T3 & T4 HO - PCBs Dioxins PBDEs _ _ Transporters Transporters Liver T4 - Gluc Biliary Excretion Plasma/Blood Free - TH Bound - TH T4 TTR/TBG Hypothalamus Pituitary Thyroid Gland TRH TSH + + T3 & T4 T3 & T4 T4 > T3 Ah - Receptor T4 UDPGTs CAR/PXR Iodine Perchlorate Thiocyanate T4 T3 Thyroperoxidase I + tyrosine T3 & T4 PTU MMI Mancozeb Pronamide Thiram HO - PCBs Dioxins PBDEs PCBs Peripheral Tissues _ _ Transporters Transporters Hepatic Target Thyroid Target <ul><li>Low Dose Effects </li></ul><ul><li>Mixtures of EDCs </li></ul><ul><li>Species Extrapolation </li></ul><ul><li>Toxicogenomics in RA </li></ul><ul><li>Biomarkers and Screening </li></ul>
  16. 16. Mixtures Of Thyroid Hormone Disruptors: Evolving Statistical Models to Predict Outcome Default Dose Additivity Model predicts effects on T4 at environmental levels of exposure. (Flippin et al., under review) Hepatic Target – Similar MOA Hepatic + Thyroid Targets – Different MOA Combined Mixture Model accurately predicts effects of mixtures with components with different MOA. Predicted Empirical (Crofton et al., 2005) Default Dose Additivity Model Combined Dose + Effect Additivity Model 18 PHAHs 18 PHAHs + 3 Pesticides R mixture = [UGT inducers] Dose Addition
  17. 17. <ul><li>Mixtures of chemicals that alter gonadal or thyroid hormones via a common MOA behave in a Dose-Additive manner </li></ul><ul><li>Regardless of the molecular MOA , c hemicals that disrupt sexual differentiation produce dose-additive responses,– i.e., need to think of common pathways of toxicity </li></ul><ul><li>Unlike carcinogenesis risk assessments, Response-Additivity Models do not fit the data , combined mixture models may prove to be the most predictive </li></ul><ul><li>Knowledge about the precise mechanism of toxicity is not necessary to predict the interactions, but extensive dose-response data are needed for each chemical </li></ul>Dose-Additivity Model Best Fits the Data and Most Consistent with Biology of Hormonal Action
  18. 18. Studies to Examine Extrapolation Across Species and Inform Screening Efforts <ul><li>Estrogen and Androgen Receptors Across Species </li></ul><ul><li>Aromatase Activity – MOA for EDCs in Rats and Fish? </li></ul><ul><li>Low Dose Effects/Animal Models </li></ul><ul><li>Mixtures of EDCs </li></ul><ul><li>Species Extrapolation </li></ul><ul><li>Toxicogenomics in RA </li></ul><ul><li>Biomarkers and Screening </li></ul>
  19. 20. Aromatase Activity – MOA for EDCs in Rats and Fish? A Case Study with Atrazine Goal: Improve species extrapolation by understanding cellular events leading to altered aromatase activity/gene expression in different target tissues & species. Reduced reproductive success with increase in brain aromatase at the lowest dose. Atrazine is major component in plasma. Egg Production Decreased Increase Brain Aromatase Atrazine metabolites are primary components in rat plasma Atrazine is primary component in fish plasma Differential Metabolism CONCLUDE: Induction of aromatase is not the primary MOA for atrazine-induced toxicity in rat or fish. Differential metabolism may underlie species- & tissue- specific effects. Increased serum estrogens are not caused by activation of aromatase or increased cyp19 gene expression in brain or testes. Major metabolites present in plasma. cyp19 Proposed MOA Atrazine Serum Estrogen Increased No Change in Aromatase Testes Aromatase Activity
  20. 21. Biomarkers, Toxicogenomics, and Screening Tools <ul><li>Biomarkers of Reproductive & Thyroid Dysfunction </li></ul><ul><ul><li>Biomarkers of Thyroid Dysfunction </li></ul></ul><ul><ul><li>Toxicogenomics in Risk Assessment </li></ul></ul><ul><ul><li>Proteomics as Bioindicators of Reproductive Toxicity </li></ul></ul><ul><li>Amphibian Model for Thyroid Hormone Disruptor </li></ul><ul><ul><li>Development of alternative biochemical and molecular screens for EDCs acting on thyroid axis </li></ul></ul><ul><li>Low Dose Effects/Animal Models </li></ul><ul><li>Mixtures of EDCs </li></ul><ul><li>Species Extrapolation </li></ul><ul><li>Toxicogenomics in RA </li></ul><ul><li>Biomarkers and Screening </li></ul>
  21. 22. Using Toxicogenomics in Risk Assessment: A Case Study with Dibutyl Phthalate <ul><li>Increasing use of ‘omics technology: </li></ul><ul><ul><li>How can this type of data be used in risk assessment? </li></ul></ul><ul><ul><ul><li>What are its limitations? </li></ul></ul></ul><ul><ul><ul><ul><li>How best can this info be interfaced with toxicity data? </li></ul></ul></ul></ul><ul><li>Recommendations for Toxicogenomics Studies: </li></ul><ul><ul><li>Parallel genomic and toxicity study design characteristics (e.g., dose, timing of exposure, tissues) </li></ul></ul><ul><ul><li>Time-course data over critical window of exposure for endpoints of interest </li></ul></ul><ul><ul><li>Increase # samples and replicates to improve study power and to permit pathway analysis. </li></ul></ul><ul><ul><li>Incorporate multiple doses and low doses to address dose-response </li></ul></ul>
  22. 23. Future Work on Reproductive & Thyroid EDCs <ul><li>Low Dose </li></ul><ul><ul><li>Biomarkers of TH disruption and neurodevelopmental outcomes </li></ul></ul><ul><ul><ul><li>Quantitative BBDR for thyroid disruption in fetus and neonate </li></ul></ul></ul><ul><ul><ul><ul><li>Reproductive and thyroid toxicity to inform mixtures studies </li></ul></ul></ul></ul><ul><li>Mixtures </li></ul><ul><ul><li>Multiple chemicals with different MOA, critical developmental periods, evaluate both genders, improved statistical models </li></ul></ul><ul><li>Extrapolation </li></ul><ul><ul><li>Animal models of human neurodevelopmental outcomes </li></ul></ul><ul><ul><ul><ul><li>AR/ER receptors – Expand chemicals, species, receptors </li></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Aromatase - ADME, low dose, brain as site of action? </li></ul></ul></ul></ul></ul><ul><li>Biomarkers </li></ul><ul><ul><li>Identify biomarkers of TH disruption, incorporate proteomics into toxicogenomic profiles of reproductive toxicants </li></ul></ul><ul><li>Screening </li></ul><ul><ul><li>AR and ER binding assays across multiple species </li></ul></ul><ul><ul><ul><li>Amphibian and mammalian models for thyroid hormone disruption </li></ul></ul></ul>
  23. 24. The NHEERL Players … <ul><li>RTD Scientists </li></ul><ul><ul><li>Ralph Cooper </li></ul></ul><ul><ul><li>Earl Gray </li></ul></ul><ul><ul><li>Phillip Hartig </li></ul></ul><ul><ul><li>Gary Klinefelter </li></ul></ul><ul><ul><li>Susan Laws </li></ul></ul><ul><ul><li>Tammy Stoker </li></ul></ul><ul><ul><li>Vickie Wilson </li></ul></ul><ul><li>NTD Scientists </li></ul><ul><ul><li>Kevin Crofton </li></ul></ul><ul><ul><li>Mary Gilbert </li></ul></ul><ul><li>MED Scientists </li></ul><ul><ul><li>Sig Diegtz </li></ul></ul><ul><ul><li>Mike Hornung </li></ul></ul><ul><ul><li>Joseph Tietge </li></ul></ul><ul><li>AED Scientists </li></ul><ul><ul><li>Lesley Mills </li></ul></ul><ul><li>ETD Scientists </li></ul><ul><ul><li>Mike DeVito </li></ul></ul>Elaine Francis, National Program Director Doug Wolf, Assistant Laboratory Director

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