Draft Detailed Review Paper State of the Science on Novel In Vitro and In Vivo Screening and Testing Methods and Endpoints for Evaluating Endocrine Disruptors Contractor: RTI International 3040 Cornwallis Road P.O. BOX 12194 Research Triangle Park, NC 27709 Draft #1 July 2011 Authors Gerald A. LeBlanc, Ph.D. Seth W. Kullman, Ph.D. Department of Environmental and Department of Environmental and Molecular Toxicology Molecular Toxicology North Carolina State University North Carolina State University Raleigh, NC Raleigh, NC David O. Norris, Ph.D. William S. Baldwin, Ph.D. Department of Integrative Physiology Department of Biological Sciences and University of Colorado Environmental Toxicology Boulder, CO Clemson University Clemson, SC Werner Kloas, Ph.D. Department of Ecophysiology and John M. Greally, Ph.D. Aquaculture IGB Department of Genetics Endocrinology at Humboldt University Albert Einstein College of Medicine Germany Bronx, NY Screening and Testing Methods and Endpoints for Evaluating Endocrine Disruptors Table of Contents 1. Introduction ......................................................................................................................................... 1-5 2. The Corticotropin-Releasing Hormone/ Vasopressin:ACTH:Glucocorticoid Signaling Pathway ............................................................................................................................................... 2-1 2.1 The HPA Axis: Hypothalamic Pituitary Adrenocortical .......................................................... 2-1 2.1.1 Corticotropin-releasing Hormone (CRH) ..................................................................... 2-2 2.1.2 Arginine Vasopressin (AVP)/Arginine Vasotocin (AVT)............................................ 2-2 2.1.3 Corticotropin (ACTH) .................................................................................................. 2-3 2.1.4 Luteinizing Hormone (LH) and Chorionic Gonadotropin (CG) ................................... 2-3 2.1.5 Glucocorticoid (GCs) ................................................................................................... 2-3 2.1.5.1 Glucocorticoid Receptors (GRs) .................................................................. 2-3 2.1.5.2 CRH and Glucocorticoid-binding Protein (transcortinprotein and glucocorticoid-binding protein [transcortin]) ............................................... 2-4 2.1.6. Neuroendocrine Regulation of the HPA Axis .............................................................. 2-4 2.2 Potential Effects of EDCs on the HPA Axis ............................................................................ 2-4 2.3 Precedent Chemicals as Potential Disruptors of the HPA Axis................................................ 2-5 2.3.1 Steroid Synthesis and Receptor Agonists and Antagonists .......................................... 2-5 2.3.2 Metals ........................................................................................................................... 2-5 2.3.3 Neuroactive Chemicals ................................................................................................. 2-6 2.3.4 Vasopressin Receptor Agonists and Antagonists ......................................................... 2-6 2.3.5 CRH Receptor Antagonists .......................................................................................... 2-6 2.3.6 Pesticides ...................................................................................................................... 2-6 2.3.7 Arylhydrocarbon Receptor (AhR) Agonists ................................................................. 2-6 2.3.8 Estrogenic and Androgenic Disruptors on the HPA Axis ............................................ 2-7 2.4 In-vitro Screening Assays ......................................................................................................... 2-7 2.4.1 Computerized Receptor Assay ..................................................................................... 2-7 2.4.2 Competitive-binding Receptor Assays ......................................................................... 2-7 2.4.2.1 Cell Culture Systems .................................................................................... 2-7 2.4.3 Gene-activation Assays ................................................................................................ 2-8 2.4.4 In-vivo Screening Assays ............................................................................................. 2-8 2.4.4.1 Fish ............................................................................................................... 2-9 2.4.4.2 Amphibians .................................................................................................. 2-9 2.4.5 Challenges and Limitations .......................................................................................... 2-9 2.4.5.1 In-vitro vs In-vivo Assays ............................................................................ 2-9 2.4.5.2 Mixtures vs. Single Chemicals ................................................................... 2-10 2.4.5.3 Other Parameters to Be Considered ........................................................... 2-10 3. The Somatotropic Axis ........................................................................................................................ 3-1 3.1 Overview .................................................................................................................................. 3-1 3.2 Consequences of Disruption by Environmental Chemicals ..................................................... 3-3 3.2.1 Estrogenic Chemicals ................................................................................................... 3-3 3.2.2 Anti-thyroid Chemicals ................................................................................................ 3-3 3.2.3 Corticosteroid Stimulants ............................................................................................. 3-3 3.3 Chemicals that Directly Disrupt the Somatotropic Axis .......................................................... 3-4 3.4 Assays ....................................................................................................................................... 3-4 3.4.1 In-vitro Assays .............................................................................................................. 3-4 3.4.2 In-vivo Assays .............................................................................................................. 3-4 3.5 Strengths, Challenges, and Limitations .................................................................................... 3-4 4. The Retinoid Signaling Pathway ......................................................................................................... 4-1 ii Screening and Testing Methods and Endpoints for Evaluating Endocrine Disruptors 4.1 Overview .................................................................................................................................. 4-1 4.1.1 Retinoic Acid Receptor (RAR) Signaling .................................................................... 4-1 4.1.2 The Retinoid X Receptor Signaling Network ............................................................... 4-1 4.1.2.1 Reproduction ................................................................................................ 4-2 4.1.2.2 Development ................................................................................................ 4-2 4.1.2.3 Lipid Homeostasis ........................................................................................ 4-2 4.2 Disruption of Retinoid Signaling by Xenobiotics .................................................................... 4-3 4.2.1 Reductions in Retinoid Levels ...................................................................................... 4-3 4.2.2 RAR Agonists ............................................................................................................... 4-3 4.2.3 RXR Agonists/Antagonists ........................................................................................... 4-3 4.3 Consequences of Disrupted Retinoid Signaling ....................................................................... 4-4 4.4 Assays ....................................................................................................................................... 4-4 4.4.1 Reporter Assays for the Assessment of Ah Receptor Agonists .................................... 4-4 4.4.2 Alterations in Retinoid Levels and Metabolism In Vivo .............................................. 4-5 4.4 3 RAR Agonists/Antagonists (In-vitro Reporter Assays)................................................ 4-5 4.4.4 RXR Agonist/Antagonists (In-vitro Reporter Assays) ................................................. 4-6 4.4.5 Adipocyte Differentiation Assay .................................................................................. 4-6 4.4.6 Microarrays ................................................................................................................... 4-7 4.4.7 Alterations in Lipid Levels and Metabolism (In vivo) ................................................. 4-7 4.5 Strengths, Challenges, and Limitations .................................................................................... 4-7 5. The Thyroid-Releasing Hormone:Thyroid Stimulating Hormone:Thyroid Hormone Signaling Pathway ..............................................................................................................................................