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 #2 November 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 Abstract ..................................................................................................................................................... A-1 1. Introduction ......................................................................................................................................... 1-1 1.1 Relevance of this DRP to Diseases and Syndromes of Contemporary Concern ....................... 1-4 2. The Hypothalamic:Pituitary:Adrenocortical (HPA) Axis ................................................................... 2-1 2.1 Overview ................................................................................................................................... 2-1 2.1.1 Corticotropin-releasing Hormone (CRH) ................................................................... 2-2 2.1.2 Arginine Vasopressin (AVP)/Arginine Vasotocin (AVT) .......................................... 2-3 2.1.3 Corticotropin (ACTH) ................................................................................................ 2-3 2.1.4 Luteinizing Hormone (LH) and Chorionic Gonadotropin (CG) ................................. 2-3 2.1.5 Glucocorticoids ........................................................................................................... 2-3 2.1.5.1 Glucocorticoid Receptors (GRs) .................................................................. 2-4 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 Consequences of Disruption ...................................................................................................... 2-5 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 .......................................................................................................................... 2-6 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 .................................................................................................................... 2-7 2.3.7 Arylhydrocarbon Receptor (AhR) Agonists ............................................................... 2-7 2.3.8 Estrogens and Androgens ........................................................................................... 2-7 2.4 In Vitro Assays .......................................................................................................................... 2-7 2.4.1 Transactivation Reporter Assays ................................................................................ 2-7 2.4.2 Microarrays ................................................................................................................. 2-7 2.4.3 Cell Culture Systems ................................................................................................... 2-8 2.4.3.1 Corticotropes ................................................................................................ 2-8 2.4.3.2 Adrenal Cortical Cells .................................................................................. 2-8 2.4.3.3 Glucocorticoid Target Cells ......................................................................... 2-8 2.5 In Vivo Assays ........................................................................................................................... 2-8 2.5.1 Mammals .................................................................................................................... 2-9 2.5.2 Fish .............................................................................................................................. 2-9 2.5.2 Amphibians ................................................................................................................. 2-9 2.6 Strengths, Challenges, and Limitations ..................................................................................... 2-9 2.6.1 Stress, the Adverse Outcome Pathway, and Assay Selection ................................... 2-10 3. Hypothalamus:Pituitary:Gonad (HPG) Axis ....................................................................................... 3-1 3.1 Overview ................................................................................................................................... 3-1 3.1.1 Structure of the HPG Axis .......................................................................................... 3-1 3.1.2. Structure and Actions of HPG hormones .................................................................... 3-1 3.1.3. Function of the HPG Axis ........................................................................................... 3-3 3.2 Consequences of Disruption ...................................................................................................... 3-3 3.2.1 (Anti)estrogens ............................................................................................................ 3-3 3.2.1.1 Reproduction ................................................................................................ 3-4 3.2.1.2 Metabolism and Growth ............................................................................... 3-4 3.2.1.3 Immune System ............................................................................................ 3-5 ii Screening and Testing Methods and Endpoints for Evaluating Endocrine Disruptors 3.2.2 (Anti)androgens .......................................................................................................... 3-5 3.2.2.1 Reproduction ................................................................................................ 3-5 3.2.2.2 Growth .......................................................................................................... 3-6 3.2.3 (Anti)gestagens ........................................................................................................... 3-6 3.2.3.1 Reproduction ................................................................................................ 3-6 3.2.3.2 Immune System ............................................................................................ 3-7 3.3 Precedent Chemicals .................................................................................................................. 3-7 3.3.1 (Anti)estrogens ............................................................................................................ 3-7 3.3.1.1 Bisphenol A (BPA) ...................................................................................... 3-7 3.3.1.2 Phthalate Esters ............................................................................................ 3-8 3.3.1.3 Polychlorinated Biphenyls (PCBs) ............................................................... 3-8 3.3.2 (Anti)androgens .......................................................................................................... 3-9 3.3.2.1 Di-(2-ethylhexyl)phthalate (DEHP) ............................................................. 3-9 3.3.2.2 Flutamide ...................................................................................................... 3-9 3.3.3 (Anti)gestagens ........................................................................................................... 3-9 3.3.3.1 Levonorgestrel (LNG) .................................................................................. 3-9 3.4 In Vitro Assays .......................................................................................................................... 3-9 3.4.1 (Anti)estrogens .......................................................................................................... 3-10 3.4.1.1 ER Transactivation Assays ......................................................................... 3-10 3.4.1.2 Membrane Receptor Binding ..................................................................... 3-10 3.4.1.3 Cell-based Microarrays .............................................................................