ENDOCRINE DISRUPTING POTENTIAL OF ENVIRONMENTAL CHEMICALS CHARACTERIZED BY HIGH-THROUGHPUT SCREENING Daniel M. Rotroff A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Environmental Sciences and Engineering. Chapel Hill 2013 Approved by: Ivan Rusyn, M.D. Ph.D. David J. Dix, Ph.D Thomas Knudsen, Ph.D. Fred Wright, Ph.D. Rebecca Fry, Ph.D. i © 2013 Daniel M. Rotroff ALL RIGHTS RESERVED ii ABSTRACT DANIEL M. ROTROFF: Endocrine Disrupting Potential of Environmental Chemicals Characterized by High-Throughput Screening (Under the direction of Dr. David J. Dix) Over the past 20 years, an increased focus on detecting environmental chemicals that pose a risk of endocrine disruption and congressional legislation have driven the creation of the U.S. EPA Endocrine Disruptor Screening Program (EDSP). Several thousand chemicals are subject to the EDSP, which will require millions of dollars and decades to process using current test batteries. In order to identify opportunities for increased chemical throughput, we initially investigated how well EPA ToxCast in vitro high-throughput screening (HTS) assays relevant for estrogen, androgen, steroidogenic, and thyroid disrupting mechanisms could identify compounds relative to in vitro and in vivo data collected from studies related to the EDSP Tier 1 screen. An iterative, balanced optimization model was implemented and indicated that ToxCast HTS assays measuring estrogen receptor (ER) and androgen receptor (AR) activation classify compounds with estrogenic and androgenic activity in guideline studies with a high degree of accuracy, respectively. The ER signaling pathway involves a wide array of molecular initiating events and cellular processes. This dissertation examined whether active chemicals in ToxCast ER transactivation assays could indicate chemical- induced upregulation of the ER pathway through ligand binding leading to induced changes in T47D growth kinetics. Considering the complex set of ER in vitro assays in toto increased the overall sensitivity of detection for ER reference chemicals. In addition, this research iii highlighted important aspects of the biological response such as non-ER specificity in the cell growth assay. These nuances are likely important considerations for the construction of a predictive model. In effort to accurately predict the estrogenic potential of environmental chemicals in a high-throughput format, multiple orthogonal in vitro ER assays were used to develop a predictive model for ~2000 chemicals. Model results indicate a high degree of predictivity for both the uterotrophic in vivo assay and ER reference chemicals. The information provided by the model will aid in understanding how environmental chemicals contribute to endocrine-related human health consequences and predict the estrogenic potential of chemicals through the use of in vitro assays, limiting the need to run more costly and animal-intensive in vivo bioassays. iv ACKOWLEDGEMENTS First and foremost I would like to express my deep gratitude to my research advisor, Dr. David Dix, Ph.D. Without his support and expertise this would not have been possible. His academic and professional guidance has been invaluable, and will continue to be so in all of my future endeavors. I also owe a great deal of appreciation to my academic advisor, Dr. Ivan Rusyn, M.D. Ph.D., who made sure that I stayed on target throughout this entire process, despite my occasional efforts to test certain boundaries. I would like to express my appreciation to the additional members of my committee, Dr. Thomas Knudsen, Ph.D., Dr. Fred Wright, Ph.D., and Dr. Rebecca Fry, Ph.D. Their diverse knowledge and experience pushed me to be a better student and a better researcher. I owe a great deal of gratitude to Dr. Richard Judson, Ph.D., who kept his door open to me and spent many hours helping me formulate new and innovative ideas. His amazing work ethic sets a high standard, and through his support and mentorship I have benefitted greatly. Lastly, I would like to show my gratefulness to my colleagues, collaborators, and managers at the U.S. Environmental Protection Agency National Center for Computational Toxicology. Their continued effort to push science forward and willingness to share from their vast knowledge and their distinct backgrounds are what have made this experience so significant. The research described here has been partially supported by the U.S. Environmental Protection Agency. The views expressed in this paper are those of the author and do not v necessarily reflect the views or policies of the U.S. Environmental Protection Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. vi TABLE OF CONTENTS LIST OF TABLES .................................................................................x LIST OF FIGURES ............................................................................. xi LIST OF ABBREVIATIONS ............................................................ xiii Chapter I. INTRODUCTION ............................................................................................................1 Endocrine Physiology ............................................................................................... 1 Human Endocrine Disruption and Pathology ........................................................... 3 Environmental Impact of Endocrine Disruption ...................................................... 6 Endocrine Disruptor Screening ................................................................................ 8 Computational Toxicology and Alternatives to Traditional Test Methods ............ 10 Estrogen Signaling Pathways in Predictive Toxicology ........................................ 13 Predictive Modeling of Endocrine Disruptors ........................................................ 17 References .............................................................................................................. 24 II. USING IN VITRO HIGH THROUGHPUT SCREENING ASSAYS TO IDENTIFY POTENTIAL ENDOCRINE-DISRUPTING CHEMICALS .........................................32 Introduction ............................................................................................................ 32 Methods .................................................................................................................. 36 Results .................................................................................................................... 41 Discussion .............................................................................................................. 44 Figures .................................................................................................................... 55 References .............................................................................................................. 59 vii III. REAL-TIME GROWTH KINETICS MEASURING HORMONE MIMICRY FOR 1816 UNIQUE TOXCAST CHEMICALS IN T-47D HUMAN DUCTAL CARCINOMA CELLS ...................................................................................................64 Introduction ............................................................................................................ 64 Methods .................................................................................................................. 66 Results .................................................................................................................... 74 Discussion .............................................................................................................. 78 Tables ..................................................................................................................... 84 Figures .................................................................................................................... 89 References .............................................................................................................. 96 IV. ENDOCRINE TESTING IN THE 21 ST CENTURY: USING IN VITRO ASSAYS TO PREDICT ESTROGEN RECEPTOR SIGNALING RESPONSES.............................101 Methods ................................................................................................................ 103 Discussion ............................................................................................................ 118 Tables ................................................................................................................... 124 Figures .................................................................................................................. 126 References ............................................................................................................ 137 V. GENERAL DISCUSSION ...........................................................................................141 Advances in In Vitro Technology ........................................................................ 141 Phenotypic Anchoring .......................................................................................... 143 Combining In Vitro Data to Test for In Vivo Effects ........................................... 145 Limitations ............................................................................................................ 148 References ...........................................................................................................