Characterization of Endocrine Disrupting Effects of Bisphenol a Or 17Α-Ethinyl Estradiol in Mouse Uterus
Total Page：16
File Type：pdf, Size：1020Kb
Characterization of Endocrine Disrupting Effects of Bisphenol A or 17α-Ethinyl Estradiol in Mouse Uterus A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology and Cell Biophysics of the College of Medicine by Jessica A. Kendziorski BA, DePauw University, 2010 Committee Chair: Scott M. Belcher, PhD ABSTRACT Bisphenol A (BPA) and 17α-ethinyl estradiol (EE) are estrogenic endocrine disrupting chemicals (EDCs) that adversely affect the structure and function of the uterus. Humans are ubiquitously exposed to BPA via consumption of contaminated food and beverage from polycarbonate packaging or food cans. Humans are exposed to EE through the use of oral contraceptives. While human exposure to these EDCs is widespread, little is known how these compounds alter physiological responses that lead to increased incidence of uterine pathology. The purpose of this dissertation was to investigate and characterize uterine pathologies and associated alterations in immune responsiveness and fibrosis. Two mouse strains were used, the CD1 and the C57Bl/6N strains. Exposure during adulthood included mating, parturition, and offspring rearing and mice were exposed for 12-15 weeks. Whole life exposure included placental transfer, as well as direct oral consumption, exposing mice until postnatal day (PND) 90. In order to closely mimic human exposure, mice were orally exposed through diet to known concentrations of control (0 ppm), BPA (0.03, 0.3, 3, 30, or 300 ppm), or EE (0.0001, 0.001, 0.01, 0.1, or 1.3 ppm), which resulted in calculated BPA doses of 0.04, 0.4, 4, 40, and 400 mg/kg/day and EE doses of 0.00002, 0.0002, and 0.001 mg/kg/day. Other exogenous estrogenic compounds from housing, bedding, and water were eliminated. Two distinct immune-related and fibrotic uterine pathologies were identified. Pyometra developed in C57Bl/6N mice exposed to BPA or EE during adulthood. An equine endometrosis- like phenotype, characterized by increased gland nests and stromal and periglandular fibrosis, naturally occurred in control C57Bl/6N mice. In CD1 mice, this fibrotic phenotype was present in the 30 ppm BPA group. Exposure to BPA significantly increased Col1a1 and Col3a1 expression and decreased Mmp2 and Timp2 expression. Expression and activity of matrix metalloproteinases, MMP2 and MMP14, were significantly decreased in both the control ii C57Bl/6N and 30 ppm BPA-exposed CD1 mice compared to control CD1 mice. However, both strains presented with an increased immune response, quantified as the percentage of F4/80- positive cells. The C57Bl/6N strain had a significant increase in endometrial macrophages at a lower dose of BPA (0.03 ppm) than the CD1 strain (30 ppm) exposed during adulthood. In both exposure models, CD1 mice had increased macrophages in the 30 ppm BPA group. This dissertation research was the first report of BPA altering the immune response or collagen accumulation in the uterus, leading to the induction of pyometra or an equine endometrosis-like phenotype. It was also the first report of strain-specific differences in sensitivity to the actions of BPA on these endpoints of interest. Finally, this research highlighted the potential for exposure to estrogenic compounds such as BPA and EE to impact the development and progression of fibrotic and immune-related uterine diseases. However, based on the differences observed between the two generations, physiological changes that occur during mating, pregnancy, and parturition may be necessary for estrogenic compounds to elicit fibrotic effects. These studies also emphasized the importance of understanding differences in sensitivity to estrogenic compounds between mouse strains. iii iv ACKNOWLEDGEMENTS This dissertation work is the product of the encouragement of several colleagues, family, and friends. First and foremost, I would like to acknowledge my advisor and mentor, Dr. Scott Belcher, for his support and assistance during my time as a graduate student. Through his invaluable mentoring and challenges, I have grown significantly in confidence as a scientist and a person. I am truly grateful for the guidance and support he has provided over the years. I would like to extend gratitude for the encouragement and constructive advice given by my dissertation committee members, Drs, Jo El Schultz, William J. Ball, and Heather Patisaul. This dissertation would not have been completed without their attention to progress and helping me to remain focused on the final product. I am gratefully indebted to the past and present members of the Belcher lab, specifically Dr. Eric Kendig, Robin Gear, Dana Buesing, Susie Christie, Dr. Vinicius Carreira, and Clifford Cookman, for their suggestions on experimental procedures, their help in conducting experiments, and their friendships in general during my time in graduate school. I would like to especially acknowledge Vini for his help with identifying pathologies, which laid the foundation for a great part of this dissertation research. I would like to acknowledge rotating graduate students, undergraduate students, summer students, and students from England for their contributions to this research and for their friendship, especially Joni Ford, Rachel Steinher, Ed Durley, and Kev Ungi. Their willingness to do whatever was asked of them was highly appreciated. I would like to thank all of the faculty, students, and staff in the Department of Pharmacology and Biophysics and the associated graduate program. I would like to specifically acknowledge Nancy Thyberg for everything that she has done, by keeping me updated on v deadlines, helping to organize committee meetings, and being the person I would go to for any questions. If she didn’t have the answer, she would find someone that could answer it. Finally, I would like to thank my family and friends for their encouragement, love, and support. Even though they had no idea what I was doing or why I was in school for 5+ years, my parents always asked how things were going. I would like to thank my sister Sarah for always being able to cheer me up if I was having a rough time. I would like to thank my friends, in particular Ian, Jamey, Natalie, Josh, Mat, Leslie, Chad, Cassie, Mason, and Kyle, for their ability to always make sure I had fun and didn’t go too crazy from being in the lab, and my boyfriend Carson for putting up with my complaining and for his constant reassurance and love. I would also like to thank Megan, Chenoa, Andrea, and Kelly for their laughs. Even though we were hundreds of miles apart, they made me smile and uplifted my spirits every time we texted or called. vi TABLE OF CONTENTS Page ABSTRACT ii ACKNOWLEDGEMENTS v TABLE OF CONTENTS vii LIST OF FIGURES AND TABLES xiv LIST OF ABBREVIATIONS xviii Chapter 1 INTRODUCTION 1 1.1 Statement of Purpose 2 1.1.1 Objective and Rationale 2 1.1.2 Central Hypothesis and Specific Aims 2 1.2 Definition of Endocrine Disrupting Chemicals 3 1.3 Prototypical Estrogenic EDC: Diethylstilbestrol 4 1.4 17α-Ethinyl Estradiol 6 1.5 Bisphenol A 7 1.5.1 Commercial Uses of BPA and Routes of Exposure 7 1.5.2 Pharmacokinetics and Toxicologic Exposure Limits of BPA 8 1.5.3 Receptor Binding of BPA 8 1.5.4 Endocrine Disrupting Effects of BPA in the Uterus 11 1.5.5 Extracellular Matrix Alterations in Response to BPA 12 1.6 Strain Susceptibility to Effects of Estrogenic Compounds 13 1.6.1 Susceptibility to Uterine Weight Increases and Morphological Alterations 15 1.6.2 Differences in Circulating Sex Hormones and ERα Levels 15 1.6.3 Quantitative Trait Loci Controlling Phenotypic Variation 16 vii 1.6.4 Differential Sensitivity of the Ovary and Vagina to Estrogenic Compounds 17 1.6.5 Differential Sensitivity of Other Tissues to Estrogenic Compounds 18 1.7 The Endocrine System 19 1.8 Nuclear Hormone Receptors: ERα and ERβ 20 1.8.1 Structure 20 1.8.2 Nuclear Transactivation, Membrane-Initiated, 22 and Rapid Signaling Mechanisms 1.8.3 Role of ERs in Uterine Structure and Reproductive Function 26 1.8.3.1 Postnatal Uterine Development 26 1.8.3.2 Impact on Gene Expression 27 1.8.3.3 ERα in the Brain and Impact on Cyclicity 28 1.8.3.4 Role of GPR30 in the Uterus 28 1.9 The Cycling and Pregnant Uterus 29 1.9.1 Circulating Hormones and Tissue Remodeling 29 1.9.2 Role of Extracellular Matrix in Uterine Structure and Function 32 1.9.3 Role of Extracellular Matrix during Pregnancy 36 1.10 Uterine Disorders 39 1.10.1 Pyometra 39 1.10.1.1 Description, Etiology, and Treatment of Pyometra 39 1.10.1.2 Role of Bacteria and the Immune System 40 in the Development of Pyometra 1.10.1.3 Involvement of Sex Hormones and Collagen Accumulation 41 in Pyometra viii 1.10.2 Endometriosis 41 1.10.2.1 Description, Symptoms, and Treatment of Endometriosis 41 1.10.2.2 Role of Extracellular Matrix in Pathogenesis of Endometriosis 42 1.10.3 Leiomyoma 43 1.10.3.1 Description, Symptoms, and Treatment of Leiomyoma 43 1.10.3.2 Etiology of Leiomyoma 45 1.10.3.3 Regulation of Collagen Accumulation in Leiomyoma 47 1.10.4 Equine Endometrosis 47 1.10.4.1 Description, Etiology, and Treatment of Equine Endometrosis 47 1.10.4.2 Progression of Equine Endometrosis 50 1.10.4.3 Characterization of Extracellular Matrix Alterations 51 in Equine Endometrosis 1.10.4.4 Fetal Foal Loss 52 1.11 Statement of Purpose 54 1.11.1 Objective and Rationale 54 1.11.2 Central Hypothesis and Specific Aims 54 Chapter 2 Methods 55 2.1 BPA and EE Exposure 56 2.1.1 Animal Housing 56 2.1.2 Exposure 56 2.1.3 Breeding