Estrogenic Activity of the Polybrominated Diphenyl
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ESTROGENIC ACTIVITY OF THE POLYBROMINATED DIPHENYL ETHER FLAME RETARDANT MIXTURE DE-71 Minerva Mercado-Feliciano Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Pharmacology and Toxicology, Indiana University February 2008 . Accepted by the Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy ________________________________________ Robert M. Bigsby, Ph.D. Chair ________________________________________ James E. Klaunig, Ph.D. Administrative Chair Doctoral Committee ________________________________________ Meei-Huey Jeng, Ph.D. Date of oral examination: October 2nd, 2007 ________________________________________ Lisa Kamendulis, Ph.D. ________________________________________ Todd Skaar, Ph.D. ________________________________________ William J. Sullivan, Jr., PhD. ii Acknowledgments To my ancestors and godparents, for my core education and curiosity. Víctor Mercado Alicea and Adoración Feliciano Reyes Fernando Feliciano Reyes and Dhalma Feliciano Reyes (godparents) Víctor Mercado Rodríguez and Santiaga Alicea Santos Pablo Feliciano Nieves and Juana Reyes Luís Mercado Colón and Domitila (Tití) Rodriguez Figueroa Manuel Alicea Loyo and Sinforosa Santos Arroyo Angel Feliciano Santiago and Petronila Nieves Febus María Reyes Reyes Jesús Mercado Maldonado and Eulalia Colón Francisco Rodríguez Santos and Paula Figueroa Otero Bartolo Alicea and Vicenta Loyo Ruíz Francisco Santos and Gregoria Arroyo Felipe Feliciano Bonilla and Eustoquia Santiago Ortíz Pablo Nieves Menéndez and Epifania Febus Colón Bautista Reyes Reyes and Virginia (Ginia) Reyes To my past and present mentors, for their guidance and inspiration. Sra. Agnes Cabrera Dr. José Molinelli Freytes Dr. Robert M. Bigsby To my husband and coach. Tomás Morales Vázquez To friends, family and colleagues for all their cheerleading. iii ABSTRACT Minerva Mercado-Feliciano ESTROGENIC ACTIVITY OF THE POLYBROMINATED DIPHENYL ETHER FLAME RETARDANT MIXTURE DE-71 Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants suspected to act as endocrine disruptors. We tested the commercial PBDE mixture DE-71 and its in vivo metabolites for estrogenic activity. MCF-7 breast cancer cells culture, ERE- luciferase gene expression, 3H-β-estradiol displacement from recombinant ERα, and ovariectomized (OVX) mice served as bioassays. Although DE-71 did not bind ERα, it was able to increase MCF-7 cell proliferation and this was prevented by the antiestrogen fulvestrant. DE-71 co-treatment reduced the effect of estradiol in MCF-7 cells. In the OVX mouse (BALB/c) 3-day assay, DE-71 administered alone had no effect on uterine or vaginal tissues but when administered subcutaneously potentiated estradiol’s effect on uterine weight in a dose-dependent manner. DE-71 administered SQ to BALB/c mice for 34 days slightly increased uterine epithelial height (UEH), vaginal epithelial thickness (VET) and mammary ductal lumen area, and attenuated the estradiol-induced increase in UEH; these effects were not seen in C57BL/6 mice. DE-71 increased liver weight in BALB/c, C57BL/6 and estrogen receptor-alpha knockout (ERαKO) mice. Liver cytochrome P450 1A (CYP1A) and CYP2B activities increased 2.5-fold and 7-fold respectively when DE-71 was administered PO, but only CYP2B increased (5-fold) after SQ treatment. Six OH-PBDE metabolites were found in mice after 34-day DE-71 treatment and all were able to bind recombinant ERα. Para-hydroxylated metabolites iv displayed a 10- to 30-fold higher affinity for ERα compared to ortho-hydroxylated PBDEs. Para-OH-PBDEs induced ERE-luciferase and produced an additive effect when coadministered with β-estradiol. DE-71 was also additive with β-estradiol. At high concentrations (≥ 5x10-5 M), ortho-OH-PBDEs were antiestrogenic in the ERE-luciferase assay. In conclusion, DE-71 behaves as a weak estrogen in both MCF-7 breast cancer cells and ovariectomized adult mice. Mice strain, treatment route and duration determined if DE-71 was estrogenic. BALB/c mice are more susceptible to DE-71 effects in estrogen target tissues than C57BL/6 mice. DE-71 increased liver weight, 5%-51% depending on mouse strain and treatment regime, independently of ERα. The observations that the DE-71 mixture does not displace 3H-β-estradiol from ERα while the hydroxylated metabolites do, suggest that the cellular and tissue effects were due to a metabolic activation of individual congeners. Robert M. Bigsby, Ph.D., Chair v Table of Contents List of Tables ................................................................................................................... xi List of Figures ................................................................................................................. xii List of Abbreviations ....................................................................................................... xv Introduction ...................................................................................................................... 1 General Estrogen Physiology ................................................................................ 2 Estrogens Role in Female Reproductive Physiology ............................................. 4 Metabolism and Regulation of Endogenous Estrogens ...................................... 12 Molecular Signaling by Endogenous Estrogens .................................................. 15 Estrogen Receptor Alpha Regulation ................................................................... 19 Physiological Role of Estrogen Receptor Alpha .................................................. 21 Models of Estrogen Action ................................................................................... 22 Estrogen Toxicology ............................................................................................ 25 Endocrine Disruption by Xenoestrogens ............................................................. 30 PBDEs As Suspected Xenoestrogens ................................................................... 35 Specific Aims of this Thesis .................................................................................. 41 Materials and Methods ................................................................................................... 43 Test Chemicals ......................................................................................................43 Cell Culture Conditions ........................................................................................44 Cell Proliferation Measurements ......................................................................... 46 Estrogen Response Element – Luciferase Assays ................................................ 49 In vitro Generation of Microsomal Metabolites .................................................. 55 vi Materials and Methods (cont.) Recombinant Estrogen Receptor Alpha Binding Assay ....................................... 56 Kinase Phosphorylation Assay ............................................................................ 56 Animal Treatments ............................................................................................... 58 Whole Mount Mammary Glands ...........................................................................61 Cytochrome P450 Activity Assay ..........................................................................61 Statistics ................................................................................................................62 Results ..............................................................................................................................65 Specific Aim 1: Determine if DE-71 exerts estrogenic or antiestrogenic effects using cell culture and animal models ............................................................... 65 1.1 DE-71 acts as a weak estrogen in breast cancer cell culture model 65 1.2 DE-71 acts as a weak estrogen in OVX mice model after 3 days of treatment ..........................................................................................70 1.3 DE-71 acts as a weak estrogen in OVX mice model after 34 days of treatment ..........................................................................................76 Specific Aim 2: Determine the role of metabolic activation in DE-71 estrogenic or antiestrogenic effects ....................................................................................... 88 2.1 Phenolic DE-71 metabolites were found in plasma of mice after 34 days of treatment ............................................................................ 88 2.2 In vitro microsomal metabolism increases estrogenic character of DE-71 ..............................................................................................91 2.3 DE-71 and its phenolic metabolites activate the estrogen response ...........................................................................................94 vii Results (cont.) Specific Aim 3: Explore the mechanisms by which DE-71 congeners or their metabolites can activate or antagonize ERα signaling ................................. 100 3.1 DE-71 phenolic metabolites found in mice displace beta-estradiol from Estrogen Receptor Alpha ......................................................100 3.2 DE-71 phenolic metabolites modify activation of estrogen response element signaling by beta estradiol .............................................. 105 3.3 DE-71