Examining the Effects of Gestational Exposure to Perfluorooctanoic Acid on the Developing and Differentiating Mammary Gland, Their Consequences, and the Possible Modes of Action by Which They are Mediated Sally Shoshana White 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 Curriculum of Toxicology. Chapel Hill 2008 Approved by: Suzanne E. Fenton, Ph.D. Kathleen K. Sulik, Ph.D. Louise M. Ball, Ph.D. Wendy N. Jefferson, Ph.D. David W. Threadgill, Ph.D. ©2008 Sally Shoshana White ALL RIGHTS RESERVED ii Abstract Sally Shoshana White: Examining the Effects of Gestational Exposure to Perfluorooctanoic Acid on the Developing and Differentiating Mammary Gland, Their Consequences, and the Possible Modes of Action by Which They are Mediated (Under the direction of Suzanne E. Fenton) Perfluorooctanoic acid (PFOA) is a persistent industrial compound, which is commonly detected in human and wildlife sera. Low-dose prenatal PFOA exposure negatively affects postnatal growth and survival with little prenatal effect, indicating the potential for PFOA to alter lactation in the nursing dam. These studies aim to understand the impact PFOA has on development and differentiation of the mammary gland (MG). To address this, timed-pregnant CD-1 mice were gestationally exposed to 0 or 5 mg PFOA/kg body weight. PFOA-treated dams exhibited significant histopathologic delays in MG lactational differentiation and alterations in milk protein gene expression, and their exposed female offspring displayed stunted mammary epithelial branching and growth. The roles of timing and route of exposure were then addressed by cross-fostering litters, and the 5 mg/kg dose, under either lactational- or intrauterine-only exposures, was determined sufficient to delay MG development as early as postnatal day (PND) 1, with effects persisting beyond PND63. The consequences to F1 lactational function and subsequent development of F2 offspring were investigated, and F1 dams exhibited delayed lactational differentiation, though no effect of early-life exposure on milk production or F2 offspring body weight was detected. Finally, the potential mode of action for the effects of PFOA on the MG was iii investigated, by treating pregnant wild-type (WT) and peroxisome proliferator-activated receptor alpha (PPAR!) knock-out (KO) mice similarly with PFOA. At weaning, PFOA treatment caused diminished lactational differentiation in WT, but not KO dams. In offspring, however, effects of PFOA exposure were apparent in both strains at weaning, suggesting a non-PPAR! mode of action may be responsible for offspring MG effects. In total, these studies defined a window of MG sensitivity in late fetal and early neonatal life, as well as the persistence of MG effects beyond the age at which serum PFOA concentrations reach background levels. This suggests a permanent, non-PPAR! mediated effect in the offspring, though without clear functional consequences. The characterization of MG effects in light of PFOA dosimetry data, as well as the discovery that MG effects are not strictly controlled by PPAR!, will greatly assist in the regulation of this compound. iv Acknowledgements I would like to acknowledge the collaborative assistance and professional support provided by the laboratories of Dr. Chris Lau and Dr. Barbara Abbott of the US EPA Reproductive Toxicology Division and Dr. Antonia Calafat of the Centers for Disease Control and Prevention, without which the studies presented here could have achieved neither their scale nor depth. In addition, the mentorship of Dr. Suzanne Fenton deserves profound acknowledgment; it is not only the formal training in the discipline, but also the recognition and acceptance of humanity put before me that shaped both my scientific and my personal maturation. Finally, the unwavering support, patience, and understanding of friends and family merits acknowledgment for its considerable role in my survival of graduate school. I would like to explicitly thank Charlotte White, Dr. Lon White, Dr. Tamara Tal, and Derek Cornish, who listened, and listened, and listened. v Table of Contents List of Tables...........................................................................................................................ix List of Figures...........................................................................................................................x Abbreviations..........................................................................................................................xii Chapter I. Introduction.....................................................................................................14 Regulatory Interest in PFOA..........................................................................14 PFOA in the Market and the Environment......................................................15 Toxicity of PFOA...........................................................................................19 PFOA Absorption, Distribution, Metabolism, and Excretion in Various Species...........................................................................................21 PFOA and Developmental Toxicity................................................................23 The Developing and Lactating Mammary Gland............................................26 Significance and Goals of the Doctoral Study................................................29 References.......................................................................................................31 II. Gestational PFOA Exposure of Mice is Associated with Altered Mammary Gland Development in Dams and Female Offspring....................39 Preface.............................................................................................................39 Abstract...........................................................................................................40 Introduction.....................................................................................................41 Materials and Methods...................................................................................44 vi Results.............................................................................................................54 Discussion.......................................................................................................68 References.......................................................................................................74 III. Effects of Perfluorooctanoic Acid on Mouse Mammary Gland Development and Differentiation Resulting from Cross-foster and Restricted Gestational Exposures........................................78 Preface.............................................................................................................78 Abstract...........................................................................................................79 Introduction.....................................................................................................80 Materials and Methods...................................................................................82 Results.............................................................................................................89 Discussion.....................................................................................................104 References.....................................................................................................110 IV. Investigating the Multigenerational Effects of Prenatal PFOA Exposure On Mouse Mammary Gland Development, Function, and Tumor Susceptibility.......................................115 Preface...........................................................................................................115 Abstract.........................................................................................................116 Introduction...................................................................................................117 Materials and Methods.................................................................................118 Results...........................................................................................................125 Discussion.....................................................................................................138 References.....................................................................................................142 V. Potential Modes of Action by Which PFOA Induces Altered Mammary Gland Development and Lactational Differentiation...................145 Preface...........................................................................................................145 vii Abstract.........................................................................................................146 Introduction...................................................................................................147 Materials and Methods..................................................................................149 Results...........................................................................................................154 Discussion.....................................................................................................162 References.....................................................................................................172 VI. Conclusions and Perspectives.......................................................................176