ABSTRACT GOETZ, AMBER KRISTINA. Toxicogenomic Study of Triazole Antifungal Modes of Action. (Under the direction of Dr. David J. Dix, Dr. Ernest Hodgson.) Common modes and mechanisms of hepatic and reproductive toxicity were characterized for a set of triazole antifungals. Wistar Han rats were fed myclobutanil, propiconazole, or triadimefon from gestation day 6 to postnatal day (PND) 120. Anogenital distance, body and organ weights, serum hormone levels, age at preputial separation, sperm morphology and motility, fertility and fecundity assays were selected to evaluate effects on development and adult reproductive function. All three triazoles increased anogenital distance, increased relative liver weights, induced hepatomegaly, increased absolute testis weights and serum testosterone levels. Myclobutanil and triadimefon impaired insemination and fertility, myclobutanil decreased pituitary weights, and triadimefon delayed puberty and decreased total serum thyroxine levels. The reproductive effects are consistent with disruption of testosterone homeostasis as a key event for triazole reproductive toxicity. To investigate common mechanisms of triazole toxicity, a toxicogenomic study was performed using liver and testis samples from PND92. Pathway and gene-level analysis of the liver highlighted biological processes affected by all three triazoles, including phase I-III; fatty acid, steroid, and xenobiotic metabolism; and lipid homeostasis. Triadimefon had a distinctive impact on sterol biosynthesis related genes in the liver. No common pathways were affected in the testis. To explore common mechanisms of action between in vivo and in vitro model systems, a series of comparative toxicogenomic studies were conducted on rat liver at multiple time points, rat and human primary hepatocytes, H295R cells, and PND92 liver and testis. Comparison between liver and rat hepatocytes showed consistent effect on fatty acid catabolism, sterol metabolism, and phase III transporters. Conserved effects between rat and human primary hepatocytes concluded triazole- and species-specific effects. The modulated genes affect a network of pathways regulating lipid and testosterone homeostasis through the constitutive androstane and pregnane X receptors. The gene expression results from this study suggest triazoles increase fatty acid catabolism, reduce bile acid biosynthesis, increase cholesterol biosynthesis, and induce steroid metabolism in the liver. These changes in the liver likely contribute to the observed disruption in testosterone homeostasis. TOXICOGENOMIC STUDY OF TRIAZOLE ANTIFUNGAL MODES OF ACTION by AMBER KRISTINA GOETZ A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirement for the Degree of Doctor of Philosophy TOXICOLOGY Raleigh, North Carolina 2007 APPROVED BY: ____________________________ Dr. Charlotte Farin ____________________________ ____________________________ Dr. Andrew Wallace Dr. Russ Wolfinger ___________________________ ____________________________ Dr. David Dix Dr. Ernest Hodgson Co-chair of Advisory Committee Co-chair of Advisory Committee DEDICATION This dissertation and all the work that has been put into it are dedicated to my parents and fiancé who have provided unconditional support and guidance throughout my studies and research. Their relentless motivation, humor, understanding, patience and prayers were always there, through the good times and the bad, the ebbs and flows. ii BIOGRAPHY I was born into an Air Force family in Ohio, moving rapidly to Florida and Maine where I attended a rural school emphasizing strong family values and involvement in community life. Throughout my early years I assisted my family in caring for a variety of farm animals and at the age of 10 decided to breed rabbits. This interest expanded to extensive volunteer work at the Caribou veterinary clinic. As a Girl Scout, I was selected to represent the State of Maine at a National training program in Animal Care and Management at the Bronx Zoological Park. Each of these experiences and opportunities led me to the Zoology program at the University of Maine Orono. At UMaine Orono I was introduced to the study of the taxonomy of cumaceans as well as work in the field of DNA analysis. Due to my interest and skill in the laboratory, I was selected as an undergraduate to participate in a graduate research program off the coast of Mexico. Following graduation, I continued to work in a veterinary clinic and than as a zoo attendant specializing in the care of domestic and exotic animals. In the fall of 2001 I entered the graduate program in Zoology at North Carolina State University with the support of the U.S. Environmental Protection Agency (EPA). My interest in research was refocused to toxicology, such that shortly after my admittance in the Zoology program I transferred into North Carolina State’s Molecular and Cellular Toxicology program. By the time I completed my masters in the fall of 2003, I had already begun designing the research project presented here. Upon completion of my doctorate, I will continue in the field of molecular toxicology with an avid interest in reproductive toxicology and toxicogenomic research. iii ACKNOWLEDGEMENTS First and foremost, thanks to Dr. David Dix for providing the opportunities to not only volunteer in his lab when I first began my journey towards graduate school in early 2001, but continued support throughout my years of research under his direction. His leadership, guidance, assiduous determination, and parallel interest in my research unremittingly drove my enthusiasm to learn new and polish existing skills. Thanks to the members of my advisory committee, the late Dr. Randy Rose, Dr. Charlotte Farin, Dr. Andrew Wallace, Dr. Ernest Hodgson, and Dr. Russ Wolfinger for providing encouragement, constructive criticism and guidance throughout. Of course, thanks to all of my professors for opening my eyes, broadening my mind and testing my abilities. iv TABLE OF CONTENTS Page LIST OF TABLES ….………………………………………………………………………………….. vi LIST OF FIGURES ……………………………………………………………………………………. ix CHAPTER ONE: INTRODUCTION …………………………………………………………………. 1 CHAPTER TWO: DISRUPTION OF TESTOSTERONE HOMEOSTASIS AS A MODE OF ACTION FOR THE REPRODUCTIVE TOXICITY OF TRIAZOLE ANTIFUNGALS IN THE MALE RAT Introduction …………………………………………………………………………………... 12 Materials and Methods ……………………………………………………………………… 13 Results ………………………………………………………………………………………... 18 Discussion ……………………………………………………………………………………. 22 CHAPTER THREE: MECHANISMS OF TRIAZOLE ANTIFUNGAL REPRODUCTIVE TOXICITY INFERRED FROM TOXICOGENOMICS Introduction …………………………………………………………………………………... 40 Materials and Methods ……………………………………………………………………… 41 Results ……………………………………………………………………………………….. 46 Discussion ……………………………………………………………………………………. 50 CHAPTER FOUR: COMMON AND CONSERVED TOXICOGENOMIC RESPONSES TO TRIAZOLE ANTIFUNGALS Introduction …………………………………………………………………………………... 73 Materials and Methods ……………………………………………………………………… 74 Results …………………………………………………………………………………......... 82 Discussion ……………………………………………………………………………………. 88 CHAPTER FIVE: CONCLUSIONS ………………………………………………………………….. 126 REFERENCES ………………………………………………………………………………………... 137 APPENDICIES APPENDIX A: Workflow for JMP Genomics Analysis of Affymetrix GeneChip® Data…. 147 APPENDIX B: Supplemental data from toxicogenomic studies ………………………… 153 v LIST OF TABLES Page CHAPTER TWO Table 1 Daily feed consumption and calculated dose levels …………………………… 26 Table 2 Weekly body weights ……………………………………………………………... 27 Table 3 Survival rates ………………………………………………………………………. 28 Table 4 Triazole dose, age and body weight at preputial separation ………………….. 29 Table 5 Liver weights ……………………………………………………………………….. 30 Table 6 Testis and accessory sex gland weights ……………………………………….. 31 Table 7 Pituitary and brain weights ……………………………………………………….. 32 Table 8 Liver histopathology ………………………………………………………………. 33 Table 9 Breeding performance of male rats exposed to triazole antifungals ………… 34 Table 10 Modes of action and potential mechanisms of triazole-induced reproductive toxicity ……………………………………………………………….. 35 CHAPTER THREE Table 1 Probe sets significantly modulated in adult liver and testis …………………… 57 Table 2 Pathways affected by triazoles in adult liver: GD6-PND92 exposure ……….. 58 Table 3 Pathways affected by triazoles in adult testis: GD6-PND92 exposure ………. 60 Table 4 Common gene expression changes by all three triazoles in the liver ……….. 61 Table 5 Gene expression changes by two or more triazoles in the testis …………….. 62 Table 6 Comparisons between microarray and relative qPCR fold changes …………. 63 CHAPTER FOUR Table 1 Probe sets significantly modulated in adult liver and primary hepatocytes …. 97 Table 2 Probe sets significantly modulated in adult testis and H295R cells ………….. 98 Table 3A Pathways consistently affected across time in liver and hepatocytes by myclobutanil ……………………………………………………... 99 Table 3B Pathways consistently affected across time in liver and hepatocytes by propiconazole ……………………………………………………. 100 Table 3C Pathways consistently affected across time in liver and hepatocytes by triadimefon ……………………………………………………….. 101 Table 3D Pathways conserved across rat and human hepatocytes by triazoles …….. 102 Table 4A Conserved pathways affected in the testis and H295R cells by myclobutanil 103 Table 4B Conserved pathways affected in the testis and H295R cells by propiconazole 104 Table 4C Conserved pathways affected in the testis and H295R cells by triadimefon 105 Table 5 Common gene