Evidence of Endocrine Disruption in Amphibians Due to Agricultural Chemical Exposure
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EVIDENCE OF ENDOCRINE DISRUPTION IN AMPHIBIANS DUE TO AGRICULTURAL CHEMICAL EXPOSURE By MARISOL MARIA GUTIERREZ A Dissertation submitted to the Graduate School-New Brunswick Rutgers, The State University of New Jersey and Graduate School of Biomedical Sciences University of Medicine and Dentistry of New Jersey in partial fulfillment of the requirements for the degree of Doctor of Philosophy Joint Graduate Program in Toxicology written under the direction of Professor Mark G. Robson and approved by _________________________ _________________________ _________________________ _________________________ _________________________ New Brunswick, NJ October 2007 ABSTRACT OF THE DISSERTATION Evidence of Endocrine Disruption in Amphibians Due to Agricultural Chemical Exposure by MARISOL MARIA GUTIERREZ Dissertation Director: Professor Mark G. Robson It is hypothesized that atrazine acts as an endocrine disruptor in amphibians, targeting male reproduction. To determine the impact of environmental atrazine on amphibians, we undertook a comprehensive study utilizing field and laboratory experimentation. In field studies utilizing Rana catesbeiana and R. clamitans melanota, we found evidence that secondary sexual traits are altered by environmental contamination. We also found that testes weights in both bullfrogs and green frogs were reduced at sites with chemical contamination compared to reference sites. At the histological level, renal and testicular dysgenesis were more prevalent at medium-contaminated sites than low or high. Ovotestes occurred most frequently (16.7%) at a site containing atrazine. The frequencies of renal parasites increased, while metacercarial cysts decreased with site contamination. Prevalence of hepatic and renal inflammation was higher in captures from medium and high contaminated sites compared to low. Bullfrog females from high ii contamination sites had reduced hematocrit than low and medium sites. In our field locations, there was evidence of endocrine disruption in frog populations. However, atrazine did not correlate well with observed effects; methoxychlor and metolachlor were associated with various endpoints. To further identify the role of atrazine in developmental effects, we exposed Xenopus laevis tadpoles to environmental atrazine concentrations (0, 1, 5, 20, 60, 120 ppb) during their larval period. Many endpoints examined presented with non- monotonic responses. Tadpole survival was reduced only at 5 ppb atrazine. At 5 and 60 ppb atrazine, tadpoles reached metamorphosis earlier than controls. Atrazine concentrations 5, 20 and 60 ppb yielded smaller metamorphs than controls, and metamorphs from 5 ppb atrazine also had shorter body lengths, limbs and abdominal girth. Malformations of the spine and limbs were highest at 5 and 20 ppb atrazine. We also observed a lowest observable adverse effect level (LOAEL) of 1 ppb atrazine for testicular dysgenesis, which is within environmental levels and supports concerns regarding the herbicide’s ability to hinder amphibian reproductive output. Overall, our research suggests that low environmentally relevant doses of atrazine have the ability to impact wild amphibians by altering endocrine processes. iii Dedication In Your name, Father, I dedicate this work in thanksgiving to the family You have given me: Mom (Yolanda), Dad (Arnaldo), Lizzette, Arnaldo, Madeline, Ciara, Coby and Richard. I ask for continual blessings upon my family through whom You have blessed me. Amen. iv Acknowledgements There have been a number of people who have meant a great deal to me throughout this process. First, I praise You, God, and thank You for all the individuals You have placed in my path, through whom I have felt Your love and support. It is through Your grace, Father, through the authority of my Savior, Your Son, and through the power of Your Spirit that all accomplishments have been made. I ask your blessing on all those who have been a blessing to me. I ask especially for those persons and groups listed herein. First, my family: Mom (Yolanda), Dad (Arnaldo), Lizzette, Arnaldo, Madeline, Ciara, Coby, Richard and extended family too numerous to name individually. You all have supported me continually and loved me unconditionally throughout the ups and downs of this journey. A special thanks to my “cutie- patooties,” who provided sufficient doses of silliness when needed. I am deeply grateful to the congregation to which the Lord brought me at Christ United Methodist Church. I covet your fellowship and prayers. Overall, I have been warmed by the v heart that I have found in this congregation and you truly are ministers of the Good News. Pastor Tabler, you are the first to be “my pastor.” Please know that your genuine care and open honesty continue to be a treasure to me. To the youth, my 5th & 6th grade (2001-2), and 7th & 8th grade (2002-7) Sunday School classes who have given me an outlet to teach and share while keeping me humble and honest, I thank you for putting up with me. To my fellow youth leaders (Millie Grey, Karen Griffith, Lee Hecker, Christina Beck Koppes, Dave Johnson, Mel Limson, Holly Redd, Adrian Rose and Lynne Stallworth): thank you for your faith and the ways it is manifest and may you continue to bear Christ to our youth. Also, to my co-teachers who have stood beside me: Sandra Donnay, Cherise Hepburn, Tim Espar and Christy Walther; I truly appreciate your commitment to Christian education. vi To the members of the numerous Bible studies in which I have participated over the years, I am grateful that you have kept me grounded and rooted in the Lord’s Word. A warm thanks to Evelyn Arroyo, Tamar Barkay, Laurene Cholish and company, the Colby Family, the Conklin Family, Carmen Cruz & the ladies of Las Comadres, Janet Forgash, Dorcas and Danielle Helberg, Pastor Jinny Hubbard, the Kanis Family, the Latham Family, the Mart Family (including Lily), the Mayers Family, Elsa Mortensen, Bernice Pisack and Joe Alva, Esther Rivera, the Robson Family, Beth Sanford-Hart, the Schibanoff Family, Nancy Sheasley, Mrs. Glenda Smith, the Stallworth Family and Lori White. Because of your kindness, I could never call myself a “starving student.” Through the shared meals that have nourished my body and the accompanied fellowship that has nourished my heart, I have known the Father’s love and care, and am deeply grateful for your hospitality. A special thanks to the Craft Group (Joyce Beitz, Janet Forgash, Sunny Merrill, Esther Putsai, Joan Reed and Sallie Trainor) who provided both stress and comic relief. To the folks in “Prayer ‘n Praise” (Elfrita Brewster, Al Brown, Chantelle Browne, Keith Cunningham, Meleita Fletcher, Rael vii Fuertes, Toni Guerrier & family, Joan Harrigan, Verina Nicholas, Ines Piraneque, Harriet Riley, Esther Rivera, Sindy Singh, Irene Spangler, Catherine Stanford, and Pastor Tabler), I thank you for the much needed mid-week charge of Spiritual power and rest. Also, to the Rutgers Graduate Christian Fellowship (RUGCF), I would like to extend my gratitude for keeping me bound to the Word and encouraging me to continue to grow in faith throughout graduate school. May the questions never cease as you continue to wrestle with and apply the Truth. I would especially like to thank those members who have shared their faith and lives with me most deeply: Monica Babes, Jennifer Knight, Katherine Nolan, and Todd and Cari Riley. Thank you to the farmers who permitted me onto their properties to collect frogs, especially those who came out to chat with me when they saw me trudging through their property and knew I needed a break (Mr. Robert Bruch and Mr. James Durr). A special thanks to Mr. Neil Robson, who always seemed to be “just driving through” when I was hunting for frogs; your care was appreciated. Your passing has saddened many of us and you will be missed. viii Thank you to the Robson family and the Robson farm for providing a “base of operations” and incentive to collect in the form of fresh fruit and vegetables. Dr. Dave Reimer, thank you for sacrificing your rats and mice to teach me the ways to a frog’s heart and for giving up an afternoon to go on a field trip to catch frogs. To Julie Colby, Jennifer Latham and Dr. Tom Ledoux, thank you for wading in the water with me. A special thanks to Dave Golden, the Pied Piper of frogs, for putting in much effort and offering your time and expertise to make my field studies a reality. To the Cooper and White laboratories (Josie Bonventre, Sean Bugel, Ana Cardoza, Dr. Keith Cooper, Lisa Domico, Jedd Hillegass, Christina Beck Koppes, Jessica McCormick, Kyle Murphy, Ashley Pettit, Caren Villano, Dr. Lori White and Margy Wintermeyer-Takacs): thank you for allowing me to have a space, for inviting me to participate in your “festivities” and for creating enough insanity to keep me sane. A special thank you to Lisa Domico for being the catapult for my involvement with this project — thank you for paying attention! ix To all the laboratories in which I have worked on this project; your guidance, assistance and willingness to host me was greatly appreciated: The Buckley Lab for chemical analysis (Dr. Brian Buckley, Maria Haffer, Willie Johnson, Robert Stiles, Bozena Winnik, Ill Yang and Min Yoon); The John-Alder Lab for hormone assays (Robert Cox, Christine Duncan, Alison Golinski, Dr. Henry John-Alder and Lukás Kratochv); The Gordon Road Labs for histological procedures (Ollie Noddi, Dr. Ken Reuhl and Kathleen Roberts Yolinski). To Dr. Ken Reuhl, thanks for the many hours spent reading slides and leaving me with most of my vision. To Clint Burgher, Laura Comerford and staff, Dr. Mike Fennell, Linda Haberl and staff, and Elaine Simpson, thank you for the time and efforts spent in obtaining space and equipment for my animals. Thank you to those who assisted in some of the grunt work: Marija Borjan, Ladan Fakhrzadeh, Joe Gardell, Paromita Hore, Nathaniel Matthews, Christopher Pai, Tony Pawlak and Dina Jo Sison.