Marshall University Marshall Digital Scholar Theses, Dissertations and Capstones 1-1-2006 Thimerosal-Induced Neuritoxicity: Apoptosis Occurs Through A Mitochondrial-Mediated Pathway Via the JNK Signaling Pathway Michelle L. Herdman Follow this and additional works at: http://mds.marshall.edu/etd Part of the Biological Phenomena, Cell Phenomena, and Immunity Commons, and the Genetic Phenomena Commons Recommended Citation Herdman, Michelle L., "Thimerosal-Induced Neuritoxicity: Apoptosis Occurs Through A Mitochondrial-Mediated Pathway Via the JNK Signaling Pathway" (2006). Theses, Dissertations and Capstones. Paper 638. This Dissertation is brought to you for free and open access by Marshall Digital Scholar. It has been accepted for inclusion in Theses, Dissertations and Capstones by an authorized administrator of Marshall Digital Scholar. For more information, please contact [email protected]. THIMEROSAL-INDUCED NEUROTOXICITY: APOPTOSIS OCCURS THROUGH A MITOCHONDRIAL-MEDIATED PATHWAY VIA THE JNK SIGNALING PATHWAY by Michelle L. Herdman Dissertation submitted to the Graduate College of Marshall University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences Approved by Kelley Kiningham, PhD, Committee Chairperson Gary Rankin, PhD Monica Valentovic, PhD Richard Niles, PhD Michael Moore, PhD Pharmacology, Physiology, and Toxicology i APPROVAL OF EXAMINING COMMITTEE ________________________________ Gary Rankin, PhD ________________________________ Monica Valentovic, PhD ________________________________ Richard Niles, PhD ________________________________ Michael Moore, PhD __________________ ________________________________ Date Kelley Kiningham, PhD Accepted by Graduate College ________________________________ Leonard Deutsch, PhD, Dean ________________________ Date ii ABSTRACT _______________________________________________________________________ THIMEROSAL-INDUCED NEUROTOXICITY: APOPTOSIS OCCURS THROUGH A MITOCHONDRIAL-MEDIATED PATHWAY VIA THE JNK SIGNALING PATHWAY by Michelle L. Herdman Thimerosal is an organic mercurial containing an ethylmercury moiety attached to the sulfur atom of thiosalicylate. Since the 1930s, thimerosal has been used as an antiseptic and a preservative in a wide variety of products, including medicinal preparations administered to children and pregnant women. Past exposures to mercurials have indicated that mercury is a neurotoxin, and can also affect the kidney, skin, eyes, and immune system. Additionally, fetuses exposed to mercurials are more susceptible to toxicity because the nervous system is continuously developing. However, despite its widespread use, thimerosal was only studied on a limited basis until the end of the 1990s. At that time, the use of thimerosal in vaccines began to concern parents and physicians because of its potential neurotoxicity, creating a controversy surrounding the question of safety. Consequently, studies with cell culture and animal models have begun to discern the mechanisms of toxicity of thimerosal. The present study hypothesized that thimerosal-induced toxicity occurs through the cJun N-terminal kinase (JNK)/Activator Protein-1 (AP-1) pathway. We used a human neuroblastoma cell line (SK-N-SH) as a model for neurotoxicity because it has characteristics that resemble the developing nervous system. SK-N-SH cells treated with thimerosal underwent apoptosis in a mitochondrial-dependent manner, as demonstrated by release of cytochrome c, activation of caspases 9 and 3, degradation of poly(ADP)-ribose polymerase (PARP), DNA condensation and fragmentation, along with release of lactate dehydrogenase (LDH), which occurs late in apoptosis. Thimerosal-treated cells also showed activation of the JNK pathway through increases in phosphorylation of JNK and cJun. However, despite increases in AP-1 transcriptional activity, use of a dominant negative to cJun (TAM67) showed that AP-1 activation is not essential to thimerosal- induced apoptosis. Use of a cell permeable JNK inhibitor (SP600125) demonstrated that JNK activation is a necessary component of thimerosal-induced apoptosis. An additional component of thimerosal toxicity is an increase in oxidative stress. Antioxidants were used to determine if the oxidative stress component was connected to the JNK pathway activation. The antioxidant Trolox and the glutathione precursor N- acetylcysteine (NAC) both protected the cells from apoptosis, but served to increase the phosphorylation of JNK, while still decreasing levels of the proapoptotic protein Bim. Additionally, the JNK inhibitor decreased levels of the stress-response protein heme oxygenase-1 (HO-1). These results indicate that while the oxidative stress pathway and the JNK pathway may be affected by the actions of the other, additional intermediates are involved. Taken together, these results present a significant increase in the cumulative information concerning the mechanism of thimerosal-induced neurotoxicity. By increasing the overall knowledge base, we provide targets for the development of methods to attenuate potential neurotoxicity in patients exposed to thimerosal. iii DEDICATION I would like to dedicate this work to my son, Ryan. To Ryan, I thank you for your sacrifices and patience through all of these years. I hope that you see that hard work and perseverance do pay off in the end, and I want you to know that even though my formal education has ended, I know that I will continue to learn from you each day. iv ACKNOWLEDGMENTS I would like to express my sincerest gratitude to my mentor and advisor, Dr. Kelley Kiningham. Her support, guidance, and especially her patience throughout this project have been invaluable. I would also like to thank Dr. Rankin, Dr. Valentovic, Dr. Niles, and Dr. Moore, who have provided valuable guidance and support for both this project and my education. Special thanks to Carla Cook and Susie Saunders for their help on this project. I would like to extend special thanks to Aileen Marcelo for all of her technical help, friendship, and moral support during this project. I would also like to thank my fellow students, especially Ava Dykes and Caroline Mills, for their support during my time at Marshall University. Finally, I would like to thank my family for all of the help they have given me during these years. I would not have been able to do this without their help. Thank you. v TABLE OF CONTENTS ABSTRACT .......................................................................................................................... iii DEDICATION ........................................................................................................................iv ACKNOWLEDGMENTS ......................................................................................................v TABLE OF CONTENTS......................................................................................................vi LIST OF FIGURES...............................................................................................................ix LIST OF TABLES.................................................................................................................xi LIST OF SYMBOLS / NOMENCLATURE .....................................................................xii CHAPTER I.............................................................................................................................1 INTRODUCTION......................................................................................................................1 1.1 Thimerosal and Neurotoxicity...............................................................................1 1.2 Hypothesis................................................................................................................2 1.3 Selection of Neurotoxicity Model .........................................................................2 CHAPTER II............................................................................................................................4 REVIEW OF LITERATURE ......................................................................................................4 2.1 Mercury .....................................................................................................................4 2.2 Thimerosal................................................................................................................9 2.3 cJun N-Terminal Kinase (JNK)/Activator Protein-1 (AP-1)...........................18 2.4 Oxidative Stress ....................................................................................................27 CHAPTER III.........................................................................................................................31 METHODS............................................................................................................................31 3.1 Cell Line..................................................................................................................31 3.2 Materials .................................................................................................................31 3.3 Characterization of Cell Line...............................................................................32 3.4 Protein Analysis.....................................................................................................35 3.5 Treatment of Cells with Thimerosal...................................................................37 3.6 Cell Viability Assays .............................................................................................37