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The Chemistry of Nitric Oxide-Induced Deamination and Cross-Linking of DNA by Jennifer L

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The Chemistry of Nitric Oxide-Induced Deamination and Cross-Linking of DNA by Jennifer L The Chemistry of Nitric Oxide-Induced Deamination and Cross-Linking of DNA by Jennifer L. Caulfield B.S. Chemistry University of Richmond, 1991 SUBMITTED TO THE DEPARTMENT OF CHEMISTRY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY September 1997 C 1997 Massachusetts Institute of Technology All rights reserved Signature of Author I"- -0 Department of Chemistry August 21, 1997 Certified by_ 'f Steven R. Tannenbaum Professor of Chemistry and Toxicology / .. Thesis Supervisor Accepted by Dietmar Seyferth Chairman, Departmental Committee on Graduate Students ~s %~~t~ This doctoral thesis has been examined by a committee of the Department of Chemistry as follows: <2 ProfesjJ6ohn M. Essigmann Chairman (-N rofessor JiAes R. Williamson d1 V Professor GeralqWýogan The Chemistry of Nitric Oxide-Induced Deamination and Cross-Linking of DNA by Jennifer L. Caulfield SUBMITTED TO THE DEPARTMENT OF CHEMISTRY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DOCTOR OF PHILOSOPHY IN CHEMISTRY Abstract Nitric oxide has long been studied as an environmental pollutant but only recently was discovered to play a major role in mammalian physiology. It is produced by numerous cell types and serves as a neurotransmitter, vasodilator, and cytostatic/cytotoxic agent in the immune system. Along with the necessary roles of NO in vivo, it can be involved in reactions that may produce cell damage. The autoxidation of nitric oxide results in formation of the nitrosating agent N20 3 which can damage DNA directly by nitrosation of primary amines on DNA bases leading to deamination. This work aims to better understand NO--induced mutations by determining relative rates of formation of the deamination products of guanine and cytosine (xanthine and uracil) in NO-treated deoxynucleosides, single and double stranded DNA, and a G-quartet oligonucleotide. A silastic membrane delivery system was used to deliver NO- at a rate of -10 nmol/ml/min for 1 hour resulting in a final -600 uM NO2 concentration. Treated samples include: 1) the deoxynucleosides 2'-deoxycytidine & 2'-deoxyguanosine, 2) the single stranded oligomer AACCCCAA & 2'-deoxyguanosine, 3) the single stranded oligomer TGTGTGTG & 2'-deoxycytidine, 4) the G-quartet oligomer TTGGGGTT & 2'- deoxycytidine, and 5) the double stranded oligomer CGCGCGCGCGCG & 2'- deoxycytidine. Using GC/MS, xanthine formation was found to be more than twice the amount of uracil formation which may have important consequences for mechanisms of NO-induced mutations. Interestingly, single stranded oligomers were significantly more reactive toward N20 3 than deoxynucleosides. However, the double stranded oligomer was less reactive suggesting that Watson-Crick base pair formation may protect DNA from deamination in agreement with previous reports. Therefore, genome integrity may be best preserved in double stranded DNA. The reactivity of a G-quartet oligonucleotide was also decreased presumably due to hydrogen bonding. Interstrand cross-linking induced by nitric oxide was investigated using an ATATCGATCGATAT oligomer labeled with a 5'-fluorescein. After treatment with nitric oxide, a late eluting peak in the region where double stranded oligomers elute was observed using an HPLC-laser induced fluorescence (HPLC-LIF) system. Quantification demonstrated that cross-link formation is dose dependent and is formed at -6% of the amount of the deamination product xanthine. The identity of the nucleus of the interstrand cross-link has not been established but most likely matches the dG-dG interstrand cross-link previously observed upon nitrous acid treatment. During the course of DNA deamination studies, it was discovered that N- nitrosation was inhibited by NaHCO 3. This inhibition was previously shown to occur with phosphate and chloride due to anion scavenging of N20 3 by formation of nitrosyl compounds which are rapidly hydrolyzed to nitrite. An analogous reaction of bicarbonate with N20 3 is significant due to the substantial concentrations of bicarbonate in physiological fluids (up to 40 mM NaHCO 3). In order to determine the rate constant for reaction of bicarbonate with N20 3, a reactor designed for the study of nitrosation kinetics was implemented. The rate constant for the bicarbonate/N 20 3 reaction relative to that for 0 N20 3 hydrolysis was found to be 9.3 X 102 M' at pH 8.9 and 25 C suggesting a strong scavenging ability of bicarbonate. The knowledge of the bicarbonate/N 20 3 rate constant will contribute to kinetic modeling of the fate of N 20 3 specifically in the calculation of the amount of N20 3 scavenged by bicarbonate in particular systems. Thesis Supervisor: Steven R. Tannenbaum Title: Professor of Chemistry and Toxicology Acknowledgments I would first like to thank my thesis advisor Professor Steven Tannenbaum for his patience and understanding throughout my graduate work. I feel privileged to have been in this laboratory and have had the opportunity to acquire even a small fraction of his knowledge. In addition, enormous debts of gratitude go out to the numerous members of the Tannenbaum group who have shared my time here. In no particular order, special thanks to Samar Burney, Teresa Wright, Can Ozbal, Olga Parkin, Jacquin Niles, Vicky Singh, Laura Kennedy, Shawn Harriman, Snait Tamir, Tim Day, Pete Wishnok, Paul Skipper, Sara Stillwell, LaCreis Kidd, Carlton SooHoo, Koli Taghizadeh, Gerald Cozier, Teresa deRojas-Walker, Don Brunson, Ching-Hung (Gene) Hsu, Jim and Tiffany Parkin for making the past several years a great experience. Extra thanks to my lunch buddies Samar Burney and Teresa Wright for critically reading this thesis. You are all great friends and I will miss you. I have had the opportunity to work with many terrific scientists through several of my projects. The bicarbonate chapter would not have been possible without the help of Vicky Singh and Professor Bill Deen. I want to acknowledge Vicky for her development of the kinetic reactor. Bill Deen was instrumental in the analysis of bicarbonate kinetics. The deamination work required numerous hours of GC/MS analysis. Pete Wishnok was always available for questions regarding the GC/MS and LC/MS. Those questions were quite frequent. Thank you, Pete, for taking the time to teach me to clean the source and for the patience to make me learn on my own. Technical thanks should be given to Joe Glogowski for the analysis of numerous nitrite/nitrate samples. Finally, the chapter of this thesis dealing with cross-linking of DNA could never have been completed without the OZ001 laser system and the expert, Can Ozbal. Thank you for all of the laughs. I must thank all of my friends in Boston and beyond for providing me with much needed diversions. Special thanks are given to my husband, Scott, who has survived this journey with me from the start. He always (well, almost always) listened to all of my frustrations and I am forever grateful. Thank you for being so patient. The members of my family - dad, Louise, Nick, Jeff, Dominic, and Socks - have always been a tremendous support. I would never have stuck with this if it weren't for my dad, Dick Caulfield. At this point, I am grateful that you pushed me even harder than I was willing to push myself. Thank you for all of your support! Finally, this thesis is dedicated to my mother, Sharon Caulfield. I wish more than anything that she could be here to share this with me. I never would be at this point if it weren't for her and the rest of my family. I love you all. Table of Contents Title Page Abstract Acknowledgments Table of Contents Abbreviations List of Figures List of Tables Chapter 1 - Background and Literature Review Chapter 2 - Bicarbonate Inhibits N-Nitrosation in Oxygenated Nitric Oxide Solutions Chapter 3 - Nitric Oxide-Induced Deamination of Cytosine 101 and Guanine in Deoxynucleosides & Oligonucleotides Chapter 4 - Laser Detection of Nitric Oxide-Induced 133 Cross-Links in DNA Chapter 5 - Conclusions 151 Chapter 1I Background and Literature Review Nitric Oxide (NO) 1-1 Biological Importance a) Vasodilation - Endothelium Derived Relaxation Factor b) Neurotransmission c) Host Defense - Macrophages 1-2 Nitric Oxide Biosynthesis - Nitric Oxide Synthase 1-3 Reactive Oxygen Species (ROS) 1-4 Nitric Oxide Chemistry a) Diffusion of Free Nitric Oxide b) NO Reaction with Superoxide Forming Peroxynitrite c) NO Reaction with Oxygen Forming Nitrous Anhydride -Reaction of N20 3 with Biological Anions 1-5 Direct and Indirect DNA Damage by Nitric Oxide a) Oxidation and Strand Breaks by Peroxynitrite b) Endogenous Formation of N-Nitroso Compounds c) Deamination of DNA Bases by N20 3 - Additional Reaction Products d) Cross-Link Formation by N20 3 - DNA-Protein Cross-Links - DNA Intrastrand Cross-Links - DNA Interstrand Cross-Links 1-6 Nitric Oxide-Induced Mutagenicity and Cytotoxicity a) Effect of NO Delivery on Cytotoxicity & Mutagenicity Chapter 2 Bicarbonate Inhibits N-Nitrosation in Oxygenated Nitric Oxide Solutions 2-1 Abstract 2-2 Introduction 2-3 Materials and Methods a) Reagents b) Reactor c) N-Nitrosation of Morpholine d) Nitrite and N-Nitrosomorpholine Analysis e) Kinetic Model and Reaction Scheme 2-4 Results a) Morpholine Concentration and pH b) Effect of Hydroxide Ion Concentration on Nitrosation Kinetics c) Effect of Phosphate on N-Nitrosation at pH 8.9 d) Effect of Bicarbonate on N-Nitrosation at pH 8.9 2-5 Discussion 2-6 References 2-7 Figure Legends Chapter 3 Nitric Oxide-Induced Deamination of Cytosine & Guanine in Deoxynucleosides
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