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Inflammatory Processes Nucleobase Deamination as a Biomarker of Inflammatory Processes by Min Dong B.S. in Pharmaceutical Science (1994) Beijing Medical University, Beijing, PR China M.S. in Biophysics (1999) Tsinghua University, Beijing, PR China SUBMITTED TO BIOLOGICAL ENGINEERING DIVISION IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THF DEGREE OF MASSACHUSETTS OF TECHNOI-OGY DOCTOR OF PHILOSOPHY FEB 2 5 at the LIBRARJIES MASSCHUSETTS INSTITUTE QF TECHNOLOGY L January 2005 © 2005 Massachusetts Institute of Technology All rights reserved Signature of Author -t oBiological EngineeringDivision January 25, 2005 Certified by Dr. Peter C. Dedon Profesr of Toxicology and Biological Engineering Am / {X7{I January 25, 2005 Acceptedby //~~~~/ ' "<~ Dr. AlanJ. Grodzinsky /m ia , Committee on Graduate Students, Biological Engineering Division AfM!CHIVES This doctoral thesis has been examined by a committee as follows: Professor Steven R. Tannenbaum Chairman / P essor Gerald N. Wogan / , Professor 'Killiam G. Thilly ...... ... ProfessofBruce Demple Dr. John S. Wishnok Professor Peter C. Dedon ............................................. Thesis Advisor -2- Nucleobase Deamination as a Biomarker of Inflammatory Processes by Min Dong SUBMITTED TO THE BIOLOGICAL ENGINEERING DIVISION IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN MOLECULAR TOXICOLOGY ABSTRACT The objective of this thesis project was to develop nucleobase deamination products as biomarkers of inflammation and to study the role of these DNA lesions in the pathophysiology of inflammation-induced carcinogenesis. The basis of this research is the epidemiological evidence that chronic inflammation is associated with an increased risk of cancer, yet the link between the inflammatory process and the development of cancer has eluded definition. Biomarker development began with the establishment of a sensitive liquid chromatography- mass spectrometry (LC-MS) method to quantify four of the nucleobase deamination products: 2'-deoxyxanthosine (dX) and 2'-deoxyoxanosine (dO) from the deamination of dG; 2'- deoxyuridine (dU) from dC; and 2'-deoxyinosine (dl) from dA. The analytical method was then validated and tested with both in vitro and in vivo studies involving quantification of nucleobase deamination products in isolated plasmid DNA and human lymphoblastoid cells exposed to nitric oxide (NO') at controlled physiological concentrations. Finally, the formation of nucleobase deamination products was analyzed in SJL/RcsX mice, an established mouse model of NO' over production. This set of studies revealed several important features of the nitrosative chemistry of NO' derivatives. The in vitro formation of dX, dl and dU was found to occur at nearly identical rates (k = 1.2 x 105M's-). Low levels of nucleobase deamination products were formed in cells exposed to NO', which suggests that cellular factors significantly influence the nitrosative chemistry. However, cellular glutathione (GSH) was found to play a smaller role than expected, considering the effects of GSH on steady-state concentrations of N203 in in vitro kinetic studies. Moderate increases (-30%) in nucleobase deamination products were observed in the SJL mice bearing the RcsX tumor, but the biological meaning of these increases awaits studies of DNA repair kinetics. dO was not detected in any study at levels above 5 per 108nt, leading to the prediction that it will not be present at significant levels in inflamed tissues in humans. -3- As a complement to the LC-MS method for the quantification of nucleobase deamination products, enzymatic probes were also developed for oxidative and nitrosative DNA lesions. These probes would not only allow differential quantification of the two types of DNA damage, but would also allow the lesions to be mapped in any DNA sequence by coupling their activity with the technique of ligation-mediated PCR. As an extension of the biomarker study, the effects of ONOO- dose and dose-rate on the DNA damage and mutations induced in the supF gene were investigated. The observations suggest that both the dose and dose-rate at which a genetic target is exposed to ONOO- substantially influence the damage and mutational response and these parameters will need to be considered in assessing the potential effects of ONOO- in vivo. Finally, an extended study using the analytical method developed in this thesis yielded results in E. coli consistent with a new paradigm: perturbations of nucleobase metabolism may lead to incorporation of the purine precursors hypoxanthine (I) and xanthine (X) into DNA. This can be regarded as another endogenous process causing DNA damage that may lead to human diseases such as cancer. In summary, this thesis work established an analytical method for the quantification of a collection of representative nucleobase deamination products. The analytical method has been applied to address a series of biologically important questions. Results obtained in this thesis contribute to the development of nucleobase deamination products as biomarkers of the inflammatory process. -4- __ ACKNOWLEDGMENTS No research endeavor is ever carried out in solitude. This thesis would not have been possible without the assistance and encouragement of many individuals to whom my deep gratitude is owed. First and foremost, I would like to thank my thesis advisor, Prof. Peter C. Dedon, for giving me an opportunity to work in his laboratory. My warmest and deepest gratitude is expressed for his scientific guidance, for his continuous support and encouragement, and for the enthusiasm and inspiration, which was always there when I needed it. His careful reviewing of draft papers and the thesis manuscript was very much appreciated. I have also been fortunate to benefit from interactions with each member of my thesis committee: Profs. Steven R. Tannenbaum, Gerald N. Wogan, Bruce Demple, William G. Thilly and Dr. John S. Wishnok. I would like to thank them for providing much appreciated insight, interest, and encouragement in this research, as well as invaluable guidance and advice on certain questions and dilemmas, which really helped shape this work into what it is now. I would also like to acknowledge the generosity of my committee members in making resources in their groups available to me. I am deeply indebted to a group of scientists for valuable contributions to this work. Dr. Chen Wang from the research group of Prof. William M. Deen contributed to the work described in Chapter 3. She was the person who developed the novel nitric oxide delivery system and performed the kinetic modeling of the in vitro formation of nucleobase deamination products. Drs. Chunqi Li, Min Young Kim and Ms. Laura J. Trudel from the research group of Prof. Gerald N. Wogan contributed the work in Chapters 4, 5 and 7. Dr. Li and Ms. Trudel provided generous assistant and valuable inputs in designing and implementing the cell experiments. Mutation study by Dr. Kim highlighted the biological significance of the DNA damage caused by peroxynitrite described in Chapter 7. Dr. Hongbin Yu from the research group of Prof. Steven R Tannenbaum kindly provided his data regarding the formation of nucleobase oxidation products for an insightful discussion of the SJL/RcsX mice study that is described in Chapter 5. For the studies described in the Appendix, Dr. Nicholas E. Burgis from the research group of Prof. Richard P. Cunningham provided the bacteria strains. Dr. Lisiane B. Meira from the research of Prof. Leona D. Samson provided the tissue sample of the Aag mice. I am also very grateful to Dr. Koli Taghizadeh and Ms. Elaine Plummer Turano from the Bioanalytical Facilities Core of the Center for Environmental Health Sciences (CEHS) at MIT. Without their expertise and generous help in using the mass spectrometry and other equipments, this thesis work could not have been carried out this smoothly. In my daily work I have been blessed with a friendly and cheerful group of fellows who have given me wonderful help during the past five years. Very special thanks go to Dr. Michael DeMott for a lot of stimulating discussions and for his tremendous amounts of time and effort devoted to reviewing and correcting a substantial portion of this thesis. Very special thanks also go to Ms. Marita Barth for her proof reading of my thesis and for other helps in various aspects. -5- Very special thanks are due to Mr. Xinfeng Zhou and Drs. Bingzi Chen, Tao Jiang and Bo Pang for sharing their ideas and expeience, and for the terridic assistance on many occasions that really expedited my progress in research. Their caring friendship throughout my graduate study will always be cherished. Special thanks go to Miss Yelena Margolin and Dr. Christiane Collins for the pleasure of being classmates and labmates with them. My specila tanks also go to Ms. Olga V. Parkin who acts as a stand-in mother figure, but of course mainly for helping out with all the things a student in our lab needs helping out with. As well as keeping me stocked with general supplies, Ms. Jacklene Goodluck Griffith has also inadvertently, and without fail, provided something much greater in all the years I've known her: a friendly smile and a hug every time we met. Ms. Debra Luchanin and Ms. Dalia Gabour, the former and current academic administrator, and Ms. Rolanda Dudley-Cowans, the administrative officer, deserved a separate acknowledgement. Their assistant and support made my life a lot easier at MIT. I would also like to thank my parents for their love and support, for instilling my love of science when I was young, and for creating an environment in which following this path seemed so natural. And last but not least Donghao, for the very special person he is, for the incredible amount of support and patience he had with me, and for being a marvelous friend whom I can always count on. This work was supported mainly by a grant from the National Cancer Institute (CA26735) and partly by the David Koch graduate fellowship in cancer research from Center of Cancer Research at MIT.
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