OXIDATIVE AND NITROSATIVE DNA DAMAGE: OCCURRENCE, MEASUREMENT AND MECHANISM LIM KOK SEONG (B.Sc. (Hons) Pharmacy, Strathclyde, UK) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2005 FOREWORD The biggest worry that I had during the past four years while I was studying abroad is my 89-year-old grandmother. “Grandma, what would happen to me if you leave?” I tearfully asked her. “Don’t worry; I will always be there for you.” Twenty years have passed, and I had unknowingly believed in what she said since I was a child. My grandmother is the one who looked after me from the day I was born, who guided me along the way in life, who shared with me her happiness and sadness at all times, whom I relied upon when I needed someone to talk to - the very one who gave me a sense of contentment and security in life. November 2004 - it began as another ordinary month - the fortieth month after I first left home for PhD study, the time finally came for me to start preparing this thesis. Just when I kept thinking that I would soon be able to go accompany my grandmother again back home, she passed away quietly, leaving me in sorrow. Four-year is not long, but too long for her. I would like to dedicate this thesis to my grandmother who left this world on the 27th November 2004. From an ignorant child to an understanding adult, I feel blessed that I had a loving grandmother who had been my friend, my mentor and my guardian in i life. For being everything she had been and helping me to become everything I can be, she has my everlasting love. ii ACKNOWLEDGMENTS Hidden between lines and figures of this thesis is a story - a story of adventure, friendship, love, gratitude and maturation - another chapter of my book of life. The story began four years ago when I approached a laboratory labeled “Oxidant and Antioxidant Laboratory”, surveyed it from a distance and imagined how a scientist would experiment behind the blue doors. Not even knowing much about a Gilson pipette (and certainly unaware that scientific research work is very much dependent on performance of this amazing gadget), I asked the “boss” about the possibility of working in the laboratory as a student. I have been in since then. Now, four years later, this story, which has been made possible by many wonderful people behind this blueturned-brown door, is coming to its end. I would like to thank all those people who made this thesis possible and an enjoyable experience for me. My deepest gratitude to my supervisor, Prof. Barry Halliwell who has given me the chance to participate in this research project. His invaluable advice, consistent support and encouragement have made the work on this thesis an inspiring, sometimes challenging, but always interesting experience. His time, understanding and patience meant a lot to me. The journey through the graduate study has been very enriching, and I am honored for the opportunity to work in his laboratory. I am very grateful to Dr. Andrew Jenner and Ms. Huang Shan Hong for sharing their invaluable experience and advice on GC/MS. My special thanks to Mr. Wang Huansong for his technical advice on HPLC in the later part of the project, and for the iii many enjoyable and fruitful discussions and sharing of insights on life as well as research, which have made my final year in graduate study a very memorable one. I would also like to express my sincere thanks to Prof. Kandiah Jeyaseelan and Dr. Arunmozhiarasi Armugam for the pleasant discussions and continuous support while I worked on the molecular biology aspect of mitochondrial DNA. I am also grateful to Dr. Matthew Whiteman and Prof. Sit Kim Ping for their ideas and advice throughout the course of the study. My appreciation to Ms. Long Lee Hua for her help in the past few years, and all other former and current members of antioxidant laboratory as well as those at “Annex” (MD 4A) for their comradeship and assistance, especially to Dr. Peng Zhaofeng, Jia Ling, Soon Yew, Yvonne, Charmian, Yimin and Dr. Jan Gruber. My most heartfelt thanks to my beloved family for always being there when I needed them most, and never once complaining about how infrequently I visit, they deserve far more credit than I can ever give them. Their encouragement has turned my journey through graduate study into a pleasure. iv TABLE OF CONTENTS Foreword i Acknowledgments iii Table of Contents v Summary xii List of Tables xiv List of Figures xv Abbreviations xx CHAPTER INTRODUCTION 1.1 Reactive Species Derived from Oxygen and Nitrogen 1.1.1. The Chemistry of Reactive Oxygen Species 1.1.2. The Sources of Reactive Oxygen Species 1.1.3. The Chemistry of Reactive Nitrogen Species v 1.1.4. The Sources of Reactive Nitrogen Species 1.2 Oxidative and Nitrosative Damage in DNA 1.2.1. DNA damage Induced by Reactive Oxygen Species 1.2.2. DNA Damage Induced by Reactive Nitrogen Species 1.3 Mutagenicity of Oxidative and Nitrosative DNA Damage Products 14 17 1.4 Methods Used in the Quantitative Study of Oxidative and Nitrosative DNA Damage 1.5 Aims of this Study 18 22 1.5.1. Oxidative Damage in Mitochondria and Nuclei 22 1.5.2. Artefacts in the Study of Oxidative Damage in Mitochondrial DNA 24 1.5.3. Detection and Measurement of DNA Deamination Products 25 CHAPTER MATERIALS AND METHODS 28 2.1 Chemicals 28 2.2 Animals 30 2.3 Isolation of Pure Mitochondrial DNA, Nuclear DNA and Total Cellular DNA 31 2.3.1. Separation of Organelles 31 2.3.2. Isolation of DNA 32 vi 2.3.3. Separation of DNA 33 2.3.4. A Modified Method 34 2.4 Characterization of the Pure Mitochondrial DNA and Nuclear DNA 35 2.4.1. Verification of Purity of mtDNA 35 2.4.2. Restriction Digestion of DNA 36 2.4.3. Agarose Gel Electrophoresis 36 2.4.4. Quantitation of DNA 37 2.5 Isolation of Coupled Mitochondria 37 2.6 Analysis of Oxidative Damage End Products 39 2.6.1. Oxidative DNA Damage 39 2.6.1.1. DNA Sample Pooling 39 2.6.1.2. GC/MS Analysis of DNA Bases 39 2.6.2. Oxidative Protein Damage 40 2.6.2.1. Preparation of Subcellular Protein Extracts 40 2.6.2.2. Determination of Protein Carbonyl Content 41 2.6.3. Lipid Peroxidation 2.6.3.1. Determination of Malondialdehyde Content 2.6.4. Time Course Study 42 42 42 2.7 Analysis of Oxygen Consumption and H2O2 Production by Mitochondria43 2.7.1. Measurement of H2O2 Produced by Mitochondria 43 2.7.2. Oxygen Consumption by Mitochondria 44 2.7.3. H2O2 Scavenging Activity in Subcellular Compartments 44 2.8 Analysis of Nitrosative DNA Damage End Products 45 2.8.1. Enzymatic Hydrolysis of DNA 45 2.8.2. Isolation and Analysis of Nucleoside using HPLC 45 2.8.3. GC/MS Analysis of Nucleosides 46 2.8.4. In vitro DNA Deamination 47 vii 2.9 Analysis of Nitric Oxide End Products 2.9.1. Griess Assay of NO2⎯ and NO3⎯ 2.10 Statistical Analysis 47 47 48 CHAPTER ANALYSIS OF OXIDATIVE DAMAGE IN MITOCHONDRIAL AND NUCLEAR DNA 3.1 Results 49 49 3.1.1. Isolation and Characterization of Pure Mitochondrial DNA 3.1.1.1. Isolation of Pure Mitochondrial DNA 49 49 3.1.1.1.1. Separation of Organelles 51 3.1.1.1.2. Separation of DNA 53 3.1.1.1.3. A Modified Protocol 55 3.1.1.2. Characterization of Pure Mitochondrial DNA 56 3.1.1.3. Verification of Purity of the DNA 59 3.1.2. Oxidative Damage in Mitochondrial and Nuclear DNA 60 3.1.2.1. Comparison between Phenol and DNAzol Method 60 3.1.2.2. Comparison between Mitochondrial and Nuclear DNA 61 3.2 Discussion 66 viii 3.2.1. Isolation and Characterization of Pure Mitochondrial DNA 66 3.2.2. Oxidative Damage in Mitochondrial and Nuclear DNA 71 3.2.2.1. Inefficient Mitochondrial DNA Repair? 75 3.2.2.2. Ex Vivo Damage to Mitochondrial DNA? 76 3.2.2.3. In Vivo Damage to Mitochondrial DNA? 77 CHAPTER IS THERE EX VIVO PRODUCTION OF HYDROGEN PEROXIDE AND OXIDATION OF BIOMOLECULES DURING ISOLATION OF MITOCHONDRIA? 4.1 Results 80 80 4.1.1. Is there Artefactual Oxidation of DNA during Isolation? 80 4.1.2. Is there Release of H2O2 from the Rat Mitochondria during Isolation? 83 4.1.3. Is there Artefactual Oxidation of Protein and Lipid during Isolation? 97 4.1.4. Protein Carbonyl in Subcellular Compartments 4.2 Discussion 101 104 ix electrochemical detection assay of 8-oxo-deoxyguanosine and 8-oxo-guanine. Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 288-293. Henner, W. D., Rodriguez, L. O., Hecht, S. M. and Haseltine, W. A. gamma Ray induced deoxyribonucleic acid strand breaks. 3' Glycolate termini. J. Biol. Chem. 258 (1983) 711-713. Herrero, A. and Barja, G. ADP-regulation of mitochondrial free radical production is different with complex I- or complex II-linked substrates: implications for the exercise paradox and brain hypermetabolism. J. Bioenerg. Biomembr. 29 (1997a) 241-249. Herrero, A. and Barja, G. Sites and mechanisms responsible for the low rate of free radical production of heart mitochondria in the long-lived pigeon. Mech. Ageing Dev. 98 (1997b) 95-111. Higuchi, Y. and Linn, S. Purification of all forms of HeLa cell mitochondrial DNA and assessment of damage to it caused by hydrogen peroxide treatment of mitochondria or cells. J. Biol. Chem. 270 (1995) 7950-7956. Hofer, T. and Möller, L. Reduction of oxidation during the preparation of DNA and analysis of 8-hydroxy-2'-deoxyguanosine. Chem. Res. Toxicol. 11 (1998) 882-887. Horai, S. and Hayasaka, K. Intraspecific nucleotide sequence differences in the major noncoding region of human mitochondrial DNA. Am. J. Hum. Genet. 46 (1990) 828-842. Huie, R. E. and Padmaja, S. The reaction of no with superoxide. Free Radic. Res. Commun. 18 (1993) 195-199. Hutchinson, F. Chemical changes induced in DNA by ionizing radiation. Prog. Nucleic Acid Res. Mol. Biol. 32 (1985) 115-154. Inoue, S. and Kawanishi, S. Oxidative DNA damage induced by simultaneous generation of nitric oxide and superoxide. FEBS Lett. 371 (1995) 86-88. References 153 Iwai-Kanai, E., Hasegawa, K., Adachi, S., Fujita, M., Akao, M., Kawamura, T. and Kita, T. Effects of endothelin-1 on mitochondrial function during the protection against myocardial cell apoptosis. Biochem. Biophys. Res. Commun. 305 (2003) 898-903. Jaruga, P., Birincioglu, M., Rodriguez, H. and Dizdaroglu, M. Mass spectrometric assays for the tandem lesion 8,5'-cyclo-2'-deoxyguanosine in mammalian DNA. Biochemistry. 41 (2002) 3703-3711. Jaruga, P., Rodriguez, H. and Dizdaroglu, M. Measurement of 8-hydroxy-2'deoxyadenosine in DNA by liquid chromatography/mass spectrometry. Free Radic. Biol. Med. 31 (2001) 336-344. Jenner, A., England, T. G., Aruoma, O. I. and Halliwell, B. Measurement of oxidative DNA damage by gas chromatography-mass spectrometry: ethanethiol prevents artifactual generation of oxidized DNA bases. Biochem. J. 331 (1998) 365-369. Jiang, D., Hatahet, Z., Blaisdell, J. O., Melamede, R. J. and Wallace, S. S. Escherichia coli endonuclease VIII: cloning, sequencing, and overexpression of the nei structural gene and characterization of nei and nei nth mutants. J. Bacteriol. 179 (1997) 3773-3782. Kamiya, H., Miura, K., Ishikawa, H., Inoue, H., Nishimura, S. and Ohtsuka, E. cHa-ras containing 8-hydroxyguanine at codon 12 induces point mutations at the modified and adjacent positions. Cancer Res. 52 (1992) 3483-3485. Kan, W., Zhao, K. S., Jiang, Y., Yan, W., Huang, Q., Wang, J., Qin, Q., Huang, X. and Wang, S. Lung, spleen, and kidney are the major places for inducible nitric oxide synthase expression in endotoxic shock: role of p38 mitogenactivated protein kinase in signal transduction of inducible nitric oxide synthase expression. Shock 21 (2004) 281-287. Karran, P. and Lindahl, T. Enzymatic excision of free hypoxanthine from polydeoxynucleotides and DNA containing deoxyinosine monophosphate residues. J. Biol. Chem. 253 (1978) 5877-5879. Karran, P. and Lindahl, T. Hypoxanthine in deoxyribonucleic acid: generation by heat-induced hydrolysis of adenine residues and release in free form by a deoxyribonucleic acid glycosylase from calf thymus. Biochemistry. 19 (1980) 6005-6011. References 154 Kennedy, L. J., Moore, K., Jr., Caulfield, J. L., Tannenbaum, S. R. and Dedon, P. C. Quantitation of 8-oxoguanine and strand breaks produced by four oxidizing agents. Chem. Res. Toxicol. 10 (1997) 386-392. Koppenol, W. H. Chemistry of peroxynitrite and its relevance to biological systems. Met. Ions Biol. Syst. 36 (1999) 597-619. Korshunov, S. S., Skulachev, V. P. and Starkov, A. A. High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 416 (1997) 15-18. Kouchakdjian, M., Bodepudi, V., Shibutani, S., Eisenberg, M., Johnson, F., Grollman, A. P. and Patel, D. J. NMR structural studies of the ionizing radiation adduct 7-hydro-8-oxodeoxyguanosine (8-oxo-7H-dG) opposite deoxyadenosine in a DNA duplex. 8-Oxo-7H-dG(syn).dA(anti) alignment at lesion site. Biochemistry. 30 (1991) 1403-1412. Kuo, K. C., McCune, R. A., Gehrke, C. W., Midgett, R. and Ehrlich, M. Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA. Nucleic Acids Res (1980) 4763-4776. Kushnareva, Y., Murphy, A. N. and Andreyev, A. Complex I-mediated reactive oxygen species generation: modulation by cytochrome c and NAD(P)+ oxidation-reduction state. Biochem. J. 368 (2002) 545-553. Kwong, L. K. and Sohal, R. S. Age-related changes in activities of mitochondrial electron transport complexes in various tissues of the mouse. Arch. Biochem. Biophys. 373 (2000) 16-22. Kwong, L. K. and Sohal, R. S. Tissue-specific mitochondrial production of H2O2: its dependence on substrates and sensitivity to inhibitors. Methods Enzymol. 349 (2002) 341-346. Lakings, D. B. and Gehrke, C. W. Analysis of base composition of RNA and DNA hydrolysates by gas-liquid chromatography. J. Chromatogr. 62 (1971) 347367. References 155 Lanni, A., Moreno, M., Lombardi, A. and Goglia, F. Biochemical and functional differences in rat liver mitochondrial subpopulations obtained at different gravitational forces. Int. J. Biochem. Cell Biol. 28 (1996) 337-343. LeDoux, S. P., Patton, N. J., Avery, L. J. and Wilson, G. L. Repair of Nmethylpurines in the mitochondrial DNA of xeroderma pigmentosum complementation group D cells. Carcinogenesis 14 (1993) 913-917. LeDoux, S. P., Wilson, G. L., Beecham, E. J., Stevnsner, T., Wassermann, K. and Bohr, V. A. Repair of mitochondrial DNA after various types of DNA damage in Chinese hamster ovary cells. Carcinogenesis 13 (1992) 19671973. Levine, R. L., Garland, D., Oliver, C. N., Amici, A., Climent, I., Lenz, A. G., Ahn, B. W., Shaltiel, S. and Stadtman, E. R. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol. 186 (1990) 464-478. Lewis, R. S., Tamir, S., Tannenbaum, S. R. and Deen, W. M. Kinetic analysis of the fate of nitric oxide synthesized by macrophages in vitro. J. Biol. Chem. 270 (1995) 29350-29355. Li, C. Q., Wright, T. L., Dong, M., Dommels, Y. E., Trudel, L. J., Dedon, P. C., Tannenbaum, S. R. and Wogan, G. N. Biological role of glutathione in nitric oxide-induced toxicity in cell culture and animal models. Free Radic. Biol. Med. 39 (2005) 1489-1498. Licht, W. R., Tannenbaum, S. R. and Deen, W. M. Use of ascorbic acid to inhibit nitrosation: kinetic and mass transfer considerations for an in vitro system. Carcinogenesis (1988) 365-372. Lindahl, T. and Nyberg, B. Heat-induced deamination of cytosine residues in deoxyribonucleic acid. Biochemistry. 13 (1974) 3405-3410. Liu, J., Head, E., Gharib, A. M., Yuan, W., Ingersoll, R. T., Hagen, T. M., Cotman, C. W. and Ames, B. N. Memory loss in old rats is associated with brain mitochondrial decay and RNA/DNA oxidation: partial reversal by feeding acetyl-L-carnitine and/or R-alpha -lipoic acid. Proc. Natl. Acad. Sci. U. S. A. 99 (2002a) 2356-2361. References 156 Liu, R. H., Baldwin, B., Tennant, B. C. and Hotchkiss, J. H. Elevated formation of nitrate and N-nitrosodimethylamine in woodchucks (Marmota monax) associated with chronic woodchuck hepatitis virus infection. Cancer Res. 51 (1991) 3925-3929. Liu, R. H., Jacob, J. R., Tennant, B. C. and Hotchkiss, J. H. Nitrite and nitrosamine synthesis by hepatocytes isolated from normal woodchucks (Marmota monax) and woodchucks chronically infected with woodchuck hepatitis virus. Cancer Res. 52 (1992) 4139-4143. Liu, S., Adcock, I. M., Old, R. W., Barnes, P. J. and Evans, T. W. Lipopolysaccharide treatment in vivo induces widespread tissue expression of inducible nitric oxide synthase mRNA. Biochem. Biophys. Res. Commun. 196 (1993) 1208-1213. Liu, Y., Fiskum, G. and Schubert, D. Generation of reactive oxygen species by the mitochondrial electron transport chain. J. Neurochem. 80 (2002b) 780-787. Loeb, L. A. and Preston, B. D. Mutagenesis by apurinic/apyrimidinic sites. Annu. Rev. Genet. 20 (1986) 201-230. Lopez-Mediavilla, C., Orfao, A., Gonzalez, M. and Medina, J. M. Identification by flow cytometry of two distinct rhodamine-123-stained mitochondrial populations in rat liver. FEBS Lett. 254 (1989) 115-120. Lopez-Mediavilla, C., Orfao, A., San Miguel, J. and Medina, J. M. Developmental changes in rat liver mitochondrial populations analyzed by flow cytometry. Exp. Cell Res. 203 (1992) 134-140. Loschen, G., Flohe, L. and Chance, B. Respiratory chain linked H(2)O(2) production in pigeon heart mitochondria. FEBS Lett. 18 (1971) 261-264. Lyras, L., Cairns, N. J., Jenner, A., Jenner, P. and Halliwell, B. An assessment of oxidative damage to proteins, lipids, and DNA in brain from patients with Alzheimer's disease. J. Neurochem. 68 (1997) 2061-2069. Lyras, L., Perry, R. H., Perry, E. K., Ince, P. G., Jenner, A., Jenner, P. and Halliwell, B. Oxidative damage to proteins, lipids, and DNA in cortical brain regions References 157 from patients with dementia with Lewy bodies. J. Neurochem. 71 (1998) 302-312. Marletta, M. A. Mammalian synthesis of nitrite, nitrate, nitric oxide, and Nnitrosating agents. Chem. Res. Toxicol. (1988) 249-257. McCloskey, J. A. Electron ionization mass spectra of trimethylsilyl derivatives of nucleosides. Methods Enzymol. 193 (1990) 825-842. Mela, L. and Seitz, S. Isolation of mitochondria with emphasis on heart mitochondria from small amounts of tissue. Methods Enzymol. 55 (1979) 3946. Melov, S., Hertz, G. Z., Stormo, G. D. and Johnson, T. E. Detection of deletions in the mitochondrial genome of Caenorhabditis elegans. Nucleic Acids Res 22 (1994) 1075-1078. Miller, V., Pacakova, V. and Smolkova, E. Gas-liquid chromatographic analysis of trimethylsilyl derivatives of pyrimidine and purine bases and nucleosides. J. Chromatogr. 119 (1976) 355-367. Mitchell, P. Vectorial chemiosmotic processes. Annu. Rev. Biochem. 46 (1977) 9961005. Miwa, S. and Brand, M. D. Mitochondrial matrix reactive oxygen species production is very sensitive to mild uncoupling. Biochem. Soc. Trans. 31 (2003) 1300-1301. Moriya, M., Ou, C., Bodepudi, V., Johnson, F., Takeshita, M. and Grollman, A. P. Site-specific mutagenesis using a gapped duplex vector: a study of translesion synthesis past 8-oxodeoxyguanosine in E. coli. Mutat. Res. 254 (1991) 281-288. Myrnes, B., Guddal, P. H. and Krokan, H. Metabolism of dITP in HeLa cell extracts, incorporation into DNA by isolated nuclei and release of hypoxanthine from DNA by a hypoxanthine-DNA glycosylase activity. Nucleic Acids Res 10 (1982) 3693-3701. References 158 Nakae, D., Mizumoto, Y., Kobayashi, E., Noguchi, O. and Konishi, Y. Improved genomic/nuclear DNA extraction for 8-hydroxydeoxyguanosine analysis of small amounts of rat liver tissue. Cancer Lett. 97 (1995) 233-239. Nedergaard, J. and Cannon, B. Overview--preparation and properties of mitochrondria from different sources. Methods Enzymol. 55 (1979) 3-28. Nguyen, T., Brunson, D., Crespi, C. L., Penman, B. W., Wishnok, J. S. and Tannenbaum, S. R. DNA damage and mutation in human cells exposed to nitric oxide in vitro. Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 3030-3034. Nicholls, D. G. and Locke, R. M. Thermogenic mechanisms in brown fat. Physiol. Rev. 64 (1984) 1-64. Nohl, H., Gille, L., Kozlov, A. and Staniek, K. Are mitochondria a spontaneous and permanent source of reactive oxygen species? Redox Rep (2003) 135-141. Nohl, H. and Hegner, D. Evidence for the existence of catalase in the matrix space of rat-heart mitochondria. FEBS Lett. 89 (1978) 126-130. Noji, H. and Yoshida, M. The rotary machine in the cell, ATP synthase. J. Biol. Chem. 276 (2001) 1665-1668. Ohshima, H., Tatemichi, M. and Sawa, T. Chemical basis of inflammation-induced carcinogenesis. Arch. Biochem. Biophys. 417 (2003) 3-11. Olinski, R., Zastawny, T., Budzbon, J., Skokowski, J., Zegarski, W. and Dizdaroglu, M. DNA base modifications in chromatin of human cancerous tissues. FEBS Lett. 309 (1992) 193-198. Packer, L., Pollak, J. K., Munn, E. A. and Greville, G. D. Effect of high sucrose concentrations on mitochondria: analysis of mitochondrial populations by density-gradient centrifugation after fixation with glutaraldehyde. J. Bioenerg. (1971) 305-316. Pflaum, M., Will, O. and Epe, B. Determination of steady-state levels of oxidative DNA base modifications in mammalian cells by means of repair endonucleases. Carcinogenesis 18 (1997) 2225-2231. References 159 Phillips, D. H. Detection of DNA modifications by the 32P-postlabelling assay. Mutat. Res. 378 (1997) 1-12. Pleshakova, O. V., Kutsyi, M. P., Sukharev, S. A., Sadovnikov, V. B. and Gaziev, A. I. Study of protein carbonyls in subcellular fractions isolated from liver and spleen of old and gamma-irradiated rats. Mech. Ageing Dev. 103 (1998) 4555. Pollak, J. K. The maturation of the inner membrane of foetal rat liver mitochondria. Biochem. J. 150 (1975) 477-488. Pryor, W. A. and Squadrito, G. L. The chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide. Am. J. Physiol. 268 (1995) L699722. Purmal, A. A., Kow, Y. W. and Wallace, S. S. Major oxidative products of cytosine, 5-hydroxycytosine and 5-hydroxyuracil, exhibit sequence context-dependent mispairing in vitro. Nucleic Acids Res 22 (1994) 72-78. Purmal, A. A., Lampman, G. W., Bond, J. P., Hatahet, Z. and Wallace, S. S. Enzymatic processing of uracil glycol, a major oxidative product of DNA cytosine. J. Biol. Chem. 273 (1998) 10026-10035. Qu, B., Li, Q. T., Wong, K. P., Ong, C. N. and Halliwell, B. Mitochondrial damage by the "pro-oxidant" peroxisomal proliferator clofibrate. Free Radic. Biol. Med. 27 (1999) 1095-1102. Radi, R., Turrens, J. F., Chang, L. Y., Bush, K. M., Crapo, J. D. and Freeman, B. A. Detection of catalase in rat heart mitochondria. J. Biol. Chem. 266 (1991) 22028-22034. Ravanat, J. L., Duretz, B., Guiller, A., Douki, T. and Cadet, J. Isotope dilution highperformance liquid chromatography-electrospray tandem mass spectrometry assay for the measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine in biological samples. J Chromatogr B Biomed Sci Appl 715 (1998) 349-356. Rehman, A., Nourooz-Zadeh, J., Möller, W., Tritschler, H., Pereira, P. and Halliwell, B. Increased oxidative damage to all DNA bases in patients with type II diabetes mellitus. FEBS Lett. 448 (1999) 120-122. References 160 Reinhart, P. H., Taylor, W. M. and Bygrave, F. L. A procedure for the rapid preparation of mitochondria from rat liver. Biochem. J. 204 (1982) 731-735. Richter, C., Park, J. W. and Ames, B. N. Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 6465-6467. Rivett, A. J. Preferential degradation of the oxidatively modified form of glutamine synthetase by intracellular mammalian proteases. J. Biol. Chem. 260 (1985a) 300-305. Rivett, A. J. Purification of a liver alkaline protease which degrades oxidatively modified glutamine synthetase. Characterization as a high molecular weight cysteine proteinase. J. Biol. Chem. 260 (1985b) 12600-12606. Roseman, J. E. and Levine, R. L. Purification of a protease from Escherichia coli with specificity for oxidized glutamine synthetase. J. Biol. Chem. 262 (1987) 2101-2110. Salazar, J. J. and Van Houten, B. Preferential mitochondrial DNA injury caused by glucose oxidase as a steady generator of hydrogen peroxide in human fibroblasts. Mutat. Res. 385 (1997) 139-149. Sambrook, J. and Russell, D. W. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press, New York, 2001 Saparbaev, M. and Laval, J. Excision of hypoxanthine from DNA containing dIMP residues by the Escherichia coli, yeast, rat, and human alkylpurine DNA glycosylases. Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 5873-5877. Saparbaev, M., Mani, J. C. and Laval, J. Interactions of the human, rat, Saccharomyces cerevisiae and Escherichia coli 3-methyladenine-DNA glycosylases with DNA containing dIMP residues. Nucleic Acids Res 28 (2000) 1332-1339. Senturker, S. and Dizdaroglu, M. The effect of experimental conditions on the levels of oxidatively modified bases in DNA as measured by gas chromatographymass spectrometry: how many modified bases are involved? Prepurification or not? Free Radic. Biol. Med. 27 (1999) 370-380. References 161 Shafirovich, V., Cadet, J., Gasparutto, D., Dourandin, A. and Geacintov, N. E. Nitrogen dioxide as an oxidizing agent of 8-oxo-7,8-dihydro-2'deoxyguanosine but not of 2'-deoxyguanosine. Chem. Res. Toxicol. 14 (2001) 233-241. Shafirovich, V., Mock, S., Kolbanovskiy, A. and Geacintov, N. E. Photochemically catalyzed generation of site-specific 8-nitroguanine adducts in DNA by the reaction of long-lived neutral guanine radicals with nitrogen dioxide. Chem. Res. Toxicol. 15 (2002) 591-597. Shapiro, R. and Klein, R. S. The deamination of cytidine and cytosine by acidic buffer solutions. Mutagenic implications. Biochemistry. (1966) 2358-2362. Shapiro, R. and Yamaguchi, H. Nucleic acid reactivity and conformation. I. Deamination of cytosine by nitrous acid. Biochim. Biophys. Acta 281 (1972) 501-506. Shibutani, S., Takeshita, M. and Grollman, A. P. Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature 349 (1991) 431-434. Shigenaga, M. K., Hagen, T. M. and Ames, B. N. Oxidative damage and mitochondrial decay in aging. Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 10771-10778. Simonetti, S., Chen, X., DiMauro, S. and Schon, E. A. Accumulation of deletions in human mitochondrial DNA during normal aging: analysis by quantitative PCR. Biochim. Biophys. Acta 1180 (1992) 113-122. Skulachev, V. P. Role of uncoupled and non-coupled oxidations in maintenance of safely low levels of oxygen and its one-electron reductants. Q. Rev. Biophys. 29 (1996) 169-202. Soong, N. W., Hinton, D. R., Cortopassi, G. and Arnheim, N. Mosaicism for a specific somatic mitochondrial DNA mutation in adult human brain. Nat. Genet. (1992) 318-323. Spencer, J. P., Jenner, A., Chimel, K., Aruoma, O. I., Cross, C. E., Wu, R. and Halliwell, B. DNA damage in human respiratory tract epithelial cells: References 162 damage by gas phase cigarette smoke apparently involves attack by reactive nitrogen species in addition to oxygen radicals. FEBS Lett. 375 (1995) 179182. Spencer, J. P., Whiteman, M., Jenner, A. and Halliwell, B. Nitrite-induced deamination and hypochlorite-induced oxidation of DNA in intact human respiratory tract epithelial cells. Free Radic. Biol. Med. 28 (2000) 1039-1050. Spencer, J. P., Wong, J., Jenner, A., Aruoma, O. I., Cross, C. E. and Halliwell, B. Base modification and strand breakage in isolated calf thymus DNA and in DNA from human skin epidermal keratinocytes exposed to peroxynitrite or 3-morpholinosydnonimine. Chem. Res. Toxicol. (1996) 1152-1158. St-Pierre, J., Brand, M. D. and Boutilier, R. G. Mitochondria as ATP consumers: cellular treason in anoxia. Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 86708674. St-Pierre, J., Buckingham, J. A., Roebuck, S. J. and Brand, M. D. Topology of superoxide production from different sites in the mitochondrial electron transport chain. J. Biol. Chem. 277 (2002) 44784-44790. Stadtman, E. R. Protein oxidation and aging. Science 257 (1992) 1220-1224. Stadtman, E. R. Protein oxidation in aging and age-related diseases. Ann. N. Y. Acad. Sci. 928 (2001) 22-38. Stadtman, E. R. and Levine, R. L. Protein oxidation. Ann. N. Y. Acad. Sci. 899 (2000) 191-208. Stadtman, E. R. and Oliver, C. N. Metal-catalyzed oxidation of proteins. Physiological consequences. J. Biol. Chem. 266 (1991) 2005-2008. Staniek, K. and Nohl, H. H(2)O(2) detection from intact mitochondria as a measure for one-electron reduction of dioxygen requires a non-invasive assay system. Biochim. Biophys. Acta 1413 (1999) 70-80. Staniek, K. and Nohl, H. Are mitochondria a permanent source of reactive oxygen species? Biochim. Biophys. Acta 1460 (2000) 268-275. References 163 Starke-Reed, P. E. and Oliver, C. N. Protein oxidation and proteolysis during aging and oxidative stress. Arch. Biochem. Biophys. 275 (1989) 559-567. Starke, P. E. and Farber, J. L. Ferric iron and superoxide ions are required for the killing of cultured hepatocytes by hydrogen peroxide. Evidence for the participation of hydroxyl radicals formed by an iron-catalyzed Haber-Weiss reaction. J. Biol. Chem. 260 (1985) 10099-10104. Starkov, A. A. and Fiskum, G. Myxothiazol induces H(2)O(2) production from mitochondrial respiratory chain. Biochem. Biophys. Res. Commun. 281 (2001) 645-650. Steenken, S. Purine bases, nucleosides, and nucleotides: aqueous solution redox chemistry and transformation reactions of their radical cations and e- and OH adducts. Chem. Rev. 89 (1989) 503-520. Stuart, J. A., Brindle, K. M., Harper, J. A. and Brand, M. D. Mitochondrial proton leak and the uncoupling proteins. J. Bioenerg. Biomembr. 31 (1999) 517-525. Suter, M. and Richter, C. Fragmented mitochondrial DNA is the predominant carrier of oxidized DNA bases. Biochemistry. 38 (1999) 459-464. Taffe, B. G., Larminat, F., Laval, J., Croteau, D. L., Anson, R. M. and Bohr, V. A. Gene-specific nuclear and mitochondrial repair of formamidopyrimidine DNA glycosylase-sensitive sites in Chinese hamster ovary cells. Mutat. Res. 364 (1996) 183-192. Talbot, D. A., Lambert, A. J. and Brand, M. D. Production of endogenous matrix superoxide from mitochondrial complex I leads to activation of uncoupling protein 3. FEBS Lett. 556 (2004) 111-115. Teebor, G. W., Frenkel, K. and Goldstein, M. S. Ionizing radiation and tritium transmutation both cause formation of 5-hydroxymethyl-2'-deoxyuridine in cellular DNA. Proc. Natl. Acad. Sci. U. S. A. 81 (1984) 318-321. Thorslund, T., Sunesen, M., Bohr, V. A. and Stevnsner, T. Repair of 8-oxoG is slower in endogenous nuclear genes than in mitochondrial DNA and is without strand bias. DNA Repair (2002) 261-273. References 164 Toyokuni, S., Mori, T. and Dizdaroglu, M. DNA base modifications in renal chromatin of Wistar rats treated with a renal carcinogen, ferric nitrilotriacetate. Int. J. Cancer 57 (1994) 123-128. Tretyakova, N. Y., Burney, S., Pamir, B., Wishnok, J. S., Dedon, P. C., Wogan, G. N. and Tannenbaum, S. R. Peroxynitrite-induced DNA damage in the supF gene: correlation with the mutational spectrum. Mutat. Res. 447 (2000) 287303. Turrens, J. F., Alexandre, A. and Lehninger, A. L. Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria. Arch. Biochem. Biophys. 237 (1985) 408-414. Turrens, J. F. and Boveris, A. Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem. J. 191 (1980) 421427. Turrens, J. F., Freeman, B. A. and Crapo, J. D. Hyperoxia increases H2O2 release by lung mitochondria and microsomes. Arch. Biochem. Biophys. 217 (1982a) 411-421. Turrens, J. F., Freeman, B. A., Levitt, J. G. and Crapo, J. D. The effect of hyperoxia on superoxide production by lung submitochondrial particles. Arch. Biochem. Biophys. 217 (1982b) 401-410. Uchida, K., Kato, Y. and Kawakishi, S. A novel mechanism for oxidative cleavage of prolyl peptides induced by the hydroxyl radical. Biochem. Biophys. Res. Commun. 169 (1990) 265-271. Uchida, K. and Stadtman, E. R. Covalent attachment of 4-hydroxynonenal to glyceraldehyde-3-phosphate dehydrogenase. A possible involvement of intra- and intermolecular cross-linking reaction. J. Biol. Chem. 268 (1993) 6388-6393. Uppu, R. M., Squadrito, G. L. and Pryor, W. A. Acceleration of peroxynitrite oxidations by carbon dioxide. Arch. Biochem. Biophys. 327 (1996) 335-343. References 165 Venditti, P., Costagliola, I. R. and Di Meo, S. H2O2 production and response to stress conditions by mitochondrial fractions from rat liver. J. Bioenerg. Biomembr. 34 (2002) 115-125. Vignais, P. V. The superoxide-generating NADPH oxidase: structural aspects and activation mechanism. Cell. Mol. Life Sci. 59 (2002) 1428-1459. von Sonntag, C. The Chemical Basis of Radiation Biology. Taylor and Francis, New York, 1987 Wallace, D. C., Ye, J. H., Neckelmann, S. N., Singh, G., Webster, K. A. and Greenberg, B. D. Sequence analysis of cDNAs for the human and bovine ATP synthase beta subunit: mitochondrial DNA genes sustain seventeen times more mutations. Curr. Genet. 12 (1987) 81-90. Wallace, S. S. Biological consequences of free radical-damaged DNA bases. Free Radic. Biol. Med. 33 (2002) 1-14. Watson, J. T. Selected-ion measurements. Methods Enzymol. 193 (1990) 86-106. Weinfeld, M. and Soderlind, K. J. 32P-postlabeling detection of radiation-induced DNA damage: identification and estimation of thymine glycols and phosphoglycolate termini. Biochemistry. 30 (1991) 1091-1097. Whiteman, M., Spencer, J. P., Jenner, A. and Halliwell, B. Hypochlorous acidinduced DNA base modification: potentiation by nitrite: biomarkers of DNA damage by reactive oxygen species. Biochem. Biophys. Res. Commun. 257 (1999) 572-576. Wink, D. A. and Ford, P. C. Nitric oxide reactions important to biological systems: A survey of some kinetics investigations. Methods: A Companion to Methods Enzymol. (1995) 14-20. Wink, D. A., Hanbauer, I., Grisham, M. B., Laval, F., Nims, R. W., Laval, J., Cook, J., Pacelli, R., Liebmann, J., Krishna, M., Ford, P. C. and Mitchell, J. B. Chemical biology of nitric oxide: regulation and protective and toxic mechanisms. Curr. Top. Cell. Regul. 34 (1996) 159-187. References 166 Wink, D. A., Kasprzak, K. S., Maragos, C. M., Elespuru, R. K., Misra, M., Dunams, T. M., Cebula, T. A., Koch, W. H., Andrews, A. W., Allen, J. S. and Keefer, L. K. DNA deaminating ability and genotoxicity of nitric oxide and its progenitors. Science 254 (1991) 1001-1003. Wink, D. A. and Mitchell, J. B. Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic. Biol. Med. 25 (1998) 434-456. Wink, D. A., Nims, R. W., Darbyshire, J. F., Christodoulou, D., Hanbauer, I., Cox, G. W., Laval, F., Laval, J., Cook, J. A., Krishna, M. C. and et al. Reaction kinetics for nitrosation of cysteine and glutathione in aerobic nitric oxide solutions at neutral pH. Insights into the fate and physiological effects of intermediates generated in the NO/O2 reaction. Chem. Res. Toxicol. (1994) 519-525. Wood, M. L., Dizdaroglu, M., Gajewski, E. and Essigmann, J. M. Mechanistic studies of ionizing radiation and oxidative mutagenesis: genetic effects of a single 8-hydroxyguanine (7-hydro-8-oxoguanine) residue inserted at a unique site in a viral genome. Biochemistry. 29 (1990) 7024-7032. Wu, J. C., Kozarich, J. W. and Stubbe, J. Mechanism of bleomycin: evidence for a rate-determining 4'-hydrogen abstraction from poly(dA-dU) associated with the formation of both free base and base propenal. Biochemistry. 24 (1985) 7562-7568. Yakes, F. M. and Van Houten, B. Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 514-519. Yao, M., Hatahet, Z., Melamede, R. J. and Kow, Y. W. Purification and characterization of a novel deoxyinosine-specific enzyme, deoxyinosine 3' endonuclease, from Escherichia coli. J. Biol. Chem. 269 (1994) 16260-16268. Yao, M. and Kow, Y. W. Interaction of deoxyinosine 3'-endonuclease from Escherichia coli with DNA containing deoxyinosine. J. Biol. Chem. 270 (1995) 28609-28616. Yermilov, V., Rubio, J., Becchi, M., Friesen, M. D., Pignatelli, B. and Ohshima, H. Formation of 8-nitroguanine by the reaction of guanine with peroxynitrite in vitro. Carcinogenesis 16 (1995a) 2045-2050. References 167 Yermilov, V., Rubio, J. and Ohshima, H. Formation of 8-nitroguanine in DNA treated with peroxynitrite in vitro and its rapid removal from DNA by depurination. FEBS Lett. 376 (1995b) 207-210. Yermilov, V., Yoshie, Y., Rubio, J. and Ohshima, H. Effects of carbon dioxide/bicarbonate on induction of DNA single-strand breaks and formation of 8-nitroguanine, 8-oxoguanine and base-propenal mediated by peroxynitrite. FEBS Lett. 399 (1996) 67-70. Yin, B., Whyatt, R. M., Perera, F. P., Randall, M. C., Cooper, T. B. and Santella, R. M. Determination of 8-hydroxydeoxyguanosine by an immunoaffinity chromatography-monoclonal antibody-based ELISA. Free Radic. Biol. Med. 18 (1995) 1023-1032. Zastawny, T. H., Dabrowska, M., Jaskolski, T., Klimarczyk, M., Kulinski, L., Koszela, A., Szczesniewicz, M., Sliwinska, M., Witkowski, P. and Olinski, R. Comparison of oxidative base damage in mitochondrial and nuclear DNA. Free Radic. Biol. Med. 24 (1998) 722-725. Zhang, C., Walker, L. M. and Mayeux, P. R. Role of nitric oxide in lipopolysaccharide-induced oxidant stress in the rat kidney. Biochem. Pharmacol. 59 (2000) 203-209. Zhao, K., Whiteman, M., Spencer, J. P. and Halliwell, B. DNA damage by nitrite and peroxynitrite: protection by dietary phenols. Methods Enzymol. 335 (2001) 296-307. References 168 [...]... study DNA damage The theory that mtDNA is more heavily damaged than nDNA therefore does not stand on firm ground As mitochondrial DNA constitutes only 1% of the total cellular DNA, we have, in this study, obtained mtDNA of desired purity in order to make a valid comparison of oxidative damage between mtDNA and nDNA Our data show that a possibility exists that mtDNA damage is not higher than nDNA damage, ... preparations 5-formyl uracil and 2-OH adenine were present at levels that are too low for detection 60 Table 2 Oxidative base damage products in mtDNA and nDNA isolated from rat liver tissue using modified DNAzol method Approximately 80 rats were sacrificed, the livers were removed for DNA extraction, and nDNA and pure mtDNA samples from 5-8 rats were pooled to give 11 DNA samples for analysis... 10255, 11065 and 11230 (B) MtDNA restriction fragments on 0.5% (w/v) agarose gel stained with ethidium bromide 58 Figure 12 nDNA (n1-6) and mtDNA (mt1-6) isolated from rat liver using the modified DNAzol method were used as templates for primers specific to the cytochrome b gene and the β-actin gene nDNA preparations contain both nDNA and mtDNA whereas mtDNA preparations contain only mtDNA ... distinct bands of DNA formed – upper (U) & lower (L) band These fractions, together with the fraction between the 2 bands (M), were used as templates for primers specific to the cytochrome b (a) and the β-actin gene (b) 54 Figure 9 Total cellular DNA was isolated using phenol method from rat liver and mtDNA separated by CsCl continuous gradient centrifugation One distinct band of DNA formed and was... previous studies on oxidative damage in mitochondrial and nuclear DNA 67 Table 5 Comparison of levels of 8-OHdG or 8-OH Guanine in mtDNA and nDNA from several studies Conversion factor used is 1nmol of 8-OHdG/mg DNA equals to 318 8-OHdG/106 DNA bases (Halliwell, 1999) 74 Table 6 Comparison of 2’-deoxyinosine levels in DNA from 2 organs in both control and LPS-treated rats... ions of (A) Fapy Guanine and (B) 8-OH Guanine in mtDNA and nDNA 62 Figure 14 Restriction fragments of nDNA, cmDNA and mtDNA on 0.5% (w/v) agarose gel stained with ethidium bromide 63 xv Figure 15 Gel electrophoresis of total cellular DNA isolated from rat liver Liver tissue homogenate was incubated for the time periods shown before DNA isolation and gel electrophoresis... 8-OH Guanine in nDNA than in mtDNA Three other lesions – Fapy Guanine, Fapy Adenine and 5-OH Cytosine were found to be statistically significantly higher in nDNA than in mtDNA In view of the great variation among the reported damage levels, several possibilities of ex vivo artefactual oxidation have been suggested to explain the observed high levels of damage in mtDNA if the mtDNA damage has been overestimated... two independent experiments 5-Formyl Uracil and 2-OH Adenine were present at levels that were too low for detection 61 Table 3 Oxidative base damage products in pure mtDNA, cmDNA and nDNA isolated from rat liver tissue using modified DNAzol method Each data point represents the mean ± sd for at least five independent preparations 5-formyl uracil and 2-OH adenine were present at levels that... Sonntag, 1987) and further reactions generate a variety of deoxyribose breakdown products, base loss and strand breaks (von Sonntag, 1987) These damages have been shown to accumulate during exposure of bacteria and mammalian cells to H2O2, O•⎯⎯, gamma 2 radiation or ozone (Birnboim and Kanabus-Kaminska, 1985; de Mello Filho and Meneghini, 1985) Unlike •OH, O• ⎯ and H2O2 per se do not react with DNA at 2... iron-nitrosyl-hemoglobin (Doyle et al., 1981) Under oxygenated conditions, however, nitrite is oxidized to nitrate by oxyhemoglobin Chapter 1 Introduction 7 1.2 Oxidative and Nitrosative Damage in DNA 1.2.1 DNA damage Induced by Reactive Oxygen Species Oxygen radicals may attack DNA at either the sugar or the base, giving rise to a large number of products (Hutchinson, 1985) Attack on deoxyribose by •OH can abstract a . 1.2 Oxidative and Nitrosative Damage in DNA 8 1.2.1. DNA damage Induced by Reactive Oxygen Species 8 1.2.2. DNA Damage Induced by Reactive Nitrogen Species 14 1.3 Mutagenicity of Oxidative and. Oxidative and Nitrosative DNA Damage Products 17 1.4 Methods Used in the Quantitative Study of Oxidative and Nitrosative DNA Damage 18 1.5 Aims of this Study 22 1.5.1. Oxidative Damage in Mitochondria. mtDNA of desired purity in order to make a valid comparison of oxidative damage between mtDNA and nDNA. Our data show that a possibility exists that mtDNA damage is not higher than nDNA damage,