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Exercise-Induced DNA Damage: Is There a Relationship with Inflammatory Responses?

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Exercise-Induced DNA Damage: Is There a Relationship with Inflammatory Responses? DISSERTATION Titel der Dissertation Effects of an Ironman triathlon on DNA stability angestrebter akademischer Grad Doktorin der Naturwissenschaften (Dr. rer.nat.) Verfasserin / Verfasser: Stefanie Reichhold Matrikel-Nummer: 0008640 Dissertationsgebiet (lt. Ernährungswissenschaften Studienblatt): Betreuerin / Betreuer: A.o. Univ.-Prof. Dr. Karl-Heinz Wagner Wien, am 05.Februar 2009 Preface Diese Dissertation ist im Rahmen meiner Tätigkeit als wissenschaftliche Mitarbeiterin in der Arbeitsgruppe von a.o. Univ.-Prof. Dr. Karl-Heinz Wagner (Emerging Field "Oxidative Stress and Oxidative DNA Damage") am Department für Ernährungswissenschaften der Universität Wien entstanden. Das Projekt wurde vom Österreichischen Wissenschaftsfonds (FWF) finanziert. Mein spezieller Dank gilt Herrn a.o. Univ.-Prof. Dr. Karl-Heinz Wagner für die Ermöglichung und auch für die kompetente und wissenschaftliche Betreuung dieser Arbeit. Er hat stets großes Vertrauen in mich gesetzt. Durch seine Unterstützung konnte ich wertvolle Erfahrungen auf nationalen und internationalen Kongressen sammeln, sowie die Resultate meiner Arbeit in angesehenen Journalen publizieren. Dadurch eröffneten sich für mich neue Horizonte und Möglichkeiten. Danke, Karl-Heinz! Danken möchte ich Herrn o. Univ.-Prof. Dr. Ibrahim Elmadfa für die Ermöglichung dieser Dissertation am Department für Ernährungswissenschaften. I would like to thank Prof. Zdeňka Ďuračková (Department of Medical Chemistry, Biochemistry and Clinical Biochemistry, Faculty of Medicine, Comenius University, Bratislava) and Prof. Helga Stopper (Department of Toxicology, University of Würzburg, Germany) for taking their time to review this thesis. Besonders möchte ich Mag. Oliver Neubauer für die unkomplizierte und gute Zusammenarbeit im Rahmen des Projekts danken. Oliver stand stets für kompetente Ratschläge und wissenschaftliche Diskussionen zur Verfügung. Ferner bedanke ich mich bei meinen ArbeitsgruppenkollegenInnen und FreundInnen Mag.a Karin Koschutnig, Mag.a Sonja Kanzler, Mag.a Elisabeth Plasser und Mag. Oliver Neubauer für ihre Freundschaft, ihre tatkräftige Mithilfe im Labor, das perfekte Arbeitsklima, sowie die lustigen „After-Work-Aktivitäten“. Ich danke a.o. Univ.-Prof. Dr. Siegfried Kansmüller (Institut für Krebsforschung der Medizinischen Universität Wien) für die gute Zusammenarbeit und die zahlreichen fachkundigen und wissenschaftlichen Gespräche. An dieser Stelle möchte ich auch Dr. Veronika Ehrlich, Mag.a Christine Hölzl und Mag.a Franziska Ferk (Institut für Krebsforschung der Medizinischen Universität Wien) für die nette und lehrreiche Einführung in den Mikrokern- und COMET- Test danken. Veronika, Christine und Franziska standen mir stets mit Rat und Tat zur Seite. Ein großes Dankeschön geht auch an alle Athleten für die Teilnahme an dieser Studie. Besonders bedanke ich mich bei meinen engsten Freundinnen (Tini, Verena, Gabi, Anja, Doris, Karin und Dani), auf die ich mich immer verlassen konnte und die mir stets zur Seite gestanden sind. Mein größter Dank gilt jedoch Bernd (ildüaoe), meinen Eltern Edeltraud und Dietmar, meinen Schwestern Christina und Teresa, sowie meinen Großeltern Josefa, Herta und Walter. Sie haben mir mein Studium ermöglicht, mich stets unterstützt und immer an mich geglaubt. Ich möchte ihnen diese Arbeit widmen. Appendix The present thesis is based on the following original articles and reviews, respectively, which are referred to in the text by Roman numerals. Paper I Reichhold S, Neubauer O, Ehrlich V, Knasmüller S, Wagner K-H No acute and persistent DNA damage after an Ironman Triathlon. Cancer Epidemiology Biomarkers and Prevention 2008; 17: 1913-1919. Paper II Reichhold S, Neubauer O, Stadlmayr B, Valentini J, Hoelzl C, Ferk F, Knasmüller S, Wagner K-H. Effects of an Ironman triathlon on oxidative DNA damage. Mutation Research/ Genetic Toxicology and Environmental Mutagenesis 2008; submitted. Review I Reichhold S, Neubauer O, Bulmer A, Knasmüller S, Wagner K- H. Endurance exercise and DNA stability: Is there a link to duration and intensity?. Mutation Research- Reviews in Mutation Research 2008; in press. Review II Neubauer O, Reichhold S, Nersesyan A, König D, Wagner K-H. Exercise-induced DNA damage: Is there a relationship with inflammatory responses?. Exercise Immunology Reviews 2008; in press. Reprints and accepted papers were published by kind permission of respective publishers. Table of Contents I CONTENTS LIST OF FIGURES .......................................................................................................V LIST OF TABLES .................................................................................................... VIII ABBREVIATIONS ...................................................................................................... IX 1. INTRODUCTION.......................................................................................................1 2. BACKGROUND .........................................................................................................3 2.1. DNA damage.........................................................................................................3 2.1.1. DNA structure, damage and repair..................................................................3 2.1.2. Risk factors of oxidative stress and DNA damage..........................................5 2.1.3. Markers of DNA damage used in studies related to exercise .........................5 2.1.3.1. Cytokinesis-block micronucleus cytome assay........................................5 2.1.3.2. Single cell gel electrophoresis assay........................................................7 2.1.3.3. Sister chromatid exchange assay..............................................................8 2.2. Exercise and DNA damage................................................................................10 2.2.1. Exercise-induced ROS formation .................................................................10 2.2.2. Exercise-induced DNA damage....................................................................11 2.2.3. Exercise-induced adaptation .........................................................................12 3. MATERIALS AND METHODS .............................................................................13 3.1. Project description and subjects.......................................................................13 3.2 Race conditions....................................................................................................14 3.3. Blood collection ..................................................................................................15 3.4. CBMN Cyt assay ................................................................................................15 3.4.1. Equipment for the CBMN Cyt assay ............................................................15 3.4.2. Reagents for the CBMN Cyt assay ...............................................................16 Table of Contents II 3.4.2.1. Manufacturing processes and storage of reagents for the CBMN Cyt assay....................................................................................................................17 3.4.3. Basic assay approach ....................................................................................18 3.4.4. Protocol for the CBMN Cyt assay ................................................................18 3.4.5. Microscopic assessment................................................................................21 3.5. SCGE assays.......................................................................................................24 3.5.1. Equipment for the SCGE assays ...................................................................24 3.5.2. Reagents for the SCGE assays......................................................................25 3.5.2.1. Manufacturing processes and storage of reagents for the SCGE assays26 3.5.3. Basic assays approach...................................................................................27 3.5.4. Protocol for the SCGE assays .......................................................................28 3.5.5. Microscopic assessment and calculation.......................................................30 3.6. SCE assay............................................................................................................30 3.6.1. Equipment for the SCE assay........................................................................31 3.6.2. Reagents for the SCE assay ..........................................................................31 3.6.2.1. Manufacturing processes and storage of reagents for the SCE assay....32 3.6.3. Basic assay approach ....................................................................................35 3.6.4. Protocol for the SCE assay............................................................................35 3.6.5. Microscopic assessment................................................................................37 3.7. Measurement of vitamin B12 and folate ...........................................................38 57 3.7.1. Equipment for SimulTRAC-SNB Radioassay Kit Vitamin B12 [ Co]/Folate [125I].........................................................................................................................38 3.7.2. Reagents for the SimulTRAC-SNB Radioassay Kit Vitamin B12 [57Co]/Folate [125I] ..................................................................................................39 Table of Contents III 3.7.2.1. Manufacturing
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