The Influence of Oxidative Stress, Carcinogens and Cloning on DNA ...

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Index 3.1 Spectrophotometeric determination <strong>of</strong> DNA concentration ..................................... 41 3.2 Determination <strong>of</strong> genome wide methylation level with CE-LIF .............................. 41 3.3 Enhancement <strong>of</strong> the sensitivity <strong>of</strong> the CE-LIF method ............................................ 45 3.3.1 Comparison <strong>of</strong> two different LIF-detectors ......................................................... 45 3.3.1.1 Determination <strong>of</strong> signal to noise ratio .......................................................... 48 3.3.1.2 Reproducibility <strong>of</strong> the measurements ........................................................... 50 3.4 Synthesis <strong>of</strong> 8-oxo-2’-deoxyguanosine-3’-monophosphate (8-oxo-dGMP) ........... 51 3.4.1 Optimisation <strong>of</strong> the synthesis ............................................................................... 51 3.4.2 Purification <strong>of</strong> 8-oxo-2’-deoxyguanosine-3’-monophosphate ............................. 56 3.4.3 Characterisation <strong>of</strong> 8-oxo-2’-deoxyguanosine-3’-monophosphate ...................... 63 3.4.4 Analysis <strong>of</strong> 8-oxo-2’-deoxyguanosine-3’-monophosphate by CE-LIF ............... 64 3.4.5 Detection <strong>of</strong> 8-oxo-dGMP <strong>and</strong> determination <strong>of</strong> methylation level in real samples with CE-LIF ......................................................................................................... 66 3.4.6 Study <strong>of</strong> the electrophoretic mobility <strong>of</strong> 8-oxo-dGMP ........................................ 69 3.4.7 Correlation between the methylation level, oxidative stress <strong>and</strong> carcinogenesis 71 3.5 <strong>Influence</strong> <strong>of</strong> carcinogens on the genomic methylation level .................................... 73 3.5.1 <strong>Influence</strong> <strong>of</strong> aristolochic acid treatment on Sprague-Dawley rats ....................... 73 3.5.1.1 Quantification <strong>of</strong> DNA in AA-dosed rat samples ........................................ 74 3.5.1.2 CE-LIF analysis <strong>of</strong> methylation level in rats dosed with AA ...................... 75 3.5.1.3 Computation <strong>and</strong> evaluation <strong>of</strong> data ............................................................. 78 3.5.2 <strong>Influence</strong> <strong>of</strong> aristolochic acid treatment on Hupki mice ...................................... 83 3.5.2.1 CE-LIF analysis <strong>of</strong> methylation level in mice dosed with AA .................... 83 3.5.2.2 Computation <strong>and</strong> evaluation <strong>of</strong> data ............................................................. 86 3.5.2.3 Effect <strong>of</strong> the length <strong>of</strong> treatment .................................................................. 87 3.5.2.4 Correlation between the adduct level <strong>and</strong> the methylation level ................. 89 3.5.3 In-vivo study <strong>of</strong> the influence <strong>of</strong> 3-nitrobenzanthrone treatment (3-NBA) on Sprague-Dawley rats ............................................................................................ 90 3.5.3.1 Quantification <strong>of</strong> DNA in 3-NBA-dosed rat samples .................................. 90 3.5.3.2 CE-LIF analysis <strong>of</strong> methylation level in rats dosed with 3-NBA dosed rats 91 3.5.3.3 Computation <strong>and</strong> statistical analysis <strong>of</strong> data ................................................ 93 3.5.3.4 Correlation between the adduct level <strong>and</strong> methylation level ....................... 94 3.5.3.5 Comparison <strong>of</strong> the effect <strong>of</strong> various exogenous carcinogens on the genomic methylation level <strong>of</strong> Sprague-Dawley rats ................................................... 96 3.5.4 In-vitro study <strong>of</strong> various carcinogens on the genomic methylation level ............ 96 2
3 Index 3.5.4.1 Quantification <strong>of</strong> DNA in Caco-2 cell samples ........................................... 96 3.5.4.2 CE-LIF analysis <strong>of</strong> methylation level in Caco-2 cells incubated with various carcinogens ................................................................................................... 97 3.5.4.3 Computation <strong>and</strong> statistical analysis <strong>of</strong> data ................................................ 99 3.6 <strong>Influence</strong> <strong>of</strong> biological reproduction on DNA methylation ................................... 101 3.6.1 Accuracy <strong>of</strong> the measurements .......................................................................... 102 3.6.2 Determination <strong>of</strong> 5-methyl cytosine level in blood cells <strong>of</strong> healthy Simmental <strong>and</strong> Holstein clones ................................................................................................... 103 3.6.2.1 Comparative study <strong>of</strong> DNA methylation status between clones <strong>of</strong> the Simmental breed <strong>and</strong> the Holstein breed .................................................... 106 3.6.3 Individual <strong>and</strong> comparative study <strong>of</strong> DNA methylation status between the nuclear mother donor <strong>and</strong> its clones in Holstein breed. .................................................. 108 3.6.4 Comparative study <strong>of</strong> DNA methylation status between clones <strong>and</strong> monozygotic twins <strong>of</strong> the Simmental breed ............................................................................. 109 3.6.5 Effect <strong>of</strong> the age, nutrition <strong>and</strong> cloning process on the epigenetic variability <strong>of</strong> clones .................................................................................................................. 110 3.6.6 Determination <strong>of</strong> 5-methyl cytosine level in liver tissue <strong>of</strong> healthy <strong>and</strong> abnormal Holstein clones ................................................................................................... 112 3.6.7 Determination <strong>of</strong> 5-methyl cytosine level in muscles <strong>of</strong> healthy <strong>and</strong> abnormal Holstein clones ................................................................................................... 113 4 Summary ........................................................................................................................ 116 5 Materials <strong>and</strong> Methods ................................................................................................... 119 5.1 Chemicals ............................................................................................................... 119 5.1.1 General laboratory chemicals ............................................................................. 119 5.1.2 Chemicals for the synthesis <strong>of</strong> 8-oxo-2’-deoxyguanosine-3’-mono-phosphate 120 5.1.3 Solvents for HPLC separation <strong>of</strong> 8-oxo-2’-deoxyguanosine-3’-mono-phosphate from its starting material .................................................................................... 120 5.2 Experimental preparations ...................................................................................... 121 5.2.1 St<strong>and</strong>ard Preparations ......................................................................................... 121 5.2.2 Preparation <strong>of</strong> washing solutions for CE ............................................................ 122 5.2.3 Specific preparation for the synthesis <strong>and</strong> characterisation <strong>of</strong> 8-oxo-2’deoxyguanosine-3’-monophosphate ................................................................... 122 5.2.4 Enzyme preparations .......................................................................................... 123 5.2.4.1 Dialysis <strong>of</strong> the spleen phosphodiesterase enzyme (SPD) ........................... 123
Page 1 and 2: The Influe
Page 3 and 4: The work at h<stro
Page 5 and 6: First, I feel always grateful to Al
Page 7: I would like to extend my thanks to
Page 11 and 12: The following acro
Page 13 and 14: FDA Food and drug
Page 15: RIA Radioimmunoassay ROS Reactive o
Page 20 and 21: Index 5.2.4.2 Dialysis of</
Page 22 and 23: Introduction 1.1.2 DNA methylation
Page 24 and 25: Introduction 1.2 The</stron
Page 26 and 27: Introduction Figure 4: Model for me
Page 28 and 29: Introduction Cancer is classified i
Page 30 and 31: Introduction DNA methylation Gene h
Page 32 and 33: Introduction 1.2.7.1 Clonin
Page 34 and 35: Introduction 1.2.8 Twins an
Page 36 and 37: Introduction chemicals, alkylating
Page 38 and 39: Introduction AP sites are not only
Page 40 and 41: Introduction 1.3.2.1 Oxidat
Page 42 and 43: Introduction Finally, flavo- <stron
Page 44 and 45: Introduction chromatography with el
Page 46 and 47: Introduction One of</strong
Page 48 and 49: Introduction facturing of</
Page 50 and 51: Introduction 320) ; but due to its
Page 52 and 53: Introduction HO O O Figure 15: Mech
Page 54 and 55: Introduction - Methylation Sensitiv
Page 56 and 57: Introduction 5‘ 5‘ 5 Figure 18:
Page 58 and 59: Introduction - Radioactive-mediated
Page 60 and 61: Aim and objectives
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Results and Discus
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Summary 4 Summary DNA methylation i
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Summary The same s
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Experimental part Sodium tetrapheny
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Experimental part � Sodium tetrab
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Experimental part framework was fas
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Experimental part propanol
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Experimental part 12.5 mU/µL SPD)
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Experimental part Condition Value D
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Experimental part 5.4 Instrumentati
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Experimental part Graduated cylinde
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Experimental part 3-Nitrobenzanthro
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Appendix In-vitro: Denoting a react
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Appendix Laboratory Document Hydrol
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Appendix BODIPY and</strong
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Appendix Description of</st
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Appendix Transmission [%] 120 100 8
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Appendix Figure 62: Mass spectrum E
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List of figures Fi
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List of tables 8 L
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Literature Literature 1. Egger, G.;
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Literature 41. Bestor, T.H.; "<stro
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Literature 78. Venitt, S.; "Mechani
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Literature 113. Sinclair, K.D.; Mce
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Literature 148. Kadan-Lottick, N.S.
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Literature cyte DNA by gas chromato
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Literature 221. Mirand</str
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Literature 257. Lingaraju, G.M.; Sa
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Literature different tissues <stron
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Literature 324. Vishwanath, B.S.; F
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Literature 359. Wei, L.; Li, R.K.;
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Literature 394. Ramsahoye, B.H.; "M
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Literature 431. Wirtz, M.; Schumann
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Literature 467. Cornelius, M.; Wort
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Literature 502. Issa, J.P.J.; Ottav
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Literature 536. Pinheiro, J. <stron
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