Protection against oxidative DNA damage by antioxidants, hormone-receptor blockers and HMG-CoA-reductase inhibitors [Elektronische Ressource] / vorgelegt von Ursula Schmid

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• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 19
• Langue: English
• Poids de l'ouvrage: 131 Mo

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Protection against oxidative DNA damage by
antioxidants, hormone-receptor blockers and
HMG-CoA-reductase inhibitors



Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Bayerischen Julius-Maximilians-Universität zu Würzburg




vorgelegt von
Ursula Schmid
aus Rottweil






Würzburg 2008



Eingereicht am: ...........................................................................................

Mitglieder der Promotionskommission:
Vorsitzender:................................................................................................
Gutachter: .................................................................................................... ............................................................................................

Tag des Promotionskolloquiums: .................................................................

Doktorurkunde ausgehändigt am: ................................................................. Index
1. INTRODUCTION _______________________________________________________________ 3
1.1. OXIDATIVE STRESS AND GENOMIC DAMAGE __________________________________________ 3
1.2. RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM (RAAS) _________________________________ 9
1.3. MODULATION OF THE RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM _______________________ 12
1.4. OXIDATIVE DNA DAMAGE CAUSED BY LIPID DISORDER _________________________________ 15
1.5. IDENTIFICATION AND QUANTIFICATION OF OXIDATIVE DNA DAMAGE ________________________ 16
1.5.1. Comet Assay __________________________________________________________ 16
1.5.2. Micronucleus frequency test ______________________________________________ 17
1.5.3. Quantification of reactive oxygen species ____________________________________ 19
2. OBJECTIVES ________________________________________________________________ 20
3. ABBREVIATIONS _____________________________________________________________ 22
4. ANTIOXIDANTS ______________________________________________________________ 25
4.1. BENFOTIAMINE EXHIBITS DIRECT ANTIOXIDATIVE CAPACITY AND PREVENTS INDUCTION OF DNA DAMAGE
IN VITRO _____________________________________________________________________ 25
4.1.1. Background ___________________________________________________________ 25
4.1.2. Experimental __________________________________________________________ 29
4.1.2.1. Material ____________________________________________________________________ 29
4.1.2.2. Cell culture _________________________________________________________________ 29
4.1.2.3. Comet Assay 29
4.1.2.4. Micronucleus frequency test ____________________________________________________ 30
4.1.2.5. Flow cytometric analysis of oxidative stress ________________________________________ 30
4.1.2.6. RNA isolation and semi quantitative reverse transcriptase PCR ________________________ 31
4.1.2.7. Ferric reducing ability of plasma assay (FRAP) _____________________________________ 31
4.1.2.8. Transketolase activity assay ____________________________________________________ 32
4.1.2.9. Statistics ___________________________________________________________________ 32
4.1.3. Results _______________________________________________________________ 33
4.1.3.1. Oxidative stress - Benfotiamine__________________________________________________ 33
4.1.3.2. Genomic damage - Benfotiamine ________________________________________________ 36
4.1.3.3. Apoptosis and Proliferation - Benfotiamine _________________________________________ 38
4.1.3.4. Transketolase expression and activity - Benfotiamine ________________________________ 38
4.1.3.5. Oxidative Stress – α-Tocopherol _________________________________________________ 40
4.1.3.6. Genomic damage – α-Tocopherol ________________________________________________ 41
4.1.3.7. Apoptosis and proliferation – α-Tocopherol ________________________________________ 42
4.1.4. Discussion ____________________________________________________________ 44
5. RECEPTOR BLOCKADE – ANGIOTENSIN II TYPE 1 RECEPTOR (AT R) ________________ 46 1
5.1. ANGIOTENSIN II-INDUCED GENOMIC DAMAGE IN RENAL CELLS CAN BE PREVENTED BY ANGIOTENSIN II
TYPE 1 RECEPTOR BLOCKADE OR RADICAL SCAVENGING ___________________________________ 46
5.1.1. Background ___________________________________________________________ 46
5.1.2. Experimental __________________________________________________________ 48
5.1.2.1. Material ____________________________________________________________________ 48
5.1.2.2. Cell culture _________________________________________________________________ 48
5.1.2.3. Comet assay ________________________________________________________________ 48
5.1.2.4. Micronucleus frequency test ____________________________________________________ 49
5.1.2.5. Quantification of apoptotic cells__________________________________________________ 49
5.1.2.6. Proliferation index ____________________________________________________________ 49
5.1.2.7. RT-PCR experiments _________________________________________________________ 49
5.1.2.8. Flow cytometric analysis of oxidative stress ________________________________________ 50
5.1.2.9. Statistical analysis 51
5.1.3. Results _______________________________________________________________ 52
5.1.4. Discussion 61
5.2. ANGIOTENSIN II INDUCES DNA DAMAGE IN THE ISOLATED PERFUSED KIDNEY _________________ 63
5.2.1. Background ___________________________________________________________ 63
5.2.2. Experimental __________________________________________________________ 65
5.2.2.1. Material ____________________________________________________________________ 65
5.2.2.2. Cell culture _________________________________________________________________ 65
5.2.2.3. Isolated perfused mouse kidneys ________________________________________________ 65
5.2.2.4. Comet Assay ________________________________________________________________ 67
5.2.2.5. RNA isolation and semi quantitative reverse transcriptase PCR ________________________ 68
5.2.2.6. Determination of formamidopyrimidine DNA glycosylase (FPG) sensitive sites _____________ 68
5.2.2.7. Detection of phosphorylated γ-H2AX sites _________________________________________ 69
5.2.2.8. Detection of abasic sites _______________________________________________________ 70 Index
5.2.2.9. Micronucleus frequency test ____________________________________________________ 70
5.2.2.10. Statistics __________________________________________________________________ 70
5.2.3. Results _______________________________________________________________ 72
5.2.3.1. Isolated perfused mouse kidney _________________________________________________ 72
5.2.3.2. Cell culture experiments _______________________________________________________ 75
5.2.4. Discussion ____________________________________________________________ 81
6. RECEPTOR BLOCKADE – MINERALOCORTICOID RECEPTOR _______________________ 84
6.1. ALDOSTERONE CAUSES DNA STRAND BREAKS AND MICRONUCLEI IN RENAL CELLS ____________ 84
6.1.1. Background ___________________________________________________________ 84
6.1.2. Experimental __________________________________________________________ 86
6.1.2.1. Material ____________________________________________________________________ 86
6.1.2.2. Cell culture _________________________________________________________________ 86
6.1.2.3. Comet assay ________________________________________________________________ 87
6.1.2.4. Micronucleus frequency test ____________________________________________________ 87
6.1.2.5. Quantification of apoptotic cells__________________________________________________ 87
6.1.2.6. Proliferation index ____________________________________________________________ 87
6.1.2.7. Vitality assay 87
6.1.2.8. RT-PCR experiments _________________________________________________________ 88
6.1.2.9. Flow cytometric analysis of oxidative stress ________________________________________ 88
6.1.2.10. Statistical analysis 88
6.1.3. Results _______________________________________________________________ 89
6.1.4. Discussion 98
6.2. ALDOSTERONE INDUCES OXIDATIVE STRESS AND GENOMIC DAMAGE IN VIVO ________________ 100
6.2.1. Background __________________________________________________________ 100
6.2.2. Experimental 102
6.2.2.1. Material ___________________________________________________________________ 102
6.2.2.2. Treatment of DOCA rats ______________________________________________________ 102
6.2.2.3. Extraction of primary kidney cells _______________________________________________ 103
6.2.2.4. Comet Assay _______________________________________________________________ 103
6.2.2.5. Micronucleus frequency test ___________________________________________________ 104
6.2.2.6. Flow cytometric analysis of oxidative stress _______________________________________ 104
6.2.2.7. Statistics __________________________________________________________________ 104
6.2.3. Results ______________________________________________________________ 105
6.2.3.1. Oxidative stress _____________________________________________________________ 105
6.2.3.2. DNA damage 106
6.2.3.3. Micronuclei, Mitoses and Apoptoses _____________________________________________ 107
6.2.4. Discussion