Oxidative and nitrosative stress induced by the mineralocorticoid aldosterone [Elektronische Ressource] : mechanism of induction and role of signal transduction pathways and transcription factors / vorgelegt von Nina Queisser

Nina Rhoades

Sujets

• Biologie

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2010
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Oxidative and nitrosative stress induced by the
mineralocorticoid aldosterone

Mechanism of induction and role of signal transduction pathways and
transcription factors









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




vorgelegt von
Nina Queisser
aus Ludwigsburg







Würzburg 2010
Eingereicht am: 28.09.2010………………………………………………………………….



Mitglieder der Promotionskommission:

Vorsitzender: Prof. Dr. Roy Gross…….……………………………………………………..

Gutachter: Prof. Dr. Helga Stopper………………………………………………………….

Gutachter: Prof. Dr. Ricardo Benavente….………………………………………………...



Tag des Promotionskolloquiums: 08.12.2010…………………………………………



Doktorurkunde ausgehändigt am: …………………………………………………………..
INDEX I
INDEX

INDEX....................................................................................................................................................... I
ABBREVIATIONS..................................................................................................................................IV
1 INTRODUCTION ............................................................................................................................ 1
1.1 Increased cancer incidence of hypertensive individuals and involvement of the renin-
angiotensin-aldosterone system ............................................................................................ 1
1.2 Aldosterone signaling............................................................................................................. 2
1.3 Aldosterone induces cardiovascular complications and renal damage................................. 4
1.4 Oxidative/nitrosative stress and DNA damage ...................................................................... 5
1.5 Prooxidant enzymes .............................................................................................................. 7
1.5.1 NAD(P)H oxidase................................................................................................................... 7
1.5.2 Nitric oxide synthase (NOS)................................................................................................... 9
1.6 Antioxidants ......................................................................................................................... 10
1.7 ROS modulation of gene expression ................................................................................... 12
1.7.1 Transcription factor Nrf2 ...................................................................................................... 13
1.7.2 Transcription factor NF- B................................................................................................... 14
1.7.3 MAP kinase/ERK pathway................................................................................................... 16
1.7.3.1 Transcription factor CREB........................................................................................... 17
1.7.3.2 STAT transcription factors........................................................................................... 18
2 OBJECTIVES ............................................................................................................................... 19
3 MATERIALS AND METHODS ..................................................................................................... 21
3.1 Materials............................................................................................................................... 21
3.1.1 Cell lines and cell culture reagents ...................................................................................... 21
3.1.2 Animals ................................................................................................................................ 21
3.1.3 Chemicals and Reagents..................................................................................................... 21
3.1.4 Antibodies ............................................................................................................................ 22
3.1.5 Oligonucleotides .................................................................................................................. 22
3.2 Methods ............................................................................................................................... 22
3.2.1 Cell culture ........................................................................................................................... 22
3.2.2 Animal treatment.................................................................................................................. 23
3.2.3 Genotoxicity tests................................................................................................................. 25
3.2.3.1 Vitality assay................................................................................................................ 25
3.2.3.2 Comet assay................................................................................................................ 25
3.2.3.3 Determination of formamidopyrimidine DNA glycosylase-sensitive sites ................... 27
3.2.3.4 Micronucleus frequency test........................................................................................ 27
3.2.3.5 Proliferation index and apoptosis ................................................................................ 29
3.2.4 Microscopy........................................................................................................................... 29
3.2.4.1 Detection of fluorescent dyes...................................................................................... 29
3.2.4.1.1 Evaluation of the cellular superoxide anion concentration.......................................... 29
3.2.4.1.2 Evaluation of intracellular calcium levels by laser scanning confocal microscopy...... 30
3.2.4.1.3 Evaluation of cellular NO by scanning laser confocal microscopy.............................. 30
3.2.4.1.4 Detection of apoptotic cells ......................................................................................... 31
3.2.4.2 Immunocytochemistry ................................................................................................. 32
3.2.4.2.1 Detection of phosphorylated -H2AX sites.................................................................. 32
3.2.4.2.2 Detection of 8-oxodG by immunofluorescent staining ................................................ 33
3.2.4.2.3 Detection of proliferation ............................................................................................. 33
3.2.4.3 Immunohistochemistry ................................................................................................ 35
kg INDEX II
3.2.4.3.1 Detection of apoptosis................................................................................................. 35
3.2.4.3.2 Detection of oxidative stress on cryosections ............................................................. 35
3.2.4.3.3 Detection of phosphorylated -H2AX sites.................................................................. 36
3.2.4.3.4 Detection of proliferation ............................................................................................. 37
3.2.5 Analytics............................................................................................................................... 38
3.2.5.1 Quantification of 8-oxodG by liquid chromatography-mass spectrometry .................. 38
3.2.5.2 Quantification of GSH by high-performance liquid chromatography........................... 39
3.2.5.3 Fluorimetric determination of GSH levels.................................................................... 40
3.2.5.4 Fluorimetric evaluation of intracellular calcium levels ................................................. 41
3.2.5.5 FRAP assay: Method to measure the antioxidant capacity of substances ................. 41
3.2.5.6 Fluorimetric quantification of intracellular oxidants ..................................................... 42
3.2.5.7 Fluorimetric quantification of intracellular NO ............................................................. 43
3.2.5.8 Flow cytometric analysis of oxidative stress ............................................................... 43
3.2.6 Proteinchemical methods..................................................................................................... 44
3.2.6.1 Electromobility shift assay........................................................................................... 44
3.2.6.2 Western blot analysis .................................................................................................. 45
3.2.7 Molecular biological methods .............................................................................................. 47
3.2.7.1 RT-PCR experiments .................................................................................................. 47
3.2.8 Statistical analysis................................................................................................................ 47
4 RESULTS ......................................................................