Toxicity of combustion condensates on human cells [Elektronische Ressource] / von Nevena Stojičić

Nevena Stojičić

Sujets

• Biologie

Informations

• Publié par: rheinische_friedrich-wilhelms-universitat_bonn
• Publié le: 01 janvier 2010
• Nombre de lectures: 25
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Toxicity of combustion condensates on human cells






Dissertation
zur
Erlangung des Doktorgrades (Dr. rer. nat.)
der
Mathematisch-Naturwissenschaftlichen Fakultät
der
Rheinischen Friedrich-Wilhelms-Universität Bonn


vorgelegt von

Nevena Stojiči ć
aus
Belgrad





Bonn 2009

Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät
der Rheinischen Friedrich-Wilhelms-Universität Bonn.
























1. Gutachter: Prof. Dr. Waldemar Kolanus

2. Gutachter: PD Dr. Ruth Hemmersbach

Tag der Promotion: 18.02.2010
Erscheinungsjahr: 2010



Table of contents
Table of contents
Table of contents……………………………………………………………………..……..I
List of figures………………………………………………………………………………IV
List of tables……….……………………………………………………………………….VI
1 INTRODUCTION................................................................................................. 1
1.1 Air pollution and health impacts...................................................................................1
1.1.1 Combustion as a source of air pollution ........................................................................ 1
1.1.2 Particulate matter in ambient air: physical and chemical characterisation.................... 2
1.1.3 Chemical composition of combustion exhaust.............................................................. 4
1.1.4 Laboratory flames as a model source of combustion generated particles.................... 6
1.1.5 Health effects of airborne particles................................................................................ 7
1.1.6 Toxicologically relevant properties of the airborne PM ............................................... 10
1.2 Toxicity..........................................................................................................................11
1.2.1 Genotoxicity................................................................................................................. 12
1.2.2 Cytotoxicity .................................................................................................................. 13
1.2.3 Test-systems for toxicity assessment.......................................................................... 13
1.3 Cell death: types and mechanisms.............................................................................13
1.4 Activation of NF- B in response to stress .................................................................15
1.5 Aims of the research....................................................................................................18
2 MATERIAL AND METHODS ............................................................................ 20
2.1 Material..........................................................................................................................20
2.1.1 Laboratory equipment ................................................................................................. 20
2.1.2 Consumable material .................................................................................................. 21
2.1.3 Chemicals.................................................................................................................... 21
2.1.4 Media, buffer and sera ................................................................................................ 22
2.1.5 Test samples ............................................................................................................... 22
2.1.6 Commercial reaction kits ............................................................................................. 23
2.1.7 Bacterial strain............................................................................................................. 23
2.1.8 Human cell lines .......................................................................................................... 23
2.2 Methods.........................................................................................................................24
2.2.1 Generation and analysis of combustion condensates................................................. 24
2.2.1.1 Generation of combustion condensates ................................................................. 24
2.2.1.2 Total organic carbon content analysis .................................................................... 26
2.2.1.3 High-resolution transmission electron microscopy ................................................. 27
2.2.1.4 Spectrophotometry.................................................................................................. 27
2.2.1.5 Ultracentrifugation................................................................................................... 27
2.2.1.6 Filtration .................................................................................................................. 28
2.2.2 Prokaryotic cell culture and SWITCH test ................................................................... 28
2.2.2.1 Standard cultivation................................................................................................. 28
2.2.2.2 SWITCH test ........................................................................................................... 28
2.2.3 Eukaryotic cell cultures................................................................................................ 30
2.2.3.1 Cultivation of A-549 cell line.................................................................................... 30
2.2.3.2 U-937 cell line ................................................................................... 31
2.2.3.3 Long-term storage of cells ...................................................................................... 31
2.2.3.4 Growth kinetics of the used cell lines...................................................................... 32
2.2.3.5 Stable transfection of U-937 with pNF-B-EGFP/Neo plasmid .............................. 32
2.2.4 Flow cytometry ............................................................................................................ 34
2.2.5 NF- B-activation assay ............................................................................................... 35
2.2.6 Apoptosis assays......................................................................................................... 35
2.2.6.1 Annexin V-PE / 7-AAD assay.................................................................................. 35
ITable of contents
2.2.6.2 DNA content and cell cycle analysis....................................................................... 36
2.2.6.3 Active caspase-3 assay .......................................................................................... 37
2.2.6.4 DAPI staining .......................................................................................................... 38
2.2.7 Crystal violet assay...................................................................................................... 39
2.2.8 MTT test ...................................................................................................................... 39
2.2.9 Molecular biological methods...................................................................................... 40
2.2.9.1 Total RNA isolation ................................................................................................. 40
2.2.9.2 Quantification and quality analysis of isolated RNA ............................................... 40
2.2.9.3 Quantitative real-time reverse transcription PCR (qRT-PCR) ................................ 40
2.2.10 Statistical analysis ....................................................................................................... 46
3 RESULTS 47
3.1 Physical and chemical analysis of combustion condensates .................................47
3.1.1 Visual inspection of the presence of soot in combustion condensates....................... 47
3.1.2 Morphological analysis of the soot present in combustion condensates .................... 48
3.1.3 TOC content of combustion condensates depends on combustion conditions .......... 50
3.1.4 UV-spectrophotometry of combustion condensates reveals a presence of
nanoparticles............................................................................................................... 51
3.2 Combustion condensates are genotoxic and cytotoxic according to the bacterial
SWITCH test..................................................................................................................53
3.2.1 Toxicity of ethylene combustion condensates depends on their TOC content ........... 54
3.2.2 Toxicity of diesel combustion condensates depends on their TOC content ............... 58
3.2.3 Prolonged storage of combustion condensates has no impact on their toxicity ......... 61
3.2.4 Toxicologically relevant constituent of the combustion exhaust is being captured in all
three cool traps ........................................................................................................... 62
3.2.5 Soot has no significant contribution to the toxicity of ethylene combustion condensates
....