Toxicological effects of Nanoparticles: In vitro studies with

1,74

MB

Titanium Dioxide

125

stron

4300

ID

Environmental Health Research Institute, University Duesseldorf

2005

rok

Contents

Abstract 1

Zusammenfassung 2

Abbreviations 3

1. Introduction 5

1.1 History 5

1.2 Air pollution and respiratory diseases 6

1.3 Particle exposure and diseases 6

1.4 Particulate Matter (PM10, PM2.5 and PM0.1) 7

1.5 Ultrafine and Nanoparticles in historical context 9

1.6 Ultrafine versus Nanoparticles 10

1.7 Hypothesis for the factors influencing the effects of UF particles 14

1.8 Hypothesis for mechanisms and mediators involved in toxicity induced by ultrafine particles 18

1.9 Particle Uptake 20

1.10 Oxidative stress in Lung 20

1.11 Inflammatory mediator: Interleukin-8 27

2. Aim and Design 30

2.1 Hypothesis and Study Questions 31

2.2 Schematic presentation of the thesis 32

3. Materials and methods 33

3.1 Particles 33

3.2 Particle morphology HRTEM 34

3.3 Modulation of the Particle surface by Methylation 34

3.4 Evaluation of endotoxin contamination by Limulus Amebocyte Lysate (LAL) Assay 36

3.5 Detection of reactive oxygen species 37

3.6 ESR in acellular system 39

3.7 Culturing and treatment of A549 cells 41

3.8 Culturing and treatment of human primary cells 42

3.9 ESR in A549 cells 43

3.10 LDH-assay 43

3.11 Measurement of total Glutathione Depletion 44

3.12 Determination of particle uptake in A549 cells by electron microscopy 45

3.13 Total RNA extraction 46

3.14 Reverse transcriptase-polymerase chain reaction 47

3.15 Agarose gel electrophoresis of PCR product 48

3.16 Interleukin-8 ELISA 48

3.17 Statistical Analysis 49

4. Results and Discussion 50

4.1 High resolution Transmission electron micrographs 50

4.2 Analysis of the methylation efficiency 52

4.3 Endotoxin contamination of the TiO2 particles (LAL Assay) 54

4.4 ROS formation by the different particle preparations in acellular system 55

4.5 Formation of ROS in A549 cells upon treatment with the different particles 59

4.6 LDH 62

4.7 Glutathione depletion 64

4.8 Release of interleukin-8 – The size effect 65

4.9 Evaluation of the role of membrane damage in IL-8 release from A549 cells 68

4.10 IL-8 mRNA expression and protein release - The methylation effect 69

4.11 Particle uptake 71

5. Summary and General Discussion 74

5.1 Background 74

5.2 TiO2 - The surface area driven effects 75

5.3 TiO2 - surface chemistry driven effects 77

5.4 Extrapolation of findings 81

6. General Conclusions 82

References 90

Acknowledgements 115

Curriculum Vitae 117

List of publications 118