ITEM METADATA RECORD
Title: Epigenetic effects of environmental stressors
Other Titles: Epigenetische veranderingen en omgevingsstressoren
Authors: Tabish, Ali; S0223213
Issue Date: 28-Apr-2015
Table of Contents: Index
1. CHAPTER 1 ................................................................................................................................................ 7
1.1. INTRODUCTION .................................................................................................................................................... 8
1.2. ENVIRONMENTAL CARCINOGENESIS ................................................................................................................. 9
1.2.1. Genetic mechanisms in carcinogenesis ............................................................................................. 9
1.2.2. Non-genetic mechanisms in carcinogenesis ................................................................................. 11
1.3. EPIGENETICS ..................................................................................................................................................... 12
1.3.1. Histone modifications ............................................................................................................................ 13
1.3.2. microRNA ................................................................................................................................................... 14
1.3.3. DNA methylation ..................................................................................................................................... 16
1.3.3.1. Regulation of DNA methylation................................................................................................... 17
1.3.3.2. DNA methylation in carcinogenesis ........................................................................................... 18
1.3.3.3. DNA methylation and environmental exposure .................................................................... 20
1.4. ENVIRONMENTAL EPIGENETICS ..................................................................................................................... 21
1.4.1. Exposure to environmental stressors .............................................................................................. 21
1.4.1.1. Exposure to hydrocarbons, halocarbons, cytostatic agents ...................................................... 21
1.4.1.2. Exposure to organic solvents ....................................................................................................................... 22
1.4.1.3. Exposure to nanomaterial ............................................................................................................................. 23
1.5. CHAPTER REFERENCES .................................................................................................................................... 25
2. CHAPTER 2 .............................................................................................................................................. 32
2.1. STUDY OBJECTIVES ........................................................................................................................................... 33
2.1.1. To investigate the epigenetic effects of environmental stressors ........................................ 33
2.1.1.1. Exposure to organic solvents, hydrocarbons, halocarbons, cytostatic agents in vitro ........... 33
2.1.1.2. Exposure to organic solvents in humans ................................................................................................. 34
2.1.2. Exposure to nanomaterial ................................................................................................................... 34
2.1.2.1. Exposure to gold nanoparticles and carbon nanotubes ...................................................................... 34
2.2. CHAPTER REFERENCES .................................................................................................................................... 37
3. CHAPTER 3 .............................................................................................................................................. 38
EPIGENETIC FACTORS IN CANCER RISK: EFFECT OF CHEMICAL CARCINOGENS ON GLOBAL DNA METHYLATION PATTERN IN HUMAN TK6 CELLS ...................................................................................................................................... 39
3.1. INTRODUCTION ................................................................................................................................................. 41
3.2. MATERIALS AND METHODS ............................................................................................................................. 42
3.2.1. Cell culture ................................................................................................................................................. 42
3.2.2. Chemicals, viability assays and dose selection ........................................................................... 43
3.2.3. DNA extraction, concentration and purity.................................................................................... 43
3.2.4. Enzymatic hydrolysis of DNA ............................................................................................................. 43
3.2.5. Calibration standards ........................................................................................................................... 44
3.2.6. LC-ESI-MS/MS instrumental analysis ............................................................................................. 44
3.2.7. Calibration curve ..................................................................................................................................... 45
3.3. STATISTICS ........................................................................................................................................................ 45
3.4. RESULTS ............................................................................................................................................................. 45
3.5. DISCUSSION ....................................................................................................................................................... 50
3.6. CHAPTER REFERENCES .................................................................................................................................... 53
4. CHAPTER 4 .............................................................................................................................................. 59
EPIGENETIC CHANGES IN LYMPHOCYTES OF SOLVENT EXPOSED INDIVIDUALS ...................................................... 60
4.1. INTRODUCTION ................................................................................................................................................. 62
4.2. POPULATION AND METHODS .......................................................................................................................... 64
4.2.1. Subjects ....................................................................................................................................................... 64
4.2.2. Exposure assessment ............................................................................................................................. 64
4.2.3. Questionnaires ......................................................................................................................................... 65
4.2.4. Genotyping ................................................................................................................................................. 65
4.2.5. Global DNA methylation analysis ..................................................................................................... 65
4.3. STATISTICS ........................................................................................................................................................ 66
4.4. RESULTS ............................................................................................................................................................. 66
4.4.1. Study population characteristics ...................................................................................................... 66
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4.4.2. Global DNA methylation in solvent exposed and CTE patients ............................................. 67
4.4.3. Gene-environment paradigm ............................................................................................................. 69
4.5. DISCUSSION ....................................................................................................................................................... 71
4.6. CHAPTER REFERENCES .................................................................................................................................... 76
5. CHAPTER 5 .............................................................................................................................................. 79
DNA METHYLATION CHANGES BY EXPOSURE TO NANOMATERIAL IN MICE BLOOD AND LUNG ............................. 80
5.1. INTRODUCTION ................................................................................................................................................. 82
5.2. METHODS........................................................................................................................................................... 83
5.2.1. Gold nanoparticles and carbon nanotubes ................................................................................... 83
5.2.2. Preparation of nanomaterial ............................................................................................................. 84
5.2.3. Animals ....................................................................................................................................................... 84
5.2.4. Experimental design .............................................................................................................................. 84
5.2.5. Bronchoalveolar lavage and bronchoalveolar lavage fluid processing ............................ 85
5.2.6. Lung cytokine measurements by cytometric bead array ........................................................ 85
5.2.7. DNA damage measurement by comet assay ................................................................................ 86
5.2.8. Oxidative stress measurement by Liquid chromatography-mass spectrometry ............ 86
5.2.9. Global DNA methylation measurement by LC-MS/MS ............................................................. 87
5.2.10. Gene specific DNA methylation measurements by bisulfite pyrosequencing ............ 87
5.3. STATISTICAL ANALYSIS .................................................................................................................................... 88
5.4. RESULTS ............................................................................................................................................................. 89
5.4.1. Nanomaterial characteristics ............................................................................................................ 89
5.4.2. Cytotoxicity of nanomaterial .............................................................................................................. 89
5.4.3. Oxidative stress and DNA damage effects ..................................................................................... 91
5.4.4. Global DNA methylation and hydroxymethylation in lungs ................................................... 91
5.4.5. Gene promoter methylation in lungs and blood ......................................................................... 92
5.5. DISCUSSION ....................................................................................................................................................... 96
5.6. CHAPTER REFERENCES .................................................................................................................................. 101
6. CHAPTER 6 ........................................................................................................................................... 112
GLOBAL METHYLATION AND HYDROXYMETHYLATION IN DNA FROM BLOOD AND SALIVA IN HEALTHY VOLUNTEERS ..................................................................................................................................................................... 113
6.1. INTRODUCTION ............................................................................................................................................... 115
6.2. MATERIAL AND METHODS ............................................................................................................................. 117
6.2.1. Study design and Population ........................................................................................................... 117
6.2.2. Sample Collection ................................................................................................................................. 118
6.2.3. DNA extraction ...................................................................................................................................... 118
6.2.4. DNA methylation and DNA hydroxymethylation analysis ................................................... 118
6.2.5. Pyrosequencing of LINE-1 and Alu elements ............................................................................ 119
6.3. STATISTICAL ANALYSIS .................................................................................................................................. 119
6.4. RESULTS ........................................................................................................................................................... 120
6.4.1. Characteristics of participants and method .............................................................................. 120
6.4.2. Results of %methylation and %hydroxymethylation ............................................................ 123
Figure 3: Correlation between %DNA methylation and % DNA hydroxymethylation in the same tissue ............................................................................................................................................................... 124
6.4.3. LINE1 and Alu methylation .............................................................................................................. 124
6.5. DISCUSSION ..................................................................................................................................................... 125
6.6. CHAPTER REFERENCES .................................................................................................................................. 130
7. CHAPTER 7 ........................................................................................................................................... 133
ASSESSMENT OF REPETITIVE ELEMENTS DNA METHYLATION BY PYROSEQUENCING .......................................... 134
7.1. INTRODUCTION ............................................................................................................................................... 136
7.2. MATERIALS ...................................................................................................................................................... 137
7.3. METHOD .......................................................................................................................................................... 138
7.3.1. PCR amplification of bisulfite-converted DNA .......................................................................... 138
7.3.2. Bisulfite pyrosequencing ................................................................................................................... 139
7.3.2.1. Sample preparation.................................................................................................................................... 139
7.3.2.2. Strand separation ........................................................................................................................................ 139
7.3.2.3. Primer annealing ......................................................................................................................................... 140
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Environment and Health - miscellaneous

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