The biological significance of chemically-induced DNA adducts in relation to background DNA damage [Elektronische Ressource] / vorgelegt von Andreas Brink

The biological significance of chemically-induced DNA adducts
in relation to background DNA damage







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








vorgelegt von
Andreas Brink
aus Ochsenfurt



Würzburg 2007




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



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Doktorurkunde ausgehändigt am: ................................................................................

1 Introduction............................................................................................................ 1
1.1 Chemical carcinogenesis .................................................................................. 1
1.2 Genotoxicity ...................................................................................................... 2
1.2.1 DNA adducts .............................................................................................. 3
1.2.2 Endogenous DNA damage......................................................................... 6
1.2.3 Measurement of DNA adducts ................................................................... 8
1.2.4 Markers of genotoxicity............................................................................... 9
1.3 Chemically-induced hepatocarcinogenesis..................................................... 12
1.4 Kupffer cells and background DNA damage ................................................... 13
2 Objectives 15
3 Analytical method development......................................................................... 17
63.1 LC-MS/MS analysis of 7-mG, O -mdGuo, 8-oxodGuo, and εdAdo ................. 17
3.2 Electrospray ionization.................................................................................... 19
3.3 Multiple reaction monitoring mode .................................................................. 19
3.4 Experimental ................................................................................................... 21
3.4.1 Reagents .................................................................................................. 21
63.4.2 Synthesis of O -mdGuo and the labeled internal standard....................... 21
3.4.3 Liquid chromatography ............................................................................. 22
3.4.4 Mass spectrometry ................................................................................... 23
3.4.5 Method validation ..................................................................................... 24
3.4.6 Animals and administration of dimethylnitrosamine.................................. 25
3.4.7 DNA isolation and DNA hydrolysis ........................................................... 25
3.5 Results and Discussion 27
63.5.1 Synthesis of O mdGuo and the internal standard..................................... 27
3.5.2 Analysis of 7-mG ...................................................................................... 29
3.5.3 Column switching LC-MS/MS method...................................................... 31
3.5.4 Column switching: Method validation ....................................................... 33
3.5.5 Analysis of adducts in rat liver .................................................................. 34
4 Comet assay and cytotoxicity ............................................................................ 39
4.1 The comet assay............................................................................................. 39
4.2 Comet assay and sodium arsenite 40
4.3 Experimental ................................................................................................... 41
4.3.1 Chemicals and reagents........................................................................... 41
4.3.2 Cell culture ............................................................................................... 42
4.3.3 Treatment of cells..................................................................................... 42
4.3.4 Comet assay ............................................................................................ 42
4.3.5 Apoptosis and cell viability........................................................................ 43
4.3.6 Analysis of comet assay data ................................................................... 43
4.4 Results............................................................................................................ 44
4.4.1 Comet assay 44
4.4.2 Apoptosis and cell viability analysis.......................................................... 47
4.4.3 Comparative analysis of comet assay and cytotoxicity tests .................... 50
4.5 Discussion....................................................................................................... 52
5 Biological significance of DNA alkylation ......................................................... 55
5.1 Background..................................................................................................... 55
5.1.1 DNA adducts: background vs. induced..................................................... 55
5.1.2 DNA adducts induced by methylating agents ........................................... 56
5.2 Materials and Methods.................................................................................... 58
5.2.1 Cell culture and treatment ........................................................................ 58
5.2.2 DNA isolation and quantification of DNA adducts..................................... 58
5.2.3 Comet assay analysis............................................................................... 59
5.2.4 Micronucleus test...................................................................................... 59
5.2.5 Mouse lymphoma thymidine kinase assay: 96-well version ..................... 59
5.3 Results............................................................................................................ 61
5.3.1 DNA methylation by MMS and MNU ........................................................ 61
5.3.2 DNA adduct profile: Relative reactivity of MMS and MNU ........................ 63
5.3.3 DNA breakage and chromosomal damage............................................... 64
5.3.4 Mouse lymphoma thymidine kinase assay 66
5.3.5 Cell viability .............................................................................................. 67
5.3.6 Oxidative DNA modifications .................................................................... 68
5.3.7 Comparison of DNA adducts with other endpoints of genotoxicity ........... 70
5.3.7.1 Induction of 7-mG versus comet assay and micronucleus test .......... 72
65.3.7.2 Induction of O -mdGuo versus mutant frequency .............................. 73
5.3.8 Extrapolation to low dose - low-effect region, doubling dose.................... 73
5.3.9 Low-dose MNU: Experimental data.......................................................... 77
5.4 Discussion....................................................................................................... 79
5.4.1 DNA methylation and biological consequences........................................ 79
5.4.2 Choice of cell line and genotoxicity assays .............................................. 82
5.4.3 Comparison of increments in different endpoints: doubling dose ............. 82
6 Biological significance of oxidative DNA damage............................................ 85
6.1 Kupffer cells and hepatocarcinogenesis.......................................................... 85
6.2 Oxidative DNA damage markers..................................................................... 87
6.3 Experimental ................................................................................................... 88
6.3.1 Mice.......................................................................................................... 88
6.3.2 Treatment ................................................................................................. 88
6.3.3 DNA isolation, hydrolysis and LC-MS/MS analysis................................... 88
6.3.4 Data analysis............................................................................................ 89
6.4 Results............................................................................................................ 90
6.5 Discussion....................................................................................................... 93
7 Discussion ........................................................................................................... 97
7.1 Analysis of DNA adducts by LC-MS/MS ...............................................