Detection, stability and factors influencing the formation of promutagenic endogenous DNA damage

Genotoxic agents derived from diet may contribute to the total human burden of potentially carcinogenic DNA damage. The major malondialdehyde (MDA) DNA adduct, malondialdehyde-Z'-deoxyguanosine (M -dG) and two adducts formed by nitrosated glycine derivatives, 06-carboxymethyl-2,-deoxyguanosine (06-CMdG) and 06-methyl-2'- deoxyguanosinc (06-McdG), were studied. The long-term stability of Mi-dG in calf thymus DNA (CT-DNA) was assessed. MDA-treated CT-DNA standards were found to be relatively stable at room temperature (RT), 4 C, -20 C and -80 C. Degradation at RT was detectable after 3 weeks of storage. CT-DNA standards were more stable in water when compared to phosphate buffer. Levels of M -dG in highly modified CT-DNA could be reduced substantially using primary amines. M -dG stability in human DNA was assessed using an immunoslot blot (ISB) assay incorporating a propidium iodide staining procedure for the correction of differences in DNA binding to the ISB filter. Studies on the stability of low levels of Mi-dG in human DNA were not fully conclusive, although changes in adduct levels were temperature independent. The ratio of the two 06-alkylguanine adducts induced by potassium diazoacetate (KDA) treatment of DNA could be modulated using different buffer systems. These findings provided a unique opportunity to investigate the contribution of 06-CMdG and 06-MedG to the p53 mutational spectrum using a yeast-based p53 functional assay. Although adduct levels differed considerably, the two spectra obtained for KDA treatments in Tris-EDTA and phosphate buffer were statistically indistinguishable. The KDA-induced mutation spectra were, however, significantly different from that induced by N-methyl-N-nitrosourea, indicating that the key features of KDA-induced p53 spectra were caused mainly by 06-CMdG. Studies on MDA-induced mutational spectra were less conclusive due to a much smaller number of mutations detectable following sequencing. The results presented in this thesis highlight some key aspects of methodology as well as the significance of DNA damage measurements in studies of diet-related cancer risk.