The supF assay for understanding DNA adduct-induced mutagenesis : traditional application and development of a site-specific version

A primary aim of this study was to establish and utilise the supF assay to investigate the mutagenicity of the cancer drug tamoxifen in target endometrial cells. In particular, the supF assay was employed to ascertain the mutations caused by two model reactive intermediates of tamoxifen, alpha-acetoxytamoxifen and 4-hydroxytamoxifen quinone methide (4-OHtamQM) in methylated pSP189 plasmid. The plasmid was methylated in vitro in order to allow application of the LwPy53 algorithm which enables in silico prediction of the G→T mutation distribution along the human p53 gene, using alpha-acetoxytamoxifen and 4-OHtamQM-induced mutation data from the supF assay.;Relative mutation frequencies increased proportionally with the adduct level for alpha-acetoxytamoxifen, up to ∼15 times the background frequency, while 4-OHtamQM failed to raise the mutation frequency above that of the untreated control. The majority of mutations in alpha-acetoxytamoxifen-treated plasmid were GC →TA transversions, while GC→AT transitions predominated in both 4-OHtamQM-treated plasmid and the untreated control. Based on the p53 G→T transversion predictions, alpha-acetoxytamoxifen induced damage resulted in two hotspots at positions 244 and 273; these mutations might be expected to occur in tamoxifen associated endometrial tumours if tamoxifen adducts play a role in cancer development.;The second part of the thesis was devoted to the development and validation of a novel site-specific assay. The assay, which is a site-specific version of the supF assay, was validated and utilized to investigate the mutagenic potential and types of mutations caused by individual O 6-MeG adducts situated in intact double stranded or gapped plasmids, within two different sequence contexts, in both E. coli and human cells.