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The mutagenicity of the reaction of DNA with genotoxic carcinogens

By Keith I.E. McLuckie


Mechanisms by which carcinogens induce mutations in human cells can be investigated using carcinogen exposed shuttle vector plasmids. In particular, the pSP189 plasmid can be treated with carcinogens in vitro and transfected into human fibroblasts. Following replication, where DNA repair or mutagenesis occurs, recovered plasmids can be screened in indicator bacteria for induced mutations in the supF gene.\ud The aim of this study was to establish and utilise assays to investigate the mutagenicity of genotoxic agents. Specifically, two model reactive intermediates of the cancer drug tamoxifen, α-acetoxytamoxifen and 4-hydroxytamoxifen quinone methide (4-OHtamQM), along with binary treatments of BPDE with UVB or UVC radiation were assessed for their mutagenic potential in human cells.\ud The quantitatively minor DNA adduct of tamoxifen formed by 4-OHtamQM is more mutagenic than the major tamoxifen adduct, formed by a-acetoxytamoxifen in Ad293 cells. The majority of mutations in α-acetoxytamoxifen treated plasmid were GC→TA transversions, while GC→AT transitions were predominant in 4-OHtamQM treated plasmid. Mutational hotspots were observed for both compounds. In GM04429 cells mutations induced by α-acetoxytamoxifen were mainly GC→TA, whereas in GM00637 fibroblasts GC→AT transitions were more common.\ud Treatment of plasmid with BPDE preferentially induced GC→TA transversions whilst UVB and UVC induced GC→AT transitions. Binary treatments were more mutagenic than single treatments, with BPDE then UV being more mutagenic than UV then BPDE. This suggests a synergistic mechanism of activation of BPDE DNA adducts by UV radiation.\ud In the final part of this work, a system for investigating the site-selectivity and consequences of mutagen reaction with DNA was developed. In the future this methodology can be used to detect mutations arising from specific DNA adducts inserted at known locations in important genes. This assay builds on the results provided by the standard supF assay and enables a more detailed investigation of the mechanisms involved in mutagenesis

Publisher: University of Leicester
Year: 2003
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