Liquid chromatography-mass spectrometry for detection and characterization of DNA biomarkers and reactive metabolites

• Online Access: http://hdl.handle.net/2047/d10016740

The focus of the research presented in this dissertation has been on the elucidation of carcinogenic pathways, including the role of reactive drug intermediates, DNA adducts and the existence of immortal DNA strands. This dissertation presents advances in liquid chromatography-mass spectrometry for the analysis of reactive metabolites, DNA adducts and the evaluation of adult stem cell mechanisms. Reactive metabolites have been closely associated with idiosyncratic or adverse drug reactions often leading to cell death and the development of cancer. These side effects are often hard to predict during the drug development phase, for this purpose reactive metabolite screening assays have been developed. For this purpose, a complementary reactive metabolite trapping assay to the glutathione trapping assay was developed, where potassium cyanide was used as the trapping agent. DNA adducts have been associated with carcinogenesis, however, the exact implications associated with specific DNA adduct levels are yet to be elucidated. In order to address these questions a sensitive multidimensional chromatography-mass spectrometric method was used for the quantification of DNA adducts of cigarette smoke constituent, 4-aminobiphenyl (4-ABP) was developed. Human bladder RT-4 cells were dosed with 4-ABP for the evaluation of chemopreventive agents in the prevention of adduct formation. Finally, unique cellular properties of ASCs, the most probable precursor to cancer, specifically the immortal DNA strand theory, are explored via LC-MS.