Microfluidic Electrochemical Arrays and LC-MS/MS Approaches for Establishing Pathways of Metabolite-Related DNA Oxidation

The xenobiotics such as drugs or environmental pollutants are often biotransformed into metabolites via bioactivation process by metabolic enzymes. However, some metabolites show higher toxicity after the activation, which react with endogenous compounds such as DNA to cause DNA damage. DNA oxidative damage is an inevitable consequence of cellular metabolism, which can be caused by pollutant and drug metabolisms in which high level of reactive oxygen species (ROS) has been produced. ROS could induce oxidative damage of DNA, leading to mutations on DNA. 8-oxo-7-hydro-2’-deoxyguanosine (8-oxodG) is the most important biomarker to indicate the extent of oxidative stress since it is the predominant base lesions formed during DNA oxidative damage. The aim of the present research is to analyze the level of 8-oxodG after the reaction between test xenobiotic compounds with DNA to investigate the chemistry metabolic pathway by using voltammetry array and LC-MS/MS. In this thesis, Chapter 2 describes the designing of an inexpensive gold electrochemical array coated with metallopolymer in a microfluidic channel to measure 8-oxodG directly in samples of intact DNA in a single-step assay. The array responses have been calibrated using LC-MS/MS. The LC-MS/MS assay has also applied on calibrating other related arrays such as electrochemiluminescent (ECL) array for fast detection DNA oxidative samples in the high-throughput way. Chapter 3 describes the development of a new sensor array with the capacity of simultaneously detecting both DNA oxidative damage and DNA adducts formation. By interpreting the sensor responses of two types of metallopolymers, a comprehensive metabolic-genotoxic chemistry profile can be provided. LC-MS/MS assays have been developed to confirm the formation of the metabolites and final DNA oxidation products. Chapter 4 focuses on the development of LC-MS/MS assay to study antibody orientation in order to improve the performance of immunoassay. Overall, these research activities have demonstrated the new analytical approaches on investigating the mechanisms of metabolites-related genotoxicity. These approaches can be used to predict the potential genotoxicity of chemical products and drug candidates. All these analytical approaches further enabled our understanding of fundamental molecular mechanisms in metabolite-related genotoxicity