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Structural Analysis of Detrimental Oxidative Products from Phenolic Carcinogens and Construction of Nucleotide Tools from Analogous Processes.

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Title: Structural Analysis of Detrimental Oxidative Products from Phenolic Carcinogens and Construction of Nucleotide Tools from Analogous Processes.
Author: Witham, Aaron
Department: Department of Chemistry
Program: Toxicology
Advisor: Manderville, Richard
Abstract: Toxicological research involves multiple cellular targets, but perhaps the most prominent is DNA. The importance of this molecule for life, and the many phenotypical consequences from its modification, necessitates the study of toxicant interaction with DNA. Phenols are ubiquitous in nature, possess positive and negative cellular effects, and are primary metabolites of several established carcinogens. Phenol toxicity is oxidatively mediated and oxidation of phenols can produce phenoxyl radicals that react directly at the C8 position of guanine to afford both carbon-linked and oxygen-linked C8-2ꞌ-deoxyguanosine (dG) adducts. Phenols also undergo oxidation to form catechols, which redox cycle to produce quinones. This process exacerbates oxidative stress in cells and promotes covalent attachment to cellular macromolecules, as quinones are susceptible to nucleophilic attack. O-linked-C8-guanine adducts were studied in DNA, expanding upon previous work in the Manderville laboratory on the simplest O-linked adduct, C8-phenoxy-2ꞌ-deoxyguanosine (PhOdG). The impact of phenyl ring expansion and chlorination of the PhOG adduct in a duplex environment was studied, and suggested that pi-stacking and lipophilicity affect adduct orientation within the helix, which may rationalize mutagenic outcomes in human DNA. Mass spectrometry was used to elucidate fragmentation pathways for effective dose biomarker detection from exposure to chlorinated phenols. Oxidative reactivity of the phenolic C-linked adduct (p-PhOH-dG) was also assessed, as this adduct possesses a lowered oxidation potential, and is susceptible to targeted oxidative attack to generate secondary oxidation products. Hydroxyl radical addition to p-PhOH-dG formed a catechol, which subsequently oxidized to a quinone adduct, and was trapped by a sulphur nucleophile. This previously unexplored oxidative pathway may contribute to phenol toxicity. Formation of reactive centers from oxidative processes on guanine adducts encouraged their use to confer covalent reactivity to functional oligonucleotides (aptamers). Furan modified aptamers for thrombin were synthesized and site selectively reacted with nucleophiles to afford covalent products. Linking aptamers to their targets allows for identification of unknown protein targets, and enhances aptamer therapeutic effects. The body of work herein provides mechanistic toxicological information for phenol adducts in DNA and uses oxidative reactivity of C8 guanine adducts to functionalize aptamers for utility in industrial applications.
Date: 2014-06
Rights: Attribution-NonCommercial-NoDerivs 2.5 Canada
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Attribution-NonCommercial-NoDerivs 2.5 Canada Except where otherwise noted, this item's license is described as Attribution-NonCommercial-NoDerivs 2.5 Canada