Degner, Amanda2020-08-252020-08-252019-08https://hdl.handle.net/11299/214986University of Minnesota M.S. thesis.August 2019. Major: Medicinal Chemistry. Advisor: Natalia Tretyakova. 1 computer file (PDF); xiii, 200 pages.Humans are exposed to a wide range of electrophilic agents, which can form covalent adducts at nucleophilic sites in biomolecules including DNA and proteins. If not repaired, DNA adducts can inhibit biological processes such as DNA replication and transcription, which can lead to mutagenesis, carcinogenesis, and toxicity. DNA repair pathways exist to remove these lesions, but the role of different pathways in the repair of different DNA adducts is not fully understood. Additionally, electrophiles can be detoxified through further metabolism such as glutathione conjugation before they are able to form adducts. Interindividual differences in the balance between detoxification, adduct formation, and repair ultimately determines the risk a person has of developing cancer after carcinogen exposure. In Chapter 2, we investigated the role of nucleotide excision repair (NER) and Fanconi Anemia (FA) repair pathways in repair of a 1,3-butadiene (BD) induced DNA-DNA interstrand crosslink (ICL), bis-N7G-BD. NanoLC-nanoESI+-MS/MS methodology was employed to quantify the amount of crosslinks present after human cell lines deficient in components of NER or FA and their isogenic controls were treated with an electrophilic metabolite of BD, 1,2,3,4-diepoxybutane (DEB), and allowed to repair for up to 72 h. Despite observing increased sensitivity to DEB treatment in repair deficient cell lines in cytotoxicity assays, no change in repair kinetics was observed between repair deficient and control cells, suggesting that in humans, there is a redundancy in DNA repair pathways. The same nanoLC-nanoESI+-MS/MS methodology was employed to measure bis-N7G-BD formation in HL60 cells, where crosslink levels correlated with cytotoxicity and micronuclei formation. In Chapter 3, we investigated the roll of a glutathione S-transferase theta 1 (GSTT1) in detoxification of another epoxide metabolite of BD, 3,4-epoxy-1-butene (EB). In a previous genome wide association study, differences in GSTT1 copy number explained a fraction of ethnic differences in BD metabolism, which could contribute to ethnic differences in smoking-induced lung cancer risk. A HPLC-ESI+-MS/MS method was developed for the quantitation of EB-GSH conjugates in cells that did or did not express GSTT1 after treatment of EB. No difference in EB-GSH conjugates or EB-GII DNA adduct levels were observed between GSTT1-/- and GSTT1+/+ cell lines. However, the expression of GSTT1 was had a protective effect against EB-induced apoptosis. In Chapter 4, an unknown electrophile-induced adduct to hemoglobin (Hb) was identified. Previous adductomic screens of human blood samples using the FIRE procedure to screen for adducts to N-terminal valine in Hb found an unknown adduct corresponding to an added mass of 106.042 Da. This adduct was identified as a 4-OHBn adduct to N-terminal valine, which was confirmed by HPLC-ESI+-HRMS analysis of authentic synthesized standard. Accurate mass, retention time, and MS/MS fragmentation patterns between the standard and unknown adduct matched. Levels of the 4OHBn-Val adduct were quantified to be 380 ± 160 pmol/g Hb in 12 human blood samples. Both 4-quinone methide and 4-hydroxybenzaldehyde were found to be capable of forming this adduct and thus were identified as possible sources.en1,3-ButadieneDNA adductElectrophileGlutathione conjugationMass SpectrometryQuinone methideThesis or Dissertation