Browsing by Subject "DNA adduct"

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    Analyses Of Detoxification And Dna Damage From The Human Carcinogens Benzene And N′-Nitrosonornicotine
    (2016-05) Zarth, Adam
    The process of chemical carcinogenesis is initiated by DNA damage. This dissertation will describe quantitative approaches to assess the detoxification and DNA damage pathways of two human carcinogens: benzene and N′-nitrosonornicotine (NNN). The aspects of carcinogenesis relevant for this work include exposure to the carcinogen, biological activation to a reactive electrophile, metabolic detoxification processes, and DNA addition product (adduct) formation upon reaction of the electrophile with DNA. This dissertation will begin by presenting a collaborative study on exposure to benzene when smoking tobacco via a hookah; a urinary biomarker of benzene exposure significantly increases after a single smoking event. Next, it will describe studies of enzyme kinetics which determined for the first time that a human enzyme, GSTP1, is a good catalyst for the detoxification of benzene oxide, the activated form of benzene. This study also provided direct biochemical confirmation that GSTT1 is an important enzyme in this detoxification process. The next chapter will present collaborative data demonstrating that sulforaphane, an active phytochemical in broccoli sprouts, can upregulate these enzymatic detoxification processes in humans exposed to benzene and other air pollutants, likely by upregulating GSTP1. The last chapter on benzene will describe data showing that the major DNA adduct arising from the reaction between benzene oxide and DNA, 7-phenylguanine, is not detectable in humans or animals exposed to benzene. Thus, 7-phenylguanine is not likely to be the etiological agent responsible for the mechanism of benzene carcinogenicity, but instead some other mechanism of carcinogenesis is more important. The final chapter of this dissertation will shift focus to the analysis of a DNA adduct arising from NNN metabolic activation. NNN can be activated via two pathways: 2′-hydroxylation and 5′-hydroxylation. 2′-Hydroxylation has been more extensively studied, as it is the major pathway in rat esophagus, a target tissue of NNN carcinogenicity. However, the work presented here demonstrates that 5′-hydroxylation of NNN by human enzymes leads to higher levels of DNA adducts than does the 2′-hydroxylation pathway, and thus, 5′-hydroxylation may be the more relevant pathway for future DNA adduct studies in humans who use tobacco products.
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    Biomonitoring Exposures to Environmental and Dietary Carcinogens by Targeted and Untargeted Mass Spectrometry
    (2022-07) Konorev, Dmitri
    Humans are exposed to a wide variety of exogenous chemicals that may be implicated in DNA damage and cancer. Typical sources of carcinogen exposure include the diet, environment, and tobacco smoke. There is an epidemiological link between smoking cancer, as well as cooked and red meat consumption, and cancer. Despite extensive study, the chemicals responsible for carcinogenesis are unconfirmed. Specific and sensitive markers of DNA damage by discrete chemicals are needed confirm existing paradigms of carcinogenesis. Chapter 1 outlines the epidemiology and mechanisms by which chemicals from the diet and tobacco can lead to colorectal cancer and bladder cancer. Tobacco smoking is a well-established cause of bladder cancer, as is occupational exposure to high levels of carcinogenic aromatic amines, such as 4-aminobiphenyl and 2-naphthylamine, which are also present in tobacco smoke at low levels. The levels of these compounds in tobacco smoke may be insufficient to bladder carcinogenesis. Other related compounds, such as alkylaniline derivatives, and structurally-related heterocyclic aromatic amines, are present at much higher levels in tobacco smoke and may be risk factors for bladder cancer. Chapter 2 outlines our methodologies to assay a nonpolar, basic fraction of tobacco smoke condensate, containing aromatic amines and heterocyclic aromatic amines, by liquid chromatography coupled to mass spectrometry (LC/MS). These compounds were measured by targeted and untargeted LC/MS as well as with the use of high field asymmetric waveform ion mobility spectrometry coupled to LC/MS to add an additional dimension of separation and reduce sample complexity. Chapter 3 presents our original research in the development of highly sensitive, validated LC/MS methods to measure DNA adducts and abasic sites from cooked meat and tobacco carcinogens in colorectal tissue. These methods were developed using a rat animal model dosed with carcinogens that form DNA adducts associated with colorectal cancer based on previous epidemiological and mechanistic evidence. In Chapter 4, we applied this methodology to human colorectal DNA samples from colorectal cancer patient biopsy samples. We did not detect DNA adducts of exogenous carcinogens but did detect endogenously-formed DNA and abasic sites in these samples. We then contextualized these results within greater paradigms of colorectal cancer carcinogenesis.
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    DNA-Protein Cross-links: Formation in Cells and Tissues, Repair, and Inhibition of DNA Transcription
    (2019-04) Park, Daeyoon
    DNA is constantly damaged by exogenous and endogenous agents, generating a range of nucleobase lesions. It is important to understand the biological consequences and repair mechanisms of DNA adducts. Cellular proteins can become covalently trapped on DNA to generate DNA-protein crosslinks (DPCs). Because of their unusually bulky nature, DPCs are anticipated to block many cellular processes including replication, transcription, and repair. However, cellular effects of DPCs have not been fully elucidated. Chapter 1 of this thesis provides background information on the formation, biological consequences, and repair pathways of DPCs studied in previous studies. In Chapter 2, we employed a quantitative nanoLC-ESI+-MS/MS assay to investigate the formation of free radical-induced DPCs between thymidine in DNA and tyrosine sidechains of proteins. This methodology was used to examine the role of SPRTN protease and immunoproteasome in DPC repair in human cells and mouse models. In Chapter 3, a mass spectrometry based CTAB assay was used to study the effects of DNA-peptide crosslinks on transcription in human cells. We constructed plasmid molecules containing DPCs between C5 of dC and lysine sidechains of polypeptides in order to mimic conjugates that form endogenously at DNA epigenetic marks (5-formylc-dC). Lesion bearing and control plasmids were transfected into human cells, and the amounts of RNA transcripts were determined using a mass spectrometry based approach. Moreover, DNA lesion bearing plasmid models were used to determine the importance of NER pathway in DPC repair. In Chapter 4, we investigated in vivo formation of DPCs in cells exposed to monofunctional alkylating agent, methyl methanesulfonate (MMS). A mass spectrometry-based TMT proteomics approach was used to characterize MMS-induced DNA-protein cross-linking in Chinese hamster lung fibroblasts (V79). utilizing Our results revealed that DPCs can be produced via nucleophilic attack of proteins at the C8 position of N7-methylguanine (MdG). Our results revealed novel DPC formation mechanisms and the toxicities of monofunctional agent induced DPCs. In summary, mass spectrometry-based quantification was used to the amounts of free radical induced DPCs in cells, providing evidence for the role of DPC proteolysis in repair, while CTAB assay demonstrated the effect of endogenously formed DPCs on transcription. Moreover, a mass spectrometry-based methodology was applied to examine a novel DPC formation mechanism following treatment with monofunctional alkylating agents.
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    Mass Spectrometry Based Analysis Of Electrophile Induced Adducts Of Biomolecules
    (2019-08) Degner, Amanda
    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.