Browsing by Subject "DNA Adduct"

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    Analysis of pyridyloxobutyl and pyridylhydroxybutyl DNA adducts in extra-hepatic tissues of rats treated with tobacco-specific nitrosamines.
    (2009-07) Zhang, Siyi
    The tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK) and N’-nitrosonornicotine (NNN) are potent carcinogens in tobacco products, and are believed to play a major role as causes of tobacco-related cancers. NNK and NNN require metabolic activation to exert their carcinogenic effects. Cytochrome P450-catalyzed α-hydroxylation of NNK and NNN generates a reactive intermediate, which alkylates DNA to form pyridyloxobutyl (POB)-DNA adducts. NNK is reduced to its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in a reversible and stereoselective manner. NNAL similarly undergoes α- hydroxylation and produces pyridylhydroxybutyl (PHB)-DNA adducts. In this thesis, we used liquid chromatography-electrospray ionization-tandem mass spectrometry (LCESI- MS/MS) to analyze POB- and PHB-DNA adducts in extra-hepatic tissues of F344 rats treated chronically with NNK and enantiomers of NNAL and NNN. POB- and PHB-DNA adduct levels were remarkably similar in NNK- and (S)-NNAL-treated rats, while distinctively different from those in (R)-NNAL-treated rats. These data indicate extensive retention of (S)-NNAL in various tissues of NNK-treated rats, and support a mechanism in which the preferential metabolism of NNK to (S)-NNAL, followed by sequestration of (S)-NNAL in the target tissues and reoxidation to NNK, is important to NNK tumorigenesis. (S)-NNN treatment produced more POB-DNA adducts in the rat oral mucosa, whereas adduct formation from (R)-NNN treatment was more favored in the nasal olfactory and respiratory mucosa. These results suggest that different mechanisms are involved in NNN metabolism and tumorigenesis in rat nasal and oral tissues, and that (S)-NNN might be an oral carcinogen in rats. In additional studies of this thesis, LC-ESI-MS/MS methods were developed for the quantitative analysis of 1, N2-propanodeoxyguanosine adducts derived from acrolein and crotonaldehyde (Acr-dGuo and Cro-dGuo). Acrolein and crotonaldehyde are widely-spread environmental pollutants, are present in cigarette smoke, and are formed endogenously through lipid peroxidation. Our methods for the analysis of Acr-dGuo and Cro-dGuo adducts are sensitive, accurate, and precise. These adducts were detected for the first time in the human lung. However, no differences were observed in adduct levels between self-reported smokers and non-smokers. The potential importance of these adducts in the human lung requires further study.
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    Metabolic Activation and DNA-Damaging Properties of Carcinogenic N-Nitrosamines
    (2019-05) Carlson, Erik
    Upon entry into a host, carcinogens are subjected to a variety of Phase I and Phase II metabolic pathways that result in bioactivation or detoxification. The bioactivation pathways are of particular importance because they often generate DNA-damaging compounds. It would stand to reason that fully understanding these activation pathways, their outcomes, and their differences amongst individuals would aid in combating cancer. This dissertation focuses on the metabolic activation of two tobacco carcinogens: 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). Concepts pertinent to this work such as tobacco carcinogenesis, chemistry of N-nitroso compounds, cytochrome P450 metabolism, and tobacco-specific nitrosamines are reviewed. The first study of this dissertation evaluates a hypothesized metabolic pathway for N-nitrosamines: processive P450 oxidation of NNK and NNN to N-nitrosamides. In this study, the three corresponding N-nitrosamides were synthesized, tested for stability, and monitored for formation in vitro. This study shows for the first time that N-nitrosamides are direct products of N-nitrosamine metabolism by cytochrome P450s. While these compounds were minor metabolites, their relative stability and DNA-damaging properties could impart biological relevance. Determining the generality of this metabolic pathway requires future work. The second study sought after the structures and abundance of stable 2'- deoxyadenosine (dAdo) damage (DNA adducts) induced by NNK bioactivation. This was accomplished by synthesizing hypothetical dAdo-adduct structures based on known reactivity and applying them to in vitro and in vivo assays. In vitro data indicates that N6- and N1-adducts are formed, however, in vivo data only shows N6-adduct formation, indicating extensive repair of N1-adducts. The relative abundance of these adducts were determined in rat liver and lung for three different treatment groups. The biological activity of these adducts requires future study. The last study measured direct biomarkers for human NNN metabolic activation for the first time by using [pyridine-D4]NNN-enriched tobacco. The deuterium-labelling allows NNN metabolites to be selectively measured by mass spectrometry and removes all interference by competing nicotine metabolites. This study is ongoing but current data suggests metabolic activation of NNN varies among individuals and is at least partially due to the activity of P450 2A6, the dominant enzyme for NNN bioactivation.