Browsing by Subject "Carcinogenesis"

Now showing 1 - 4 of 4
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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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    Elevated levels of 1-hydroxypyrene and NOe-Nitrosonornicotine in the urine of smokers with head and neck cancer: a matched control study
    (2014-04) Khariwala, Samir Suresh
    Background: Head and neck squamous cell carcinoma (HNSCC) is associated with tobacco use. Still, most smokers do not develop HNSCC. The mechanisms of varying susceptibility to HNSCC are poorly studied to date. Tobacco metabolite research provides insight regarding the innate metabolism and excretion of carcinogens. Methods: Smokers with HNSCC (cases) were compared to smokers without HNSCC (controls) in a matched cohort. The tobacco metabolites studied are: 1-hydroxypyrene (1-HOP), Nf-nitrosonornicotine (NNN), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). Results: In 33 subjects, mean 1-HOP was 1.82 pmol/mg creatinine vs 1.08 pmol/mg creatinine (p=0.004) and mean NNN was 0.10 pmol/mg creatinine vs 0.04 pmol/mg creatinine (p=0.01) in cases and controls, respectively. NNAL did not differ between groups. Conclusions: Smokers with HNSCC have elevated urinary levels of 1-HOP and total NNN compared to matched controls suggesting an increased effective exposure to these carcinogens. Tobacco constituent metabolites may be useful in understanding tobacco-related carcinogenesis in HNSCC.
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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.
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    Regulatory roles of calprotectin in head and neck squamous cell carcinogenesis.
    (2011-07) Khammanivong, Ali
    Malignant transformation in squamous cell carcinomas (SCC) such as those of the head and neck (HNSCC) remains an enigmatic process that results in abnormal cellular differentiation, loss of growth and cell cycle regulation, gain of replicative immortality and resistance to apoptosis, activation of cellular migration and invasion, increase in energy metabolism, and evasion of immune destruction. These abnormalities in cell functions emerged as the hallmarks of cancer. Calprotectin, a heterodimeric protein complex of calcium regulating S100A8 and S100A9 encoded by genes mapped to the chromosomal locus 1q21.3 of the epidermal differentiation complex (EDC), may play essential roles in the regulation of cell differentiation, cell cycle progression, cellular survival and cell migration that are part of the cancer hallmarks. While highly upregulated in a variety of cancers, calprotectin is down-regulated in squamous cell carcinomas of the cervix, esophagus and the head and neck. Using microarray analysis for gene expression we found that the S100A9 subunit of calprotectin was significantly down-regulated along with other EDC genes in human primary HNSCC cases, suggesting a loss of functional S100A8/A9 protein complex and differentiation during carcinogenesis. Expression of S100A9 correlated strongly with a set of HNSCC downregulated genes putatively involved in loss of cytodifferentiation and control of cell cycle. To probe its role in carcinogenesis, S100A8/A9 was stably expressed in a calprotectin-negative human carcinoma cell line (KB cells). Expression of S100A8/A9 in KB cells up-regulated differentiation and cell-cell contact growth inhibition signaling pathways, re-established epithelial actin microfilament cytoskeletal structures and v cellular adhesion to the extracellular matrix, down-regulated anti-apoptosis gene networks, and suppressed anchorage-independent survival and stress-induced (by serum starvation) cell migration. Calprotectin appeared to induce growth suppression by signaling a reactivation of G2/M cell cycle checkpoint regulators, Chk1 and PP2A, and inactivation of mitotic activators Cdc25C and Cdc2. As a result, we saw a marked increase in Cdc2-PThr14/Tyr15 phosphorylation and down-regulation of cyclin B1, suggesting an inactivation of the mitotic entry promoting Cdc2/cyclin B1 complex, resulting in cell cycle and mitotic arrest at the G2/M checkpoint. When inoculated into nude mice, KB cells producing calprotectin showed reduced tumor growth when compared to sham-transfected control KB cells. Using shRNA, silencing of S100A8/A9 expression in the TR146 human HNSCC cell line increased carcinoma growth and survival and reduced Cdc2 phosphorylation at Thr14/Tyr15. Calprotectin expression in KB and TR146 cells also down-regulated expression of putative HNSCC marker genes, INHBA, PTGS2 (Cox-2) and SULF1, found to be expressed only in HNSCC samples. Calprotectin-mediated control of cellular differentiation and G2/M cell cycle checkpoint is, therefore, a likely suppressive mechanism in human squamous cell carcinomas and may point to new molecular targets for therapy.