Browsing by Subject "NNK"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    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.
  • Thumbnail Image
    Item
    The metabolism of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone [NNK] and the enantiomers of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol [NNAL] in the isolated perfused rat lung system.
    (2010-08) Maertens, Laura A.
    4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent carcinogen found specifically in tobacco products. It has been shown to be a lung-specific carcinogen in rodents, and may play a critical role in the formation of lung cancer in smokers. One of the enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a metabolite of NNK, may be important to the selective pulmonary carcinogenicity of NNK. The objective of the current research was to better characterize the pulmonary metabolism of NNK, (S)-NNAL, and (R)-NNAL using the isolated perfused rat lung (IPRL) system to elucidate the mechanisms behind the lung-specific nature of NNK. This research examined metabolite formation, distribution of the metabolites between the perfusate and tissue, the formation of individual DNA adducts in the tissue, and the effects of concentration and the chemopreventive agent PEITC. The results showed that NNK was readily metabolized and DNA adducts were detected in the tissue at the end of the 180 min perfusions. Both an increase in NNK concentration and the co-administration of PEITC were shown to inhibit NNK metabolism. PEITC was also shown to significantly reduce the formation of DNA adducts. The results obtained for the NNK perfusions were in agreement with previously published results. (S)-NNAL and (R)-NNAL were not metabolized as extensively by the lung as NNK. The metabolism of the two enantiomers was similar, which was in contrast to previous in vitro and in vivo results. The only observed difference between the two enantiomers was the formation of low levels of a pyridyloxobutyl (POB)-DNA adduct in the (S)-NNAL perfusions, which indicated reoxidation to NNK. The unexpected results for the NNAL enantiomers may be a result of diffusional barriers to the preformed metabolites that do not exist when the enantiomers are formed from NNK in the tissue. This work showed that the IPRL system was a valid system for examining the pulmonary metabolism of NNK and the formation of DNA adducts, but it may have some limitations for more polar compounds that cannot penetrate the diffusional barriers of the lung and the cells to gain access to the enzymatic sites responsible for metabolism.
  • Thumbnail Image
    Item
    Metabolism of nicotine and the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK): genetic and phenotypic variation.
    (2009-11) Berg, Jeannette Zinggeler
    Nicotine is the addictive agent in tobacco and differences in nicotine metabolism may affect tobacco use, and consequently exposure to tobacco carcinogens. A lung procarcinogen in tobacco is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its carcinogenic effect is dependent on metabolic activation and is counter-balanced by metabolic detoxification. Nicotine and NNK are structurally related and both are metabolized by cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), and flavin-monoxygenase (FMO) enzymes. The goal of this thesis research was to explore variation in nicotine metabolism in vivo and to probe specific enzyme-catalyzed reactions of NNK in vitro. Low nicotine and cotinine glucuronidation were observed among African Americans compared to Europeans, and among individuals with a variant UGT2B10 genotype. In a controlled dose study of ethnic differences in nicotine metabolism (n= 93 smokers), African Americans excreted 30-40 % less nicotine and cotinine as their glucuronide conjugates than European Americans. This difference in glucuronidation explained the higher free cotinine concentrations observed in African Americans compared to European Americans. The most efficient in vitro catalyst of nicotine and cotinine glucuronidation is UGT2B10. We demonstrated that UGT2B10 contributes to in vivo nicotine metabolism in a genotype-phenotype analysis of 325 smokers. Individuals who were heterozygous for the UGT2B10 Asp67Tyr allele excreted less nicotine or cotinine as their glucuronide conjugates than wild-type; the ratio of cotinine glucuronide:cotinine was decreased by 60 %, while increases in urinary and plasma cotinine and trans-3'-hydroxycotinine were observed. Strikingly, a robust biomarker of nicotine intake, nicotine equivalents, were lower among Asp67Tyr heterozygotes compared to individuals without this allele; 58.2 nmol/ml (95 % CI, 48.9 - 68.2) versus 69.2 nmol/ml (95 % CI, 64.3 - 74.5). Individuals with low activity UGT2B10 may smoke less intensely, as reported for individuals with CYP2A6 polymorphisms that cause decreased nicotine C-oxidation. In contrast to nicotine, NNK is a carcinogen. It is metabolized to reactive intermediates that can form DNA and protein adducts, or it is detoxified by glucuronidation. P450 2A13 is the most efficient catalyst of NNK oxidation. We explored the effect of an active site mutant, Asn297Ala, on enzyme function and found that loss of hydrogen bonding to substrate in the active site affected substrate orientation and product formation. The orphan P450 2A7 was considered as a potential catalyst for NNK oxidation, but expression of wild-type or two naturally-occurring variants failed to yield protein with a P450 spectra and no appreciable activity towards P450 2A substrates was observed. Preliminary experiments were conducted to search for the glucuronide conjugate formed from the unstable oxidation product alpha-hydroxymethyl NNK, which has not been identified in any human system. The extent to which variation in metabolism mediates smoking behavior and cancer risk warrants consideration. The enzymes involved are potential drug targets for smoking cessation pharmacotherapy and cancer chemoprevention.
  • Thumbnail Image
    Item
    Two barriers to Ras mediated oncogenesis: translational control checkpoint and proliferative block by autophagy.
    (2009-06) Kim, Yong Yean
    According to the American Cancer Society, cancer is the second leading cause of mortality in the United States, accounting for more than half a million deaths per year. Cancer is a complex disease with multiple factors influencing its genesis, maintenance, growth, and invasion which makes the treatment and prevention of the disease challenging. Despite the high mortality and morbidity associated with cancer, it is a disease of the old, with the median age for cancer incidence being 66 years old according to the National Cancer Institute. Therefore, the human body is remarkably adept at protecting itself from malignant transformation. In my thesis, I explore the oncogene Ras which is represented in approximately 30% of all human cancers, making Ras one of the most commonly activated oncogenes. I analyzed the innate barriers to oncogenic Ras (RasV12) induced transformation in the cell in an attempt to better understand tumor defense systems which can be mimicked for novel cancer therapy. My results confirmed one putative barrier which had good experimental grounding and identified a completely new barrier. One putative barrier to RasV12 oncogenesis was the existence of a translational control check point in tumor defense. Activation of translation initiation has been shown to be on the causal pathway to cancer and is activated by Ras via two different pathways. The rate limiting translation initiation factor 4E (eIF4E) is up regulated in many cancers and over expression of eIF4E confers cells with transformed phenotypes. Therefore, if activation of translation is oncogenic, then it is reasonable to posit the existence of a translational control checkpoint in tumor defense. The logical guardians of this checkpoint would be the primary negative regulators of translational initiation, the eIF4E binding protein (4E-BP) family of proteins. I show that translational control checkpoint does indeed exist in tumor defense and that the 4E-BPs are the guardians of this checkpoint. Mice lacking two of the three 4E-BPs (4ebp1-/-/4ebp2-/-) were more sensitive to tobacco carcinogen NNK induced lung tumors and showed tumors with increased vascularity. Also, 4ebp1-/-/4ebp2-/- genotype was associated with a skewing of the genome wide translational profile towards growth and proliferation even before NNK treatment indicating a cancer primed state. Lastly, I showed that the cytochrome P450 2A5, the protein that metabolizes NNK to its carcinogenic product, was translationally up regulated increasing the carcinogenic potency of NNK. The second barrier to RasV12 oncogenesis was an unexpected discovery. In an effort to determine the mechanism of RasV12 oncogenesis and its defense, I discovered that RasV12 triggered proliferative block even in cells which have bypassed the senescence barriers. This was unexpected since previous reports had shown that in this setting, RasV12 actually caused anchorage independent growth and invasion in vitro. The nature of the proliferative block was not senescence although it has many of the characters of senescence. Due to a striking phenotypical change where large vacuoles accumulate, I explored the possibility that autophagy was playing a role in the proliferative block. I show that RasV12 expressing cells displayed hallmarks of autophagy such as double membraned vacuoles with pieces of organelles, acidic nature of the vacuoles, and positivity for early and late markers of autophagy. My study validates the current efforts to develop targeted therapy against the translation initiation complex and provides autophagy as a new potential target for caner therapy. By learning from the cell's innate cancer barriers, I hope that we will be able to develop more effect therapies for cancer.