Thesis (Ph. D.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains viii, 147 p. : ill. Vita. Includes abstract. Includes bibliographical references.

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Thesis (Ph.D.)--Medical University of Ohio, 2006. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Michael A. Pereira. Includes abstract. Document formatted into pages: v, 152 p. Title from title page of PDF document. Title at ETD Web site: Involvement of DNA methylation and CpG endonuclease activity in environmental carcinogenesis and cancer chemoprevention. Bibliography: pages 65-66, 90-92, 123-125, 137-150.

The nucleus may be the critical site for the activation of chemical carcinogens, and subsequently the initiation of neoplasia. However, isolated nuclei may be contaminated with endoplasmic reticulum, the major site of the drug metabolizing enzymes. One of the objectives of the present study was to determine whether the enzymes in isolated rat hepatic nuclei were of nuclear origin and, if so, to compare these enzymes with those in the microsomal fraction. The selective manipulation of nuclear enzymes would be a useful tool in determining their role in cellular toxicity. Recentrifugation experiments, with aryl hydrocarbon hydroxylase (AHH) activity as a marker, showed that isolated nuclei were not contaminated with endoplasmic reticulum in the form of microsomes formed upon homogenization. However, small "tags" of endoplasmic reticulum, continuous with the nuclear membrane, and indiscernable in electron micrographs, could remain following centrifugation and account for all of the measurable enzyme activity in the isolated nuclei. It was reasoned that if endoplasmic reticulum accounted for all of the activity, then the ratio of nuclear to microsomal activity for all enzymes determined should be the same. The ratios of epoxide hydrolase and AHH were found to differ in the two fractions. The simplest interpretation of these data was that drug metabolizing enzymes existed in the nuclei. However, the distribution of drug metabolizing enzymes throughout the endoplasmic reticulum is known to be heterogeneous and these "tags" could differ from the total endoplasmic reticulum (microsomes) in their enzyme make-up. Whether these enzymes are in the nuclear membrane, nucleoplasm, or as "tags" of endoplasmic reticulum, they represent activities in close proximity to potential target sites in the nucleus. The inhibition, induction, and activation characteristics of nuclear and microsomal enzymes were studied with the goal of selective manipulation of nuclear enzymes. The enzymes in the nuclear and microsomal fractions were found to differ' only in quantitative inducibility, and were identical in all other respects. Therefore, the selective manipulation of nuclear enzymes was not achieved. The induction of hepatic drug metabolizing enzymes is a measure of altered genetic expression in the liver. Inducers of drug metabolizing enzymes have also been shown to promote neoplasia in the liver. Therefore, studying the induction of such enzymes may lead to a further understanding of the mechanism of tumour promotion. Phenobarbital, 3-methylcholanthrene and pregnenolone-16α-carbonitrile produce three distinct induction responses. In the present study, spironolactone and trans-stiIbene oxide were shown to produce distinct induction responses, also. Spironolactone was shown to be a different inducer based on the protein band patterns observed following SDS-polyacrylamide gel electrophoresis of liver microsomes. trans-Stilbene oxide was found to produce a significantly different maximal level of AHH activity. The observation of five distinct induction responses suggests at least five recognition sites (receptors) mediating the pleiotropic actions of exogenous compounds in the liver. / Pharmaceutical Sciences, Faculty of / Graduate

Eleven subjects completed a clinical trial to determine the safety/tolerability of freeze-dried black raspberries (BRB) and to measure, in plasma and urine, specific anthocyanins-cyanidin-3-glucoside, cyanidin-3-sambubioside, cyanidin-3-rutinoside, and cyanidin-3- xylosylrutinoside, as well as ellagic acid. Subjects were fed 45 g of freeze-dried BRB daily for 7 days. Blood samples were collected predose on days 1 and 7 and at 10 time points postdose. Urine was collected for 12 hours predose on days 1 and 7 and at three 4-hour intervals postdose. Maximum concentrations of anthocyanins and ellagic acid in plasma occurred at 1 to 2 hours, and maximum quantities in urine appeared from 0 to 4 hours. Overall, less than 1% of these compounds were absorbed and excreted in urine. None of the pharmacokinetic parameters changed significantly between days 1 and 7. In conclusion, 45 g of freeze-dried BRB daily are well tolerated and result in quantifiable anthocyanins and ellagic acid in plasma and urine.

Indiana University-Purdue University Indianapolis (IUPUI) / Since exposure to carcinogens can occur in the environment from various point sources, cancer risk assessment attempts to define and limit potential exposure such that the risk of developing cancer is negligible. While cancer risk assessment is widely used with certain methodologies well accepted in the scientific literature and regulatory guidances, there are still gaps which increase uncertainties when assessing risk including: (1) mixtures of genotoxins, (2) genotoxic metabolites, and (3) nongenotoxic carcinogens. An in silico model was developed to predict the cancer risk of a genotoxin which improved methodology for a single compound and mixtures. Monte Carlo simulations performed with a carcinogenicity potency database to estimate the overall carcinogenic risk of a mixture of genotoxic compounds showed that structural similarity would not likely increase the overall cancer risk. A cancer risk model was developed for genotoxic metabolites using excretion material in both animals and humans to determine the probability not exceeding a 1 in 100,000 excess cancer risk. Two model nongenotoxic compounds (fenofibrate and methapyraline) were tested in short-term microarray studies to develop a framework for cancer risk assessment. It was determined that a threshold for potential key events could be derived using benchmark dose analysis in combination with well developed ontologies (Kegg/GO), which were at or below measured tumorigenic and precursor events. In conclusion, informatics was effective in advancing toxicology-based cancer risk assessment using databases and predictive techniques which fill critical gaps in its methodology.

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Thesis (D.Tech.: Biotechnology)-Dept. of Biotechnology, Durban Institute of Technology, 2005 xx, 175, [14] leaves : ill. ; 30 cm / Previous studies indicate that a mutation in the non-oncogenic p53 gene is epidemiologically linked to human HCC (Ozturk, 1991; Chan et al., 2003). Hsu et al. (1991) found this link in Chinese, South African and Asian patients and Hollstein et al. (1993) found the same gene mutation in Taiwanese patients. The incidence of these aberrations is reported to be about 20- 50% in HCC’s (Kishimoto et al., 1997). There is sufficient evidence to indicate that carotenoids in addition to their well known antioxidant properties (Paiva and Russel, 1999), also affect intercellular communication, immune responses, neoplastic transformations and growth control, and cellular levels of enzymes that detoxify carcinogens (Zhang et al., 1991; Brockman et al., 1992; Pryor et al., 2000). To date studies carried out have used the rat (Foote et al., 1970; Gradelet et al., 1998) and the mule duckling model (Cheng et al., 2001) to show the protective effect of these carotenoids against AFB1 exposure. Of the well known carotenoids, lycopene and beta- carotene occur in abundance in fruits and vegetables and are safe for human consumption. Aflatoxin B1 frequently induces mutations of the p53 gene which is linked to HCC. Although there is much evidence from epidemiological studies linking the beneficial aspects of carotenoids to the prevention of cancer, the cellular and molecular mechanisms need to be understood in order to implement large scale intervention strategies to prevent AFB1 induced carcinoma. The use of chemical or dietary interventions to alter the susceptibility of humans to the actions of carcinogens and to block, retard or reverse carcinogenesis is an emerging chemoprotective strategy for disease prevention (Abdulla and Gruber, 2000; Kensler et al., 2003; Bingham and Riboli, 2004). Chemoprotection by natural products involves maintaining cellular integrity, preventing DNA alterations, activation of p53 suppressor protein and apoptosis. The aim of this study was thus to investigate the cellular and molecular mechanisms by which beta-carotene and lycopene may prevent the AFB1-induced toxic changes in human hepatocytes. In order to achieve this aim, the following objectives were set out: i. To optimise an in vitro system for the evaluation of AFB1 damage to cultured hepatocytes. ii. To determine the biochemical protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by measuring the mitochondrial activity, cell viability and ROS levels using appropriate enzyme assays and flow cytometry. iii. To determine the cellular protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by studying the morphological changes at the structural and ultrastructural levels using phase contrast light and electron microscopy respectively. iv. To determine the molecular protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by detecting apoptotic bodies as genomic markers and measuring the levels of p53 protein and AFB1-N7-guanine adducts produced.

In the context of the somatic mutation theory of chemical carcinogenesis, mutations are thought to arise during the replication of DNA past carcinogen-DNA adducts. The work described in this thesis deals with the testing of a hypothetical mechanism whereby mammalian cells are able to replicate their DNA past polycyclic aromatic hydrocarbon DNA adducts. The second objective of this thesis work was to develop both in vivo and in vitro models to study the induction of mutations in a target human gene by chemical carcinogens from two different classes, polycyclic aromatic hydrocarbons and nitrosamines. To approach the hypothetical mechanism of bypass replication in mammalian cells, synchronized Chinese hamster ovary cells were treated with the ultimate carcinogenic form of benzo (a) pyrene, 7β, 8α-dihydroxy-9α, 10α-epoxy-7,8,9,10-tetrahydrobenzo (a) pyrene (BPDE I). Using the pH step alkaline elution assay, it was found that the reduced rate of S phase progression was due to a delay in the appearance of multiple replicon size nascent DNA. It was determined using agarose gel electrophoresis that the ligation of Okazaki size replication intermediates was blocked in BPDE I-treated, synchronized CHO cells. The data obtained were, therefore, supportive of the 'block-gap' model of DNA bypass replication in carcinogen damaged mammalian cells. To study mutagenesis of a specific sequence induced by chemical carcinogens, the human c-Ha-ras proto-oncogene was transfected into the mouse fibroblast cell line, NIH 3T3. Transfected NIH 3T3 cell lines (HHRN 1-4) were isolated that had a low copy number of the human c-Ha-ras proto-oncogene and a non-transformed phenotype. It was determined that the integrated human c-Ha-ras gene was hypomethylated, and expressed at the messenger level. The human c-Ha-ras protein, p21, was also detected in these transfected cell lines. Treatment of the HHRN cell lines with the nitrosamine, N-methyl-N-nitroso-N'-nitroguanidine (MNNG) resulted in transformed NIH 3T3 foci. In vitro MNNG treatment of the plasmid, z-6, and transfection into NIH 3T3 cells led to the isolation of transformed cell lines. Screening of the in vitro and in vivo treated, transformed cell lines by RNA:RNA duplex mismatch analysis led to the detection of no mutations within the first exon of the human c-Ha-ras oncogene.

The purpose of this thesis is to explore differences in lifetime excess cancer risk (LECR) for Canadians from intake of contaminants in food and beverages based on geographic location, gender and income levels. A probabilistic risk assessment approach (Monte Carlo simulation) was used to estimate the range and frequency of possible daily contaminant intakes for Canadians, and associate these intake levels with lifetime excess cancer risk. Monte Carlo risk simulation was applied to estimate probable contaminant intake and associated lifetime excess cancer risk from arsenic, benzene, lead, polychlorinated biphenyls (PCBs) and tetrachloroethylene (PERC) in 60 whole foods from the dietary patterns of 34,944 Canadians from 10 provinces, as derived from Health Canada’s Canadian Community Health Survey, Cycle 2.2, Nutrition (2004)1. These results were compared to the current Health Canada guideline that suggests that 10 extra cancers per one million people is a negligible risk. Of the 5 contaminants tested in my model arsenic showed the greatest difference between urban and rural estimated lifetime excess cancer risk, although extra cancers in both rural and urban Canada were predicted from exposure to PCB and benzene. Lifetime excess cancer risk is estimated to be higher for men in Canada for all five contaminants, with an emphasis on males in British Columbia compared to females from the dietary intake of arsenic. When based on income level, my model predicts extra cancers higher for low and middle incomes from dietary exposures to arsenic, benzene, lead and PERC, however, high income populations are more likely to have extra cancers from dietary intake of PCBs. / Graduate