by Leung Yuet Kin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 86-100). / Abstract also in Chinese. / List of Figures --- p.ix / List of Tables --- p.xii / List of Abbreviations --- p.xiii / Chapter Chapter One: --- Introduction / Chapter 1.1 --- Carcinogenicity of the tobacco products --- p.1 / Chapter 1.2 --- Biochemical Pathway involved in NNK metabolism --- p.5 / Chapter 1.2.1 --- Metabolic activation of NNK --- p.6 / Chapter 1.2.2 --- Detoxification of NNK --- p.8 / Chapter 1.3 --- Modulation of NNK carcinogenesis --- p.9 / Chapter 1.4 --- Mediation of NNK oxidation : chemoprevention by isothiocyanates --- p.11 / Chapter 1.5 --- Aim of study --- p.13 / Chapter 1.5.1 --- Experimental approaches --- p.15 / Chapter Chapter Two : --- Modulation of α-carbon hydroxylations in NNK metabolism / Chapter 2.1 --- Background --- p.17 / Chapter 2.2 --- Materials and Methods --- p.17 / Chapter 2.2.1 --- Chemicals --- p.17 / Chapter 2.2.2 --- Methods --- p.18 / Chapter 2.2.2.1 --- Preparation of rat microsomes --- p.18 / Chapter 2.2.2.2 --- Assay of NNK metabolism --- p.18 / Chapter 2.2.2.3 --- Determination of solvent extraction --- p.19 / Chapter 2.2.2.4 --- Determination of detergent effects --- p.19 / Chapter 2.2.2.5 --- HPLC analysis of NNK metabolites --- p.20 / Chapter 2.2.2.6 --- Study of strain differences between SD rats and F344 rats --- p.20 / Chapter 2.3 --- Results --- p.21 / Chapter 2.3.1 --- HPLC analysis of NNK metabolites --- p.21 / Chapter 2.3.2 --- Determination of solvent extraction --- p.22 / Chapter 2.3.3 --- Effects of detergents --- p.25 / Chapter 2.3.4 --- Study of strain differences using F344 rats and SD rats --- p.26 / Chapter 2.4 --- Discussion --- p.27 / Chapter Chapter Three : --- Modulation by potentiation of detoxification process in NNK metabolism / Chapter 3.1 --- Background --- p.30 / Chapter 3.2 --- Materials and Methods --- p.31 / Chapter 3.2.1 --- Chemicals --- p.31 / Chapter 3.2.2 --- Methods --- p.32 / Chapter 3.2.2.1 --- Preparation of rat microsomes --- p.32 / Chapter 3.2.2.2 --- Analysis of NNK metabolism --- p.32 / Chapter 3.2.2.3 --- UDP-glucuronosyltransferase Assay --- p.33 / Chapter 3.2.2.4 --- Cytochrome P450 2E1 Assay --- p.34 / Chapter 3.3 --- Results --- p.34 / Chapter 3.3.1 --- Screening tests of the effects of vitamins and drugs --- p.34 / Chapter 3.3.1.1 --- Male liver microsomes --- p.35 / Chapter 3.3.1.2 --- Female liver microsomes --- p.37 / Chapter 3.3.1.3 --- Male lung microsomes --- p.39 / Chapter 3.3.1.4 --- Female lung microsmes --- p.41 / Chapter 3.3.2 --- Kinetic analysis of vitamin C-palmitate on NNK reduction --- p.43 / Chapter 3.3.3 --- Effects of vitamin C-palmitate on UDP- glucuronosyltransferase --- p.47 / Chapter 3.3.4 --- Effects of vitamin C-palmitate on cytochrome P4502E1 (CYP2E1) --- p.48 / Chapter 3.4 --- Discussion --- p.50 / Chapter Chapter Four: --- Purification of carbonyl reductase from rat liver microsomes / Chapter 4.1 --- Background --- p.58 / Chapter 4.2 --- Materials and Methods --- p.58 / Chapter 4.2.1 --- Chemicals --- p.58 / Chapter 4.2.2 --- Methods --- p.59 / Chapter 4.2.2.1 --- Preparation of male rat liver microsomes --- p.59 / Chapter 4.2.2.2 --- Purification of carbonyl reductase from rat liver microsomes --- p.59 / Chapter A. --- Solubilization of microsomes --- p.59 / Chapter B. --- Chromatographic separation by octyl-Sepharose CL-4B --- p.60 / Chapter C. --- Chromatographic separation by DEAE-cellulose --- p.60 / Chapter 4.2.2.3 --- SDS polyacrylamide gel electrophoresis --- p.61 / Chapter 4.3 --- Results --- p.61 / Chapter 4.4 --- Discussion --- p.63 / Chapter Chapter Five : --- Summary and Discussion / Chapter 5.1 --- Summary --- p.66 / Chapter 5.2 --- Discussion --- p.67 / Chapter 5.2.1 --- Anticarcinogenesis of vitamin C in NNK-induced cancer through smoking --- p.67 / Chapter 5.2.2 --- Future studies on microsomal carbonyl reductase --- p.68 / Chapter 5.2.3 --- Future studies on UDP-glucuronidation of NNAL --- p.72 / Appendix A Effects of atropine on tolbutamide metabolism --- p.74 / Appendix B Recovery of keto acid and keto alcohol from the assay mixture ( without solvent extraction ) --- p.75 / Appendix C Calibration Curve of NNAL --- p.76 / Appendix D Effects of methanol on NNK carbonyl reduction --- p.77 / Appendix E Time course study of NNAL production at various concentrations of vitamin C-palmitate --- p.78 / Appendix F Time course assay of UDP-glucuronosyltransferase at various concentrations of vitamin C-palmitate --- p.80 / Appendix G Calculation of specific activity of UDP-Glucuronosyl transferase --- p.82 / Appendix H Calculation of specific activity of cytochrome P450 2E1 --- p.84 / References --- p.86

Thesis (M.S.)--University of Wisconsin--Madison, 1983. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

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.