by Wong Wing-yee, Felice. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 144-166). / Abstract also in Chinese. / Acknowledgements --- p.i / List of Abbreviations --- p.ii / Abstract --- p.iv / Abstract (Chinese Version) --- p.vi / Table of Contents --- p.viii / List of Tables --- p.xii / List of Figures --- p.xiv / List of Appendices --- p.xvi / Chapter Chapter I --- Literature Review / Chapter 1. --- Introduction --- p.1 / Chapter 2. --- Background of Cytochrome P450 --- p.3 / Chapter 2.1 --- Discovery --- p.3 / Chapter 2.2 --- Tissue Distribution --- p.3 / Chapter 2.3 --- Structure and Functions --- p.7 / Chapter 2.4 --- Nomenclature of the P450 Superfamily --- p.10 / Chapter 3. --- Cytochrome P450 2E1 (CYP2E1) --- p.11 / Chapter 3.1 --- Discovery --- p.11 / Chapter 3.2 --- Tissue Distribution --- p.12 / Chapter 3.3 --- Substrates and Inducers --- p.13 / Chapter 3.4 --- Toxicological Role of CYP2E1 --- p.15 / Chapter 4. --- CYP2E1-knockout Mouse Model --- p.17 / Chapter Chapter II --- Carbon Tetrachloride (CC14) Study / Chapter 1. --- Introduction --- p.19 / Chapter 1.1 --- General Properties and Usage of CC14 --- p.19 / Chapter 1.2 --- Toxicological Aspects of CC14 --- p.19 / Chapter 1.3 --- Mechanism of CCl4-induced Hepatotoxicity --- p.20 / Chapter 1.4 --- Role of CYP2E1 in CCl4-induced Hepatotoxicity --- p.23 / Chapter 1.5 --- Objectives of the Study --- p.27 / Chapter 2. --- Materials and Methods --- p.29 / Chapter 2.1 --- Chemicals and Materials --- p.29 / Chapter 2.2 --- Animals --- p.29 / Chapter 2.3 --- Acute CC14 Treatment --- p.29 / Chapter 2.4 --- Preparation of Microsomal Fractions --- p.30 / Chapter 2.5 --- Determination of Microsomal Protein Concentration --- p.31 / Chapter 2.6 --- Determination of Serum Aminotransferase Activities --- p.31 / Chapter 2.7 --- Liver Histology --- p.32 / Chapter 2.8 --- Hepatic Microsomal CYP2E1 Activity -p-nitrophenol Assay --- p.34 / Chapter 2.9 --- SDS-PAGE and Western Blot Analysis --- p.35 / Chapter 2.10 --- Detection of Lipid Peroxidation in vitro and in vivo --- p.35 / Chapter 2.10.1 --- In vitro Lipid Peroxidation - 2-Thiobarbituric Acid (TBA) assay --- p.35 / Chapter 2.10.2 --- In vivo Lipid Peroxidation - Microsomal Conjugated Dienes Detection --- p.36 / Chapter 2.11 --- Hepatic Lipid Fatty Acid Composition Analysis --- p.39 / Chapter 2.11.1 --- Lipid Extraction --- p.39 / Chapter 2.11.2 --- Thin Layer Chromatography --- p.39 / Chapter 2.11.3 --- Methylation --- p.40 / Chapter 2.11.4 --- Gas Chromatography --- p.40 / Chapter 2.12 --- Statistical Analysis --- p.41 / Chapter 3. --- Results --- p.42 / Chapter 3.1 --- "Mortality, Liver Weight and Liver Color" --- p.42 / Chapter 3.2 --- Hepatotoxicity --- p.42 / Chapter 3.2.1 --- Serum ALT and AST activities --- p.42 / Chapter 3.2.2 --- Liver Histology --- p.45 / Chapter 3.3 --- CYP2E1-catalysed PNP Activities and CYP2E1 Protein Levels --- p.49 / Chapter 3.3.1 --- CYP2El-catalyzed PNP Activities --- p.49 / Chapter 3.3.2 --- CYP2E1 Protein Levels --- p.52 / Chapter 3.4 --- Lipid Peroxidation --- p.52 / Chapter 3.4.1 --- In vitro Lipid Peroxidation --- p.52 / Chapter 3.4.2 --- In vivo Lipid Peroxidation --- p.54 / Chapter 3.5 --- Hepatic Lipid Fatty Acid Composition --- p.56 / Chapter 3.5.1 --- Fatty Acid Composition in Hepatic Phospholipid --- p.56 / Chapter 3.5.2 --- Fatty Acid Composition in Hepatic Microsomal Phospholipid --- p.59 / Chapter 3.5.3 --- Fatty Acid Composition in Hepatic Triglyceride --- p.61 / Chapter 4. --- Discussion --- p.63 / Chapter 4.1 --- CYP2E1 is Required in CCl4-mediated Hepatotoxicity --- p.63 / Chapter 4.2 --- CYP2E1 is Degraded following CC14 Exposure --- p.65 / Chapter 4.3 --- CYP2E1 is Required in CCl4-induced Lipid Peroxidation --- p.67 / Chapter 4.4 --- CYP2E1 is Required in CCl4-induced Hepatic Phospholipid Depletion --- p.70 / Chapter 4.5 --- CYP2E1 is Required in CCl4-induced Hepatic Triglyceride Accumulation --- p.72 / Chapter 5. --- Conclusion --- p.76 / Chapter Chapter III --- Chronic Ethanol Consumption Study / Chapter 1. --- Introduction --- p.77 / Chapter 1.1 --- Multiple Metabolic Pathways for Ethanol Metabolism --- p.77 / Chapter 1.2 --- Metabolism of Ethanol by the Microsomal Ethanol Oxidizing System --- p.79 / Chapter 1.3 --- Role of CYP2E1 in Ethanol Metabolism --- p.82 / Chapter 1.4 --- Role of CYP2E1 in Alcoholic Liver Disease and Associated Oxidative Stress --- p.84 / Chapter 1.5 --- Objectives of the Study --- p.89 / Chapter 2. --- Materials and Methods --- p.90 / Chapter 2.1 --- Chemicals and Materials --- p.90 / Chapter 2.2 --- Animals --- p.90 / Chapter 2.3 --- Chronic Ethanol Treatment --- p.90 / Chapter 2.3.1 --- Ethanol Diet Composition --- p.90 / Chapter 2.3.2 --- Ethanol Feeding --- p.90 / Chapter 2.4 --- Monitoring of Blood Ethanol Levels --- p.96 / Chapter 2.5 --- Preparation of Microsomal Fractions --- p.96 / Chapter 2.6 --- Determination of Microsomal Protein Concentration --- p.97 / Chapter 2.7 --- Determination of Serum Aminotransferase Activities --- p.98 / Chapter 2.8 --- Liver Histology --- p.98 / Chapter 2.9 --- SDS-PAGE and Western Blot Analysis --- p.99 / Chapter 2.10 --- Hepatic Fatty Acid Composition Analysis --- p.100 / Chapter 2.10.1 --- Lipid Extraction --- p.100 / Chapter 2.10.2 --- Thin Layer Chromatography --- p.101 / Chapter 2.10.3 --- Methylation --- p.101 / Chapter 2.10.4 --- Gas Chromatography --- p.102 / Chapter 2.11 --- Statistical Analysis --- p.103 / Chapter 3. --- Results --- p.104 / Chapter 3.1 --- Average Food Consumption --- p.104 / Chapter 3.2 --- Average Ethanol Consumption for Ethanol Liquid Diet Feeding Group --- p.104 / Chapter 3.3 --- Body Weight Gain --- p.104 / Chapter 3.4 --- Blood Ethanol Levels --- p.108 / Chapter 3.5 --- "Mortality, Liver Weight and Liver Color" --- p.108 / Chapter 3.6 --- Serum ALT and AST Activities --- p.110 / Chapter 3.7 --- Liver Histology --- p.114 / Chapter 3.8 --- Western Blot Analysis --- p.119 / Chapter 3.9 --- Hepatic Lipid Fatty Acid Composition --- p.119 / Chapter 3.9.1 --- Fatty Acid Composition in Hepatic Phospholipid --- p.119 / Chapter 3.9.2 --- Fatty Acid Composition in Hepatic Triglyceride --- p.123 / Chapter 4. --- Discussion --- p.126 / Chapter 4.1 --- Nutrients Displacement after Chronic Ethanol Consumption --- p.126 / Chapter 4.2 --- Varied Blood Ethanol Levels after Chronic Ethanol Consumption --- p.127 / Chapter 4.3 --- Increase in CYP2E1 Levels after Chronic Feeding of Ethanolin WT mice --- p.127 / Chapter 4.4 --- Lack of Evidence Indicating the Development of Ethanol- Induced Liver Injury --- p.129 / Chapter 4.4.1 --- No Elevations in Serum ALT and AST Activities --- p.129 / Chapter 4.4.2 --- Normal Liver Histology --- p.130 / Chapter 4.4.3 --- Lack of Triglyceride Accumulation --- p.131 / Chapter 4.4.4 --- Elevations in Hepatic PL --- p.132 / Chapter 4.5 --- Possible Reasons for the Absence of Liver Damage after Chronic Ethanol Consumption in our Mouse Model --- p.134 / Chapter 5. --- Conclusion --- p.137 / Chapter Chapter IV --- Concluding Remarks / Chapter 1. --- A Comparison between Acute CC14 Study and Chronic Ethanol Consumption Study --- p.139 / Chapter 1.1 --- Regulation of CYP2E1 Expression --- p.139 / Chapter 1.2 --- Free Radical Production Involved in CC14- and Chronic Ethanol Consumption-Mediated Liver Injury --- p.140 / Chapter 1.3 --- An Overall Comparison between CC14 study and Chronic Ethanol Consumption Study --- p.140 / Chapter 2. --- Future Studies --- p.142 / Chapter 2.1 --- Acute CC14 Study --- p.142 / Chapter 2.1.1 --- Calcium Homeostasis Studies --- p.142 / Chapter 2.1.2 --- Spin Trapping Studies --- p.142 / Chapter 2.2 --- Chronic Ethanol Study --- p.142 / Chapter 2.2.1 --- "Generation of a Heterozygous ""Ethanol-Sensitive"" Mouse Strain (SV/129/ter x C57BL/6)" --- p.143 / Chapter 3. --- Concluding Remarks --- p.143 / References --- p.144 / Appendix --- p.167