Meng Jie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references. / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Table of Contents --- p.vi / Abbreviation --- p.x / List of Figures --- p.xi / List of Tables --- p.xiii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Black Mustard and Sinigrin --- p.2 / Chapter 1.2 --- Hepatocellular Carcinoma --- p.5 / Chapter 1.2.1 --- Different Stages of HCC --- p.6 / Chapter 1.2.2 --- Risk Factors --- p.8 / Chapter 1.2.3 --- Treatments of HCC --- p.10 / Chapter 1.3 --- Biomarkers Used to Evaluate Effects of Sinigrin on HCC --- p.12 / Chapter 1.3.1 --- AST & ALT --- p.12 / Chapter 1.3.2 --- Glutathione S Transferase -p (GST-p) --- p.13 / Chapter 1.4 --- Tumor Suppressor Genes and Oncogenes --- p.14 / Chapter 1.4.1 --- "p53, the Tumor Suppressor Gene" --- p.15 / Chapter 1.4.2 --- p53-dependent pathway --- p.15 / Chapter 1.4.2.1 --- Mdm2 --- p.16 / Chapter 1.4.2.2 --- Bax and Bcl-2 --- p.17 / Chapter 1.4.2.3 --- PCNA and p21wAF1/CIP1 --- p.18 / Chapter 1.5 --- Aim of the Project --- p.19 / Chapter Chapter 2: --- Materials and Methods --- p.20 / Chapter 2.1 --- In vitro Studies --- p.21 / Chapter 2.1.1 --- Neutral Red Assay --- p.21 / Chapter 2.1.1.1 --- Chemicals and Reagents --- p.21 / Chapter 2.1.1.2 --- Liver Cells --- p.23 / Chapter 2.1.1.3 --- Neutral Red Assay --- p.24 / Chapter 2.1.2 --- Flow Cytometery --- p.24 / Chapter 2.1.2.1 --- Chemicals and Reagents --- p.25 / Chapter 2.1.2.2 --- Flow Cytometery Analysis --- p.25 / Chapter 2.1.3 --- DNA Fragmentation --- p.26 / Chapter 2.1.3.1 --- Chemicals and Reagents --- p.26 / Chapter 2.1.3.2 --- DNA Extraction --- p.28 / Chapter 2.1.3.3 --- DNA Agarose Gel Electrophoresis --- p.29 / Chapter 2.1.4 --- cDNA Microarray --- p.29 / Chapter 2.1.4.1 --- Chemicals and Reagents --- p.30 / Chapter 2.1.4.2 --- RNA Extraction --- p.33 / Chapter 2.1.4.3 --- RNA Quantity and Quality Control --- p.34 / Chapter 2.1.4.4 --- RT-PCR --- p.35 / Chapter 2.1.4.5 --- cRNA Convention and Purification --- p.36 / Chapter 2.1.4.6 --- Hybridization --- p.37 / Chapter 2.1.4.7 --- Washing and Detection --- p.37 / Chapter 2.1.4.8 --- Data Analysis --- p.38 / Chapter 2.2 --- In vivo Studies --- p.39 / Chapter 2.2.1 --- Animal Treatment --- p.39 / Chapter 2.2.1.1 --- Chemicals and Reagents --- p.39 / Chapter 2.2.1.2 --- Chemical Carcinogens --- p.40 / Chapter 2.2.1.3 --- Promotion Stage --- p.41 / Chapter 2.2.1.4 --- Progression Stage --- p.44 / Chapter 2.2.2 --- Measurement of Serum ALT and AST Activities --- p.46 / Chapter 2.2.2.1 --- Chemicals and Reagents --- p.46 / Chapter 2.2.2.2 --- Activity Assay --- p.46 / Chapter 2.2.3 --- Histological Analysis --- p.47 / Chapter 2.2.3.1 --- Chemicals and Reagents --- p.47 / Chapter 2.2.3.2 --- Preparation of Slides --- p.49 / Chapter 2.2.3.3 --- H&E Staining --- p.49 / Chapter 2.2.3.4 --- GST-p Immuno-staining --- p.50 / Chapter 2.2.4 --- Semi-Quantitative RT-PCR Analysis of mRNA Expression --- p.53 / Chapter 2.2.4.1 --- Chemicals and Reagents --- p.53 / Chapter 2.2.4.2 --- Extraction of total RNA from rat liver --- p.53 / Chapter 2.2.4.3 --- Quantity and Quality Control of RNA --- p.53 / Chapter 2.2.4.4 --- RT-PCR (Reverse Transcription) --- p.54 / Chapter 2.2.4.5 --- PCR --- p.54 / Chapter 2.2.4.6 --- DNA gel electrophoresis --- p.55 / Chapter 2.2.4.7 --- Data Analysis --- p.56 / Chapter 2.2.5 --- Western Blot Analysis for Biomarkers --- p.56 / Chapter 2.2.5.1 --- Chemicals and Reagents --- p.56 / Chapter 2.2.5.2 --- Extraction of the Cytosol Protein --- p.60 / Chapter 2.2.5.3 --- Extraction of the Nuclear protein --- p.61 / Chapter 2.2.5.4 --- SDS Gel Electrophoresis --- p.61 / Chapter 2.2.5.5 --- Western Blot --- p.62 / Chapter 2.2.5.6 --- Interaction with Antibodies --- p.63 / Chapter 2.2.5.7 --- ECL Detection --- p.63 / Chapter 2.2.5.8 --- Data Analysis --- p.64 / Chapter Chapter 3: --- Results --- p.65 / Chapter 3.1 --- In vitro Studies --- p.66 / Chapter 3.1.1 --- Cell Viability test and IC50 --- p.66 / Chapter 3.1.2 --- Cell Cycle Analysis --- p.68 / Chapter 3.1.3 --- DNA Fragmentation --- p.71 / Chapter 3.1.4 --- Effects of Sinigrin on Gene Expression --- p.73 / Chapter 3.2 --- In vivo Studies --- p.77 / Chapter 3.2.1 --- Effects of Sinigrin on HCC Development (Promotion stage) in Rats --- p.77 / Chapter 3.2.1.1 --- Direct Observation --- p.77 / Chapter 3.2.1.2 --- Relative Liver / Body Weight Ratio --- p.79 / Chapter 3.2.1.3 --- AST/ALT Assay --- p.81 / Chapter 3.2.1.4 --- Basic Structure of Hepatocytes --- p.83 / Chapter 3.2.1.5 --- GST-p Foci Area --- p.85 / Chapter 3.2.1.6 --- mRNA Expression of p53 and Mdm2 --- p.88 / Chapter 3.2.1.7 --- Protein Expression of Biomarkers --- p.90 / Chapter 3.2.2 --- Effects of Sinigrin on HCC Development (Progression stage) in Rats --- p.97 / Chapter 3.2.2.1 --- Direct Observation --- p.97 / Chapter 3.2.2.2 --- Relative Liver / Body Weight Ratio --- p.99 / Chapter 3.2.2.3 --- AST/ALT Assay --- p.101 / Chapter 3.2.2.4 --- Basic Structure of Hepatocytes --- p.103 / Chapter 3.2.2.5 --- GST-p Foci Area --- p.105 / Chapter 3.2.2.6 --- mRNA Expression of p53 and Mdm2 --- p.108 / Chapter 3.2.2.7 --- Protein Expression of Biomarkers --- p.110 / Chapter Chapter 4: --- Discussion --- p.116 / Chapter 4.1 --- Protective and Therapeutic Benefits of Sinigrin --- p.117 / Chapter 4.1.1 --- Effects of SIN on Cancer and Normal Cells --- p.117 / Chapter 4.1.2 --- Effective Tumor Induction by DEN-CC14 Treatment --- p.118 / Chapter 4.1.3 --- Protective Effect of SIN in the Promotion Stage of HCC --- p.118 / Chapter 4.1.4 --- Therapeutic Effect of SIN in the Progression Stage of HCC --- p.119 / Chapter 4.2 --- Biological Activities of SIN --- p.121 / Chapter 4.3 --- Summary --- p.134 / References --- p.xiv

Lymphomas and leukemias are the most common cancer in children and young adults and in utero exposure to carcinogens may contribute to the etiology of these cancers. A polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DBP), was administered to pregnant mice (15 mg/Kg b.w., gavage) on gestation day 17. Significant mortalities in young offspring were observed due to T-cell lymphoma. Lung and liver tumors also were observed in survivors at 10 months of age. To assess the role of the Aryl Hydrocarbon Receptor (AHR), we utilized crosses of B6129SF1/J (responsive) mice with strain 129S1/SvImJ (non-responsive). Offspring born to AHR non-responsive mothers had greater susceptibility to lymphoma, irrespective of offspring genotype. Responsive offspring displayed increased mortality if the mother was responsive. Lung adenomas showed Ki-ras mutations and exhibited a 50% decrease and a 35-fold increase in expression of Rb and p19/ARF mRNA, respectively. To examine the risk/benefit of maternal dietary phytochemical treatment against transplacental cancer, 2000 ppm indole-3-carbinol (I3C) was given to pregnant mice through diet from gestation day 9 till weaning. I3C significantly lowered mortality caused by lymphomas regardless of the maternal genotype, and also reduced lung tumor multiplicity in offspring born to AHR [superscript b-l/d] dams. Distribution of I3C in most maternal and fetal tissues was quantified following a single gavage of [¹⁴C]-I3C to the pregnant mice. DBP-DNA adducts were observed in both maternal and fetal tissues by ³³P postlabeling and HPLC analysis and were modulated by I3C and AHR genotype. I3C also modulated phase I and phase II enzyme protein expression in dams and gene expression in newborn thymus. I3C chemoprotection may involve modification of the bioavailability of DBP to the fetus and/or modulation of gene expression in the fetus as well. This is the first demonstration that transplacental exposure to an environmental PAH can induce a highly aggressive lymphoma in mice. These results raise the possibility that PAH exposures to pregnant women could contribute to similar cancers in children and young adults and, that the addition of chemoprotective agents to the maternal diet may reduce cancer risk among offspring. / Graduation date: 2006