This investigation evaluated the potential protective role of Zn, through modulation of MT, against the teratogenic effects of isotretinoin (ITR), a dermatological drug which causes oxidative damage, in CD-1 mouse embryos. Significant induction of MT by Zn supplementation was observed in mouse embryos both in vivo and in vitro. On gestational day (GD) 6.5, pregnant mice received a subcutaneous Zn injection of 0 (saline), 20 and 40 mg/kg, resulting in embryonic MT concentrations of 12.5, 54.5 and 93.4 $ mu$g/g protein, respectively, after 48h. Embryos were extracted at GD 8.5 and incubated with 0 (saline) and 15 $ mu$M Zn for 48h. There was a six-fold increase in MT expression in the Zn treatment group, resulting in MT concentration of 688 $ mu$g/g protein. Pre-induction of MT by Zn exposure was found to offer protection against subsequent exposure to ITR in vivo and in vitro. Zn injections of 40 and 20 mg/kg to pregnant mice on GD 8.5 and 9.5, respectively, alleviated fetal damage caused by three intragastric intubations of 100 mg ITR/kg on GD 10.5. Zn pre-treatment significantly increased fetal and placental masses, totally eliminated the incidence of cleft palate and lessened the frequency of post-partum mortality by 74%. Pre-treatment of GD 8.5 embryos with a 24h culture period with 15 $ mu$M Zn improved growth and totally restored normal embryonic development altered by a 24h culture with 17 $ mu$M ITR. Zn decreased the frequencies of unfused mid brain and fore brain by 100%, and the incidence of abnormal flexion by 74%. A reduction in MT concentrations was observed in groups exposed to ITR, indicating that MT may have been oxidized by ITR-derived toxic FR. (Abstract shortened by UMI.)

No description available.

No description available.

No description available.

The synthesis and degradation of metallothionein (MT) was studied in streptozotocin-induced diabetic rats and monolayer cultures of adult rat hepatocytes. Elevated levels of MT-I and MT-II were identified in the liver and kidney of untreated diabetic rats. The relative rates of hepatic and renal MT synthesis were significantly higher in STZ-diabetic rats than in controls. The changes in the relative rate of MT synthesis were maximal by 4 and 10 days in liver and kidney, respective, after administration of streptozotocin. The relative rate of cytoplasmic MT turnover was also increased in liver, but largely unaffected in the kidney, of diabetic rats. The altered metabolism of hepatic MT in diabetic rats was attributed primarily to chronic changes in the levels of pancreatic and adrenal hormones in plasma. In contrast, increased synthesis of renal MT in the diabetic rat was due largely to accumulation of excessive dietary Cu in the kidney. Critical analysis of in vivo studies with diabetic rats and other literature revealed that cytoplasmic turnover of MT may not reflect actual degradation of this protein. Therefore, the characteristics of MT degradation in primary cultures of hepatocytes were investigated in subsequent studies. Hepatocytes were incubated in medium containing ³⁵S-cysteine and 100 uM zn overnight to induce MT synthesis. The level of ³⁵S-MT was quantified in heat stable extracts of cell homogenates by Fast Protein Liquid Chromatography (FPIC). When Zn was removed from medium, the rate of ³⁵S-MT turnover (t_1/2= 7 hours) was four times faster than general ³H-protein (t_1/2= 29 hours). This decrease in cellular MT level reflected degradation since less than 1% of cellular MT was secreted. The rate of MT degradation was inversely proportional to cellular Zn status. Cycloheximide, chloroquine and tosyl-lysine chloromethyl ketone (TLCK) inhibited ³⁵S-MT degradation by 33, 65 and 50%, respectively, without affecting cellular Zn status. Degradation of ³H-protein was inhibited by 41, 41 and 16% in the presence of cycloheximide, chloroquine and TI.CK, respectively. Removal of insulin increased ³H-protein degradation by 30%, but did not alter ³⁵S-MT degradation. Together, these data suggest that hepatic MT degradation (a) is primarily regulated by cellular Zn status and (b) occurs in both lysosomal and cytoplasmic compartments. / Ph. D.

Au, Yee Man Candy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 128-144). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xiii / Chapter 1. --- Chapter One Introduction / Chapter 1.1 --- Homeostasis and detoxification of metal ions --- p.1 / Chapter 1.2 --- Biochemistry of metallothionein --- p.3 / Chapter 1.2.1 --- Structure of metallothionein --- p.4 / Chapter 1.2.2 --- Isoforms of metallothionein --- p.5 / Chapter 1.2.3 --- Roles of metallothionein --- p.6 / Chapter 1.2.4 --- Structure of metallothionein gene --- p.9 / Chapter 1.2.5 --- Metal responsive element (MRE) --- p.10 / Chapter 1.2.6 --- Regulation of MT gene --- p.11 / Chapter 1.3 --- Metal responsive element-binding transcription factor 1 (MTF-1) --- p.16 / Chapter 1.3.1 --- Structure of MTF-1 --- p.16 / Chapter 1.3.2 --- Target genes of MTF-1 --- p.18 / Chapter 1.4 --- Teleost MT and MTF-1 --- p.20 / Chapter 1.5 --- Tilapia --- p.26 / Chapter 1.6 --- Tilapia MT and MTF-1 --- p.26 / Chapter 1.7 --- Aims of study --- p.30 / Chapter 2. --- Chapter Two Materials and Methods / Chapter 2.1 --- Quantification of MTF-1 isoforms and MT mRNA levels in tilapia and Hepa-Tl cells by real-time PCR --- p.32 / Chapter 2.1.1 --- Heavy metal exposure on tilapia --- p.32 / Chapter 2.1.1.1 --- Animals --- p.32 / Chapter 2.1.1.2 --- Heavy metal exposure --- p.32 / Chapter 2.1.1.3 --- Total RNA extraction --- p.33 / Chapter 2.1.1.4 --- Reverse Transcription --- p.35 / Chapter 2.1.2 --- Heavy metal exposure on Hepa-Tl cells --- p.36 / Chapter 2.1.2.1 --- Cell Culture --- p.36 / Chapter 2.1.2.2 --- Metal treatment on Hepa-Tl cells --- p.37 / Chapter 2.1.3 --- SYBR green --- p.39 / Chapter 2.1.3.1 --- Primer Design --- p.39 / Chapter 2.1.3.2 --- Validation of cycling condition --- p.41 / Chapter 2.1.3.3 --- Determination of relative amount of target gene present in the samples --- p.43 / Chapter 2.1.3.4 --- Statistical analysis --- p.44 / Chapter 2.1.4 --- TaqMan probes --- p.44 / Chapter 2.1.4.1 --- Primer Design --- p.44 / Chapter 2.1.4.2 --- Validation of cycling condition --- p.45 / Chapter 2.2 --- Localization study of MTF-1 isoforms --- p.46 / Chapter 2.2.1 --- Amplification of the full length tilapia MTF-1 isoforms --- p.46 / Chapter 2.2.2 --- Preparation of Escherichia coli competent cells --- p.48 / Chapter 2.2.3 --- Transformation --- p.49 / Chapter 2.2.4 --- Confirmation of the insert of the ligation products --- p.50 / Chapter 2.2.5 --- Cloning of MTF-1-L and MTF-1-S gene into phrGFPII-1 vector --- p.51 / Chapter 2.2.6 --- Transient transfection of plasmids to Hepa-Tl cells --- p.54 / Chapter 2.2.7 --- Staining of the nucleus by Hoechst 33342 --- p.55 / Chapter 2.2.8 --- Metal treatment on Hepa-Tl cells --- p.55 / Chapter 2.3 --- Electrophoretic mobility shift assay (EMSA) --- p.56 / Chapter 2.3.1 --- Preparation of Hepa-Tl whole-cell protein extract --- p.56 / Chapter 2.3.2 --- In vitro transcription/translation of tilapia MTF-1 isoforms --- p.57 / Chapter 2.3.3 --- Annealing of the tiMREg oligonucleotides --- p.58 / Chapter 2.3.4 --- Labeling of the annealed tiMREg oligonucleotides --- p.58 / Chapter 2.3.5 --- Electrophoretic mobility shift assay --- p.59 / Chapter 3. --- Chapter Three Results / Chapter 3.1 --- Quantification of MTF-1 isoforms and MT mRNA levels in tilapia and Hepa-Tl cells by real-time PCR --- p.62 / Chapter 3.1.1 --- Validation of primers for real-time PCR --- p.62 / Chapter 3.1.2 --- Tissue distribution of MTF-1 isoforms in tilapia and Hepa-Tl cell-line --- p.63 / Chapter 3.1.3 --- Effect of metal treatment on MTF-1 isoforms and MT gene expression level in different tissues of tilapia and Hepa-Tl cell-line --- p.68 / Chapter 3.2 --- Localization study of MTF-1 isoforms --- p.82 / Chapter 3.2.1 --- Cloning of MTF-1 isoforms into phrGFPII-1 vector --- p.82 / Chapter 3.2.2 --- Transient transfection of phrGFPII-1 plasmids to Hepa-Tl cells --- p.82 / Chapter 3.3 --- Electrophoretic mobility shift assay (EMSA) --- p.96 / Chapter 4. --- Chapter Four Discussion / Chapter 4.1 --- Tissue distribution of MTF-1 isoforms --- p.104 / Chapter 4.2 --- Effect of metal stress on the mRNA expression level of MT and MTF-1 isoforms --- p.106 / Chapter 4.3 --- In vitro study of the localization of the MTF-1 isoforms --- p.114 / Chapter 4.4 --- DNA binding of MTF-1 synthesized by in vitro transcription/translation method --- p.121 / Chapter 4.5 --- Conclusion --- p.125 / Chapter 5. --- REFERENCES --- p.128

Lam Kwok Lim. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 107-126). / Abstract also in Chinese. / Acknowledgments --- p.i / Presentations Derived from the Present Thesis Work --- p.ii / Abstract --- p.iv / Abbreviations --- p.vii / Abbreviation Table for Amino Acids --- p.ix / List of Figures --- p.x / List of Tables --- p.xii / Contents --- p.xiii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Metallothionein (MT) --- p.1 / Chapter 1.1.1 --- Classification of MT --- p.1 / Chapter 1.1.2 --- Structure of MT --- p.2 / Chapter 1.1.3. --- Structure of MT Genes --- p.4 / Chapter 1.1.4 --- Function of MT --- p.5 / Chapter 1.1.5 --- Regulation of MT Expression --- p.7 / Chapter 1.1.6 --- Fish MT --- p.9 / Chapter 1.1.7. --- Aims and Rationale of the Present Study --- p.12 / Chapter 2 --- MT mRNA Induction of Tilapia After Intraperitoneal Injection of Metal --- p.18 / Chapter 2.1 --- Introduction --- p.18 / Chapter 2.1.1. --- Specific Aims of This Chapter --- p.19 / Chapter 2.2 --- Materials and Methods --- p.20 / Chapter 2.2.1 --- Regents --- p.20 / Chapter 2.2.1.1 --- Purification of Total RNA --- p.20 / Chapter 2.2.1.2 --- Denaturing Gel and Vacuum Blotting of RNA (Northern Blotting) --- p.20 / Chapter 2.2.1.3 --- Hybridization --- p.21 / Chapter 2.2.2 --- Methods --- p.21 / Chapter 2.2.2.1 --- Purification of Total RNA --- p.21 / Chapter 2.2.2.2 --- Vacuum Blotting of Total RNA (Northern Blotting) --- p.22 / Chapter 2.2.2.3 --- Radioactive Labeling of Nucleic Acid Probes --- p.22 / Chapter 2.2.2.4 --- Hybridization --- p.22 / Chapter 2.2.2.5 --- Densitometric Analysis --- p.23 / Chapter 2.2.2.6 --- Calculation of MT mRNA Levels and Analysis of Results --- p.23 / Chapter 2.2.3 --- Endogenous MT mRNA Expression of Juvenile Tilapia and Carp --- p.23 / Chapter 2.2.4 --- Induction of MT mRNA Juvenile Tilapia and Carp Injected with Metals --- p.24 / Chapter 2.3 --- Results --- p.25 / Chapter 2.3.1 --- Endogenous Levels of MT mRNA in Tilapias in Normal Conditions --- p.25 / Chapter 2.3.2 --- Induction of MT mRNA Levels in Juvenile Tilapia Injected with Metals --- p.25 / Chapter 2.3.1.1 --- Copper Injection --- p.25 / Chapter 2.3.1.2 --- Zinc Injection --- p.25 / Chapter 2.3.1.3 --- Cadmium Injection --- p.26 / Chapter 2.3.3 --- Induction of MT mRNA Levels in Juvenile Carp with Zinc Injection --- p.26 / Chapter 2.4 --- Discussion --- p.26 / Chapter 2.4.1 --- MT mRNA Expression of Tilapia and Carp Injected with Metals --- p.26 / Chapter 2.5 --- Conclusions --- p.29 / Chapter 3 --- Induction Level of MT mRNA in Tilapia After Aqueous Exposure to Metals --- p.35 / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.1.1 --- Specific aims of this chapter --- p.36 / Chapter 3.2 --- Material s and Methods --- p.36 / Chapter 3.2.1 --- 96hours LC-50 values for zinc and copper --- p.36 / Chapter 3.2.2 --- Induction of MT mRNA in Juvenile Tiapias under Metal Aqueous Exposures --- p.37 / Chapter 3.2.3 --- Calculation of Fold Induction of MT mRNA and Analysis of Results --- p.38 / Chapter 3.2.4 --- Metal Analysis --- p.38 / Chapter 3.3 --- Results --- p.38 / Chapter 3.3.1 --- LC-50 values of metals for Juvenile Tilapia --- p.38 / Chapter 3.3.2 --- Induction of MT mRNA in Juvenile Tilapia under Metal Aqueous Exposures --- p.39 / Chapter 3.3.2.1 --- Aqueous Exposure to Copper --- p.39 / Chapter 3.3.2.2 --- Aqueous Exposure to Zinc --- p.40 / Chapter 3.3.2.3 --- Aqueous Exposure to Cadmium --- p.41 / Chapter 3.3.3 --- Induction of MT mRNA in Juvenile Carp after Aqueous Exposures to Metal --- p.41 / Chapter 3.3.3.1 --- Aqueous Exposure to Cadmium --- p.41 / Chapter 3.3.4 --- Metal Concentrations of Water Samples from the Aquaria in the Metal Exposure Test of Tilapia and Carp --- p.42 / Chapter 3.4 --- Discussion --- p.42 / Chapter 3.4.1 --- LC-50 values of Metals for Tilapia --- p.42 / Chapter 3.4.2 --- MT mRNA Expression of Tilapias under Metal Aqueous Exposure --- p.44 / Chapter 3.4.3 --- Normalization of the Signals of Northern Blot Analysis --- p.47 / Chapter 3.5 --- Conclusions --- p.48 / Chapter 4 --- Field Study --- p.58 / Chapter 4.1 --- Introduction --- p.58 / Chapter 4.1.1 --- Specific Aims of this Chapter --- p.59 / Chapter 4.2 --- Materials and Methods --- p.59 / Chapter 4.2.1 --- Sampling Sites --- p.59 / Chapter 4.2.2 --- Data Analysis --- p.60 / Chapter 4.2.3 --- Harvest of Feral Tilapia --- p.60 / Chapter 4.2.4 --- Determination of Metal Concentration of Metal Concentration in the Tissues of Feral Tilapia --- p.60 / Chapter 4.2.5 --- Endogenous MT mRNA Levels Using Northern Blot Analysis --- p.61 / Chapter 4.2.6 --- Calculation of MT mRNA Levels and Analysis of Results --- p.61 / Chapter 4.3 --- Results --- p.62 / Chapter 4.3.1 --- Metal Concentrations in the Tissues of Feral Tilapia --- p.62 / Chapter 4.3.2 --- Comparison of Metal Concentrations Among Different Tissues of Feral Tilapia --- p.62 / Chapter 4.3.3 --- MT mRNA Levels in the Tissues of Feral Tilapia --- p.63 / Chapter 4.3.4 --- Correlation Between Metal Concentrations and Endogenous MT mRNA Levels in the Tissues of Feral Tilapia --- p.63 / Chapter 4.4 --- Discussion --- p.64 / Chapter 4.4.1 --- Bioaccumulation of Metals --- p.64 / Chapter 4.4.2 --- Endogenous Levels of MT mRNA in the Feral Tilapia --- p.67 / Chapter 4.5 --- Conclusions --- p.68 / Chapter 5 --- Cloning of Tilapia MT Genes --- p.86 / Chapter 5.1 --- Specific Aims of This Chapter 、 --- p.86 / Chapter 5.2 --- Materials and Methods --- p.87 / Chapter 5.2.1 --- Regents --- p.87 / Chapter 5.2.1.1 --- Preparation of Plasmid DNA --- p.87 / Chapter 5.2.1.2 --- Preparation of Genomic DNA --- p.87 / Chapter 5.2.1.3 --- Restriction Enzyme Digestion --- p.88 / Chapter 5.2.1.4 --- Vacuum Blotting of DNA (Southern Blotting) --- p.88 / Chapter 5.2.1.5 --- Polymerase Chain Reaction --- p.89 / Chapter 5.2.1.6 --- Transformation of E.coli Competent Cells --- p.89 / Chapter 5.2.1.7 --- Nucleotide Sequence Determination --- p.89 / Chapter 5.2.1.8 --- List of Primers --- p.90 / Chapter 5.2.1.8.1 --- Primers for Nucleotide Sequence Determination --- p.90 / Chapter 5.2.1.8.2 --- Tilapia MT Specific Primers for PCR --- p.90 / Chapter 5.2.2 --- Methods --- p.91 / Chapter 5.2.2.1 --- Preparation of Plasmid --- p.91 / Chapter 5.2.2.2 --- Preparation of Genomic DNA --- p.91 / Chapter 5.2.2.3 --- Preparation of Enzyme Digestion --- p.92 / Chapter 5.2.2.4 --- Vacuum Blotting of Genomic DNA (Southern Blotting) --- p.92 / Chapter 5.2.2.5 --- Radioactive Labeling of Nucleic Acid Probes --- p.92 / Chapter 5.2.2.6 --- Hybridization --- p.93 / Chapter 5.2.2.7 --- Polymerase Chain Reaction --- p.93 / Chapter 5.2.3 --- Southern Blot Analysis of Tilapia Genomic DNA --- p.93 / Chapter 5.2.4 --- Analysis of the Sequences of Tilapia MT Genes --- p.94 / Chapter 5.2.4.1 --- Amplification of MT Genes Using PCR --- p.94 / Chapter 5.2.4.2 --- Cloning of the MT Genes --- p.94 / Chapter 5.2.4.3 --- Transformation of E.coli Competent Cell --- p.94 / Chapter 5.2.4.4 --- Nucleotide Sequence Determination --- p.95 / Chapter 5.3 --- Results --- p.95 / Chapter 5.3.1 --- Southern Blot Analysis of Tilapia Genomic DNA --- p.95 / Chapter 5.3.2 --- Amplification of MT Gene Fragments Using PCR --- p.95 / Chapter 5.3.3 --- Analysis of the Sequences of Tilapia MT Genes --- p.96 / Chapter 5.4 --- Discussion --- p.96 / Chapter 5.4.1 --- Fish MT Genes --- p.96 / Chapter 5.5 --- Conclusions --- p.98 / Chapter 6 --- General Discussion --- p.104 / References --- p.107

Shiu Ka Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 176-182). / Abstract also in Chinese. / Acknowledgments --- p.i / Presentations Derived from the Present Thesis Work --- p.ii / Chinese Abstract --- p.iii / English Abstract --- p.iv / List of Abbreviations --- p.v / Abbreviation for Amino Acids and Nucleotides --- p.vii / List of Figures --- p.viii / List of Tables --- p.vi / Contents / Chapter Chapter.1 --- Literature Review --- p.1 / Chapter 1.1 --- Transcriptional Regulation of Gene Expression --- p.1 / Chapter 1.2 --- MT: A Brief Review --- p.4 / Chapter 1.3 --- Transcriptional Regulation of MT --- p.15 / Chapter 1.4 --- MT Promoter Organization and Function --- p.18 / Chapter 1.5 --- Fish MT Genes --- p.29 / Chapter 1.6 --- Aim and Rationale of Present Studies --- p.32 / Chapter Chapter 2 --- PCR Cloning of Common Carp MT Gene --- p.34 / Chapter 2.1 --- Introduction --- p.34 / Chapter 2.1.1 --- The Biology of Common Carp --- p.34 / Chapter 2.1.2 --- The Study of Common Carp MT --- p.35 / Chapter 2.2 --- Materials and Methods --- p.39 / Chapter 2.2.1 --- Materials --- p.39 / Chapter 2.2.1.1 --- Polymerase Chain Reaction (PCR) --- p.39 / Chapter 2.2.1.2 --- Agarose Gel Electrophoresis --- p.39 / Chapter 2.2.1.3 --- Gene Clean by Sephaglas´ёØ BandPrep Kit (Pharmacia) --- p.40 / Chapter 2.2.1.4 --- TA Cloning --- p.40 / Chapter 2.2.1.5 --- Transformation of Plasmid Vector into Competent Cell (Heat Shock Method) --- p.41 / Chapter 2.2.1.6 --- Preparation of Plasmid DNA --- p.41 / Chapter 2.2.1.7 --- DNA Sequencing --- p.42 / Chapter 2.2.1.7.1 --- Template Denaturation and Primer Annealing --- p.42 / Chapter 2.2.1.7.2 --- Labeling and Termination Reaction --- p.42 / Chapter 2.2.1.7.3 --- DNA Sequencing Electrophoresis --- p.43 / Chapter 2.2.1.8 --- Total RNA Extraction --- p.43 / Chapter 2.2.1.9 --- PolyA RNA Extraction --- p.44 / Chapter 2.2.1.10 --- Micro Bio-Spin Chromatography --- p.44 / Chapter 2.2.1.11 --- Analysis of the Transcription Start Site --- p.45 / Chapter 2.2.2 --- Methods --- p.46 / Chapter 2.2.2.1 --- Polymerase Chain Reaction (PCR) --- p.46 / Chapter 2.2.2.2 --- Gene Clean by Sephaglas ´ёØ BandPrep Kit (Pharmacia) --- p.46 / Chapter 2.2.2.3 --- TA Cloning --- p.47 / Chapter 2.2.2.4 --- Transformation of Plasmid Vector into Competent Cell (Heat Shock Method) --- p.47 / Chapter 2.2.2.5 --- Transformation of Plasmid Vector into Competent Cell (Heat Shock Method) --- p.48 / Chapter 2.2.2.6 --- Preparation of Plasmid DNA --- p.48 / Chapter 2.2.2.6.1 --- Small Scale Alkali Preparation of Plasmid DNA --- p.48 / Chapter 2.2.2.6.2 --- Large Scale Preparation of Plasmid DNA using Wizard Maxiprep Kit (Promega) --- p.49 / Chapter 2.2.7 --- DNA Sequencing --- p.50 / Chapter 2.2.2.7.1 --- Template Denaturation and Primer Annealing --- p.50 / Chapter 2.2.2.7.2 --- Labeling and Termination Reaction --- p.51 / Chapter 2.2.2.7.3 --- DNA Sequencing Electrophoresis --- p.51 / Chapter 2.2.2.8 --- Total RNA Extraction --- p.52 / Chapter 2.2.2.9 --- PolyA RNA Extraction --- p.53 / Chapter 2.2.2.10 --- Analysis of the Transcription Start Site --- p.55 / Chapter 2.3 --- Results --- p.56 / Chapter 2.3.1 --- PCR Cloning of the MT Gene --- p.56 / Chapter 2.3.2 --- Identification of the Transcriptional Start Site --- p.57 / Chapter 2.4 --- Discussion --- p.60 / Chapter 2.4.1 --- PCR Cloning of the MT Gene --- p.60 / Chapter 2.4.2 --- Comparison of Common Carp MT Promoter with Other --- p.60 / Chapter 2.4.3 --- Identification of the Transcriptional Start Site --- p.62 / Chapter 2.5 --- Conclusion --- p.63 / Chapter Chapter 3. --- Functional Assay of Common Carp MT Promoter --- p.64 / Chapter 3.1 --- Introduction --- p.64 / Chapter 3.1.1 --- Fish MT Promoters --- p.64 / Chapter 3.2 --- Materials and Methods --- p.68 / Chapter 3.2.1 --- Materials --- p.68 / Chapter 3.2.1.2 --- Micro Bio-Spin Chromatography --- p.68 / Chapter 3.2.1.3 --- Construction of Deletion Mutants --- p.68 / Chapter 3.2.1.4 --- Isolation of Hepatocytes --- p.69 / Chapter 3.2.1.5 --- Determination of LC50 Values for Common Carp Hepatocytes --- p.69 / Chapter 3.2.1.6 --- Transfection by LipofectAMINE´ёØ (Gibco) --- p.70 / Chapter 3.2.1.9 --- Determination of the Amount of Protein by BCA Protein Assay --- p.70 / Chapter 3.2.1.10 --- β-galactosidase Analysis --- p.71 / Chapter 3.2.2 --- Methods --- p.72 / Chapter 3.2.2.1 --- Subcloning of 5' Flanking Region of Common Carp MT Gene into Reporter Gene --- p.72 / Chapter 3.2.2.2 --- Micro Bio-Spin Chromatography (Bio-rad) --- p.72 / Chapter 3.2.2.3 --- Creating Deletion Mutants --- p.73 / Chapter 3.2.2.4 --- Isolation of Hepatocytes --- p.73 / Chapter 3.2.2.5 --- Determination ofLC50 Values for Common Carp Hepatocytes --- p.74 / Chapter 3.2.2.6 --- Transfection with LipofectAMINE´ёØ (Gibco BRL) --- p.75 / Chapter 3.2.2.7 --- Optimization of Incubation Time of Cells with LipofectAMINE´ёØ --- p.75 / Chapter 3.2.2.8 --- Optimization of Amount of DNA for Transfection --- p.76 / Chapter 3.2.2.9 --- Determination of Protein Concentration by --- p.76 / Chapter 3 2.2.10 --- β-galactosidase Analysis --- p.77 / Chapter 3.2.2.11 --- Fluorescence Measurement --- p.77 / Chapter 3.2.2.12 --- Dose-Response Curve of Different Metals on Transfected Cells --- p.77 / Chapter 3.2.2.13 --- "Fold-Induction of Different Metals, LPS and H202" --- p.78 / Chapter 3.3. --- Result --- p.79 / Chapter 3.3.1 --- Deletion Mutants --- p.79 / Chapter 3.3.2 --- LC50 of Common Carp Hepatocytes --- p.80 / Chapter 3.3.3 --- Optimization of Transfection --- p.81 / Chapter 3.3.4 --- Dose Response Curve --- p.85 / Chapter 3.3.5 --- Deletion Mutants with Different Treatments --- p.95 / Chapter 3.4 --- Discussion --- p.109 / Chapter 3.4.1 --- LC50 Values of Metal Toxicity in Different in vitro Fish Cells Studies --- p.109 / Chapter 3.4.2 --- Dose Response Curve (Figure 3.9 to 3.16) --- p.110 / Chapter 3.4.3 --- Fold Induction in Deletion Mutants --- p.111 / Chapter 3.5 --- Conclusion --- p.128 / Chapter Chapter 4. --- MRE-Binding Proteins --- p.129 / Chapter 4.1 --- Introduction --- p.129 / Chapter 4.1.1 --- MTF-1 --- p.129 / Chapter 4.1.1.1 --- Structure of MTF-1 --- p.129 / Chapter 4.1.1.2 --- MTF-1 is a Zinc Dependent Factor --- p.130 / Chapter 4.1.1.3 --- Band-shift Assay of MTF-1 --- p.132 / Chapter 4.1.1.4 --- MTF-1 is Essential for Both Basal and Metal-Induced MT Transcription --- p.133 / Chapter 4.1.2 --- MBP-l --- p.134 / Chapter 4.1.3 --- MBF-l l --- p.35 / Chapter 4.1.4 --- Rat Zinc Activated Protein --- p.135 / Chapter 4.1.5 --- MREBF-1 and MREBF-2 --- p.136 / Chapter 4.1.6 --- Human Zinc Regulatory Factor --- p.136 / Chapter 4.1.7 --- MREBP --- p.137 / Chapter 4.1.8 --- Aim of This Chapter --- p.138 / Chapter 4.2 --- Materials and Methods --- p.139 / Chapter 4.2.1 --- Materials --- p.139 / Chapter 4.2.1.1 --- Preparation of Nuclear Extract from Common Carp Liver Tissue --- p.139 / Chapter 4.2.1.2 --- Preparation of the Double-Stranded Oligonucleotides --- p.139 / Chapter 4.2.1.3 --- Binding Reaction of Protein and DNA --- p.141 / Chapter 4.2.1.4 --- Gel-Shift Mobility Electrophoresis --- p.142 / Chapter 4.2.1.5 --- Screening of Expression Library --- p.142 / Chapter 4.2.1.5.1 --- Preparation of Labeled DNA Probe --- p.142 / Chapter 4.2.1.5.2 --- Plating of the Library --- p.142 / Chapter 4.2.1.6. --- Isolation of Positive Clones In Vivo Excision --- p.143 / Chapter 4.2.2 --- Methods --- p.144 / Chapter 4.2.2.1 --- Gel Mobility-Shift Assays --- p.144 / Chapter 4.2.2.1.1 --- Preparation of Nuclear Extract from Common Carp Liver Tissue --- p.145 / Chapter 4.2.2.1.2 --- Preparation of the Double-Stranded Oligonucleotides --- p.145 / Chapter 4.2.2.1.3 --- Binding Reaction of Protein and DNA --- p.146 / Chapter 4.2.2.1.4 --- Gel-Shift Mobility Electrophoresis --- p.146 / Chapter 4.2.2.2 --- Screening of Expression Library --- p.146 / Chapter 4.2.2.2.1 --- Preparation of Labeled DNA Probe --- p.147 / Chapter 4.2.2.2.2 --- Plating of the Library --- p.148 / Chapter 4.2.2.2.3 --- Isolation of Positive Clones --- p.150 / Chapter 4.3 --- Results --- p.150 / Chapter 4.3.1 --- Gel Mobility-Shift Assays --- p.150 / Chapter 4.3.2 --- Expression Library Screening --- p.163 / Chapter 4.4 --- Discussion --- p.166 / Chapter 4.4.1 --- Gel Mobility-Shift Assays --- p.166 / Chapter 4.4.2 --- Expression Library Screening --- p.171 / Chapter 4.5 --- Conclusion --- p.172 / Chapter Chapter 5 --- Conclusion --- p.173 / Chapter 5.1 --- Conclusion --- p.173 / Chapter 5 2 --- Model of MT Gene Transcription --- p.174 / Chapter 5.3 --- Future Direction --- p.175 / references --- p.176

The prevalent and ongoing exposures to polychlorinated biphenyls (PCBs) demands an understanding of the threat they pose and also a means in which to mitigate their potential toxicity. This thesis set out to investigate a phenomenon associated with a specific PCB congener, 3,3',4,4',5-pentachlorobiphenyl (PCB126), for the underpinnings of its mechanism, and also its usefulness as a toxin against which to establish a mitigative strategy. The phenomenon in particular is the disruption of hepatic trace elements, specifically an increase in copper and decreases in zinc, selenium, iron, and manganese in the liver. Four questions were posed to address the overarching goals: 1) When does micronutrient disruption occur in the context of liver pathology? 2) What metal transporters or chaperones are involved? 3) Can the previously shown beneficial micronutrient, zinc, alter the disruption and improve outcome? 4) What is occurring spatially within the liver acinus where micronutrients are distributed? By answering these four questions, a fundamental understanding of this occurrence will be ascertained. A chronology of PCB126-hepatotoxicity showed onset of liver pathology at 36 hours and later alterations in micronutrients at 3 days, suggesting disruption of hepatic trace elements is likely the result of liver degeneration. In addition, a key metal transport protein, metallothionein, was induced by PCB126. Utilizing a double knockout animal model, metallothionein was shown to abrogate some toxicity but had little involvement of micronutrient perturbation. Previous investigations have suggested the unique property of zinc in rescuing/preventing hepatic damage by a variety of toxic agents. Dietary zinc had a modest effect in ameliorating PCB126 hepatotoxicity and preserving micronutrient homeostasis. This suggests that the mitigative potential of zinc supplementation on PCB126 exposure is limited. Finally, a fine spatial investigation of the liver acinus was conducted to establish the levels of trace elements from the portal triad to the central vein. In addition, novel findings of high concentrations of extracellular zinc were discovered. In all, this dissertation has shown that disruption of hepatic micronutrients caused by PCB126 are likely the result of liver degeneration by means of disturbing the spatial trace element gradients and provides appropriate context for therapeutic/preventive strategies against PCBs.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 170-185).