Firstly, a cell line derived from the liver of tilapia, Hepa-T1, was used as a model and exposed to two sub-lethal concentrations of waterborne copper for 96 h. The proteins expressed in Hepa T1 were investigated by differential protein profiling using two-dimensional gel electrophoresis (2-DE). It was found that Cu2+ (120 microM and 300 microM) caused differential expression of 93 different proteins, 18 of which were further verified by real-time quantitative polymerase chain reaction (PCR) analysis. Following analysis with ingenuity pathway software, several proteins were found to be involved in lipid metabolism, tissue connective development and cell cycle control, thus indicating that copper toxicity affects these cellular functions. / Fourthly, to further reveal the mechanism of copper tolerance and sensitivity in tilapia and zebrafish, two important copper transporters (ATP7A & B) and metallothionein (MT) were chosen for studying. Until now, a full length of ATP7A and partial length of ATP7B were obtained in tilapia. Then a real time quantitative PCR was conducted to study the different regulations of these three genes in tilapia and zebrafish. It was found that Cu2+ could induce more MT and ATP7A & B in tilapia than zebrafish both in vivo and in vitro. These results help us to understand that the copper tolerance of tilapia is possibly due to higher expression level of both copper transporters and MT. / Last but not least, I also compared the toxicity and biomarker gene expression in zebrafish exposed to Cu2O nanoparticle (NP) and CuCl2, respectively. It was found that the toxicity of CuCl2 is much higher than that of Cu2O NP. Then seven genes, including MT, ATP7A & B, copper transporter 1 (Ctr1), metal regulatory transcription factor 1 (MTF1), glutathione sulfur transferase (GST), Cu/Zn superoxide dismutase (Cu/Zn SOD), were chosen for studying. It was found that both Cu2O NP and CuCl 2 up-regulated the mRNA levels of MT, Cu/Zn SOD, and Ctr1, ATP7A & 7B, but down-regulated the mRNA levels of GST. Interestingly, the inductions of MT, Ctr1, ATP7A & B in the Cu2O NP exposure groups were much higher than that of CuCl2 exposure groups in vivo . Furthermore, as determined by using Ctr1, ATP7A and ATP7B gene expression, the no observable effect levels (NOELs) of CuCl2 and nano-Cu2O were 11 ppb and 50 ppb, whereas the lowest observable effect levels (LOELs) of CuCl2 and nano-Cu2O were 43 ppb and 125 ppb. (Abstract shortened by UMI.) / Secondly, the high copper contents in the liver of the tilapia make this fish a suitable model for the study of copper binding proteins. Liver was dissected from tilapia injected with Cu2+ and cytosolic fractions were separated by using Superdex 75 column chromatography followed with atomic absorption spectrometry. Fractions containing copper-binding proteins were found in two major peaks, analyzed using differential proteomic approaches, and loaded on a Cu chelating ion-immobilized affinity column (Cu-IMAC). Of the 113 differentially expressed proteins in these two peaks, some well-characterized copper binding proteins were found, including copper transporter ATP7A, cytochrome c oxidase, metallothionein, collagen, catalase, and vitellogenin. These proteins are mainly involved in endocrine disruption, mitochondria dysfunction, ion competition, lipid metabolism, copper transfer, and cytoskeleton disruption. In addition, a more concrete image about copper transportation pathway was hypothesized according to the function of the novel copper binding proteins identified. / The aims of this study are to identify some novel copper binding proteins and proteins related to Cu2+ toxicity or detoxification mechanisms in the tilapia (Oreochromis niloticus) and the zebrafish (Danio rerio) using a proteomic approach, and to reveal the mechanism of copper tolerance and copper sensitivity by comparing the different biochemical responses to copper exposures between the two model species. / Thirdly, zebrafish liver cell line (ZFL) was also used as a model to study the mechanism of copper toxicity. After processing similar experimental procedures of previous Hepa T1 experiment, 72 different proteins were identified to be regulated by Cu2+ (100 microM and 200 microM). More than 50 % of these proteins were also found differentially expressed in the tilapia. The results suggested that the toxicity mechanism between zebrafish and tilapia was generally conserved. Although, in ZFL, the regulation of several proteins, related to ROS effect, mitochondrion copper transportation, and stress response, was quite different from that in tilapia. / Chen, Dongshi. / Adviser: Chun Ung Ming. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 173-190). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Szeto Tsz Kwan Leo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 112-120). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Tables --- p.ix / List of Figures --- p.x / Abbreviations --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Heavy metals contaminations in Shing Mun River --- p.1 / Chapter 1.1 --- Importance of copper regulation and role of liverin copper metabolism --- p.6 / Chapter 1.1.1 --- Role of copper --- p.6 / Chapter 1.1.2 --- Toxicity due to unbalanced copper regulation --- p.7 / Chapter 1.1.3 --- Function of liver in copper detoxification --- p.9 / Chapter 1.2 --- Aims and rationale of this research --- p.11 / Chapter Chapter 2 --- Heavy metal concentrations of tilapia samples collected from Shing Mun River --- p.12 / Chapter 2.1 --- Introduction --- p.12 / Chapter 2.1.1 --- Sampling sites - Fo Tan and Siu Lek Yuen Nullah --- p.12 / Chapter 2.1.2 --- Tilapia samples collected from the sites --- p.16 / Chapter 2.1.3 --- Tilapia as a study model --- p.18 / Chapter 2.1.4 --- Bioavailability of heavy metals in water --- p.19 / Chapter 2.1.5 --- Metal content in liver --- p.20 / Chapter 2.1.6 --- Aim of this chapter --- p.20 / Chapter 2.2 --- Materials and Methods --- p.22 / Chapter 2.2.1 --- Collection of control and field samples --- p.22 / Chapter 2.2.2 --- Heavy metal concentrations determination --- p.23 / Chapter 2.2.3 --- Homogenization of liver cells --- p.24 / Chapter 2.2.4 --- Subcellular fractionation --- p.24 / Chapter 2.2.5 --- Determination of copper and zinc content in each subcellular fraction --- p.253 / Chapter 2.3 --- Results --- p.27 / Chapter 2.3.1 --- Physical data --- p.27 / Chapter 2.3.2 --- Metal concentrations in liver and muscle --- p.27 / Chapter 2.3.3 --- Copper and zinc subcellular distribution in liver cell --- p.33 / Chapter 2.4 --- Discussion --- p.36 / Chapter 2.4.1 --- Difference in metal concentration between sites --- p.36 / Chapter 2.4.2 --- Copper contamination in water and fish organ (muscle and liver) from the Shing Mun River --- p.36 / Chapter 2.4.3 --- Comparison of metal content in muscle and liver at Fo Tan site with previous studies --- p.39 / Chapter 2.4.4 --- Copper and zinc concentrations in the liver of tilapia --- p.42 / Chapter 2.4.5 --- Copper and zinc sebcellular distribution in the liver of tilapia --- p.43 / Chapter Chapter 3 --- Column chromatography of hepatic proteins from tilapias --- p.44 / Chapter 3.1 --- Transport of metals from circulatory system to liver --- p.44 / Chapter 3.1.1 --- Copper transporting plasma proteins in vertebrates --- p.44 / Chapter 3.1.2 --- Copper uptake into hepatocytes --- p.45 / Chapter 3.1.3 --- Intracellular metabolism of copper --- p.48 / Chapter 3.1.4 --- Mechanism of copper toxicity following excess accumulation --- p.49 / Chapter 3.1.5 --- Aim of this chapter --- p.50 / Chapter 3.2 --- Materials and Methods --- p.51 / Chapter 3.2.1 --- Purification of liver cytosolic proteins by gel-filtration column chromatography --- p.51 / Chapter 3.2.2 --- Copper content detection in elution --- p.52 / Chapter 3.2.3 --- Analysis of peaks from elution profile using tricine gel SDS PAGE --- p.53 / Chapter 3.3 --- Results --- p.55 / Chapter 3.3.1 --- Gel-filtration liquid chromatography elution profiles --- p.55 / Chapter 3.3.2 --- SDS PAGE analysis of peaks in elution profiles --- p.51 / Chapter 3.4 --- Discussion --- p.54 / Chapter 3.4.1 --- Comparison of gel filtration profiles of sample liver cytosol between sites and sexes --- p.64 / Chapter 3.4.2 --- Possible proteins in peaks found in the gel filtration profiles --- p.64 / Chapter 3.4.3 --- Common copper-indeced proteins --- p.67 / Chapter 3.5 --- Conclusion --- p.70 / Chapter Chapter 4 --- Two-dimensional electrophoresis of hepatic cutosol of tilapias caught from Shing Mun River and copper-treated HEPA T1 cell --- p.72 / Chapter 4.1 --- Introduction --- p.72 / Chapter 4.1.1 --- The need of ´بin vitro' experiment --- p.72 / Chapter 4.1.2 --- Choice of cell line --- p.73 / Chapter 4.1.3 --- Aim of this chapter --- p.74 / Chapter 4.2 --- Materials and Methods --- p.76 / Chapter 4.2.1 --- HEPA T1 cell cultivation --- p.76 / Chapter 4.2.2 --- Copper exposure of HEPA T1 cell --- p.77 / Chapter 4.2.3 --- Subcellular protein extraction of the copper-treated HEPA T1 cells --- p.77 / Chapter 4.2.4 --- Bicinchoninic Acidic (BCA) Protein Assay --- p.79 / Chapter 4.2.5 --- Two-dimensional gel electrophoresis --- p.79 / Chapter 4.3 --- Results --- p.83 / Chapter 4.3.1 --- Graphical presentation of spots observed on 2-dimensional gel of field samples and copper-injected samples --- p.33 / Chapter 4.3.2 --- Graphical presentation of spots detected on 2-dimensional gel of HEPAT1 cells --- p.84 / Chapter 4.3.3 --- Comparison of matched spots on 2-dimensional gels among control and copper-treated HEPAT1 cells --- p.97 / Chapter 4.4 --- Discussion --- p.105 / Chapter 4.4.1 --- Comparison of the spot patterns between field sample and copperOtreated HEPA T1 cells --- p.105 / Chapter 4.5 --- Conclusion --- p.107 / Chapter Chapter 5 --- General Discussions --- p.108 / Chapter 5.2 --- Research Overview --- p.108 / Chapter 5.2 --- Characterization of metal binding proteins from the cytosol of liver of tilapia --- p.109 / REFERENCES --- p.112