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

Ph.D. / The current study evaluated a bio-monitoring technique developed in the USA by Adams, Brown and Goede, 1993. This project was sponsored by the Department of Water Affairs and Forestry (DWAF), to enable testing of the Health Assessment Index (HAI) under South African conditions. Testing took place in the Olifants River system, one of the most polluted river systems . in South Africa. Initially two river points were tested using Oreochromis mossambicus (Robinson, 1996), Clarias gariepinus (Marx, 1996) and Labeo rosae (Luus-Powell, 1997). The current study re-tested the HAI at the same two sample sites, namely Mamba and Balule in the Kruger National Park, using 0. mossambicus and C. gariepinus respectively. Two additional sites were tested in the upper catchment area, namely Loskop Dam and Bronkhorstspruit Dam. The current study further enabled the comparison of HAI results collected during drought and flood conditions. Results obtained after deployment of the HAI were corroborated using chemical analysis of water, sediment and biota. Water and sediment analysis was carried out by the Institute for Water Quality Studies using standard techniques. Bio-accumulation of aluminium, copper, iron, lead, manganese, nickel, strontium and zinc was assessed in the gills, liver, skin and muscle tissue of sample fish using standard Atomic Absorption Spectrometry techniques. Modifications made to the original HAI involved the inclusion of variable ranking in the assessment of fish parasites, with endo- and ectoparasites evaluated separately. Testing of this parasite hypothesis lead to the development of a Parasite Index component to the HAI. Assessment of water, sediment and fish tissue determined that the Olifants River system is indeed exposed to macro and heavy metal pollutants, which negatively affect aquatic health. Constituents posing the greatest threat are chlorides, fluorides, phosphates, total dissolved solids, copper and iron concentrations. Testing the HAI and parasite hypothesis using C. gariepinus, provided the most meaningful results. During testing of the parasite hypothesis both endo- and ectoparasite numbers conformed to the suggested idea that higher endoparasite numbers will occur at highly impacted areas, whereby ectoparasite numbers will be low. This was particularly evident in the lower catchment area, whereby comparisons between drought and flood conditions were carried out. Subsequent decreases in water quality directly after the flood were noted using water and sediment analysis. This observation reflects the results gathered using the HAT and during testing of the parasite hypothesis at all four sample sites. During statistical analysis of the HAI, using logistic regression analysis, parasite numbers, more specifically endoparasite numbers, were the most indicative of fish health. Environmental stressors (flood conditions) result in immunological responses observed in fish, and are reflected statistically using the HAI as changes in WBC %. It is suggested that endoparasites and WBC % provide the best overall assessment of fish condition. These variables should thus not be eliminated, in order to streamline the HAI evaluation procedures. Testing of this bio-monitoring technique under South African conditions provided meaningful results. This indicates that the HAI can be used to assess water quality, with existing water monitoring programmes further benefiting from its incorporation.