Metal Contamination and Studies of Copper-Binding Proteins from Tilapia Collected from Shing Mun River
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Metal Contamination and Studies of Copper-Binding Proteins from Tilapia Collected from Shing Mun River \ V； Szeto Tsz Kwan Leo A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Philosophy in Biochemistry ©The Chinese University of Hong Kong August, 2005 The Chinese University of Hong Kong holds the copyright of this thesis. Any person(s) intending to use a part or whole of the materials in the thesis in a proposed publication must seek copyright release from the Dean of the Graduate School. I If 6 OCT 2i jlj U:JIVEnSITY jMjj ‘SYSTEM y^K/ I I Abstract Shing Mun River is a river relatively highly contaminated by heavy metals. The heavy metal contents are high at Fo Tan according to government's record. Siu Lek Yuen is another area might be contaminated by industrial discharge as well as illegal discharges into the rainstorm nullah. In order to study the heavy metals uptake of organisms in the river, Tilapia is chosen because of its abundance in the Shing Mun River and availability to residence in the region. Thirty-five (17 male, 18 female) and thirty-two (11 male, 21 female) tilapias were collected in 2001 from the two sites respectively; while twelve (7 male, 5 female) were collected from Fo Tan and sixteen control (all female) were bought from Wo Che Wet Market in 2002. Livers were removed for metal extraction and protein analysis as they were found to be of highest metal accumulation among other tissues from previous studies. The total metal concentrations of six heavy metals: Zn, Cu, Cd, Pb, Cr and Ni were detected by using Flame AAS. Zn and Cu were found to be of highest concentration among the six and thus were chosen for analysis. Fractions of cytosol, nucleus, microsome, mitochondria and plasma membrane from the livers were obtained by high speed and ultra-centrifugation. Their Zn and Cu contents were also determined by using Flame AAS and found highest in cytosolic fractions. i Separation of cytosolic fraction by gel-filtration chromatography revealed the presence of four major peaks under UV254nm. The first three peaks (the three with highest molecular size) were found to correlate with corresponding high copper content. This suggested that they contain copper-binding proteins. The second peak (fraction no: 34-41, 14kDa) was recognized as metallothionein (MT). Further purification of the peaks was carried out with SDS PAGE and 2D gel electrophoresis. HEPA T1 cell was employed for the in-vitro experiment of copper treatment in contrast to the in-vivo one using field samples. The 2D gel results of in-vitro experiment were more consistent and thus could be further analyzed with the ImageMaster™ 2D Platinum. Twenty-six spots were induced and seventeen were suppressed. Further identifications were needed to be done on 2D gel of higher resolution. ii • ^ 城門河是一條受重金屬污染的河流，相對於其他溪流，城門河的污染程 度屬於嚴重。根據政府的紀錄，火炭一帶的重金屬含量尤爲偏高。而小瀝源 渠亦是另一個可能受污染程度較高的位置，因爲附近的工廠區所排放的污 水，以及非法傾倒的垃圾，也可能直接流進該渠’造成污染。由於非洲鰂是 城門河中的常見魚類，也是常見的食用魚類，故此有硏究其吸取重金屬的機 制的需要。於二零零一年，由火炭捕獲的非洲鰂共有三十五條(十七條雄性， 十八條雌性）’而由小瀝源捕獲的共有三十二條(十一條雄性，二十一條雌 性）；而於由二零零二年在火炭捕獲的則有十二條(七條雄性，五條雌性），另 外還於禾輋街市買了十六條(全是雌性)作爲對照樣本。 從過往的實驗結果得知肝臟乃積聚了最多金屬的內臟，故所有捕獲樣本 的肝臟都會切割下來以供量度金屬含量及分析蛋白之用。總共有六種金屬： 鋅、銅、鎘、絡、鎳以火焰原子吸收分光光度計量度，其中含量最高的乃辞 和銅，故鋅和銅被選作進一步的分析。高速離心機和極速離心機把肝臟細胞 分離爲五個從屬細胞級分，包括細胞質、細胞核、微粒體、線粒體及細胞膜。 以上的從屬細胞級分中的鋅和銅含量會以火焰原子吸收分光光度計量度。 膠體過爐色譜分析法把cytosolic級分中的成分再加以細分，並在紫外 線254奈米的監察下發現四個主要的峰値。其中三個峰値（擁有最大的分子 體積的三個）能配對上相對的銅含量峰値，這暗示當中含有黏結銅的蛋白。 iii 第三個峰値（溜分號碼：三十四至四十一，十四千道爾頓）經確認爲金屬硫 蛋白（Metallothionein，MT)。十二院基硫酸鈉聚丙儲酸胺凝膠電泳(SDS PADE)及二次元膠體電泳用作以上峰値的進一步分析°在試管內(in-vitro)的 銅暴露實驗應用了 HEPAT1細胞，以和應用了捕獲樣本的在活的有機體內 (in-vivo)的實驗作對比。二次元膠體電泳的結果顯示在試管內的實驗結果比 在活的有機體內的實驗結果爲一致，所以在試管內的實驗會用來以 ImageMasterTM 2D Platinum作出分析。分析後得知有二十六個斑點爲經銅暴 露後誘發的，而抑制的則有十七點。進一步的蛋白鑑定需要於較高解像度的二 次元膠體上進行。 iv Acknowledgement Very sincere and special thanks to my supervisor, Professor K. M. Chan, for his patience guidance and providing critical comments whenver necessary throughout the course of this study. My appreciation also goes to all the administrative staff and technicians of the Biochemistry Department, for their assistance. I am also very thankful to colleagues in the Biochemistry Department, especially to those working in BMSB RM513. Special thanks are due to Mr. Wai Lung Alfred Tang, Mr. Chit Chow, Ms. Chui Ling Margaret Tse and Ms. Kathy Kong for their assistance on fish sacrifice and all the helpful suggestion. Last but not least, I wish to give my sincere appreciation and thanks to my family, especially my parents, and friends for their invaluable support, patience and encouragement throughout my study years. vii Table of Contents Abstract i '、 摘要 iii Acknowledgements v Table of Contents vi List of Tables ix List of Figures x Abbreviations xii Chapter 1 Introduction 1 1.1 Heavy metals contaminations in Shing Mun River 1 1.1 Importance of copper regulation and role of liver in copper metabolism 6 1.1.1 Role of copper 6 1.1.2 Toxicity due to unbalanced copper regulation 7 1.1.3 Function of liver in copper detoxification 9 1.2 Aims and rationale of this research 11 Chapter 2 Heavy metal concentrations of tilapia samples collected from Shing Mun River 12 2.1 Introduction 12 2.1.1 Sampling sites - Fo Tan and Siu Lek Yuen Nullah 12 2.1.2 Tilapia samples collected from the sites 16 2.1.3 Tilapia as a study model 18 2.1.4 Bioavailability of heavy metals in water 19 2.1.5 Metal content in liver 20 2.1.6 Aim of this chapter 20 2.2 Materials and Methods 22 2.2.1 Collection of control and field samples 22 2.2.2 Heavy metal concentrations determination 23 2.2.3 Homogenization of liver cells 24 2.2.4 Subcellular fractionation 24 2.2.5 Determination of copper and zinc content in each subcellular fraction... 253 vi 2.3 Results 27 2.3.1 Physical data 27 2.3.2 Metal concentrations in liver and muscle 27 2.3.3 Copper and zinc subcellular distribution in liver cell 33 2.4 Discussion 36 2.4.1 Difference in metal concentration between sites 35 2.4.2 Copper contamination in water and fish organ (muscle and liver) from the Shing Mun River 36 2.4.3 Comparison of metal content in muscle and liver at Fo Tan site with previous studies 39 2.4.4 Copper and zinc concentrations in the liver of tilapia 42 2.4.5 Copper and zinc sebcellular distribution in the liver of tilapia 43 Chapter 3 Column chromatography of hepatic proteins from tilapias 44 3.1 Transport of metals from circulatory system to liver 44 3.1.1 Copper transporting plasma proteins in vertebrates 44 3.1.2 Copper uptake into hepatocytes 45 3.1.3 Intracellular metabolism of copper 43 3.1.4 Mechanism of copper toxicity following excess accumulation 49 3.1.5 Aim of this chapter 59 3.2 Materials and Methods 51 3.2.1 Purification of liver cytosolic proteins by gel-filtration column chromatography 5I 3.2.2 Copper content detection in elution 52 3.2.3 Analysis of peaks from elution profile using tricine gel SDS PAGE 53 3.3 Results 55 3.3.1 Gel-filtration liquid chromatography elution profiles 55 3.3.2 SDS PAGE analysis of peaks in elution profiles 51 3.4 Discussion 54 3.4.1 Comparison of gel filtration profiles of sample liver cytosol between sites and sexes 54 3.4.2 Possible proteins in peaks found in the gel filtration profiles 54 3.4.3 Common copper-indeced proteins 3.5 Conclusion 7q vii Chapter 4 Two-dimensional electrophoresis of hepatic cutosol of tilapias caught from Shing Mun River and copper-treated HEPA T1 cell 72 4.1 Introduction. 72 4.1.1 The need of ‘in vitro' experiment 72 4.1.2 Choice of cell line 73 4.1.3 Aim of this chapter 74 4.2 Materials and Methods 76 4.2.1 HEPA T1 cell cultivation 75 4.2.2 Copper exposure of HEPA T1 cell 77 4.2.3 Subcellular protein extraction of the copper-treated HEPATl cells 77 4.2.4 Bicinchoninic Acidic (BCA) Protein Assay 79 4.2.5 Two-dimensional gel electrophoresis 79 4.3 Results 33 4.3.1 Graphical presentation of spots observed on 2-dimensional gel of field samples and copper-injected samples 33 4.3.2 Graphical presentation of spots detected on 2-dimensional gel of HEPATl cells 34 4.3.3 Comparison of matched spots on 2-dimensional gels among control and copper-treated HEPATl cells 97 4.4 Discussion 1Q5 4.4.1 Comparison of the spot patterns between field sample and copperOtreated HEPA T1 cells 1 q5 4.5 Conclusion 1Q7 Chapter 5 General Discussions 108 5.2 Research Overview IQg 5.2 Characterization of metal binding proteins from the cytosol of liver of 109 tilapia REFERENCES 112 viii List of Tables Table 1.1 Metal concentrations(ug/L) in local rivers measured by the EPD 4 Table 2.1 Heavy metals concentrations (ug/L) among the monitoring sites 15 along Shing Mun River during the summer (June to August) of 2001 to 2003 Table 2.2 LC50 of copper and zinc in Common Carp and Tilapia 18 Table 2.3 Mean concentrations (ug/g, wet weight) of Zn, Cu, Cd, Ni, Pb, 21 Cr in Tissues of Tilapias Collected from Shing Mun River Sites Table 2.4 Heavy metals content (ug/g) in Liver and Muscle of Tilapia 30 samples collected in summer of 2001 Table 2.5 Concentrations (ug/g) of cadmium, copper, nickel, lead and zinc 31 in muscle and liver of the samples collected during summer of 2001 and 2002 Table 2.6 Subcellular distribution of (a) copper content and (b) zinc 35 content in hepatocytes of tilapia caught from Fo Tan and Siu Lek Yuen Table 2.7 Recommended daily allowance of copper by USEPA 38 Table 2.8 Copper content (ug/g) in different seafood found by USEPA 39 Table 2.9 Concentrations (ug/g) of Zn, Cu, Pb, Ni and Cd in tissues of 41 tilapias collected from Shan Pui River in Nam Sang Wai and the adjacent fish pond Table 3.1 Corresponding fraction number of the peaks present in Fig.