臺灣博碩士論文加值系統

摘要

本研究探討重金屬於環境中可能出現濃度下對斑馬魚胚胎中DNA傷害辨識作用之影響。
研究方法以凝膠電泳阻滯實驗檢測鎘、三價砷及二價汞處理斑馬魚胚胎九小時後，胚胎中辨識紫外線扭曲DNA與DNA錯誤配對活性之改變。胚胎在二價汞於1與3μM濃度下作用後，紫外線傷害產生之正環丁烷嘧啶雙體及(6-4)光合產物之辨識活性能力下降，此活性於3μM汞處理胚胎中降至未處理組之30%以下，而鎘處理並未產生明顯影響。三價砷於5至20μM濃度下未對紫外線傷害辨識作用產生清楚效應，但此金屬卻使胚胎中辨識寡核酸環錯誤配對的活性上升40%左右，卻也輕微抑制G-T錯誤配對活性，顯示三價砷對辨識不同錯誤配對之蛋白有差異性影響。全覆式原位雜交分析僅觀察到三價砷輕微抑制斑馬魚錯誤配對的辨識蛋白MSH6之基因表現，但仍需後續工作加以確認。

ABSTRACT


DNA Mismatch repair (MMR) and Nucleotide Excision Repair (NER) are the two DNA repair pathways that respectively corrects base mispairs generated during replication and removes helix distorting lesions such as UV- induced dipyrimidine photoproducts. Arsenic (As), Mercury (Hg) and Cadmium (Cd) are well known genotoxicants present in the aquatic environment. Inhibition of DNA damage repair has been proposed as one of the mechanisms of metal-induced genotoxicity. Because the potentials of Cd and Hg (II) to inhibit MMR in zebrafish embryos by disturbing the step of DNA mismatch recognition have been shown, this study explored the effects of Arsenite (AsIII) on DNA mismatch binding activities in Zebrafish embryos. Effects of the three metals on UV-damaged-DNA binding activities were also studied. Compared to Cd at 1 to 3 µM, both cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct (6-4 PP) binding activities in zebrafish embryos were found to be much more sensitive to Hg (II) at the same concentrations, suggesting that Hg (II) is a strong NER inhibitor. Conversely, both CPD and 6-4 PP binding activities were not affected by As(III) unless in embryos exposed to 10 to 20 µM of this ion, which are within the typical levels found in drinking water in contaminated areas. As (III) exposure failed to inhibit G-T and 2-loop DNA mismatch binding activities in zebrafish embryos and As (III) even caused a weak up-regulation of loop-specific binding capacity. Analysis of the gene expression of MutS homolog 6(MSH6), an important eukaryotic DNA mismatch binding factor, in zebrafish tissues by whole mount in situ hybridization suggested a weak inhibition of gene activity in embryos exposed to 10 to 20 µM As(III). Whether As (III) inhibits MSH6 gene expression in zebrafish embryos, however, awaits further investigation.

CONTENTS
Page
Contents…………………………………………………………………..2
Abstract ……………………………………………………………..........3
CHAPTER I Introduction………………………………………………...4
DNA Damage…………………………………………………………….4
1. DNA Repair……………………………………………………………....4
2. DNA Repair Pathways…………………………………............................5
3. Nucleotide Excision Repair………………………………………………6
4. MSH 6 Gene……………………………………………………………...6
5. UV Radiation…………………………………………………………......7
6. Mercury……………………………………………………………….......7
7. Arsenic………………………………………………………………...….7
8. The objectives of this research …………………………………………...8
CHAPTER II Materials and Methods…………………………………….9
1. Materials…………………………………………………....9, 10, 11, 12
2. Methods…………………………………………………………….....12
1. Culturing the shrimp for Zebrafish……………………………………....12
2. Zebrafish feeding………………………………………………………...12
3. Collection of developing zebrafish embryos………………………….....12
4. Treatment of Zebrafish embryos at sub lethal levels of Arsenic and Mercury……………………………………………………………….…13
5. Preparation of zebrafish embryos extracts…………………………........14
6. Zebra fish protein extraction………………………………………….....14
7. BCA Protein Assay………………………………………………...........14
8. Preparation of UV damaged oligonucleotides……………………….14,15
9. Electrophoretic Mobility Shift Assay……………………………......14,15
10. IN SITU Hybridization…………………………………………….……15
CHAPTER III RESULTS…………………………………………
1. Differential inhibitory effects of inhibition of UV- Damaged – DNA binding activities by Cd and Hg (II at different concentrations………...16
2. Hg (II) binding in lower concentrations on UV Induced DNA …………………………………………………………………....16
3. Dose Dependent Binding of Arsenic on CPD
And 6- 4 Photoproducts...........................................................................17
4. Mismatch Binding in Different Concentrations of As (III)…………..17
5. MSH 6 Gene Expression in Zebrafish Embryos……………………...18
6. Discussion…………………………………………………...........32, 33
7. References………………………………………………...34, 35, 36, 37

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