簡易檢索 / 詳目顯示

回結果列表
研究生： 楊賾翰
Yang, Tse-Han
論文名稱： rec assay於台灣南部河川底泥基因毒性測試之應用
Application of rec assay to investigate the genotoxicity of estuarine sediments of southern Taiwan
指導教授： 周佩欣
Chou, Pei-Hsin
學位類別： 碩士
Master
系所名稱： 工學院 - 環境工程學系
Department of Environmental Engineering
論文出版年： 2011
畢業學年度： 99
語文別： 中文
論文頁數： 121
中文關鍵詞： 基因毒性物質 、基因重組枯草桿菌基因毒性試驗法 、河川底泥
外文關鍵詞： Genotoxicity, rec assay, Sediment
相關次數： 點閱：83  下載：7 
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報

    • 基因毒物是一種會破壞DNA完整性的毒化物質，來源可能來自於自然界中，也可能來自於人類活動所產生的污染。由於生活、畜牧與工業污水的排放流入，河川常常是人為污染最為嚴重的地方，而許多污染物質會隨著砂土顆粒沉降累積在底泥之中，則形成持續性的污染情況。
    　　本研究利用基因重組枯草桿菌基因毒性試驗法（rec assay）檢測台灣南部河川底泥中基因毒性反應的強弱，並以生物試驗法分析高效能液相層析儀不同分段樣品的基因毒性，期望能藉此找出造成底泥中具有基因毒性之主要物質。
    　　研究結果顯示，阿公店溪底泥的基因毒性較高（R50=2.7~3.2，平均2.95，標準差0.24，n=3），其次為二仁溪底泥（R50=1.0~3.8，平均2.12，標準差0.62，n=21），高屏溪底泥的基因毒性最低（R50=1.0~2.4，平均1.50，標準差0.59，n=6）。
    　　二仁溪之分段樣品以永寧橋（R50=2.0~4.0，平均2.90，標準差1.01，n=3）、石安橋（R50=3.5~5.2，平均4.33，標準差0.85，n=3）和通地溝（R50=2.1~2.6，平均2.37，標準差0.25，n=3）的基因毒性反應最高，阿公店溪之分段樣品則以阿公店橋的基因毒性反應最高（R50=2.1）。二仁溪石安橋二月份底泥樣品經多次液相層析儀分離與生物試驗法分析後，成功找到具有基因毒性的未知化合物。
    　　目前已知此化合物之紫外光/可見光吸收光譜中的最大吸光波長為205 nm、282 nm和428 nm。此外，以酵素免疫分析法（ELISA）檢測二仁溪底泥中之戴奧辛含量，發現其支流三爺溪的戴奧辛濃度較高，鯽潭橋與永寧橋的濃度介於22.6~48.2 pg-TEQ/g-sediment之間。研究結果顯示，二仁溪底泥中的戴奧辛含量與基因毒性強弱並無相關性（R2=0.0077）。
    　　本研究亦針對受戴奧辛污染廠址之土壤進行基因毒性測試，在草叢區、單一植被區以及五氯酚工廠的土壤樣品中，僅有五氯酚工廠的土壤具有些微的基因毒性，而其他兩個區域的土壤並未檢測到基因毒性。

    Genotoxicants are chemicals causing damage to genetic material and affecting its integrity. They may be released from natural sources or human activities, and accumulate in river sediments by deposition to cause persistent contamination.
    The objectives of this study are to detect the genotoxic contamination and to identify the major genotoxicants in the esturine sediments of southern Taiwan. The rec assay was applied to investigate the genotoxicity, and high performance liquid chromatography (HPLC) was used to separate samples. The genotoxicity of HPLC fractions was also analyzed.
    The highest genotoxicity (R50=2.7~3.2, mean=2.95±0.24, n=3), Er-ren River also showed significant genotoxicity (R50=1.0~3.8, mean=2.12±0.62, n=21), and Gao-ping River showed lower genotoxicity than the other two rivers (R50=1.0~2.4, mean=1.50±0.56, n=6).
    For the HPLC fractions of sediment samples taken at Yung-ning Bridge (ER3, R50=2.0~4.0, mean=2.90±1.01, n=3), Shih-an Bridge (ER5, R50=3.5~5.2, mean=4.33±0.85, n=3) and Tung-ti Gutter (ER7, R50=2.1~2.6, mean=2.37±0.25, n=3) showed higher genotoxicity, and the genotoxicity was much higher in wet season than in dry season. For A-gon-dian River, only A-gon-dian Bridge showed higher genotoxicity (AGD2, R50=2.1).
    Since the sediment samples collected from Shih-an Bridge (ER5) showed higher genotoxicity, they were further separated by HPLC and analyzed by rec assay, and a suspected genotoxicant was found. According to its UV-Vis spectrum, the λmax of this unknown compound is 205 nm, 282 nm and 428 nm.
    Enzyme linked immunosorbent assay (ELISA) was also used to analyze dioxin concenteration in the sediment samples of Er-ren River. The highest dioxin concentrations (22.6 and 48.2 pg-TEQ/g-sediment) were detected in the samples of Chi-tan Bridge (ER1) and Yung-ning Bridge (ER3), which are located at the tributary of Er-ren River (San-yeh River).
    This study also used rec assay to analyze the genotoxicity of dioxin-contaminated soil samples collected from the grass area, vegetation area and PCP factory area of Chinese Petrochemical An-shun site. The results showed that only low genotoxicity was detected in PCP factory area.

    摘要 I Abstract III 誌謝 V 總目錄 VII 表目錄 XII 圖目錄 XVI 第一章 前言 1 1-1 研究動機 1 1-2 研究目的 2 第二章 文獻回顧 3 2-1 二仁溪流域 3 2-1-1 地理概述 3 2-1-2 水文 3 2-1-3 氣象 3 2-1-4 污染源 4 2-2 高屏溪流域 5 2-2-1 地理概述 5 2-2-2 水文 5 2-2-3 氣象 5 2-2-4 污染源 6 2-3 阿公店溪流域 7 2-3-1 地理概述 7 2-3-2 水文 7 2-3-3 氣象 7 2-3-4 污染源 8 2-4 基因毒性 8 2-4-1 DNA損傷 9 2-4-2 DNA損傷途徑 10 2-4-2-1 氧化 10 2-4-2-2 烷化 10 2-4-2-3 去胺基化 11 2-4-2-4 DNA鍵結物 12 2-4-3 基因毒性物質 12 2-4-3-1 多環芳香烴 13 2-4-3-2 鉛 14 2-4-3-3 六價鉻 15 2-4-3-4 黃麴毒素 15 2-4-4 戴奧辛 16 2-5 基因毒性生物試驗法 20 2-5-1 基因重組枯草桿菌試驗法 20 2-5-1-1 UvrABC修復系統 20 2-5-1-2 RecE pathway 21 2-5-1-3 rec assay 21 2-5-2 沙門氏菌突變試驗 23 2-5-3 彗星試驗 24 2-5-4 MutatoxTM基因毒性試驗 25 2-5-5 細胞培養染色體變異試驗 25 2-5-6 各種基因毒性生物試驗法之比較 27 第三章 實驗步驟與方法 28 3-1 採樣設備 28 3-2 採樣方式與地點 28 3-3 實驗材料與設備 32 3-3-1 實驗材料 32 3-3-2 溶液配置 33 3-3-3 實驗設備 33 3-4 實驗流程 35 3-5 樣品前處理 36 3-5-1 乾燥與均質化 36 3-5-2 樣品萃取 36 3-5-3 樣品濃縮與溶劑置換 36 3-5-4 樣品保存 37 3-6 rec Assay 37 3-6-1 枯草菌株之培養 37 3-6-2 樣品、標準品與菌株之共培養 37 3-6-3 菌株生存率之計算 38 3-6-4 基因毒性之估算 38 3-6-4-1 生存率差距 39 3-6-4-2 R20、R50、R80 40 3-6-4-3 毒性估算法之比較 41 3-6-5 標準品試驗分析 42 3-6-6 溶劑空白試驗 45 3-7 高效能液相層析儀分析 46 3-8 酵素免疫分析法 48 第四章 結果與討論 51 4-1 河川底泥之基因毒性分析 51 4-1-1 生存率差距 51 4-1-1-1 二仁溪 51 4-1-1-2 高屏溪 57 4-1-1-3 阿公店溪 59 4-1-2 R20、R50、R80 62 4-1-3 毒性估算法結果 65 4-2 HPLC結合生物試驗法分析結果 69 4-2-1 二仁溪 70 4-2-2 阿公店溪 84 4-2-3 小結 86 4-2-4 HPLC分離前後之基因毒性比較 87 4-2-5 HPLC結合生物試驗法分析結果（1分鐘） 91 4-2-6 HPLC結合生物試驗法分析結果（10秒鐘） 95 4-3 二仁溪底泥之戴奧辛濃度分析 101 4-4 戴奧辛污染土壤之基因毒性分析 103 第五章 結論與建議 108 5-1 結論 108 5-2 建議 110 參考文獻 111

    1. 勇興台，南部地區河川污染整治推動、輔導及評析計畫，京華工程顧問股份有限公司，2009
    2. 經濟部水利署 [http://www.wra.gov.tw/]
    3. 交通部中央氣象局 [http://www.cwb.gov.tw/]
    4. 台南市環境保護局，台南市三爺溪、二仁溪段非法棄置不明廢棄物緊急調查及清理規劃計畫，2008
    5. 台灣省環境保護署全國環境水質監測資訊網[http://wq.epa.gov.tw/]
    6. 錢紀銘，二仁溪底泥多氯聯苯污染分佈之研究，嘉南學報，32：54－63，2006
    7. 吳先琪，受污染底泥評估基準之理論基礎探討，台灣土壤及地下水環境保護協會簡訊，16：13－21，2005
    8. 環佑實業有限公司，屏東縣高屏溪流域河川水質改善及污染削減具體行動計畫 期末報告書，2010
    9. Doong RA, Lee SH, Lee CC, Sun YC, Wu SC: Characterization and composition of heavy metals and persistent organic pollutants in water and estuarine sediments from Gao-ping River, Taiwan. Marine Pollution Bulletin 2008, 57(6-12):846-857.
    10. Barlow S, Schlatter J: Risk assessment of carcinogens in food. Toxicology and Applied Pharmacology 2010, 243(2):180-190.
    11. Berenblum I, Shubik P: A new quantitative approach to the study of the stages of chemical carcinogenesis in the mouse skin. Cancer 1947, 1:383-390.
    12. Anne Roulston, Richard C. Marcellus, Philip E. Branton: Viruses and apoptosis. Annual Review of Microbiology 1999, 53:577-628.
    13. H L: Molecular Biology of the Cell, 5th edn; 2004.
    14. Nordberg J, Arnér ESJ: Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radical Biology and Medicine 2001, 31(11):1287-1312.
    15. Tiana L, Caib Q, Wei H: Alterations of Antioxidant Enzymes and Oxidative Damage to Macromolecules in Different Organs of Rats During Aging. Free Radical Biology and Medicine 1998, 24(9):1477-1484.
    16. Drabløs F, Feyzi E, Aas PA, Vaagbø CB, Kavli B, Bratlie MS, Peña-Diaz J, Otterlei M, Slupphaug G, Krokan HE: Alkylation damage in DNA and RNA--repair mechanisms and medical significance. DNA Repair 2004, 3(11):1389-1407.
    17. Eaton D, Gallagher E: Mechanisms of aflatoxin carcinogenesis. Annual Review of Pharmacology and Toxicology 1994, 34:135-172.
    18. Zegura B, Heath E, Cernosa A, Filipic M: Combination of in vitro bioassays for the determination of cytotoxic and genotoxic potential of wastewater, surface water and drinking water samples. Chemosphere 2009, 75(11):1453-1460.
     
    19. G. E. Kisby, M. Standley, T. Park, A. Olivas, S. Fei, T. Jacob, A. Reddy, X. Lu, P. Pattee, Nagalla SR: Proteomic Analy the Genotoxicant Methylazoxymethanol (MAM)-Induced Changes in the Developing Cerebellum. Proteome Research 2006, 5:2656-2665.
    20. Kohl C, Morgan P, Gescher A: Metabolism of the genotoxicant 2-nitropropane to a nitric oxide species. Chemico-Biological Interactions 1995, 97(2):175-184.
    21. IARC: Evaluation of the carcinogenic risk of chemicals to humans: polynuclear aromatic compounds. IARC Monograph No. 35 1983.
    22. Kamata R, Shiraishi F, Nakajima D, Takigami H, Shiraishi H: Mono-hydroxylated polychlorinated biphenyls are potent aryl hydrocarbon receptor ligands in recombinant yeast cells. Toxicology in Vitro 2009, 23(4):736-743.
    23. Xue W, Warshawsky D: Metabolic activation of polycyclic and heterocyclic aromatic hydrocarbons and DNA damage: A review. Toxicology and Applied Pharmacology 2005, 206(1):73-93.
    24. Delgado-Rodriguez A, Ortíz-Marttelo R, Graf U, Villalobos-Pietrini R, Gómez-Arroyo S: Genotoxic activity of environmentally important polycyclic aromatic hydrocarbons and their nitro derivatives in the wing spot test of Drosophila melanogaster. Mutation Research/Genetic Toxicology 1995, 341(4):235-247.
    25. Tarantini A, Maître A, Lefèbvre E, Marques M, Rajhi A, Douki T: Polycyclic aromatic hydrocarbons in binary mixtures modulate the efficiency of benzo[a]pyrene to form DNA adducts in human cells. Toxicology 2011, 279(1-3):36-44.
    26. Yang J-L, M. MV, J. MJ: Kinds of mutations formed when a shuttle vector containing adducts of (+)-7, 8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene replicates in human cells. Sci USA 1987, 84:1987.
    27. García-Lestón J, Méndez J, Pásaro E, Laffon B: Genotoxic effects of lead: An updated review. Environment International 2010, 36(6):623-636.
    28. Manerikar RS, Apte AA, Ghole VS: In vitro and in vivo genotoxicity assessment of Cr(VI) using comet assay in earthworm coelomocytes. Environmental Toxicology and Pharmacology 2008, 25(1):63-68.
    29. 水污染防治法－放流水標準，台灣省行政院環境保護署，2010
    30. 水污染防治法－地面水體分類及水質標準，台灣省行政院環境保護署，1998
    31. 李宜娟，以職場勞工與動物試驗探討鉻(VI/III)暴露下尿中鉻之排除情況與動力學，國立成功大學環境醫學研究所碩士論文，2003
    32. Gorelick NJ.: Risk assessment for Aflatoxin: I. Metabolism of Aflatoxin B1 by different species. Risk Analysis 1990, 10: 539–559.
    33. Raney, K.: Oxidation of Aflatoxins and sterigmatocystin by human liver microsomes: significance of Aflatoxin Q1 as a detoxication product of Aflatoxin B1. Chenical Research in Toxicology 1992, 5: 202–210.
     
    34. Liu, L.: Bioactivation of Aflation B1 by lipoxygenase, prostaglandin H synthase and cytochrome P450 monooxygenase in guinea-pig tissues. Carcinogenesis 1992, 13: 533–539.
    35. 土壤污染整治－土壤污染管制標準，台灣省行政院環境保護署，2011
    36. 飲用水管理－飲用水水質標準，台灣省行政院環境保護署，2007
    37. Dragan YP: Animal studies addressing the carcinogenicity of TCDD (or related compounds) with an emphasis on tumour promotion. Food additives and contaminated 2000, 17: 289–302.
    38. Hankinson O: The aryl hydrocarbon receptor complex. Annual Review of Pharmacology and Toxicolog 1995, 35: 307–340.
    39. Lehvis GP, Bradfield CA: Ahr null alleles: distionctive or different? Biochemical Pharmacology 1998, 56: 781–787.
    40. 吳家驊，不同捲菸配方產生之二手菸的毒性比較，國立成功大學環境工程研究所碩士論文，2005
    41. Van Houten B, Croteau DL, DellaVecchia MJ, Wang H, Kisker C: 'Close-fitting sleeves': DNA damage recognition by the UvrABC nuclease system. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 2005, 577:92-117.
    42. Waters TR, Eryilmaz J, Geddes S, Barrett TE: Damage detection by the UvrABC pathway: Crystal structure of UvrB bound to fluorescein-adducted DNA. FEBS Letters 2006, 580(27):6423-6427.
     
    43. Takahashi M, Bertrand-Burggraf E, Fuchs RPP, Nordén B: Structure of UvrABC excinuclease--UV-damaged DNA complexes studied by flow linear dichroism DNA curved by UvrB and UvrC. FEBS Letters 1992, 314(1):10-12.
    44. Sancar A, Rupp WD: A novel repair enzyme: UVRABC excision nuclease of Escherichia coli cuts a DNA strand on both sides of the damaged region. Cell 1983, 33(1):249-260.
    45. Grossman L, Yeung AT: The UvrABC endonuclease system of Escherichia coli -- A view from Baltimore. Mutation Research/DNA Repair 1990, 236(2-3):213-221.
    46. Kowalczykowski SC, Dixon DA, Eggleston AK, Lauder SD, Rehrauer WM: Biochemistry of homologous recombination in Escherichia coli. Microbiol 1994, 58:401-465.
    47. Clark AJ: Rec genes and homologous recombination proteins in Escherichia coli. Biochimie 1991, 73:523-532.
    48. Takahashi N, Yoshikura H, Kobayashi I: An Escherichia coli strain, BJ5183, that shows highly efficient conservative (two-progeny) DNA double-strand break repair of restriction breaks. Gene 2003, 303:89-97.
    49. Tsuneaki A, Tokio K: The RecF Pathway of Homologous Recombination Can Mediate the Initiation of DNA Damage-Inducible Replication of the Escherichia coli Chromosome. Bacteriology 1994, 176:7113-7114.
     
    50. Jane RG, David KW, Alvin JC: Genetic Analysis of the RecE Pathway of Genetic Recombination in Escherichia coli K-12. Bacteriology 1981, 145:521-532.
    51. Keim P, Lark KG: The RecE recombination pathway mediates recombination between partially homologous DNA sequences: Structural analysis of recombination products. Journal of Structural Biology 1990, 104(1-3):97-106.
    52. Takigami H, Matsui S, Matsuda T, Shimizu Y: The Bacillus subtilis rec-assay: a powerful tool for the detection of genotoxic substances in the water environment. Prospect for assessing potential impact of pollutants from stabilized wastes. Waste Management 2002, 22(2):209-213.
    53. Grabinska-Sota E, Wisniowska E, Kalka J, Scieranka B: Genotoxicological effects of some phenoxyherbicides and their metabolites on Bacillus subtilis M45 Rec- and H17 Rec+ strains. Chemosphere 2002, 47(1):81-85.
    54. Caldini G, Trotta F, Cenci G: Inhibition of 4-nitroquinoline-1-oxide genotoxicity by Bacillus strains. Research in Microbiology 2002, 153(3):165-171.
    55. Sharma MK, Sobti RC: Rec effect of certain textile dyes in Bacillus subtilis. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 2000, 465(1-2):27-38.
     
    56. Bruce N. Ames FDL, William E. Durston: An Improved Bacterial Test System for the Detection and Classification of Mutagens and Carcinogens. Proceedings of the National Academy of Sciences 1973, 70(3):782-786.
    57. Pavlica M, Klobucar GIV, Mojas N, Erben R, Papes D: Detection of DNA damage in haemocytes of zebra mussel using comet assay. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 2001, 490(2):209-214.
    58. Ritter D, Knebel J: Genotoxicity testing in vitro - Development of a higher throughput analysis method based on the comet assay. Toxicology in Vitro 2009, 23(8):1570-1575.
    59. Kumaravel TS, Jha AN: Reliable Comet assay measurements for detecting DNA damage induced by ionising radiation and chemicals. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 2006, 605(1-2):7-16.
    60. Békaert C, Rast C, Ferrier V, Bispo A, Jourdain MJ, Vasseur P: Use of in vitro (Ames and Mutatox tests) and in vivo (Amphibian Micronucleus test) assays to assess the genotoxicity of leachates from a contaminated soil. Organic Geochemistry 1999, 30(8, Part 2):953-962.
    61. Cleveland L, Little EE, Petty JD, Johnson BT, Lebo JA, Orazio CE, Dionne J, Crockett A: Toxicological and chemical screening of Antarctica sediments: Use of whole sediment toxicity tests, microtox, mutatox and semipermeable membrane devices (SPMDs). Marine Pollution Bulletin 1997, 34(3):194-202.
    62. Jarvis AS, Honeycutt ME, McFarland VA, Bulich AA, Bounds HC: A Comparison of the Ames Assay and Mutatox in Assessing the Mutagenic Potential of Contaminated Dredged Sediment. Ecotoxicology and Environmental Safety 1996, 33(2):193-200.
    63. Hauser B, Schrader G, Bahadir M: Dependence of Genotoxicity of Benzo[a]pyrene Suspensions in Mutatox Test on Dissolved Concentration and S9 Addition. Ecotoxicology and Environmental Safety 1997, 38(3):224-226.
    64. Hartmann A, Plappert U, Poetter F, Suter W: Comparative study with the alkaline Comet assay and the chromosome aberration test. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 2003, 536(1-2):27-38.
    65. Kopjar N, Garaj-Vrhovac V: Assessment of DNA damage in nuclear medicine personnel - comparative study with the alkaline comet assay and the chromosome aberration test. International Journal of Hygiene and Environmental Health 2005, 208(3):179-191.
    66. Saburo M: The Bacillus subtilislMicrosome Rec-Assay for the Detection of DNA-Damaging Substances in Waters of a Night Soil Treatment Plant. Toxicity Assessment 1988, 3:173-193.
    67. Emine Oksuzoglu: A study on the genotoxic activities of some newbenzoxazoles. Medicinal Chemistry Research 2007, 16:1-14.
    68. US EPA: Perforance of the CAPE Technologies DF1 Dioxin/Furan Immunoassay Kit for Soil and Sediment Sample 2002.
     
    69. Y.I. Korenman and A.A. Gorokhov: Distribution of Diphenylolpropane between Certain Organic Solvents and Water. Russian Journal of Applied Chemistry 1973, 46(11):2597-2599.
    70. US EPA: National primary drinking water regulation – Atrazine. 1995.
    71. Tridib Chakraborty: Vanadium inhibits the development of 2-acetylaminofluorene-induced premalignant phenotype in a two-stage chemical rat hepatocarcinogenesis model. Life Sciences 2006, 78:2839-2851.
    72. M. Kuster: Evaluation of two aquatic passive sampling configurations for their suitability in the analysis of estrogens in water. Talanta 2010, 83:493-499.
    73. Lai, K.M., Johnson, K.L., Scrimshaw, M.D., and Lester, J.N.: Binding of waterborne steroid estrogens to solid phases in river and estuarine systems. Environmental Science & Technology 2000, 34:3890-3894.
    74. Hansch C, Hoekman D, Leo A, Zhang LT, Li P.: The expanding role of quantitative structure-activity relationships (QSAR) in toxicology. Toxicology Letters 1995, 79:45-53.
    75. Migaku Kawaguchi: Stir bar sorptive extraction and trace analysis of alkylphenols in water samples by thermal desorption with in tube silylation and gas chromatography–mass spectrometry. Journal of Chromatography A 2005, 1062:23-29.
     
    76. LUBA VASILUK: BENZO[a]PYRENE BIOAVAILABILITY FROM PR67. Michael Schwarz: Carcinogenic risks of dioxin: Mechanistic considerations. Regulatory Toxicology and Pharmacology 2005, 43: 19–34.
    77. SciFinder [https://scifinder.cas.org]
    78. Neil Glagovich: Woodward's Rules for Conjugated Carbonyl Compounds. Central Connecticut State University 2007.
    79. 潘復華，二仁溪、高屏溪底泥樣品中戴奧辛及平面狀毒性多氯聯苯濃度現況背景調查，環境檢驗所環境檢驗年報，9：89－110，2002
    80. Michael Schwarz: Carcinogenic risks of dioxin: Mechanistic considerations. Regulatory Toxicology and Pharmacology 2005, 43: 19–34.

    下載圖示 校內：2013-08-31公開
    校外：2013-08-31公開
    QR CODE