Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/5566
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dc.contributor.advisor林伯雄zh_TW
dc.contributor.advisorPo-Hsiung Linen_US
dc.contributor.author侯國隆zh_TW
dc.contributor.authorHou, kuo-Lungen_US
dc.date2003zh_TW
dc.date.accessioned2014-06-06T06:35:05Z-
dc.date.available2014-06-06T06:35:05Z-
dc.identifier.urihttp://hdl.handle.net/11455/5566-
dc.description.abstract多氯聯苯是環境中常見之污染物質,且為一已知之動物致癌物質,然而其致癌之機制仍不明確,但氧化壓力可能扮演一重要之角色。多氯聯苯誘發氧化壓力主要之途徑包括:含氯數低之多氯聯苯在代謝之過程藉由氧化還原循環增加活潑性氧分子以及DNA damage,而高含氯數之多氯聯苯誘發氧化壓力可能與其改變基因之表現有關。本研究之主旨在於探討多氯聯苯於人類乳癌細胞株 (MCF-7 cells)及calf thymus (ct-DNA)中誘發基因表現失衡及核酸氧化損壞作用機制。研究結果顯示,2,2’,5-trichloro-3’,4’-dihydroxybiphenyl (Cl3-BPhCAT) (1μM)於過渡性金屬銅離子Cu(II) (20μM)及NADPH (100μM)存在之環境下能對ct-DNA誘發DNA醛基損壞 (aldehydic DNA lesions , ADL)之生成。ROS移除劑,例如氫氧自由基移除劑DMSO、甲醇,超氧自由基移除劑superoxide dismutase (SOD)對ADL之產生,均無抑制作用,但過氧化氫酵素 (catalase)卻具有抑制效應,顯示其中實際參與核酸損害作用之活潑性氧 (reactive oxygen species, ROS)分子為H2O2。同時,Cu(I)螯合劑,bathocuproinedisulfonic acid共同反應情形下,能抑制Cu(II)與Cl3-BPhCAT誘發的核酸損害作用,此一結果顯示Cu(II)/Cu(I)的氧化還原循環作用為一重要步驟,且Cu(I)與活潑性氧分子中之H2O2所結合之錯合物,Cu(I)OOH,有可能為實際參與核酸損害作用之物種。生物體內小分子物質glutathione (GSH),可移除ROS,抑制Cu(II)與Cl3-BPhCAT所誘發的DNA斷鏈,此一研究證據顯示,GSH可能是與Cl3-BPhCAT結合後中斷其氧化還原之循環,終止ROS生成,進而抑制核酸損害作用;同時GSH亦有可能是直接與ROS反應,直接移除ROS。此外,GSH亦有可能抑制Cu(I)與H2O2反應而達到保護DNA之作用。實驗結果亦顯示,Cl3-BPhCAT所誘發之ADL約有74%為putrescine可切除之ADL,此一結果證實,Cl3-BPhCAT是經由氧化還原循環產生ROS進而誘發ADL。本研究中亦發現ct-DNA氧化損壞能力與含氯取代基的數目及位置有關,與其它PCB鄰位醌類活性代謝物, 例如2,5-dichloro-3’,4’-dihydroxybiphenyl (Cl2-BPhCAT)、4-phenylcatechol (BPhCAT)之相較,含氯數越低者造成ct-DNA斷裂能力越強,此一研究結果與五氯酚 (pentachlorophenol)之quinonoid類衍生物之研究結果相符。由實驗結果推測,PCB之quinonoid類衍生物為一具活性之化物可經由不斷之氧化還原循環 (redox cycling)生成H2O2,並對DNA造成氧化損害作用,同時除氯作用對PCB之quinonoid衍生物具有增強其核酸氧化損壞作用。而人類乳癌細胞實驗結果顯示,PCB 52於MCF-7 cells中無法誘發明顯之ADL產生,而PCB 126 (10μM)和PCB 153 (50μM)於MCF-7 cells中反應2小時後即能誘發明顯之ADL產生,由RT-PCR之結果顯示,PCB 126和PCB 153誘發CYP 1A1、glutathione peroxide (GPx)、glutathione reductase (GR)、MnSOD、8-oxo-guanine DNA glycosylase 1 (hOGG1)等基因表現量增加,而對於Cu/ZnSOD和human AP endonuclease (hAPE)並無明顯之改變。由半定量RT-PCR得知,PCB 126誘發CYP 1A1、GPx、GR、MnSOD、hOGG1與控制組相比分別增加約2 - 4倍。由實驗結果推測,PCB 126誘發MCF-7產生ADL可能與CYP 1A1之活化導致「無效循環」而產生ROS有關,而DNA BER修補過程中hAPE基因之失衡亦是造成ADL累積之原因,且其實際參與之ROS物種應包括superoxide anion和hydrogen peroxide。此外PCB 153能誘發CYP 1A1、GPx、GR、MnSOD、hOGG1之基因表現,與控制組相較分別增加約2倍。因此推測PCB 153誘發MCF-7產生ADL少部分可能是因為CYP 1A1之「無效循環」所致,而DNA BER修補過程中hAPE基因之失衡亦是造成ADL累積之原因,且其實際參與之ROS物種可能包括superoxide anion和hydrogen peroxide。綜合以上之實驗結果顯示:多氯聯苯之醌類代謝物會經由氧化還原循環產生ROS,而高含氯數之多氯聯苯會藉由改變基因之表現而導致氧化壓力及核酸損壞作用。zh_TW
dc.description.abstractPolychlorinated biphenyls (PCBs) are known environmental pollutants. Evidence indicates are carcinogenic in rodent and possibly in human. To date, the mechanisms by which PCBs induce carcinogenesis are not clear thus far, but induction of the aryl hydrocarbon (Ah)-receptor signal transduction pathway, metabolism, oxidative stress, and tumor promotion may be associated with the development of PCB-induced liver tumors. Evidence suggests the formation of redox-active quinonoids by low chlorinated PCBs congeners as well as induction of imbalances in gene expression by higher chlorinated PCBs congeners as sources of oxidative stress. The objective of this research is to confer the induction of imbalances in gene expression and oxidative DNA damage by polychlorinated biphenyls (PCBs) in calf- thymus DNA (ct-DNA) and in human breast cancer cell line (MCF-7 cells). Results indicated that with the addition of NADPH (100 mM) and Cu(II) (20 mM), 2,2',5-trichloro-3',4'-dihydroxybiphenyl (Cl3-BPhCAT) induced significant increases in the number of aldehydic DNA lesions (ADL) over control (p < 0.05) in ct-DNA. Further investigation indicated that the ADL induce by Cl3-BPhCAT plus Cu(Ⅱ) and NADPH was inhibited by the additions of catalase, copper(Ⅰ)-specific chelator, and glutathione whereas superoxide dismutase and hydroxyl radical scavenger were ineffective. This finding supports the notion that formation of Cu(Ⅰ) and hydrogen peroxide via redox cycling of PCBs catechols contribute to the subsequent induction of oxidant-mediated ADL. The data also indicated that the ADL induced by Cl3-BPhCAT with the presence of Cu(Ⅱ) and NADPH contain 74% putrescine-excisable ADL in ct-DNA. These results suggest that the ADL induced by PCBs catechols in ct-DNA is likely to derive from oxidative events rather than depurination/depyrimidination of labile PCB quinone-DNA adducts. Additionally, the ADL induced by various degree of chlorination of PCB catechols decrease in the order 3,4-dihydroxybiphenyl (BPhCAT) > 2,5-dichloro-3',4'-dihydroxybiphenyl (Cl2-BPhCAT) > Cl3-BPhCAT. These data demonstrate that PCB catechols may induce significant oxidative modifications in genomic DNA in the presence of Cu(Ⅱ) and NADPH and that dechlorination process may potentiate the redox-cycling capacities of chlorinated ortho-quinonoid. Results from the analyses of PCBs-induced ADL in intact cells indicated that 2,2',5,5'-tetrachlorobiphenyl (PCB 52) did not induce significant increases in the number of ADL in MCF-7 cells with or without the addition of glutathione-depleting reagent, N-ethylmaleimide (NEM). In contrast, both PCB 126 (10μM for 2h) and PCB 153 (50μM for 2h) induced significant increases in the number of ADL over control in MCF-7 cells. Additionally, results from the semi-quantitative RT-PCR analyses indicated that both PCB 126 and PCB 153 induce significant increases (2 — 4 folds) in the expression of genes including cytochrome P450 1A1 (CYP 1A1), glutathione peroxide (GPx), glutathione reductase (GR), Mn-containing superoxide dismutase (MnSOD) and 8-oxo-guanine DNA glycosylase 1 (hOGG1) whereas Cu- and Zn-containing superoxide dismutase (Cu/ZnSOD) and apurinic endonuclease (hAPE) were ineffective. Using semi-quantitative RT-PCR method to measure the gene expression, signification induction of CYP 1A1, GPx, GR, MnSOD and hOGG1 gene expression were observed in MCF-7 cells exposed to PCB 126 induced 3.37, 2.78, 2.19, 3.87 and 3.37 -fold compared with control, respectively. These data suggest that PCB 126 and PCB153 may induced DNA damage in intact cells via “futile cycling” and the subsequent in induction of ROS. Further, the induction of imbalances in the expression of DNA base excision repair genes may also contribute to the accumulation of ADL in cells. Overall, our investigation confirmed that quinonoid derivatives of PCB can induce significant oxidative modifications in ct-DNA via redox cycling and that PCB 126 and PCB 153 appear to drive oxidative stress- and DNA repair-sensitive alterations in gene regulation and activation.en_US
dc.description.tableofcontents第一章 前言..................................................1 1.1 研究緣起.................................................1 1.2 研究目的.................................................1 第二章 文獻回顧..............................................3 2.1 多氯聯苯 (Polychlorinated Biphenyls, PCBs)...............3 2.1.1 多氯聯苯之化學結構與物理化學特性......................3 2.1.2 多氯聯苯之環境傳輸及健康效應..........................7 2.1.3 多氯聯苯之重大危害事件................................8 2.2 多氯聯苯之代謝途徑及環境中之分解.......................9 2.2.1 多氯聯苯在環境中之分解................................9 2.2.2 多氯聯苯之代謝途徑...................................10 2.2.3 多氯聯苯誘發核酸氧化損害機制.........................11 2.2.4 多氯聯苯調控體內基因表現.............................13 (A) 共平面之多氯聯苯同屬物................................13 (B) 非共平面之多氯聯苯同屬物..............................15 2.3 活潑性氧分子(reactive oxygen species, ROS)和活潑性氮氧分子(reactive nitrogen oxide species, RNOS)....................16 2.3.1 活潑性氧分子及活潑性氮氧分子之主要類型:.............16 (A) 超氧陰離子自由基 (superoxide anion radical, •O2-):..16 (B) 過氧化氫 (hydrogen peroxide, H2O2):..................17 (C) 氫氧自由基 (hydroxyl radical, •OH):..................17 (D) 一氧化氮 (nitric oxide,NO):.........................17 (E) 過氧化亞硝酸根負離子(peroxynitrite,ONOO —):........18 (F) 單分子氧 (Singlet oxygen,1O2):......................18 2.3.2 活潑性氧分子及活潑性氮氧分子之主要來源:.............19 2.3.3 氧化壓力(oxidative stress)...........................20 2.3.4 氧化壓力之影響.......................................21 (A) 核酸氧化損害 (oxidative DNA damage)...................21 (B) 脂質過氧化反應 (lipid peroxidation)...................21 (C) 蛋白質的氧化 (Protein oxidation)......................22 2.3.5 生物體之抗氧化防禦系統...............................22 (A) 酵素性防禦系統:......................................22 (B) 非酵素性防禦系統:....................................23 2.3.6 DNA醛基損壞 (Aldehydic DNA Lesions, ADL).............26 2.3.7 DNA氧化破壞及修補作用................................27 2.4 氧化壓力及外來物質調控基因之表現........................29 2.5 氧化壓力和致癌作用之關係................................32 2.5.1 Initiation...........................................32 2.5.2 Promotion............................................32 2.5.3 Progression..........................................32 第三章 實驗材料與方法.......................................33 3.1 實驗材料................................................33 3.1.1 水...................................................33 3.1.2 化學藥品.............................................33 3.2 實驗設備................................................34 3.3 實驗及分析方法..........................................35 3.3.1 製備低ADL背景值之ct-DNA(ct-DNA/MX).................35 3.3.2 DNA定量(spectrophtometer定量法)....................35 3.3.3 RNA定量(spectrophtometer定量法)....................35 3.3.4 細胞之培養 (Cell culture)............................35 3.3.5 DNA萃取..............................................35 3.3.6 RNA萃取..............................................36 3.3.7 細胞存活率之測定 (Trypan blue assay).................36 3.3.8 DNA醛基損壞分析方法 (Aldehyde Reactive Probe-Slot-Blot assay, ASB assay)............................................37 3.3.9 Putrescine cleavage assay............................37 3.3.10 反轉錄酶鏈鎖反應(RT-PCR)分析法....................38 3.3.11 半定量RT-PCR (semi-quantitative RT-PCR)方法.........39 3.4 實驗流程................................................40 3.4.1 In ct-DNA............................................40 3.4.2 In intact cells......................................42 (A) 細胞內ADL之偵測.......................................42 (B) 基因表現之分析........................................42 3.4 數據分析................................................42 第四章 實驗結果.............................................43 4.1 In ct-DNA:.............................................43 4.1.1 金屬離子及NADPH之影響................................43 4.1.2 ROS移除劑及Cu(Ⅰ)螯合劑之影響........................44 4.1.3 還原態麩胱甘肽 (Glutathione,GSH)及抗氧化劑 (BHT)存在之影響.........................................................44 4.1.4 不同含氯數之多氯聯苯醌類代謝物之比較.................45 4.1.5 Putrescine cleavage分析..............................46 4.2 MCF-7 cells 之實驗結果:................................46 4.2.1 多氯聯苯與人類乳癌細胞株MCF-7 cells反應..............46 4.2.2 細胞毒性測試.........................................47 4.2.3 多氯聯苯誘發基因表現失衡之結果.......................48 第五章 討論.................................................65 第六章 結論與建議...........................................73 6-1 結論....................................................73 6-1-1 In ct-DNA:..........................................73 6-1-2 In intact cells:....................................74 6-2 未來研究方向及建議......................................75 第七章 參考文獻.............................................76zh_TW
dc.language.isoen_USzh_TW
dc.publisher環境工程學系zh_TW
dc.subject多氯聯苯zh_TW
dc.subjectPolychlorinated biphenyls(PCB)en_US
dc.subjectDNA醛基損壞zh_TW
dc.subject氧化壓力zh_TW
dc.subject基因表現失衡zh_TW
dc.subjectAldehydic DNA Lesions(ADL)en_US
dc.subjectoxidative stressen_US
dc.subjectimbalances of gene expressionen_US
dc.title多氯聯苯誘發基因表現失衡及核酸氧化損壞作用之研究zh_TW
dc.titleInduction of imbalances in gene expression and oxidative DNA damage by polychlorinated biphenyls (PCBs)en_US
dc.typeThesis and Dissertationzh_TW
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