Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/5077
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dc.contributor.advisor林伯雄zh_TW
dc.contributor.advisorPo-Hsiung Linen_US
dc.contributor.author黃景隆zh_TW
dc.contributor.authorHuang, Ching-Lungen_US
dc.date2005zh_TW
dc.date.accessioned2014-06-06T06:33:58Z-
dc.date.available2014-06-06T06:33:58Z-
dc.identifier.urihttp://hdl.handle.net/11455/5077-
dc.description.abstract本研究之主旨在於探討低氯數之多氯聯苯同屬物(Polychlorinated biphenyls, PCBs)對人類乳腺癌細胞, 包括ERα(++)/MCF-7, ERα(+)/T47D 及 ERα(-)/MDA-MB-231,經由代謝活化過程及改變特定基因表現,能否進而誘發細胞DNA損害及細胞毒性之機制。細胞毒性測試結果顯示,三株人類乳腺癌細胞(MCF-7, T47D, MDA-MB-231)與2,2,5,5-tetrachlorinated biphenyl (PCB52)或3,3,4,4-tetrachlorinated biphenyl (PCB77)反應,皆產生顯著之細胞毒性,且細胞毒性效應隨反應時間與添加之劑量成正相關(time- and concentration-dependent effects),且PCB52所產生之細胞毒性明顯大於PCB77,且細胞凋亡(apoptosis)為導致細胞毒性的主要途徑。若T47D 及 MDA-MB-231細胞進一步以CYP1A(α-Naphthoflavone, resveratrol)及CYP2B抑制劑(metyrapone)預處理後,可保護細胞免於受PCB52及PCB77所誘發之細胞毒性,但於MCF-7細胞中,CYP1A/2B抑制劑則無法抑制由PCB52所誘發之細胞毒性,且此一結果可能與ERα存在與否有關。除此, PCB52及PCB77與MCF-7,T47D及MDA-MB-231 細胞反應後,皆可誘發細胞內ROS (reactive oxygen species)量增加,且以MDA-MB-231細胞內產生之ROS量最多,而細胞以CYP1A/2B抑制劑預處理後皆可分別減少PCB77及PCB52所誘發之ROS量,顯示ROS之累積為細胞毒性主要來源之一。另外,以無細胞毒性之PCBs濃度與細胞反應,觀察細胞內NAD(P)H消耗情形,經由PARP-1活化分析結果顯示,PCB52 (1 μM)及PCB77 (10 μM)及兩者混合皆不能誘發MCF-7 細胞之NAD(P)H下降。而在T47D及MDA-MB-231細胞中,單獨PCB52及PCB77皆可誘發NAD(P)H及 NAD+下降,且三種PARP抑制劑皆能有效抑制此一效應,但於PCB52及 PCB77混合條件下,並無明顯之NAD(P)H及NAD+下降,此一結果顯示,單獨PCB52及PCB77皆可誘發T47D及MDA-MB-231細胞DNA股斷鏈,導致PARP-1活化,但於PCB52及 PCB77混合條件下,具相互拮抗效應。進一步由半定量之反轉錄-聚合酶連鎖反應(semi-quantitative RT-PCR)結果顯示,於MCF-7細胞中,PCB77(10 μM)可誘發CYP1A1、hAPE及XRCC1 mRNA之基因表現量增加;而PCB52(1 μM)可誘發hOGG1、hAPE及XRCC1 mRNA之基因表現量增加;而混合效應可誘發hOGG1及XRCC1 mRNA之表現量增加。MDA-MB-231細胞中,PCB77(10 μM)及PCB52(10 μM)皆可誘發CYP1A1及XRCC1 mRNA之表現量增加;而混合效應可增加CYP1A1 mRNA之表現量。由以上結果顯示,於人類乳腺癌MCF-7細胞中,PCB52及PCB77皆能明顯誘發基因表現失衡,而導致細胞內ROS之產生,並直接引發細胞凋亡;相反地,人類乳腺癌MDA-MB-231細胞中,ROS之主要來源可能為PCB52或PCB77於代謝活化過程,經由氧化還原循環(redox cycling)及無效循環(futile cycling)所產生,進一步誘發DNA股斷鏈,而最終導向細胞凋亡。除此,本實驗結果也顯示,ERα於調控多氯聯苯所誘發的DNA損害及細胞毒性上扮演著重要角色。zh_TW
dc.description.abstractThe objective of this research is to examine the effects of metabolic activation and altered gene expression on the induction of oxidative stress, DNA damage, and cell toxicity by polychlorinated biphenyls (PCBs), including PCB52 and PCB77, in human ERα(++)/MCF-7, ERα(+)/T47D and ERα(-)/MDA-MB-231 breast cancer cells. Results indicated that both PCB52 and PCB77 induced concentration- and time-dependent increases in cytotoxic response in MCF-7, T47D, and MDA-MB-231 cells. The extent of cytotoxic response induced by these two PCB congeners in human breast cancer cells was greater for PCB52 than for PCB77. Additionally, the reduction in the number of viable cells exposed to PCBs was primarily mediated by apoptosis. Further, α-naphthoflavone, resveratrol, and metyrapone blocked the PCB52-induced cell toxicity in T47D and MDA-MB-231 cells, but not in MCF-7 cells. The data also showed that PCB52 and PCB77 induced significant increases in intracellular levels of reactive oxygen species (ROS) in MCF-7, T47D, and MDA-MB-231 cells, particularly in MDA-MB-231 cells. In addition, when cells were exposed to PCBs at non-cytotoxic concentration, we observed that PCB52 (1 mM) and PCB77 (10 mM) alone or mixture did not induce decreases in intracellular NAD(P)H in MCF-7 cells. In contrast, both PCB52 and PCB77 induced decreases in intracellular NAD(P)H and NAD+ through formation of DNA strand breaks and poly(ADP-ribose)polymerase-1 activation in T47D and MDA-MB-231 cells. Antagonism was detected in T47D and MDA-MB-231 cells exposed to PCB52/PCB77 mixture. Further, results from semi-quantitative reverse-transcription polymerase chain reaction (semi-quantitative RT-PCR) analyses indicated that exposure to PCB77 (10 mM) induced increases in the expression of CYP1A1, hAPE, and XRCC1 whereas PCB52 (1 mM) alone induced increases in the expression of hOGG1, hAPE, and XRCC1. Similar observation was also detected in MCF-7 cells exposed to PCB52/PCB77 mixture. On the other hand, both PCB52 (10 mM) and PCB77 (10 mM) induced increases in the expression of CYP1A1 and XRCC1 genes whereas PCB52/PCB77 mixture induced increases in the expression of CYP1A1 in MDA-MB-231 cells. Overall, this evidence indicates that both PCB52 and PCB77 may induce ROS formation and imbalances in the expression of genes responsible for the DNA repair process and apoptosis in MCF-7 cells. In contrast, PCB52 and PCB77 are likely to mediate the induction of ROS formation, DNA strand breaks, and cell toxicity through futile cycling and/or redox cycling in MDA-MB-231 cells. The data also suggests that the status of estrogen receptor a may play a role in modulating the PCB-induced DNA damage and cytotoxic response in human breast cancer cells.en_US
dc.description.tableofcontents摘要 I ABSTRACT III ABBREVIATIONS V 圖目錄 XIII 表目錄 XVII 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 4 2.1 多氯聯苯 4 2.1.1多氯聯苯結構與IUPAC命名系統 4 2.1.2多氯聯苯之物化特性 6 2.1.3多氯聯苯之環境流佈與健康效應 7 2.1.4台灣初產婦女乳汁中多氯聯苯含量 8 2.1.5多氯聯苯與乳癌相關性 10 2.2 多氯聯苯之代謝活化 11 2.2.1 cytochrome P450s之誘發 11 2.2.2 共平面多氯聯苯同屬物之代謝途徑及毒性機轉 12 2.2.3 非共平面多氯聯苯同屬物之代謝途徑及毒性機轉 14 2.2.4 多氯聯苯同屬物之混合效應 17 2.3 氧化壓力 18 2.3.1多氯聯苯誘發之氧化壓力來源 18 2.3.2 活性氧分子(Reactive oxygen species, ROS)之防禦機制 20 (A)抗氧化酵素之參與 20 (B)麩胱甘肽過氧化酵素 (Glutathione peroxidase)之參與 22 2.3.3麩胱甘肽(Glutathione)扮演之角色 22 2.4 氧化DNA損害 24 2.4.1 DNA損害反應 24 2.4.2 氧化鹼基損害之誘發機制 25 2.4.3 氧化DNA損害之修復種類 26 2.4.4 鹼基切除修復(Base excision repair, BER)程序 28 2.4.5 鹼基切除修復相關基因多型性 30 (一)hOGG1 31 (二)hAPE 31 (三)XRCC1 32 2.5 DNA股斷鏈誘發PARP-1之活性 33 2.5.1 PARP種類 33 2.5.2 PARP-1之特徵及功能 35 2.5.3 PARP-1及細胞內之能量動力 36 2.5.4 PARP-1於細胞死亡過程扮演之角色 38 2.6 細胞週期停滯或細胞凋亡產生 39 2.6.1 p53之調控 39 2.6.2 多氯聯苯與細胞週期(cell cycle)之關係 40 2.6.3 細胞凋亡(apoptosis)程序 40 2.6.4 細胞之抗氧化潛力受雌性激素受體α(ERα)調控 42 第三章 實驗材料與方法 44 3.1 實驗材料 44 3.1.1 化學藥品 44 3.1.2 細胞株來源 45 3.1.3 實驗設備 45 3.2 實驗方法 46 3.2.1 細胞培養 46 3.2.2 試藥 46 3.2.3 細胞存活率測試 (Trypan blue dye exclusion) 47 3.2.4 細胞毒性測試 (Sulforhodamine B assay, SRB assay) 47 3.2.5 螢光分析細胞內ROS之生成量 (Fluorescence assay) 47 3.2.6 PARP-1 activation assay 50 3.2.7 核酸醛基損害分析方法 (Aldehyde Reactive Probe-Slot-Blot assay, ASB assay) 51 3.2.8 Determination of intracellular NAD+ 53 3.2.9 反轉錄-聚合酶鏈鎖反應分析法(RT-PCR assay) 54 3.2.10 DAPI staining assay 56 3.2.11統計分析 58 第四章 實驗結果 60 4.1 細胞毒性測試 (SULFORHODAMINE B ASSAY, SRB ASSAY) 60 4.1.1 ERα(++)/MCF-7細胞暴露於PCB52及PCB77之測試結果 60 4.1.2 ERα(+)/T47D細胞暴露於PCB52及PCB77之測試結果 60 4.1.3 ERα(-)/MDA-MB-231細胞暴露於PCB52及PCB77之測試結果 61 4.1.4 Effects of ROS modulators and transition metals chelators 62 4.1.5 Protective roles of CYP1A/2B antagonists 62 4.1.6 ERa調控PCB52所誘發的MCF-7細胞毒性 63 4.1.7 PCB52/PCB77混合效應之測試 63 4.2 DAPI染色分析(DAPI STAINING ASSAY) 64 4.2.1 ERα(++)/MCF-7細胞 64 4.2.2 ERα(+)/T47D細胞 64 4.2.3 ERα(-)/MDA-MB-231細胞 64 4.3 螢光分析(FLUORESCENCE ASSAY)活性氧分子生成量 65 4.3.1 ERα(++)/MCF-7細胞 65 4.3.2 ERα(+)/T47D細胞 66 4.3.3 ERα(-)/MDA-MB-231細胞 66 4.4 PARP-1 ACTIVATION ASSAY 68 4.4.1 ERα(++)/MCF-7細胞 68 4.4.2 ERα(+)/T47D細胞 69 4.4.3 ERα(-)/MDA-MB-231細胞 70 4.5 DETERMINATION OF INTRACELLULAR NAD+ 70 4.5.1 ERα(+)/T47D細胞 70 4.5.2 ERα(-)/MDA-MB-231細胞 71 4.6 醛基核酸損害分析 (ASB ASSAY) 71 4.6.1 PCBs於ERα(+)/T47D細胞誘發ADL之能力 71 4.7反轉錄-聚合酶鏈鎖反應分析(RT-PCR ASSAY) 72 4.7.1 PCBs誘發ERα(++)/MCF-7細胞之基因表現 72 4.7.2 PCBs誘發ERα(-)/MDA-MB-231細胞之基因表現 73 第五章 討論 112 5.1 ERΑ(++)/MCF-7細胞 112 5.2 ERΑ(+)/T47D細胞 114 5.3 ERΑ(-)/MDA-MB-231細胞 116 5.4 總結 119 5.4.1 PCB52與PCB77對人類乳腺癌細胞之毒性潛力比較 119 5.4.2人類乳腺癌細胞之敏感度比較 120 5.4.3 雌性激素受體 (ER) 120 5.4.4 XRCC1之影響 121 5.4.5 多氯聯苯於乳腺癌細胞之代謝活化機制 122 (A)PCB52之代謝活化 122 (B)PCB77之代謝活化 124 第六章 結論 126 第七章 參考文獻 131zh_TW
dc.language.isoen_USzh_TW
dc.publisher環境工程學系zh_TW
dc.subjecthuman breast cancer cellsen_US
dc.subject人類乳腺癌細胞zh_TW
dc.subjectMCF-7 cellsen_US
dc.subjectT47D cellsen_US
dc.subjectMDA-MB-231 cellsen_US
dc.subjectPCB52en_US
dc.subjectPCB77en_US
dc.subjectoxidative stressen_US
dc.subjectDNA damageen_US
dc.subjectapoptosisen_US
dc.subjectERen_US
dc.subject多氯聯苯zh_TW
dc.subject氧化壓力zh_TW
dc.subjectDNA損害zh_TW
dc.subject細胞凋亡zh_TW
dc.subject雌性激素受體zh_TW
dc.title代謝活化及基因表現變異對多氯聯苯誘發人類乳腺癌細胞氧化壓力、DNA損害及細胞毒性作用之影響zh_TW
dc.titleEffects of metabolic activation and altered gene expression on the induction of oxidative stress, DNA damage, and cell toxicity in human breast cancer cells by polychlorinated biphenyls (PCBs)en_US
dc.typeThesis and Dissertationzh_TW
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