臺灣博碩士論文加值系統

空氣污染是存在於許多開發中國家之重要議題，而空氣細懸浮微粒PM2.5因其體積小之特性造成肺部及心血管等多種健康危害，在流行病學與細胞及動物毒理學領域已發表多篇研究文獻證實，使其在2013由世界衛生組織國際癌症研究機構(International Agency for Research on Cancer，IARC)在致癌物質分類中歸類為「Group 1」第一級致癌物。在台灣，機車密度登上全球之冠，而特色夜市文化亦榮登旅客在臺遊覽景點排名第一，然而這些在地文化發展的同時伴隨而來的是PM2.5排放量上升。本研究參考過去文獻之萃取方法針對台灣交通排放來源、夜市烹調油煙以及境外長程傳輸等污染源所採集之PM2.5進行化學分析以及毒性暴露物質萃取，分別有水溶性之超音波震盪萃取、金屬之微波消化萃取與有機之甲苯索式萃取，並進一步以化學成分含量比對以A549肺腺癌細胞以及Salmonella typhimurium所進行之毒性測試效應差異。
境外長程傳輸、本土交通排放源以及夜市烹調油煙之PM2.5樣品質量濃度分別為39.0 μg/m3、42.9 μg/m3、28.3 μg/m3，以交通排放源之環境濃度最高，採樣時間最短之夜市烹調油煙來源最低，根據細胞暴露之細胞存活率MTT試驗結果發現夜市烹調油煙樣品之金屬及有機成分萃取樣品產生之毒性在三個污染源中效應最強，而交通污染源之三種萃取樣品則無明顯毒性影響。為深入了解造成其毒性效應差異之原因，藉由數理統計發現在金屬元素Zn (r=-0.577, p=0.008)、Mo (r=0.480, p=0.032)、Cd (r=-0.565, p=0.009)、Sb (r=-0.526, p=0.017)以及V (r=-0.606, p=0.005)暴露濃度增加時，對金屬萃取物暴露之細胞具有顯著之影響，水溶性分析成分中則與水溶性萃取物暴露組之細胞存活率無顯著相關性。Reactive oxygen species (ROS)氧化壓力指標試驗中，以本土交通污染源之水溶性萃取物與金屬萃取物暴露結果之氧化壓力最強，分別為135%以及146%。Salmonella typhimurium TA1535/pSK1002及TA1535/NM2009菌株進行之umu基因毒性測試中針對有機萃取物質進行基因毒性之測試，發現在PM2.5濃度為5 μg以及20 μg之暴露下，烹調油煙樣品暴露組相對於其他兩組污染源樣品除了具有較高之基因毒性(Induction ratio=1.55)，亦出現抑制細菌生長之情形，而其有機物質在經過CYP1A1代謝前比其代謝物誘導之毒性效應更強，且隨濃度上升而增加。但在本研究中所測試之污染源樣品在5 μg以及20 μg之暴露濃度下皆未達具有基因毒性之標準。
而針對各污染來源之毒性效應，在境外長程傳輸PM2.5樣品中，有機萃取成分在細胞存活率以及暴露濃度為20 μg之基因毒性試驗中皆出現毒性效應，基因毒性測試中其萃取物質經CYP1A1代謝前出現抑菌之效應。交通污染源之三種萃取樣品中，在細胞存活率、氧化壓力試驗以及umu基因毒性試驗中均無出現顯著之毒性效應。烹調油煙樣品之金屬以及有機萃取物之細胞存活率試驗以及氧化壓力試驗中，皆為三個污染源間不良效應最強之組別，而有機萃取物又在基因毒性試驗中效應最為突出。
整體而言，雖然在試驗中發現相對毒性效應，但其結果均不顯著，其影響因素可能為暴露濃度過低或暴露時間不足。本研究所使用之毒性暴露樣品萃取方法皆可萃取出目標萃取物質，但其中未知成分之含量仍不明確，因此在毒性暴露溶液之定性及定量應納入相關研究之探討依據。

Fine particulate matters (PM2.5) induce a lot of adverse health effects on the human body, especially the respiratory and cardiovascular system, based on the numerous of epidemiological and toxicological researches, PM2.5 was delimited to be a carcinogen and listed in “Group 1” by International Agency for Research on Cancer (IARC). In Taiwan, high densities of traffic activity (378 vehicle/km2 in 2017), and specific culture, such as Night Market is the most interested attraction for tourists.
In this research, PM2.5 samples were collected from long range transport event (LRT), traffic emission activity and cooking smoke fume of the night market. And tried to extract the ions, metals, and organic compounds on PM2.5, used an in vitro experiment to evaluate the cytotoxic potential of PM2.5 composition on human lung carcinoma cell line A549, and genotoxic potential on Salmonella typhimurium.
The PM2.5 mass concentration of three anthropogenic emission ambient are 39.0 μg/m3 on LRT, 42.9 μg/m3 in traffic emission area, and 28.3 μg/m3 in the night market with cooking smoke source. Based on the results of MTT assay for cell viability, metal and organic extracts from the cooking smoke source exposed on A549 cells cause the strongest effect. We used statistics analysis to evaluate the key factor for cell viability on the PM2.5 extracts. The results indicated that the metal elements, Zn (r=-0.577, p=0.008)、Mo (r=0.480, p=0.032)、Cd (r=-0.565, p=0.009)、Sb (r=-0.526, p=0.017), and V (r=-0.606, p=0.005) have a significant relationship with cell viability. The results of reactive oxygen species (ROS) detection of ion and metal extracts by traffic emission and LRT source samples show the highest effects in all the exposed groups. On umu test for genotoxicity for organic extracts, the organic extracts obtained from cooking smoke source cause the highest genotoxicity of the three anthropogenic emission, and the organic components in the extracts induced higher bacterial inhibition than the components which was metabolized by CYP1A1, and the situation getting stronger when the higher concentration (20 μg) exposed.
Overall the LRT organic extracts with the concentration of 20 μg cause cytotoxicity on cell viability and genotoxicity, and the bacterial inhibition by the chemical compositions were metabolized by CYP1A1. The extracts from traffic emission did not show any significant effect. The metal and organic extracts of cooking smoke source cause the strongest cytotoxicity on cell viability and ROS determination, and the organic extracts also induce the strongest effect on umu genotoxicity test.
Even if the extracts of different pollutant sources caused some effects on cell viability, ROS detection and umu genotoxicity test, all the effects did not achieve the threshold of toxic define, which may be attributed to less concentration or exposure time.

目錄
摘要-I
Abstract-III
目錄-Ⅴ
表目錄-VII
圖目錄-VIII
第一章 前言-1
1.1 研究緣起-1
1.2 研究目的-3
第二章 文獻回顧-4
2.1 細懸浮微粒特性及化學組成-4
2.2 細懸浮微粒及臺灣特徵人為活動-6
2.3 細懸浮微粒流行病學研究-9
2.4 細懸浮微粒毒性效應-10
2.4.1 細懸浮微粒動物毒性測試-11
2.4.2 細懸浮微粒胞毒性測試-12
2.5 細懸浮微粒體外毒性測試方法-13
2.5.1 懸浮微粒成分萃取方法-14
2.5.2 體外細胞毒性測試方法-14
第三章 研究方法-18
3.1 研究架構及流程-18
3.2 採樣地點及設備-19
3.2.1 採樣地點選擇-19
3.2.2 採樣設備及方法-20
3.3 樣品前處理及化學分析方法-23
3.3.1 水溶性離子分析-23
3.3.2 重金屬分析-24
3.3.3 有機成分析-27
3.4 體外毒性測試樣品萃取-28
3.4.1 水溶性離子 萃取-29
3.4.2 有機成分萃取-30
3.4.3 無機成分萃取-31
3.5 體外細胞毒性測試-32
3.5.1 細胞培養-32
3.5.2 細胞存活率分析-33
3.5.3 細胞氧化壓力測試-35
3.5.4 基因毒性試驗-36
3.6數據處理與統計分析-37
第四章 實驗結果與討論-38
4.1 樣品之化學成分析結果-38
4.1.1 水溶性離子成 分組-40
4.1.2 金屬元素組成-41
4.1.3 戴奧辛成分析-43
4.2 細胞存活率試驗-47
4.3 細胞氧化壓力測定-55
4.4 基因毒性測試-59
4.4.1 TA1535/pSK1002菌株試驗結果-59
4.4.2 TA1535/NM2009菌株試驗結果-64
4.5 PM2.5成分萃取及毒性測試方法建立 成分萃取及毒性測試方法建立-72
4.5.1 水溶性成分萃取-72
4.5.2 金屬成分萃取-72
4.5.3 有機成分萃取-73
4.5.4 細胞存活率測定方法-73
4.5.5 細胞氧化壓力測定-73
4.5.6 基因毒性測定-74
4.6 萃取方法應用之 PM2.5萃取物毒性效應-74
第五章 結論與建議-78
5.1 結論-78
5.2 建議-80
參考文獻-81

表目錄
表2-1 各污染排放源之指標元素-5
表2-2 1999-2016年全國機動車輛登記數及密度彙整-8
表3-1 樣品採集資訊-19
表3-2 水溶性離子偵測極限-24
表3-3 金屬元素分析之儀器偵測極限-26
表3-4 細胞萃取使用材料-29
表4-1 本研究中污染源PM2.5採樣地點之氣象資訊-39
表4-2 本研究中污染源PM2.5之樣品質量濃度-39
表4-3 不同污染源PM2.5之水溶性離子濃度-40
表4-4 不同污染來源PM2.5樣品之金屬元素成分組成-42
表4-5 戴奧辛之毒性當量因子-43
表4-6 不同污染來源PM2.5之戴奧辛毒性當量濃度-44
表4-7 暴露10 μg PM2.5之水溶性萃取液對A549細胞之氧化壓力影響，境外傳輸污染來源結果-56
表4-8 暴露10 μg PM2.5之水溶性萃取液對A549細胞之氧化壓力影響，本土交通污染來源結果-56
表4-9 暴露10 μg PM2.5之水溶性萃取液對A549細胞之氧化壓力影響，夜市烹調油煙污染來源結果-57
表4-10 暴露10μg PM2.5之金屬萃取液對A549細胞之氧化壓力影響-57
表4-11 暴露10μg PM2.5之有機萃取液對A549細胞之氧化壓力影響-57
表4-12 國人活動期間呼吸通氣量-77

圖目錄
圖2-1不同懸浮微粒粒徑對人體呼吸系統影響-6
圖2-2 BEAS-2B細胞暴露都市交通PM2.5及PM10上可萃取有機物之細胞存活率-13
圖2-3 DCFDA氧化壓力測定原理-15
圖2-4 umu試驗中SOS反應之作用原理-17
圖3-1 研究架構圖-18
圖3-2 採樣點富貴角測站、中山空品測站以及新北市夜市測站位置圖-20
圖3-3 BGI PQ200 - PM2.5採樣器-21
圖3-4 歐盟高流量採樣器Analitica-22
圖3-5 Sibata日規PM2.5採樣器-23
圖3-6 Bioruptor® Standard Cat. No. UCD-200 超音波震盪機-29
圖3-7將PTFE濾紙裁成小塊進行超音波震盪-30
圖3-8 樣品濾紙索氏萃取及降溫中之萃取液-31
圖3-9 A549細胞生長貼附之高密度及低密度情況-33
圖3-10 以DMSO溶於MTT處理過後之細胞成紫色-34
圖3-11 未經處理之A549細胞及MTT形成紫色結晶後之A549細胞-35
圖4-1 境外長程傳輸PM2.5樣品中戴奧辛之質量濃度分布-45
圖4-2 本土交通污染來源PM2.5樣品中戴奧辛之質量濃度分布-45
圖4-4 PM2.5樣品之水溶性萃取液暴露對A549細胞株誘導之細胞存活率變異-48
圖4-5 PM2.5樣品之金屬萃取液暴露對A549細胞株誘導之細胞存活率變異-49
圖4-6 PM2.5樣品之有機萃取液暴露對A549細胞株誘導之細胞存活率變異-50
圖4-7 Spearman相關係數檢定測得與細胞存活率顯著相關之金屬元素毒性效應分布-52
圖4-8 三個污染源PM2.5水溶性、金屬及有機萃取物質暴露之A549細胞存活率比較-54
圖4-9 境外長程傳輸、交通污染源、烹調油煙三種PM2.5來源之水溶性、金屬以及有機萃取物暴露下之自由基螢光值測定-58
圖4-10 以Salmonella typhimurium TA1535/pSK1002菌株進行umu試驗之境外長程傳輸來源暴露結果-60
圖4-11 以Salmonella typhimurium TA1535/pSK1002菌株進行umu試驗之交通污染源測試結果-62
圖4-12 以Salmonella typhimurium TA1535/pSK1002菌株進行umu試驗之烹調油煙來源測試結果-63
圖4-13 以Salmonella typhimurium TA1535/NM2009菌株進行umu試驗之境外長程傳輸來源樣品測試結果-65
圖4-14 以Salmonella typhimurium TA1535/NM2009菌株進行umu試驗之交通污染源樣品測試結果-66
圖4-15 以Salmonella typhimurium TA1535/NM2009菌株進行umu試驗之烹調油煙樣品測試結果-67
圖4-16 umu試驗之TA1535/pSK1002菌株存活率，各採樣點比較-69
圖4-17 umu試驗之NM2009菌株存活率，各採樣點比較-70
圖4-18 境外傳輸、交通污染源以及烹調油煙之樣品經CYP1A1代謝，暴露於TA1535/pSK1002菌株後細菌之β-galactosidase表達量Induction Ratio比較-71
圖4-19 境外傳輸、交通污染源以及烹調油煙之樣品經CYP1A1代謝，暴露於TA1535/NM2009菌株後細菌之β-galactosidase表達量Induction Ratio比較-71

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