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研究生:蔡隆宏
研究生(外文):Lung-Hung
論文名稱:在人類氣管上皮細胞比較烹飪油煙中有毒成分之基因毒性
論文名稱(外文):Genotoxicity of cooking oil fumes components in human bronchial cells BEAS-2B
指導教授:蔡菁華林嬪嬪林嬪嬪引用關係
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:醫學分子毒理學研究所
學門:醫藥衛生學門
學類:其他醫藥衛生學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:71
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許多研究結果顯示,肺癌的發生與暴露在環境中的致癌物或致突變物有關。根據衛生署統計,肺癌為台灣男性及女性癌症死因第二及第一位。流行病學研究結果顯示,台灣女性肺癌的發生可能與長期暴露烹飪油煙有關。烹飪油煙中含有許多醛類及多環芳香烴類化合物。分別以trans, trans-2,4-decadienal (tt-DDE) 的致突變性最高和Benzo[a]pyrene (BaP) 的致癌性最高。在多環芳香烴類化合物中,含量最多為naphthalene (Nap),且被國際癌症研究中心IARC歸類為可能為致癌物 (Group 2B)。基因毒性物質會以DNA作為攻擊的目標,造成DNA斷裂以及與DNA共價鍵結產生鍵結物。之前研究結果顯示處理tt-DDE在人類氣管上皮細胞BEAS-2B上會誘發其產生自由基 (ROS),而自由基會攻擊DNA造成DNA傷害。為了提供更多科學證據以了解tt-DDE、BaP及Nap與女性肺癌的關係,本論文目的為探討tt-DDE、BaP及Nap分別在人類氣管上皮細胞 (BEAS-2B) 所產生的DNA損傷及自由基在此作用所扮演的角色。首先利用MTT assay分析三者的細胞毒性,然後經由慧星試驗 (Comet assay) 發現處理tt-DDE 4小時會產生DNA 斷裂,而BaP及Nap分別處理24小時及8小時會產生DNA斷裂。接下來我們在Comet assay過程中,添加Endo III及Fpg等修復氧化性DNA損傷的酵素來評估是否產生氧化性DNA損傷。由結果中得知於BEAS-2B細胞處理tt-DDE 及Nap並沒有明顯增加氧化性DNA損傷,而在BaP則明顯增加氧化性DNA損傷。使用抗氧化劑 (N-acetylcysteine、superoxide dismutase及catalase) 可以抑制由tt-DDE所造成的DNA 斷裂,superoxide dismutase可以抑制由Nap所造成的DNA 斷裂,superoxide dismutase與catalase可以抑制由BaP所造成的DNA 斷裂。接下來我們將從廚房收集的油煙樣品,於醛類萃取物中發現含有tt-DDE,於BEAS-2B細胞處理樣品濃度稀釋至0.1 M tt-DDE,結果發現會增加氧化性DNA損傷。本研究結果主要發現烹飪油煙中三種成分,tt-DDE、Nap及BaP都會造成DNA斷裂,雖然只有BaP會造成氧化性DNA損傷,但抗氧化劑superoxide dismutase可以預防這三種成分所造成的DNA斷裂。進而推測使用superoxide dismutase應可以降低烹飪油煙對細胞及生物體的危害。

Epidemiological studies have demonstrated that exposure to cooking oil fumes (COF) is the major risk factor for female lung cancer in Taiwan. COF contains many toxic components, such aldehydes and polycyclic aromatic hydrocarbons (PAH). Trans,trans-2,4-decadienal (tt-DDE) is the most toxic and mutagenic aldehyde identified in COF. Naphthalene (Nap), classified as group 2B carcinogen, is the most abundant PAH identified in COF. Nevertheless, bezno[a]pyrene (BaP), classified as group 2A carcinogen, is the most well-studied PAH identified in COF. Previously, we reported that tt-DDE increased oxidative stress in human bronchial epithelial cells BEAS-2B. tt-DDE, Nap, and BaP are demonstrated to induce DNA damage and/or DNA adducts. The objective of the present study is to evaluate the relationship of oxidative stress and genotoxicity (DNA strand breaks) of tt-DDE, Nap, and BaP in BEAS-2B cells. We also examined genotoxicity of COF extracts in BEAS-2B cells. The extent of DNA strand breaks was determined with Comet assay. Oxidative DNA damages were detectable by pre-incubating cells with repair enzymes for oxidative DNA damages (Formamidopyrimidine-DNA glycosylase and Endonuclease III) before performing Comet assay. When BEAS-2B cells were treated with non-cytotoxic doses of tt-DDE, Nap or BaP, the maximum levels of DNA strand breaks were detected respectively at 4, 8 or 24 hr later. Co-treatment with superoxide dismutase reduced tt-DDE, Nap and BaP induced DNA strand breaks. But co-treatment with catalase only reduced tt-DDE and BaP induced DNA strand breaks. By incubating with repair enzymes, only BaP induced DNA strand breaks were significantly increased, suggesting that only BaP induced oxidative DNA damages. But oxidative stress might enhance tt-DDE and BaP-induced DNA strand breaks. COF were collected in the kitchen and extracted with dichloromethane, which contained tt-DDE. The COF extracts induced DNA strand breaks and oxidative DNA damages in BEAS-2B cells. In conclusion, co-treatment with antioxidants, such as superoxide dismutase, may prevent COF-induced genotoxicity.

壹、 中文摘要 1
貳、 英文摘要 3
參、 縮寫表 4
一、 台灣肺癌之流行病學 5
(一)、 肺癌 5
二、 肺癌分類及其危險因素 6
(一)、 肺癌分類: 6
(二)、 肺癌之危險因素 6
三、 油煙與不抽煙女性肺癌之相關性 7
四、 油煙之來源與基因毒性 8
(一)、 油煙之產生 8
(二)、 油煙之成分與基因毒性 8
五、 DNA 傷害與癌症之形成 12
(一)、 內生性DNA傷害 13
(二)、 外生性DNA傷害 13
(三)、 DNA修復機制 15
六、 抗氧化劑 16
七、 Comet assay 17
八、 研究動機 19
九、 本論文之研究架構 20
伍、 實驗材料 (Materials) 21
陸、 實驗方法 (Methods) 22
一、 細胞培養 (cell culture) 22
二、 繼代培養 (sub-culture cell) 22
三、 冷凍細胞 (frozen cell) 22
四、 解凍細胞 (defrozen cell) 23
五、 MTT assay 23
六、 單細胞電泳法 24
七、 廚房油煙萃取 25
八、 統計分析 26
柒、 實驗結果 27
一、 處理tt-DDE對人類氣管上皮細胞株 (BEAS-2B) 生長之影響 27
二、 短期處理tt-DDE對BEAS-2B細胞之基因毒性 27
三、 抗氧化劑在tt-DDE對BEAS-2B所造成基因毒性之影響 28
四、 短期處理Nap對BEAS-2B細胞之基因毒性 28
五、 短期處理BaP對BEAS-2B細胞基因毒性之影響 28
六、 抗氧化劑在Nap與BaP對BEAS-2B所造成基因毒性之影響 29
七、 短期處理廚房油煙萃取之tt-DDE樣品對BEAS-2B細胞基因毒性之影響 29
捌、 結論 30
玖、 討論 31
壹拾、 圖表 33
圖一、 tt-DDE處理在BEAS-2B細胞之細胞毒性 33
圖二、 tt-DDE處理在BEAS-2B 細胞中產生DNA斷裂之時間與劑量反應 34
圖三、 tt-DDE 處理在BEAS-2B細胞造成氧化性DNA損傷之情形 35
圖四、 探討抗氧化劑NAC與tt-DDE共同處理在BEAS-2B細胞對DNA斷裂之影響 36
圖五、 探討抗氧化劑SOD及CAT與tt-DDE共同處理在BEAS-2B細胞對DNA斷裂之影響 37
圖六、 Nap處理在BEAS-2B 細胞之細胞毒性 38
圖七、 Nap處理在BEAS-2B細胞中產生DNA斷裂之時間關係 39
圖八、 BaP處理在BEAS-2B 細胞之細胞毒性 40
圖九、 BaP處理在BEAS-2B細胞中產生DNA斷裂之時間關係 41
圖十、 Nap及BaP 處理在BEAS-2B細胞造成氧化性DNA損傷之情形 42
圖十一、 探討抗氧化劑SOD及CAT與Nap或BaP共同處理在BEAS-2B細胞對DNA斷裂之影響 43
圖十二、 從廚房收集並萃取之tt-DDE樣品處理在BEAS-2B細胞產生氧化性DNA損傷之情形 44
壹拾壹、 附圖表 45
附圖一、 行政院衛生署2005年公布衛生統計之國人癌症死亡率趨勢圖 45
附圖二、 Substrates of Endo III 46
附圖三、 Substrates of Fpg 48
附圖四、 Nap之代謝路徑 49
附圖五、 BER修復路徑 50
附圖六、 GSH之抗氧化路徑 51
附圖七、 BER、NER相關酵素 52
附圖八、 油煙收集萃取流程圖 53
附圖九、 tt-DDE在體外與DNA鍵結之情形 54
附圖十、 Comet assay 55
附圖十一、 BaP之代謝路徑 56
附表一、 烹調食用油加熱所產生的揮發性醛類化合物成分分析 57
附表二、 IARC之致癌物質分類 58
壹拾貳、 參考文獻 59


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