- 學位論文
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
Genotoxicity of cooking oil fumes components in human bronchial cells BEAS-2B
摘要
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。
並列摘要
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.