Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/51842
標題: 煎魚油煙誘發人類肺腺癌CL-3細胞產生之DNA氧化性傷害及槲皮酮之保護機轉
DNA Oxidative Damage Induced by Cooking Oil Fumes in Human Lung Adenocarcinoma CL-3 Cells and Protection Mechanisms of Quercetin
作者: 林淑瑗
Lin, Shwu-Yuann
關鍵字: cooking oil fumes
煎魚油煙
oxidative stress
quercetin
cyclooxygenase-2
氧化緊迫
槲皮酮
環氧合酶
-2
出版社: 食品科學系
摘要: 本研究主要以人類肺腺癌CL-3細胞為研究模式,探討煎魚油煙誘發之DNA傷害,並探討天然抗氧化物 - 槲皮酮之保護分子機轉。首先為了解煎魚油煙是否造成DNA傷害,並確定此DNA傷害以氧化性傷害或是以多環芳香烴 (polycyclic aromatic hydrocarbons, PAHs)類DNA傷害為主,將人類肺腺癌CL-3細胞分別以不同濃度的煎魚油煙和相當量的BaP (benzo[a]pyrene)處理,結果發現煎魚油煙造成的慧尾長度較BaP為長,顯示煎魚油煙能使肺癌細胞產生較多的DNA單股斷裂。由此推測煎魚油煙中含有非BaP的成分所造成的DNA傷害。 為了解煎魚油煙誘發的DNA傷害除了bulky DNA 傷害之外,是否還有氧化性DNA傷害,而以各種活性氧捕捉劑與煎魚油煙同時處理人類肺腺癌CL-3細胞,並以流式細胞儀分析細胞內過氧化物生成量之變化,結果顯示活性氧捕捉劑能減少煎魚油煙誘發的DNA單股斷裂,並且煎魚油煙亦能增加胞內過氧化物的生成量,顯示煎魚油煙確能誘發人類肺腺癌CL-3細胞產生活性氧,而造成氧化性DNA傷害。已知活性氧能透過增強轉錄因子-kB (nuclear factor-kappa B, NF-kB) 與DNA鍵結能力,而活化NF-kB轉移至細胞核內以調控下游基因表現。因此推測煎魚油煙亦能活化NF-kB而誘發環氧合酶-2 (cyclooxyhenase-2, COX-2) 基因的表現。以不同濃度的煎魚油煙處理CL-3細胞,結果呈現濃度線性地增強NF-kB與DNA鍵結能力、細胞色素 P-450 1A1 (cytochrome P-450 1A1, CYP 1A1) mRNA以及COX-2 蛋白的表現。0-500 g/ml煎魚油煙和相當量的BaP比較下,煎魚油煙引起之COX-2蛋白的表現顯著較BaP為高。當各種活性氧捕捉劑與煎魚油煙同時處理人類肺腺癌CL-3細胞時,四種活性氧捕捉劑對於煎魚油煙誘發的COX-2蛋白的表現量具有不同程度抑制效果,其中又以超氧化物歧化酶 (superoxide dismutase, SOD) 和甘露糖醇 (mannitol) 之抑制效果較強,顯示參與煎魚油煙誘發COX-2蛋白表現的活性氧可能主要是超氧陰離子和羥自由基。 除此之外,煎魚油煙誘發的COX-2蛋白表現與丙二醛 (malondialdehyde; MDA)生成量及前列腺素(prostaglandin E2, PGE2)分泌量成正相關性,其相關係數分別為r2= 0.816 及r2= 0.804,顯示參與煎魚油煙誘發COX-2蛋白表現、MDA及8-OH-dG的形成以超氧陰離子和羥自由基較重要。綜合得知,煎魚油煙會促使CL-3細胞產生活性氧而造成氧化緊迫,並誘發NF-kB訊號路徑,而促使COX-2表現,因而產生PGE2而可能造成免疫微環境 (immune microenvironment) 的改變。因此油煙暴露不僅會造成bulky DNA以及氧化性DNA傷害,而引起染色體不穩定 (chromosome instability)。同時也引起免疫反應失常,這或許與經常暴露烹調油煙之台灣女性肺癌的形成有關。 為探討槲皮酮是否能減少煎魚油煙誘發的DNA氧化性傷害,以彗星分析法得知,1- 25 M槲皮酮與100 g/ml煎魚油煙同時處理人類肺腺癌CL-3細胞時,槲皮酮可呈現劑量反應的抑制煎魚油煙增強的慧尾長度,且槲皮酮之保護效果顯著高過於其他的特異性抑制劑,包括活性氧捕捉劑-NaN3、CYP1A1特異性抑制劑-NF 和COX-2特異性抑制劑-NS398,其IC50分別為5.2 M 、175.9 M、9.9 M和 13.5M。已知CYP 1A1和COX-2參與煎魚油煙誘發的DNA傷害,因此再以RT-PCR和西方墨點分析法 (Western Blot) 分析槲皮酮對煎魚油煙誘發的CYP 1A1 mRNA及COX-2蛋白表現之影響,結果發現,1- 25 M槲皮酮顯著地抑制煎魚油煙誘發的CYP 1A1 mRNA及COX-2蛋白表現,且呈劑量反應關係。 煎魚油煙產生活性氧能增強NF-kB與DNA之鍵結能力。為探討槲皮酮是否會經由多環芳香烴感受器路徑及NF-kB活化作用而調控煎魚油煙誘發的CYP1A1和COX-2的基因表現,由凝膠遲滯分析(gel retardation assay)的結果獲知,槲皮酮不但有效抑制煎魚油煙誘發的AhR-Arnt複合體與DRE的鍵結作用,也減少NF-kB與DNA鍵結能力,因而達到抑制CYP1A1和COX-2基因的表現,進而減少DNA傷害。以上結果顯示,槲皮酮有效減少NF-kB 及AhR:Arnt/ DRE之DNA鍵結能力,進而抑制CYP1A1和COX-2基因的表現,是促使槲皮酮對煎魚油煙所造成之DNA傷害的保護效果顯著高於NF、 NS398等特異性抑制劑或活性氧捕捉劑-NaN3的原因。這結果顯示槲皮酮或許能化學預防油煙引起之台灣女性肺癌之危險性。
The aim of this study is to investigate the DNA damage induced by cooking oil fumes (COF) and the possible protection mechanisms of natural antioxidant-quercetin in human lung adenocarcinoma CL-3 cells. By comet assay, it was shown that crude COF, compared to pure benzo(a)pyrene (BaP) with a quantitative equal amount in such crude COF, posses a more potent capability of single strand breakage in CL-3 cells. To verify whether reactive oxygen species (ROS) was generated in COF-treated CL-3 cells to contribute to the oxidative DNA damage, fluorescence assay was used to determine the intracellular ROS generation of COF. The results showed a linear-dose response of intracellular peroxides formation induced by COF ranged from 0 to 200 μg/ml and strongly suggested that intracellular ROS were indeed generated by COF in CL-3 cells. To explore further, four ROS scavengers, including sodium azide (NaN3), mannitol, superoxide dismutase (SOD) and catalase, were added simultaneously with COF to CL-3 cells. Our data showed that the DNA damage induced by COF was significantly inhibited by these four scavengers. ROS has been shown to involve in the induction of cyclooxygenase-2 (COX-2), therefore, the effect of these four scavengers in the COX-2 protein expression induced by COF was also evaluated by western blot. The result showed that mannitol and SOD were stronger inhibitors for COX-2 protein expression than the other two scavengers. In addition, COX-2 protein expression levels were correlated with the amounts of prostaglandin E2(PGE2) (r2= 0.804) and malondialdehyde (MDA)( r2= 0.816). These results appear to reveal that hydroxyl radical and superoxide anion may be more important in induction of COX-2 and the formation of MDA and 8-hydroxydeoxyguanosine (8-OH-dG) in CL-3 cells treated by COF. In conclusion, COF may contribute to ROS-induced oxidative stress and also the alteration of immune microenvironment mediated by PGE2 production through COX-2 induction. We thus suspect that COF-induced oxidative stress may play a more important role than bulky DNA damage in lung cancer development among Taiwanese women nonsmokers. We suspect that quercetin may be a potent inhibitor for reduction of COF-induced DNA damage, and prevention of lung cancer development. In this study, comet assay was used to evaluate the DNA damage induced by a relatively low dose of COF (100 μg/ml) in human lung adenocarcinoma CL-3 cells. To understand whether quercetin has the most potent protective effect on COF-induced DNA damage, the IC50 value of COF-induced DNA damage of quercetin was compared with those of αNF, NS-398, and NaN3, which are specific inhibitors, or scavenger of CYP1A1, COX-2, and ROS, respectively. The IC50 value of quercetin was only one half, one third and one thirty fifth of that of αNF, NS-398 and NaN3, respectively. Clearly, quercetin was the most effective inhibitor of COF-induced DNA damage followed sequentially by αNF, NS-398, and NaN3. To further elucidate whether COF-induced DNA damage inhibition of quercetin is mediated through the inhibition of COX-2 gene expression by altering NF-kB pathway, COX-2 mRNA and its protein expressions induced by COF were evaluated by RT-PCR and western blot, respectively. Our data showed that COX-2 mRNA and protein levels were significantly repressed by the addition of quercetin in a dose dependent manner. Moreover, the gel retardation assay showed that NF-kB DNA binding activity induced by COF was significantly inhibited by quercetin. From our previous and present studies, it is revealed that co-expression of COX-2 and CYP1A1 caused by COF may contribute to genomic instability in lung cancer development. Thus, quercetin may act as a potent chemopreventive agent of lung cancer for nonsmoking Taiwanese women.
URI: http://hdl.handle.net/11455/51842
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