DNA Oxidative Damage Induced by Cooking Oil Fumes in Human Lung Adenocarcinoma CL-3 Cells and Protection Mechanisms of Quercetin
博士 === 國立中興大學 === 食品科學系 === 90 === 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...
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博士 === 國立中興大學 === 食品科學系 === 90 === 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.
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author2 |
Shun-Jen Tsai, Ph.D. |
author_facet |
Shun-Jen Tsai, Ph.D. Lin Shwu-Yuann 林淑瑗 |
author |
Lin Shwu-Yuann 林淑瑗 |
spellingShingle |
Lin Shwu-Yuann 林淑瑗 DNA Oxidative Damage Induced by Cooking Oil Fumes in Human Lung Adenocarcinoma CL-3 Cells and Protection Mechanisms of Quercetin |
author_sort |
Lin Shwu-Yuann |
title |
DNA Oxidative Damage Induced by Cooking Oil Fumes in Human Lung Adenocarcinoma CL-3 Cells and Protection Mechanisms of Quercetin |
title_short |
DNA Oxidative Damage Induced by Cooking Oil Fumes in Human Lung Adenocarcinoma CL-3 Cells and Protection Mechanisms of Quercetin |
title_full |
DNA Oxidative Damage Induced by Cooking Oil Fumes in Human Lung Adenocarcinoma CL-3 Cells and Protection Mechanisms of Quercetin |
title_fullStr |
DNA Oxidative Damage Induced by Cooking Oil Fumes in Human Lung Adenocarcinoma CL-3 Cells and Protection Mechanisms of Quercetin |
title_full_unstemmed |
DNA Oxidative Damage Induced by Cooking Oil Fumes in Human Lung Adenocarcinoma CL-3 Cells and Protection Mechanisms of Quercetin |
title_sort |
dna oxidative damage induced by cooking oil fumes in human lung adenocarcinoma cl-3 cells and protection mechanisms of quercetin |
publishDate |
2002 |
url |
http://ndltd.ncl.edu.tw/handle/28397928090589836734 |
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ndltd-TW-090NCHU02530402016-06-27T16:09:33Z http://ndltd.ncl.edu.tw/handle/28397928090589836734 DNA Oxidative Damage Induced by Cooking Oil Fumes in Human Lung Adenocarcinoma CL-3 Cells and Protection Mechanisms of Quercetin 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 Lin Shwu-Yuann 林淑瑗 博士 國立中興大學 食品科學系 90 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. Shun-Jen Tsai, Ph.D. Huei Lee, Ph.D. 蔡順仁 李輝 2002 學位論文 ; thesis 173 zh-TW |