DNA Oxidative Damage Induced by Cooking Oil Fumes in Human Lung Adenocarcinoma CL-3 Cells and Protection Mechanisms of Quercetin

• Main Authors: Lin Shwu-Yuann, 林淑瑗
• Other Authors: Shun-Jen Tsai, Ph.D.
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/28397928090589836734

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.