Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/5777
標題: Benzo[a]pyrene於人類乳腺腫瘤細胞株誘發氧化壓力及核酸損壞作用之研究
Investigation of the induction of oxidative stress and DNA damage in human breast tumoral cells by Benzo[a]pyrene
作者: 饒佳文
Jao, Chia-Wen
關鍵字: oxidative stress
乳癌細胞
DNA damage
breast tumoral cells
benzo[a]pyrene
氧化壓力
核酸損壞
多環芳香族碳氫化物
出版社: 環境工程學系所
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摘要: Benzo[a]pyrene (BaP)於人類及動物實驗已被認為是致癌物質,主要是碳氫化合物不完全燃燒反應產生。動物實驗及流行病學研究指出暴露於BaP之環境,會誘發不良健康效應,例如:氧化壓力(oxidative stress)累積、癌症等。本研究之主旨在於探討環境中之BaP對雌性激素(17β-estrodiol, E2)於人類乳腺腫瘤細胞(MCF-7、MDA-MB-231),誘發細胞毒性、氧化壓力、基因表現失衡及核酸損壞作用之研究。 本研究第一部分,將針對BaP於人類乳癌細胞所誘發之氧化壓力及核酸損壞進行探討,結果顯示,MCF-7與MDA-MB-231細胞所誘發之細胞毒性皆具有時間及劑量之效應,且MCF-7細胞對BaP較為敏感,細胞毒性較明顯。BaP所誘發之氧化壓力隨反應時間增加而有持續累積之現象。由Comet assay分析結果顯示,BaP於MCF-7 與MDA-MB-231細胞皆能誘發核酸損壞,且於BaP濃度為1、10 μM,誘發核酸損壞程度較明顯,具有劑量效應。 為了瞭解芳香族碳氫化物受體(aryl hydrocarbon receptor, AhR)與雌性激素受體(estrogen receptor, ER)在核酸損壞扮演之角色,觀察MCF-7細胞中利用BaP分別與AhR、ER拮抗劑共同添加之結果,BaP與AhR拮抗劑共同添加可明顯抑制AhR所導引之基因表現量,使其核酸損壞程度降低;反之,MCF-7因具有雌性激素受體(estrogen receptor α, ERα),對細胞有某種程度之保護作用,當BaP分別與ER拮抗劑共同添加時,其保護作用喪失,核酸損壞程度較單獨BaP嚴重。另外,利用MCF-7細胞單獨暴露BaP與共同添加Catalase(CAT)、Superoxide dismutases(SOD),結果顯示可降低核酸損壞作用。BaP暴露亦能使得MCF-7與MDA-MB-231細胞色素P450(cytochrome)CYP1A1表現量上升,但以MCF-7細胞其CYP1A1表現量更為明顯。 本研究第二部分,主要針對BaP預處理及ERα存在與否對雌性激素(17β-estrodiol, E2)於人類乳癌細胞所誘發之氧化壓力及核酸損壞進行探討。結果顯示,於細胞毒性結果無顯著差異,且於氧化壓力生成測試亦無顯著之上升。而Comet assay中,單獨E2 (10 nM)即可誘發MDA-MB-231細胞核酸損壞之效應,但經BaP (10 μM, 72 h)預處理後,此一效應將完全被抑制。相反的結果在MCF-7細胞中,BaP預處理條件能促進E2所誘發核酸損壞效應。進一步研究證實BaP預處理條件,能透過改變細胞色素P450(cytochrome) CYP1A1表現,而影響E2於MCF-7與MDA-MB-231細胞之核酸損壞效應。透過上述結果,證實BaP於MCF-7與MDA-MB-231細胞,能藉由調控與E2相關代謝活化酵素表現失序,進而導致E2代謝失衡及核酸損壞之效應。此外在乳癌細胞中,ERα於E2所誘發之核酸損害效應上,扮演一個保護細胞的角色。
Benzo[a]pyrene (BaP) is classified as a carcinogen in humans and in laboratory animals. It is generated through incomplete combustion of hydrocarbons. Animal studies and epidemiological surveys have demonstrated that BaP induces adverse biological effects, including induction of oxdative stress, DNA damage, and cancer. The objective of this research is to examine the effects of one of the polyaromatic hydrocarbons (PAHs), i.e. BaP, in modulating the estrogen-induced induction of cytotoxic response, oxidative stress, altered gene expression, and DNA damage in human MCF-7 and MDA-MB-231 breast cancer cells. The first part of this research was to investigate whether the BaP induces oxdative stress and DNA damage in human breast cancer cells. Results indicated that BaP induced concertration- and time-dependent increases in cytotoxic response in human breast cancer cells and that the degree of cytotoxic response induced by BaP was greater in MCF-7 cells than in MDA-MB-231cells. Further, BaP induced ROS formation in both MCF-7 and MDA-MB-231cells after exposure for up to 72 h. Additionally, the extent of DNA strand breaks were determined by the Comet assay. After 72h of exposure, significant increases in DNA strand breaks were detected in MCF-7 and MDA-MB-231 cells exposed to BaP at concertrations 1 and 10 μM. This finding suggests that BaP induced concentration- and time-dependent increases in the extent of DNA strand breaks in both MCF-7 and MDA-MB-231 cells. To determine the effects of AhR and ERα status on the induction of DNA strand breaks by BaP, we incubated MCF-7 cells with BaP for 72h in the presence of an AhR or an ERα inhibitor. Results clearly showed that in the presence of a AhR inhibitor significant decreases in DNA strand breaks were detected in MCF-7 cells treated with BaP for 72h. Co-treatment of BaP with a ERα inhibitor further enhanced the extent of DNA strand breaks in MCF-7 cells. Furthermore, the addition of Catalase and Superoxide dismutases to the incubate reduced the BaP-mediated induction of DNA strand breaks in MCF-7 cells. We noticed that treatment of cells with BaP resulted in significant increase in the expression of CYP1A1 protein level in MCF-7 whereas slight increase CYP1A1 protein level was observed in BaP-treated MDA-MB-231 cells. The second part of this research was to uncover the effect of BaP pretreatment and ERα status on the induction of oxdative stress and DNA damage by 17 beta -estradiol (E2) in human MCF-7 and MDA-MB-231 breast cancer cells. Pretreatment of BaP did not modulate the extent of the cytotoxic response and oxidative stress induced by estrogen in MCF-7 and MDA-MB-231 cells. In contrast, we observed that E2 (10 nM) alone induced DNA strand breaks in MDA-MB-231 cells as measured by the Comet assay. Further investigation indicated that the DNA-damaging and repairing effects induced by E2 in MDA-MB-231 cells were completely blocked by pretreatment of BaP (10 μM for 72h ). On the contrary, with BaP pretreatment, significant increases in DNA strand breaks and CYP1A1 expression were detected in MCF-7 cells exposed to E2. Overall, this evidence suggests that similar to dioxins, BaP is capable of inducing imbalances in the expression of enzymes responsible for the bioactivation of estrogen leading to the subsequent accumulation of DNA damage in MCF-7 and MDA-MB-231 cells. Furthermore we confirmed that ERα plays a protective role in modulating the induction of DNA damage by E2 in human breast cancer cells.
URI: http://hdl.handle.net/11455/5777
其他識別: U0005-2707201012384000
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2707201012384000
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