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標題: 戴奧辛誘發人類乳癌細胞株(MCF-7及MDA-MB-231 cells)細胞毒性及核酸氧化損壞作用之研究
Induction of cytotoxicity, oxidative stress and DNA damage in human MCF-7 and MDA-MB-231 breast cancer cells by 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD)
作者: 黃群真
Huang, Chuan-Chen
關鍵字: TCDD;戴奧辛;breast cancer;oxidative stress;DNA damage;乳癌細胞;氧化壓力;DNA損害
出版社: 環境工程學系所
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本研究之主旨在於探討環境中之持久性有機污染物(persistent organic pollutants, POPs)中毒性最大之戴奧辛2,3,7,8-四氯戴奧辛(2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD),於人類乳癌細胞株ERα(+)/MCF-7 與 ERα(-)/MDA-MB-231細胞,誘發細胞毒性、氧化壓力及核酸氧化損害作用之研究。研究結果顯示,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。TCDD於短時間內即可使MCF-7與MDA-MB-231細胞累積明顯之ROS,但其所誘發之ROS並未隨反應時間之增加而有持續累積之現象。實驗中添加NEM(glutathione移除劑)可增加TCDD於細胞內ROS之累積量。TCDD可於短時間內降低此兩株細胞中GSH含量,但延長反應時間後GSH含量則恢復正常,此顯示細胞中ROS累積量之變化可能與GSH含量之增減有關。將MCF-7細胞於ROS試驗中添加4-OHT(ERα之antagonist),其所得之ROS累積量與MDA-MB-231細胞約略相同,其顯示ERα確實會影響由TCDD誘發之相關效應。MDA-MB-231細胞中添加α-NF(AhR之antagonist)可抑制由TCDD所誘發之ROS生成,因而推論TCDD是經由與AhR反應,進而增加細胞內ROS之累積。上述之實驗結果顯示TCDD於MCF-7細胞中誘發細胞毒性之能力較強,而其於MDA-MB-231細胞中誘發ROS累積及氧化核酸損害之程度皆較MCF-7細胞敏感。在TCDD誘發細胞中DNA損害方面,皆可使MCF-7與MDA-MB-231細胞誘發ADL之生成(p≦0.01),但此種類之核酸損害並不會持續的累積於細胞中,且於細胞所誘發之ADL大部分可被putrescine所切除。本實驗亦證實TCDD會造成MCF-7與MDA-MB-231細胞中NAD(P)H及NAD+之下降(約70 ~ 80%),而PRPA-1 〔poly(ADP-ribose)polymerase-1〕抑制劑之存在,可以抑制此一現象。此一結果顯示,TCDD之暴露可誘發MCF-7與MDA-MB-231細胞之核酸股斷鏈(DNA strand breaks)。綜合上述結果,TCDD可於人類乳癌細胞株誘發不同程度之細胞毒性、ROS形成及氧化核酸損害,而ERα可能於其中扮演重要之角色。

The objective of this research is to examine the induction of oxidative stress, DNA damage, and cell toxicity by one of the most toxic persistent organic pollutants (POPs), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in human ERα(+)/MCF-7 and ERα(-)/MDA-MB-231 breast cancer cells. Results indicated that TCDD induced concentration- and time-dependent increases in cytotoxic response in MCF-7 and MDA-MB-231 cells. Further, we detected that a 4–fold increase in ROS formation was detected in MDA-MB-231 cells exposed to TCDD (10 nM) when compared to control (p≦0.05). Similar observation was also detected in MCF-7 cells. Additionally, we confirmed that TCDD induced a decrease in intracellular GSH in both MDA-MB-231 and MCF-7 cells. The data also indicated that TCDD induced an increase in the number of aldehydic DNA lesions (ADLs) in MDA-MB-231 and MCF-7 cells. Further characterization confirmed that the ADL induced by TCDD in MCF-7 and MDA-MB-231 cells was predominantly putrescine-excisable ADL. Overall, the extent of cytotoxic response was greater in MCF-7 cells than in MDA-MB-231 cells whereas the extent of ROS formation, GSH depletion, and ADL formation were greater in MDA-MB-231 cells than in MCF-7 cells. In addition, when cells were exposed to TCDD at non-cytotoxic concentration, we observed that TCDD (1 nM) alone induce decreases in intracellular NAD(P)H and NAD+ in MDA-MB-231 and MCF-7 cells. Further investigation indicated that the TCDD-induced depletion of NAD(P)H in MDA-MB-231 and MCF-7 cells was completely blocked by three types of PARP-1〔poly(ADP-ribose)polymerase-1〕inhibitors. This evidence suggests that TCDD induced decreases in intracellular NAD(P)H and NAD+ through PARP-1 activation mediated by formation of DNA strand breaks. In conclusions, this evidence suggests that TCDD may induce ROS formation, oxidative DNA lesions, and cell death in human breast cancer cells. The data also suggests that the status of estrogen receptor α may play a role in modulating the TCDD-induced oxidative DNA damage and cell death in human breast cancer cells.
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