Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/5455
標題: 戴奧辛與多氯聯苯所調控之基因表現改變對雌性激素於人類乳癌細胞誘發核酸損壞及修補作用之影響
Effects of TCDD- and PCB-mediated altered gene expression on the estrogen-induced DNA damage and repair in human breast cancer cells
作者: 莊明傑
Chuang, Ming-Chieh
關鍵字: dioxins
戴奧辛
PCBs
estradiol DNA
damage
多氯聯苯
雌性激素
核酸損壞
出版社: 環境工程學系所
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摘要: 多氯聯苯(polychlorinated biphenyls, PCBs)與戴奧辛(dioxins)已知能於實驗動物與人體中誘發廣泛之不良健康效應,包括內分泌衡定性之失序、氧化壓力(oxidative stress)累積及癌症等。PCBs與dioxins於生物體所誘發之危害效應機制,初步認為是透過非直接致基因性途徑(epigenetic)所導致。然而,PCBs與dioxins之暴露與特定基因表現失序、核酸損壞與細胞死亡之關連性仍未完全確立,且其作用機制仍屬未知。因此本研究主旨在於探討PCBs和dioxins之暴露與生物體內核酸結構損壞作用之相關性。並運用相關之生物指標檢測方法(biomarkers),釐清PCBs和dioxins之暴露對誘發雌性激素代謝相關之易感基因失序與後續的核酸氧化損害之影響。本研究之假設為暴露PCBs及dioxins之環境,將透過代謝酵素基因表現失序與內生性雌性激素代謝失衡,促使生物體內生成oxidative stress與進一步的誘發間接致核酸損害作用發生。 本研究中第一個部分,將針對PCBs與2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD)於人類乳癌細胞所誘發核酸損害與核酸修補啟動進行探討。由慧星法(comet assay)分析結果顯示,低氯數2,2’,5,5’-tetrachlorobiphenyl (PCB52)與3,3’,4,4’-tetrachlorobiphenyl (PCB77)和高氯數2,2’,4,4’,5,5’-hexachlorobiphenyls (PCB153) 與 3,3’,4,4’,5-pentachlorobiphenyls (PCB126)及TCDD皆能於ERα(-)/MDA-MB-231細胞中誘發核酸損害。而TCDD亦能於ERα(+)/MCF-7細胞中誘發核酸損害生成。 進一步由西方墨點(western blotting)分析,能於核酸損害細胞中觀察到經活化之poly(ADP-ribose)polymerase-1所催化形成之polymers of ADP-ribose-modified PARP-1蛋白質生成。透過上述結果顯示,TCDD與PCBs能誘發核酸損壞,並透過NAD(P)H與 NAD+消耗活化PARP-1核酸修復程序。此外, MCF-7與MDA-MB-231細胞之ERα存在與否將影響PCBs與TCDD之核酸損害效應。且在MDA-MB-231細胞中,PCB52與PCB77混合條件所誘發核酸損壞具有拮抗效應。綜合上述結果, TCDD與PCBs於乳癌細胞所誘發的核酸氧化損壞與修補效應,可能是PCBs與TCDD促腫瘤發生的風險之一。 本研究第二個部分,主要針對PCB153與TCDD預處理條件及ERα之存在與否對17β-estradiol (E2)於MCF-7及MDA-MB-231細胞所誘發之核酸損壞進行探討。結果顯示,單獨E2 (10 nM)即可誘發MDA-MB-231細胞核酸損壞與修補之效應,但經PCB153 (1 μM, 2 h ) 與 TCDD (10 nM, 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,PCB153與TCDD預處理條件能促進E2所誘發核酸損壞與修補效應。進一步研究證實TCDD預處理條件,能透過改變細胞色素P450(cytochrome) CYP 1A1與CYP 1B1表現,而影響E2於MCF-7與MDA-MB-231細胞之核酸損害效應。透過上述結果,證實TCDD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與修補效應之啟動。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。
PCBs and Dioxins are known to induce a broad-spectrum of adverse biological effects, including disruption of endocrine homeostasis, induction of oxidative stress, and cancer in laboratory animals and human. The mechanisms by which PCBs and Dioxins exert their actions are believed to mediate via epigenetic pathways to cellular malignancy. However, the link between PCBs and Dioxins exposure and deregulation of specific gene expression involving DNA damage and cell death is not conclusive, and any mechanism responsible remains unknown. The objective of this research is to address the issues of whether exposure to specific congeners of PCBs and Dioxins is associated with increased levels of structural damage to genomic DNA. Secondly, we applied biomarkers to assist in understanding the roles of PCBs and Dioxins in modulating expressions of the susceptible genes responsible for the disposition of endogenous estrogen and the subsequent induction of oxidative DNA damage. We hypothesize that exposure to PCBs and Dioxins may induce oxidative stress and DNA damage via alteration of the normal profiles of metabolizing enzymes and the disposition of endogenous estrogen. The first part of this research is to investigate whether PCBs and 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) induce DNA damage in human estrogen receptor α (ERα)(+)/MCF-7 and (ERα)(-)/MDA-MB-231 breast cancer cells and whether this is accompanied by the initiation of DNA repair events. Results indicated that treatment with lower chlorinated PCBs, including 2,2',5,5'-tetrachlorobiphenyl (PCB52) and 3,3',4,4'-tetrachlorobiphenyl (PCB77), and higher chlorinated PCBs, including 2,2',4,4',5,5'-hexachlorobiphenyls (PCB153) and 3,3',4,4',5-pentachlorobiphenyls (PCB126), and TCDD resulted in DNA damage in MCF-7 and MDA-MB-231 breast cancer cells as measured by the single-cell gel electrophoresis (Comet) assay. Further investigation indicated that the catalytic activation of PARP-1 in cells treated with TCDD and PCBs was confirmed by detection of the presence of polymers of ADP-ribose-modified PARP-1 using Western blotting. Overall, this evidence confirms that PCBs and TCDD are capable to induce decreases in intracellular NAD(P)H and NAD+ through PARP-1 activation mediated by formation of DNA strand breaks. In addition, we demonstrated that the extent of DNA damage was different between MCF-7 and MDA-MB-231 cells, with a strong correlation to ERα status. Antagonism was observed between PCB52 and PCB77 for the effect on induction of DNA strand breaks in MDA-MB-231 cells. In conclusions, our findings add further support to the theme that the induction of oxidative DNA damage and repair in human breast cancer cells exposed to PCBs and TCDD may, in part, contribute to PCB- and TCDD-induced carcinogenesis. The second part of this study is to uncover the effects of PCB153 and TCDD pretreatment and ERα status on the induction of DNA damage by 17β-estradiol (E2) in human MCF-7 and MDA-MB-231 breast cancer cells. Results indicated that E2 (10 nM) alone induced DNA strand breaks and DNA repair in MDA-MB-231 cells as measured by the Comet assay and by Western blotting. Further investigation indicated that the DNA-damaging and repairing effects induced by E2 in MDA-MB-231 cells were completely blocked by pretreatment of PCB153 (1 μM for 2 h ) and TCDD (10 nM for 72 h). In contrast, with PCB153 and TCDD pretreatment, significant increases in DNA strand breaks, and PARP-1 activation were detected in MCF-7 cells exposed to E2. We demonstrated that TCDD modulated the differential induction of DNA damage by estrogen in MDA-MB-231 and MCF-7 cells primarily through alteration of the expression of cytochrome P450 1A1 and 1B1. Overall, this evidence suggests that TCDD is capable of inducing imbalances in the expression of enzymes responsible for the bioactivation of estrogen leading to the subsequent accumulation of DNA damage and initiation of DNA repair in MDA-MB-231 and MCF-7 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/11455/5455
其他識別: U0005-2108200821174500
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2108200821174500
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