Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/5080
標題: 以人類乳癌細胞研究多環與鹵化芳香族碳氫化合物之雌性激素干擾效應
Investigation of the (anti)estrogenicity induced by polycyclic and halogenated aromatic hydrocarbons in human breast carcinoma cells
作者: 李美質
Lee, Mei-chih
關鍵字: polyaromatic hydrocarbons
多環芳香族碳氫化合物
estrogen
aryl hydrocarbon receptor
estrogen receptor
雌性激素
芳香族碳氫化物受體
人類乳癌細胞T47D
雌性激素受體
出版社: 環境工程學系
摘要: 多環芳香族碳氫化合物 (polyaromatic hydrocarbons, PAHs) 為常見的環境污染物質,且為已知之人類之致癌物質。累積研究證據顯示,PAHs亦為荷爾蒙干擾物質,分別具有促進雌性激素 (estrogenic) 及抗拮雌性激素 (anti-estrogenic) 之特性,本研究目的為探討PAHs物質,包括naphthalene (Nap)、7,12-dimethylbenz[a]anthracene (DMBA)、以及鹵化芳香族碳氫化合物,2,3,7,8-tertachlorodibenzo-p-dioxin (TCDD),於人類乳癌細胞T47D細胞中對芳香族碳氫化物受體 (aryl hydrocarbon receptor, AhR) 與雌性激素受體 (estrogen receptor, ERα) 間之訊息交叉 (cross-talk) 之影響進行探討,利用細胞增生及基因表現分析,來比較各種PAHs及TCDD於不同之環境曝露濃度對其雌激素干擾效應之影響。由研究結果顯示,Nap、DMBA、與TCDD於10-13 ~ 10-7 M濃度範圍內單獨存在下,均不會誘發人類乳癌細胞T47D增生,而於同時處理雌性激素E2作用下,與AhR低鍵結力之Nap皆無雌性激素干擾效應,而DMBA與TCDD則於高濃度下具有抑制雌性激素效應,透過與AhR鍵結進而產生抑制雌性激素效應,且DMBA雌性激素干擾效應,均可由同時添加AhR agonist/antagonist, α-naphthoflavone (α-NF) 及CYP1A1抑制劑resveratrol而被抑制,此一結果顯示,DMBA之雌性激素干擾效應會因AhR所調控。由半定量RT-PCR結果顯示,細胞同時曝露於低濃度之DMBA及E2情況下,並不會改變CYP1A1基因之表現量,而PR基因表現量則有明顯增加之情形,若再同時添加AhR agonist, α-NF (10-5 M) ,其CYP1A1基因表現量明顯地被誘發;相反地,細胞曝露於高濃度DMBA及E2情況下,CYP1A1之基因表現量明顯地增加,但PR基因表現量則有被抑制之情形,若同時添加AhR antagonist, α-NF (10-8 M),其CYP1A1基因表現量明顯地被抑制,且PR基因表現量則有誘發之現象,此一結果顯示,DMBA之高低濃度所造成之雌性激素干擾效應,透過ERE-AhR訊息交叉所造成。此外,DMBA於低濃度與E2同時存在下,ERα基因表現量亦明顯地被抑制,於同時添加AhR agonist, α-NF (10-5 M) 下,其抑制ERα基因表現量則消失;反之DMBA於高濃度與E2同時存在下,ERα基因表現量相較於控制組則無改變,但BRCA1基因則明顯被抑制,若再額外添加AhR antagonist, α-NF (10-8 M) ,其ERα基因表現量將被抑制。綜合以上研究結果可確認,於本研究中所使用的PAHs物質與TCDD,皆於10-13 ~ 10-7 M濃度暴露範圍內,單獨存在下並無促進T47D細胞增生作用。而於與雌性激素同時存在下,與AhR親和力較大之物質,能於較高暴露濃度下有抑制雌性激素之作用。本研究亦確認DMBA於高濃度下所造成之抗雌性激素作用,應為透過ERE-AhR訊息交叉而導致增加雌性激素之代謝,以及透過BRCA1基因表現量改變所導致。
The primary purpose of this research is to examine the potential of polycyclic aromatic hydrocarbons (PAHs), including naphthalene (Nap), 7,12-dimethylbenz[a]anthracene (DMBA), and the halogenated aromatic hydrocarbons, 2,3,7,8-tertachlorodibenzo-p-dioxin (TCDD), to mediate the estrogen receptor-dependent induction of abnormal cell proliferation in human T-47D breast cancer cells and to investigate the effects of aryl hydrocarbon receptor (AhR) and estrogen receptor (ERα) cross-talk on the ER-dependent altered gene expression. Results indicated that Nap, DMBA, and TCDD did not induce cell proliferation in T47D cells at concentrations ranging from 10-13 to 10-7 M. Co-treatment of Nap (lower binding affinity with AhR) and E2 did not alter the estrogen-induced cell proliferation in T47D cells. In contrast, co-treatment of DMBA, TCDD, and E2 (higher binding affinity with AhR) exhibited antiestrogenic effects in T47D cells. The (anti)estrogenic effects of these compounds correlate with their respective AhR binding affinity. In addition, the antiestrogenic effects of DMBA in T47D cells were completely blocked with the addition of an AhR antagonist, α-naphthoflavone (α-NF) and a CYP1A1 inhibitor, resveratrol. Results from the semi-quantitative RT-PCR analyses confirmed that exposure to DMBA (10-12 M) plus E2 (10-8 M) did not change the expression of CYP1A1 whereas increases in the expression of progesterone receptor (PR) gene were detected in cells. No significant induction of the expression of CYP1A1 gene was detected in cells exposed to DMBA (10-12 M) plus E2 (10-8 M). In contrast, DMBA (10-7 M) plus E2 (10-8 M) induced increases in the expression of CYP1A1 gene and inhibited the expression of PR gene whereas inclusion of α-NF (10-8 M) in the medium abolished these effects. Additionally, co-treatment of DMBA (10-7 M) and E2 did not alter the expression of ERα gene whereas depression of BRCA-1 gene expression was detected. In conclusions, the anti-estrogenic effect of the model compounds selected in this study correlates with their respective AhR binding affinity. The mechanisms by which DMBA exerts its anti-estrogenic effects in human T47D breast cancer cells are likely to mediate via ER-AhR cross-talk and deregulation of BRCA-1 gene expression.
URI: http://hdl.handle.net/11455/5080
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