Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/5535
標題: 多環芳香族碳氫化合物(PAHs)誘發細胞毒性、核酸損壞及雌激素干擾效應之研究
Induction of cytotocixity, DNA damage, and (anti)estrogenicity by polycyclic aromatic hydrocarbons (PAHs)
作者: 陳亞嵐
Chen, Ya-Lan
關鍵字: PAHs
多環芳香族碳氫化合物
DNA damage
(anti)estrogenicity
核酸損壞
雌激素干擾效應
出版社: 環境工程學系
摘要: 摘要 多環芳香族碳氫化物(polyaromatic hydrocarbons, PAHs)為常見的環境污染物質,且為已知之人類致癌物質,包括肺癌及乳癌在內。本研究目的為瞭解PAH萘醌類代謝物是否具有誘發生成DNA氧化損害作用之能力,以及不同PAHs之環境曝露濃度,對人類乳癌細胞不正常增生以及相關基因表現改變之機制。由本實驗結果中發現,萘之對位醌類活性代謝物NHQ (0.01-10 μM)與Cu(II) (20 μM)及NADPH (100 μM)同時存在下,於生理條件下反應後,可誘發核酸醛基損壞作用(aldehydic DNA lesions, ADL)之生成,並可透過累積生成ROS之途徑而誘發小牛胸腺DNA核酸醛基損壞之能力,並對人類乳癌細胞MCF-7產生細胞毒性,並於不具明顯細胞毒性情況下,單獨添加NCAT (10 μM)及NHQ (4 μM)與MCF-7反應5小時後,均可造成NAD(P)H含量下降約50%,而在經預處理PRPA-1 〔poly(ADP-ribose)polymerase-1〕抑制劑,可以抑制此一現象(NAH(P)H下降由50%回升至87% (P<0.001))。此一研究結果可證明,當MCF-7細胞處於NCAT環境下,可誘發細胞之核酸單股斷鏈(DNA single strand breaks,SSBs)生成。但同樣實驗條件下,預處理PRPA-1 〔poly(ADP-ribose)polymerase-1〕抑制劑並無法有效提升原本由NHQ造成NAD(P)H下降之程度。由此得知,於人類乳癌細胞MCF-7當中,可經由內生性之DNA修補機制,以PAPR酵素對ROS所造成之ADL進行DNA repair。 本研究續以三種典型的PAHs物質,分別為Benzo[a]pyrene (B[a]P)、Benzo[a]anthracene (B[a]A)以及7,12-Dimethylbenz[a]anthracene (DMBA),藉以比較各種PAHs於不同之環境曝露濃度,其雌性激素干擾效應及對雌激素干擾效應之影響。由研究結果顯示,B[a]P (10-10M)、B[a]A (10-8 M)、以及DMBA (10-12M)均可有效誘發MCF-7細胞增生約達控制組的1.5倍(P<0.05)。且當B[a]P與E2共同存在下,可進一步的誘發細胞增生達控制組的4.5倍(P<0.001),且上述細胞增生現象,均可由同時添加具AhR抑制劑α-naphthoflavone以及CYP1A1抑制劑Resveratrol,而受到抑制 (p<0.001)。本研究更進一步證實,當B[a]P之添加濃度達10-8M以上時,並不具有誘發細胞增生的能力,且與雌激素共同添加時,反而具有雌激素抑制效應(P<0.001)。 由半定量之RT-PCR結果顯示,受ERE所調控之基因表現之結果顯示,於ER(+)之MCF-7細胞,單獨添加B[a]P(10-10 M)反應6天後,PR的基因表現量可達控制組的1.4倍(P<0.01),且於共同添加E2(10-7M)及B[a]P (10-10M)情況下,PR的基因表現量可達控制組的3.6倍(P<0.01);而若將B[a]P濃度提升至10-8 M,則可明顯抑制ER基因表現達控制組0.4倍,並當E2 (10-7 M)及B[a]P (10-8 M) 共同添加之實驗條件下,將無法誘發PR之基因表現;此外,α-NF並不會影響ERα,但可明顯誘發CYP1A1之基因表現,並可抑制經由共同添加E2 (10-7M)及B[a]P (10-10M)所誘發之PR的基因表現量。綜合以上之研究結果可確認,於較低之環境曝露濃度(10-10M),B[a]P具有雌激素效應。但於較高之環境曝露濃度(10-8M),則顯示為抗雌激素效應。
Abstract Polycyclic aromatic hydrocarbons (PAHs) are important classes of chemical carcinogens that are widespread in the environment. PAHs had been proven as inducers of human cancers including lung and breast cancer. The primary purpose of this research is to examine the potential of PAHs in mediating the induction of aldehydic DNA lesions (ADLs) by quinonoid derivatives of naphthalene and the mechanisms by which PAHs mediates the concentration-dependent induction of abnormal cell proliferation and altered gene expression in human MCF-7 breast cancer cells. Results indicated that with the addition of the transition metal copper (II) and NADPH, increases in the amount of oxidant-mediated ADLs were detected in ct-DNA exposed to quinonoid derivatives of naphthalene over the corresponding control. The data also indicated that the depletion of NAD(P)H in MCF-7 cells exposed to NCAT at 10 M (5 h) was blocked by a PARP-1 inhibitor, 3-aminobenzamide (3-AB). In contrast, 3-AB did not prevent the depletion of NAD(P)H in MCF-7 cells exposed to NHQ (4 M). Overall, this evidence suggests that while NHQ is more efficient in cell killing, NCAT is prone to induce an imbalance in DNA repair and the accumulation of ADL and DNA single strand breaks in living cells. In addition, three kinds of proto-typical PAHs compounds, including Benzo[a]pyrene (B[a]P), Benzo[a]anthracene (B[a]A), and 7,12-Dimethylbenz[a]anthracene (DMBA) were used in this study to explore the concentration-dependent induction of estrogenic/antiestrogenic effects in human breast cancer cells. Results from cell proliferation assay indicated that B[a]P (10-10M), B[a]A (10-8 M), and DMBA (10-12 M) induced a 1.5-fold increase in the number of viable cells in human MCF-7 breast cancer cells when compared to the corresponding control (p<0.05). Co-treatmentment of B[a]P and E2 induced an additional increase (4.5-fold) in the number of viable cells when compared to the corresponding control (p<0.001). The cell proliferation induced by B[a]P plus E2 was inhibited with the addition of the AhR inhibitor, α-naphthoflavone, and a Cyp1A1 inhibitor, resveratrol (p<0.001). Additionally, results from the semi-quantitative RT-PCR analyses indicated that 6 days exposure to B[a]P (10-10M) induces a 1.4-fold increase in the expression of progesterone receptor (PR) gene in ER(+)/MCF-7 cells when compared to the control (p<0.01). Co-treatment of B[a]P and E2 further induced PR gene expression (3.6-fold) over the corresponding control (p<0.01). The data also indicated that B[a]P at concentration at or above 10-8M significantly suppressed PR gene expression (~40% of control) where co-treatment of B[a]P (10-8M) and E2 (10-8M) did not alter the expression of PR gene. Further investigation indicated that α-NF significantly inhibited the PR gene expression induced by E2 (10-8M) plus B[a]P(10-10M). Overall, our data suggest that while B[a]P exhibited estrogenic effects at concentration at or below 10-10M, it displayed antiestrogenic effects at or above 10-8M when co-treated with E2 (p<0.001).
URI: http://hdl.handle.net/11455/5535
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