含硒化合物及高硒青花椰菜萃取液對砷所誘導細胞毒性之影響

Effect of selenocompounds and selenium-enriched broccoli extracts on As2O3-induced cytotoxicity in porcine endothelial cell

林肇翔(Chao-Hsaing Lin)

指導教授 ： 葉貞吟

亞洲大學/健康學院/生物科技學系碩士班/碩士(2012年)

摘要

砷為一種致癌物並具有細胞毒性，人們長期暴露在砷的環境下，會對健康產生影響，如烏腳病與癌症，硒為人類與動物生長繁殖所必需的元素，硒能影響砷誘導的細胞毒性，藉由調節細胞週期與抗氧化作用達到保護的作用，硒亦可以抑制多重抗藥性的癌細胞生長，但是對多重抗藥性傳輸子的機制仍然不清楚。本研究目的為探討含硒化合物及高硒青花椰菜萃取液對砷所引起細胞毒性之影響及其可能機制，以豬的血管內皮細胞 (porcine aortic endothelial cells; PECs) 為模式， 添加1 μM 含硒化合物 (sodium selenite, sodium selenate, selenomethionine, selenomethylselenocysteine) 或高硒花椰菜萃取液 (H-SeB) 前處理24 小時，再加入20 或40 μM 三氧化二砷 (arsenic trioxide; As2O3) 處理，探討其細胞毒性、細胞週期變化、細胞凋亡之情形、抗氧化酵素活性及抗藥性輸送子之基因表現。結果顯示前處理 1 μM 硒化合物24 小時，可顯著保護PEC 免於20 μM As2O3 之細胞的毒性，含硒化合物對於20 μM As2O3 所誘導之細胞凋亡並無影響，但含硒化合物顯著增加細胞質液麩胱甘肽過氧化酵素 (cellular glutathione peroxidase; cGPX) 酵素活性、含硒蛋白W (selenoprotein W; SeW)和麩胱苷肽S 轉移酵素A1 (glutathione-S-transferase A1; GSTA1) mRNA 表現，因此含硒化合物保護PEC 免於20 μM As2O3 之細胞毒性機制，應是經由增加抗氧化酵素活性及相關基因表現而降低砷所誘導的細胞毒性。本研究中1 μM H-SeB 前處理24 小時顯著保護PEC免於20 μM As2O3 細胞毒性，結果顯示H-SeB 減少20 μM As2O3 所引起細胞凋亡之二次壞死細胞數，且增加20 μM As2O3 所導致之cGPX酵素活性及SeW, GSTA1, MDR1 和MRP2 基因表現，因此H-SeB 保
護細胞減少20 μM As2O3 所引起細胞毒性之機制，可能是藉由調節細胞凋亡、抗氧化酵素活性及抗氧化基因與相關抗藥性輸送子之基因表現，來增加活細胞數目、細胞的抗氧化能力及其對砷的排除作用，以達到保護細胞或降低其受到砷毒性的損害。

關鍵字

硒 ； 三氧化二砷 ； 豬血管內皮細胞

並列摘要

Arsenic (As) is a human carcinogen and endothelial toxin. Exposures
to arsenic cause various health effects in humans and animals, such as Blackfoot disease and cancers. Selenium (Se) is an essential trace element for growth and reproduction in animals and humans. Selenium affect in arsenic-induced endothelial toxicity through antioxidative protection, and modulation of cell proliferation, and cell cycle progression. Although Se can inhibit the growth of drug-resistant cancer cells, the function of Se on multidrug resistance transporter is still unclear. The objective of this study was to investigate the effect and the possible mechanisms of selenocompounds and selenium-enriched broccoli extract (H-SeB) on
As-induced toxicity in porcine aortic endothelial cells (PECs). The PECs were pretreated with 1 μM of selenite, selenate, selenomethionine, selenomethylselenocysteine or H-SeB for 24 hr, and then treated with 20 or 40 μM arsenic trioxide (As2O3) for investigation of cytotoxicity, cell cycle progression, apoptosis event, antioxidative enzyme activity and the gene expression of multidrug resistance associated-transpoters. The results showed that all selenopounds at 1 μM significantly protected PECs from 20 μM As2O3-induced cytotoxicity, but did not affect apoptosis. The cellular glutathione peroxidase (cGPX) activity, as well as
selenoprotein W (SeW) and glutathione-S-transferase A1 (GSTA1) mRNA expressions were increased by selenocompoumds, therefore, the possible mechanism for selenocompounds to protect PECs from 20 μM
As2O3-induced cytotoxicity may be due to the increase in antioxidative enzyme activity and related gene expression. The results in this study indicated that 1 μM H-SeB pretreated for 24 hr significantly protected PECs from 20 μM As2O3-induced cytotoxicity with reduced secondary necrosis due to apoptosis. In addition, the As-induced cGPX activities as well as gene expressions of SeW, GSTA1, MDR1 and MRP2 were increased by H-SeB pretreated. Thus, the possible mechanism for H-SeB to protect PECs in order to increase live cells, antioxidative ability and As exclusion from 20 μM As2O3-induced cytotoxicity may be through the modulations of apoptosis, antioxidative as well as the related gene expressions of antioxidative enzyme and multidrug resistance associated transporters.

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