鄰苯二甲酸二(2-乙基己基)酯 (di(2-ethylhexyl) phthalate) 對中樞神經細胞毒性機轉之探討

Mechanism of Di-(2-ethylhexyl) Phthalate (DEHP) Induced Apoptosis in Neuro-2A Cells

鍾宜倫(Yi-Lun Chung)

指導教授 ： 林壯澔

高雄醫學大學/醫學院/生理及分子醫學研究所/碩士(2011年)

摘要

鄰苯二甲酸二（2-乙基己基）酯類 （di（2-ethylhexyl） phthalate, DEHP）為工業上廣泛使用的塑化劑，DEHP 常被用於製造PVC 材質物品例如：食品包裝物、衣物、兒童玩具及醫藥用品等。DEHP 主要藉由三種途徑: 吸入、食入和皮膚接觸進入生物體中。DEHP進入人體後75%的DEHP被代謝成MEHP等代謝物經由尿液排出體外， 但仍有25％的DEHP 殘留於體內，因此在重複的暴露下可能造成累積作用，並對生物體產生影響。目前已有研究指出 DEHP 會干擾內分泌統系統。動物實驗亦證實雄性大鼠食入大量 DEHP 後會影響其生殖能力。若對懷孕母鼠餵食DEHP則會造成胎兒死亡或畸形，顯示 DEHP 可透過母親的胎盤影響胎兒及新生兒的發育；另一方面，在胚胎發育過程暴露DEHP會造成胎鼠腦部重量降低，顯示DEHP對中樞神經細胞可能具有毒性。因此本研究利用細胞培養模式探討 DEHP 對中樞神經系統造成細胞毒性的可能性及其作用機轉。實驗方法為將 Neuro-2A 細胞培養於各種不同 DEHP 的濃度（vehicle, 10μM~80μM）之培養液中培養24至48小時。實驗結果發現 DEHP 給予後造成Neuro-2A細胞存活率下降，且呈現時間及劑量的相關性， 顯示 DEHP 會造成神經細胞毒性。另以流式細胞儀觀察 DEHP 對細胞週期的影響，則發現 DEHP 會造成細胞週期 G2/M 期阻滯，sub G1期則有上升的情形，表示DEHP對細胞造成損傷。西方墨點法進一步證實高劑量 DEHP 的暴露後增加 Bax 蛋白質在粒線體的表現，反之細胞質中的 Bax 蛋白質表現量則降低。相對的，DEHP 亦導致細胞質中 cytochrome c 蛋白質增加，減少粒線體中cytochrome c 蛋白質表現。上述實驗結果顯示 DEHP 具有神經毒性並造成細胞凋亡。DEHP 會促使 Bax 由細胞質轉位到粒線體上，造成 cytochrome c 的釋出，導致 caspase-3 活化，造成細胞凋亡，顯示內在路徑為 DEHP 造成細胞毒性的主要機轉。
關鍵字：DEHP、Apoptosis、Bax、Bcl-2、細胞週期

關鍵字

塑化劑 ； 細胞凋亡

並列摘要

The phthalate ester di-(2-ethylhexyl)-phthalate (DEHP) is widely used as plasticizer to make polyvinyl chloride（PVC）in industry. DEHP is also widely used in clothing, food packaging, children’s toys, and medical devices. Human are exposed to DEHP though ingestion, inhalation, and dermal contact. When DEHP enters human body, about 75％ of DEHP is rapidly metabolized to MEHP and is excreted in the urine via Kidney, but there are 25％ of residual DEHP in body. Therefore, repeat exposure of DEHP cause DEHP accumulation in human body and affect normal function of body. It has DEHP been proved that DEHP interfere the normal function of the endocrine system, reproductive system. Recent evidences also show that central nervous system development of rats is obviously suppressed after prenatal or fetal exposure to DEHP. This finding implies DEHP alters the development of central nervous system, but its mechanism is unclear. Therefore, this study will investigate the possible toxic mechanism of DEHP using in vitro cell culture model.
The neuroblastoma Neuro-2A cells were treated with DEHP at various concentrations (vehicle, 10~80μM) for 24 and 48 hours, the result of cell viability showed that DEHP induced cytotoxicity in dose and time-dependent manners. In addition, cell cycle analysis of Neuro-2A cells by flow cytometry showed sub-G1 phase increased and G2/M phase arrest with dose dependent after DEHP treatment for 24 hours. Moreover, Bax translocation to mitochondria was also increased after DEHP treatment for 24 hours. The cytochrome c release from mitochondria to cytosol was also increased after DEHP treatment for 48 hours. These data suggested that apoptosis may play a role in DEHP-induced cytotoxicity, and mitochondria dysfunction may be involved in DEHP-induced apoptosis.
Keywords: DEHP, Apoptosis, Bax, Bcl-2, cell cycle

並列關鍵字

DEHP ； apoptosis

參考文獻

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