活性氧分子在鎘及汞誘發人類正常肺細胞 MRC-5 凋亡之作用探討 Study on the Effects of ROS during Apoptosis Triggered by Cadmium and Mercury in Human Normal Lung Cell MRC-5
中文摘要
流行病學證據顯示：鎘及汞的污染，可能導致肺部的病變。而文獻指出，鎘及汞 可誘發細胞凋亡，並可能影響胞內氧化還原狀態，但其機轉仍未完全明瞭。因此，
本論文以人類正常肺細胞MRC-5 為細胞模式，探討鎘及汞是否會誘發活性氧分
子(reactive oxygen species, ROS)的生成，改變胞內氧化壓力，並說明何種 ROS 在 MRC-5 細胞凋亡過程扮演重要角色。首先利用多種抗氧化劑或抗氧化
酵素合併鎘或汞處理細胞，瞭解ROS 是否參與鎘及汞之細胞毒性。其中包括廣
泛性的抗氧化劑N-acetylcysteine (NAC)與 pyrrolidine dithiocarbamate (PDTC)、天然的抗氧化劑 ascorbic acid、GPx 的活性中心金屬 selenium (Se)、.O2-的特異性清除劑 tiron、.OH 的特異性清除劑 mannitol、H2O2 的 抗氧化酵素catalase 及.O2-的抗氧化酵素 SOD；結果發現，PDTC 與 NAC 可 有效抑制鎘之細胞毒性，而tiron 具有部份抑制作用；於汞方面，NAC 與 tiron
可抑制其細胞毒性，但PDTC 則無作用。因此，ROS 應參與鎘及汞之細胞毒性
作用。此外，以流式細胞儀偵測得鎘及汞分別可造成66 %及 11 %的 MRC-5
細胞凋亡，顯示鎘可明顯的引發MRC-5 細胞凋亡，導致細胞死亡。
為了直接偵檢鎘及汞是否誘發胞內ROS 的生成，因此本論文分別以 2＇,7＇
-dichlorodihydrofluorescein diacetate (DCFH-DA)，dihydro- ethidium (HEt)和 dihydrorhodamine 123 (DHR123)配合流式細胞儀檢測
H2O2，.O2-和粒線體之 H2O2。結果發現，鎘及汞分別可造成 3 倍及 20 倍的 H2O2 產生，而僅有汞可造成 3.5 倍的.O2-生成。另一方面，汞可造成粒線體
之H2O2 下降一半，但鎘則沒有差異，可見汞可能經由影響粒線體的作用，造
成細胞毒性。根據上述實驗結果，本論文推測鎘導致細胞凋亡及細胞毒性作用過 程，ROS 應扮演重要角色，且 ROS 亦部分影響汞所造成之細胞毒性作用。
英文摘要
Epidemiological evidence suggested that cadmium (Cd) and mercury (Hg) exposure might cause pulmonary damage. However, the mechanisms were not clear yet. Cd or Hg had been reported to induce apoptosis in some cell lines and might correlate with redox signaling. In our research, a normal human fetal lung fibroblast cell line (MRC-5) was used as a cell model to investigate the effects of reactive oxygen species (ROS) during apoptosis triggered by Cd or Hg. Among numerous antioxidant compounds or enzymes, N- acetylcysteine (NAC)、pyrrolidine dithiocarbamate (PDTC)、ascorbic acid、selenium (Se)、tiron、mannitol、catalase and SOD, NAC and
PDTC would suppress the Cd-induced cytotoxicity efficiently and the .O2- scavenger, tiron, showed partial protective ability. NAC and tiron also exhibited partial protective ability of Hg-induced cytotoxicity in MRC-5 cell. By flow cytometric analysis with propidium iodide (PI) staining, the sub-G1 content was raised from basal level to 66
% or 11 % after treatment with Cd or Hg, respectively. These results indicated that the cytotoxicity of Cd might mediate MRC-5 cells to apoptosis. To monitor the generation of ROS after treatment of Cd or Hg, 2’,7’- dichlorodihydrofluorescein diacetate (DCFH-DA), dihydroethidium (HEt) and dihydrorhodamine 123 (DHR123) were used to detect H2O2, .O2- and mitochondrial H2O2, respectively. Three-fold (Cd treatment) and twenty- fold (Hg treatment) elevation of H2O2 were detected.
Nevertheless, only three-fold of .O2-.was increased by Hg treatment, but there was no any effect after Cd treatment. On the other hand, the mitochondrial H2O2 was
decreased half after Hg treatment, but no change after treatment with Cd. These findings indicated that ROS might play pivotal roles during apoptosis and cytotoxicity induced by Cd, and partial participated in Hg-induced cytotoxicity..
