順鉑導致斑馬魚胚胎離子細胞氧化壓力與細胞凋亡

Abstract

順鉑為現今廣泛使用之化療藥物，卻伴隨腎毒性、神經毒性和耳毒性等副作用，其中主要限制施予劑量的因素為腎毒性。順鉑可經由銅離子運輸蛋白與有機陽離子運輸蛋白進入腎臟上皮細胞，造成腎小管損傷，目前哺乳動物細胞研究模式已知氧化壓力生成是順鉑造成細胞損傷的主要原因之一。斑馬魚是廣泛使用於毒理學研究與藥物測試的模式動物，其仔魚表皮分布的五型離子細胞與哺乳動物腎臟上皮細胞有許多相似之處，因直接暴露於環境，好操作且易觀察。本研究以斑馬魚仔魚表皮離子細胞作為研究順鉑腎毒性之工具，使用活體螢光染色觀察順鉑對離子細胞的影響，來證實順鉑會導致離子細胞氧化壓力生成、粒線體損傷和細胞凋亡。本實驗將斑馬魚胚胎浸泡於不同濃度的順鉑（0、50、100、300、500 或 1000 μM）進行長時間（4-100 hpf）或短時間（96-98 hpf）處理，再使用活體螢光染劑單染或共染的方式，標定斑馬魚仔魚卵黃囊上具粒線體活性離子細胞（Rhodamine 123/MitoTracker）與凋亡細胞（AcridineOrange），並探討當中活性氧化物的產生（CellROX/ MitoSOX）。斑馬魚胚胎分別在順鉑處理 96小時及 2 小時後，Rhodamine 123 標定具粒線體活性離子細胞數目均顯著下降，且凋亡細胞數目顯著上升；斑馬魚胚胎分別在順鉑處理 96 小時及 1 小時後，產生活性氧化物的離子細胞數目或 CellROX/MitoSOX 的螢光亮度均顯著上升。此外，將斑馬魚胚胎進行抗氧化劑 NAC（0、100、300、500 或 1000 μM）與順鉑的長時間共處理，發現 NAC 能降低胚胎的死亡率，並減緩順鉑對離子細胞所導致的氧化壓力與損害。由以上結果可證實順鉑會導致離子細胞氧化壓力生成和粒線體損傷，並引起細胞凋亡，而抗氧化劑 NAC 可作為順鉑毒性的保護劑。

Cisplatin is a widely used chemotherapeutic drug, but usually causes side-effects including nephrotoxicity, neurotoxicity and ototoxicity. Cisplatin therapy is primarily limited by severe nephrotoxicity. Cisplatin can enter the renal epithelial cells via copper transporter 1 (CTR1) and organic cation transporter 2 (OCT2), and causes renal tubular damage. It is generally accepted that cisplatin-induced oxidative stress is one of the main causes of the cytotoxicity. Zebrafish embryo is a widely used animal model for toxicology and drug testing. Mitochondia-rich ionocytes in the skin of zebrafish embryos are functionally similar to mammalian renal tubular cells, and they can be observed and examined in vivo. Using the fluorescent vital staining, this study attempted to demonstrate that cisplatin can cause oxidative stress, mitochondria damage, and apoptosis in ionocytes of zebrafish embryos. Zebrafish embryos were exposed to cisplatin (0, 50, 100, 300, 500 or 1000 μM) for 96 h (4-100 hpf) or 0.5-2 h (96-98 hpf), and then they were single- or double-stained with fluorescent dyes to reveal mitochondria activity (Rhodamine123/MitoTracker), apoptosis (Acridine Orange) and oxidative stress (CellROX/MitoSOX) in ionocytes. The results showed that both 96 h and 2 h cisplatin exposure decreased Rhodamine 123-labeled ionocytes and increased apoptotic cells in a dose-depedent manner. Oxidative stress in ionocytes was induced in both 96 h and 1 h cisplatin exposed embryos. In addition, the embryos were co-treated with cisplatin and an antioxidant, NAC (0, 100, 300, 500 or 1000 μM) for 96 h. The results showed that NAC could effectively protect embryos from cisplatin-induced oxidative stress, mitochondria damage, and decrease the mortality of embryos. In conclusion, this study demonstrated that cisplatin exposure could induce oxidative stress, mitochondria damage and apoptosis in ionocytes of zebrafish embryos, and NAC could be used to protect cisplatin-induced injury.