金屬奈米顆粒對斑馬魚仔魚的影響

Abstract

近年來奈米科技日新月異，也成為炙手可熱的科技產業之一，但是我們也需要關注金屬奈米顆粒可能對環境及生物造成的風險，在過去的研究中大多是探討金屬奈米顆粒對動物的死亡率、胚胎發育、細胞染色觀察、行為測量、基因表現，較少有更深入的發現。本篇研究目的是利用斑馬魚仔魚為動物模式，探討奈米銅(CuNP)、奈米銀(AgNP)與傳統的金屬離子硫酸銅(CuSO4)、硝酸銀(AgNO3)對仔魚的傷害。主要利用掃描式離子選擇電極技術（SIET）測量細胞的功能，結果顯示毛細胞浸泡在CuSO4、CuNP、AgNO3和AgNP 4小時後，鈣離子流入量下降，而離子細胞的氫離子梯度顯著下降，這說明了毛細胞與離子細胞功能明顯下降。利用FM1-43 及Rhodamine123標定側線毛細胞、離子細胞，結果顯示仔魚浸泡CuSO4、CuNP、AgNO3和AgNP 4小時後，毛細胞數目顯著下降，而離子細胞密度顯著減少。利用qPCR定量分析離子細胞上參與排酸蛋白的基因，結果顯示仔魚浸泡CuSO4、CuNP、AgNO3、AgNP 24小時後，nhe3b的mRNA表現量有顯著提升，表示仔魚可能對Na+吸收與H+排出受到影響，所以SIET測量到H+排出減少可能與此有關。利用CellROX標定產生reactive oxygen species (ROS)的離子細胞，結果顯示離子細胞在CuSO4 (0.5 ppm)、CuNP ( ppm)、AgNO3 (50 ppm)和AgNP (0.1 ppm) ROS有顯著上升，這可能是造成細胞損傷的原因之一。仔魚浸泡在CuSO4、CuNP、AgNO3及AgNP 4小時後，逆流行為顯著下降，最大游泳速度結果顯示只有CuSO4 (0.5 ppm)、CuNP (0.5 ppm)組有顯著下降，在活動力的測量結果發現仔魚只有在AgNO3 (50 ppm) 及AgNP (1.5 ppm)有顯著降低。綜合以上結果證實CuNP、AgNP除了會造成行為異常、細胞產生ROS及基因表現量改變害之外，另外也發現細胞的功能有受到影響，然而金屬奈米顆粒造成細胞的傷害機制仍需進一步研究。Recently, the nanotechnology has become one of the most important and exciting science. However, the effects of metal nanoparticles (NP) on environmental ecology are still needed to be studied. Previous studies showed the effects of NP on mortality, development and behavior of animals. In this present study, we further used zebrafish larvae to examine the effect of NP on hair cells and ionocytes. The toxicity of copper nanoparticles (CuNP), silver nanoparticles (AgNP), CuSO4 and AgNO3 were observed in 4-dpf zebrafish larvae. By using scanning ion-electrode techniques (SIET), Ca2+ influx of hair cells and H+ gradient of ionocytes are significantly suppressed after treatment of CuSO4, CuNP, AgNO3 and AgNP. FM1-43 and Rhodamine123 were used to labeled hair cells and ionocytes, respectively. The results showed that the number of hair cells and cell density of ionoctyes are significantly decreased after CuSO4, CuNP, AgNO3 and AgNP treatments. Gene expression nhe3b mRNA in zebrafish larvae was up-regulated after CuSO4, CuNP and AgNP treatment. In addition, we found that reactive oxygen species (ROS) activity ionocytes were significant increased after those treatments. In behavioral analysis, the maximal velocity of larva was significantly decreased after CuSO4 and CuNP treatments. The rheotaxis time was reduced in larva after CuSO4, CuNP, AgNO3 and AgNP treatments. In swimming activity experiments, the total swimming distance of larva was shortened in AgNO3 and AgNP treating groups. Taken together, we suggest that CuNP and AgNP increased the ROS activity and decreased the function of hair cells and ionocytes. Thereafter the larvae showed abnormal behaviors. However, the detail mechanisms of cytotoxicity of NPs are required to be investigated in the future.