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研究生: 方鏡雅
Fang, Ching-Ya
論文名稱: 金屬奈米顆粒對斑馬魚仔魚的影響
Effects of metallic nanoparticles on zebrafish larvae (Danio rerio)
指導教授: 林豊益
Lin, Li-Yih
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2017
畢業學年度: 105
語文別: 中文
論文頁數: 75
中文關鍵詞: CuNPAgNP斑馬魚毛細胞離子細胞
英文關鍵詞: copper nanoparticles, silver nanoparticles, zebrafish, hair cell, ionocyte
DOI URL: https://doi.org/10.6345/NTNU202202018
論文種類: 學術論文
相關次數: 點閱:152下載:0
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  • 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.

    致謝 2 摘要 3 Abstract 5 研究背景 6 研究目的 15 實驗設計 16 研究材料與方法 20 結果 31 討論 38 結論 49 References 50 圖表附件 55

    Al-Bairuty, G.A., Shaw, B.J., Handy, R.D., Henry, T.B., 2013. Histopathological effects of waterborne copper nanoparticles and copper sulphate on the organs of rainbow trout (Oncorhynchus mykiss). Aquat Toxicol 126, 104-115.

    Asharani, P.V., Lian Wu, Y., Gong, Z., Valiyaveettil, S., 2008. Toxicity of silver nanoparticles in zebrafish models. Nanotechnology 19, 255102.

    AshaRani, P.V., Low Kah Mun, G., Hande, M.P., Valiyaveettil, S., 2009. Cytotoxicity and genotoxicity of silver nanoparticles in human cells. ACS Nano 3, 279-290.

    Bahadar, H., Maqbool, F., Niaz, K., Abdollahi, M., 2016. Toxicity of Nanoparticles and an Overview of Current Experimental Models. Iran Biomed J 20, 1-11.

    Bai, W., Tian, W., Zhang, Z., He, X., Ma, Y., Liu, N., Chai, Z., 2010. Effects of copper nanoparticles on the development of zebrafish embryos. J Nanosci Nanotechnol 10, 8670-8676.

    Bilberg, K., Hovgaard, M.B., Besenbacher, F., Baatrup, E., 2012. In Vivo Toxicity of Silver Nanoparticles and Silver Ions in Zebrafish (Danio rerio). J Toxicol 2012, 293784.

    Boyle, D., Al-Bairuty, G.A., Ramsden, C.S., Sloman, K.A., Henry, T.B., Handy, R.D., 2013. Subtle alterations in swimming speed distributions of rainbow trout exposed to titanium dioxide nanoparticles are associated with gill rather than brain injury. Aquat Toxicol 126, 116-127.

    Carrillo, S.A., Anguita-Salinas, C., Pena, O.A., Morales, R.A., Munoz-Sanchez, S., Munoz-Montecinos, C., Paredes-Zuniga, S., Tapia, K., Allende, M.L., 2016. Macrophage Recruitment Contributes to Regeneration of Mechanosensory Hair Cells in the Zebrafish Lateral Line. J Cell Biochem 117, 1880-1889.

    Chen, T.H., Lin, C.Y., Tseng, M.C., 2011. Behavioral effects of titanium dioxide nanoparticles on larval zebrafish (Danio rerio). Mar Pollut Bull 63, 303-308.

    Coffin, A.B., Ou, H., Owens, K.N., Santos, F., Simon, J.A., Rubel, E.W., Raible, D.W., 2010. Chemical screening for hair cell loss and protection in the zebrafish lateral line. Zebrafish 7, 3-11.
    Donini, A., O'Donnell, M.J., 2005. Analysis of Na+, Cl-, K+, H+ and NH4+ concentration gradients adjacent to the surface of anal papillae of the mosquito Aedes aegypti: application of self-referencing ion-selective microelectrodes. The Journal of experimental biology 208, 603-610.

    Evans, D.H., Piermarini, P.M., Choe, K.P., 2005. The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physiol Rev 85, 97-177.

    Goodrich, L.V., 2005. Hear, hear for the zebrafish. Neuron 45, 3-5.

    Griffitt, R.J., Hyndman, K., Denslow, N.D., Barber, D.S., 2009. Comparison of molecular and histological changes in zebrafish gills exposed to metallic nanoparticles. Toxicol Sci 107, 404-415.

    Griffitt, R.J., Luo, J., Gao, J., Bonzongo, J.C., Barber, D.S., 2008. Effects of particle composition and species on toxicity of metallic nanomaterials in aquatic organisms. Environ Toxicol Chem 27, 1972-1978.

    Griffitt, R.J., Weil, R., Hyndman, K.A., Denslow, N.D., Powers, K., Taylor, D., Barber, D.S., 2007. Exposure to copper nanoparticles causes gill injury and acute lethality in zebrafish (Danio rerio). Environ Sci Technol 41, 8178-8186.

    Guh, Y.J., Lin, C.H., Hwang, P.P., 2015. Osmoregulation in zebrafish: ion transport mechanisms and functional regulation. EXCLI J 14, 627-659.

    Hernandez, P.P., Moreno, V., Olivari, F.A., Allende, M.L., 2006. Sub-lethal concentrations of waterborne copper are toxic to lateral line neuromasts in zebrafish (Danio rerio). Hear Res 213, 1-10.

    Hua, J., Vijver, M.G., Ahmad, F., Richardson, M.K., Peijnenburg, W.J., 2014. Toxicity of different-sized copper nano- and submicron particles and their shed copper ions to zebrafish embryos. Environ Toxicol Chem 33, 1774-1782.

    Hwang, P.P., 2009. Ion uptake and acid secretion in zebrafish (Danio rerio). J Exp Biol 212, 1745-1752.

    Hwang, P.P., Lee, T.H., 2007. New insights into fish ion regulation and mitochondrion-rich cells. Comp Biochem Physiol A Mol Integr Physiol 148, 479-497.

    Kim, K.T., Zaikova, T., Hutchison, J.E., Tanguay, R.L., 2013. Gold nanoparticles disrupt zebrafish eye development and pigmentation. Toxicol Sci 133, 275-288.

    Klein, S.G., Hennen, J., Serchi, T., Blomeke, B., Gutleb, A.C., 2011. Potential of coculture in vitro models to study inflammatory and sensitizing effects of particles on the lung. Toxicol In Vitro 25, 1516-1534.

    Lee, K.J., Nallathamby, P.D., Browning, L.M., Osgood, C.J., Xu, X.H., 2007. In vivo imaging of transport and biocompatibility of single silver nanoparticles in early development of zebrafish embryos. ACS Nano 1, 133-143.

    Li, J.J., Hartono, D., Ong, C.N., Bay, B.H., Yung, L.Y., 2010. Autophagy and oxidative stress associated with gold nanoparticles. Biomaterials 31, 5996-6003.

    Linbo, T.L., Stehr, C.M., Incardona, J.P., Scholz, N.L., 2006. Dissolved copper triggers cell death in the peripheral mechanosensory system of larval fish. Environ Toxicol Chem 25, 597-603.

    McNeil, P.L., Boyle, D., Henry, T.B., Handy, R.D., Sloman, K.A., 2014. Effects of metal nanoparticles on the lateral line system and behaviour in early life stages of zebrafish (Danio rerio). Aquat Toxicol 152, 318-323.

    Meyers, J.R., MacDonald, R.B., Duggan, A., Lenzi, D., Standaert, D.G., Corwin, J.T., Corey, D.P., 2003. Lighting up the senses: FM1-43 loading of sensory cells through nonselective ion channels. J Neurosci 23, 4054-4065.

    Morgan, T.P., Wood, C.M., 2004. A relationship between gill silver accumulation and acute silver toxicity in the freshwater rainbow trout: support for the acute silver biotic ligand model. Environ Toxicol Chem 23, 1261-1267.

    Muth-Kohne, E., Sonnack, L., Schlich, K., Hischen, F., Baumgartner, W., Hund-Rinke, K., Schafers, C., Fenske, M., 2013. The toxicity of silver nanoparticles to zebrafish embryos increases through sewage treatment processes. Ecotoxicology 22, 1264-1277.

    Nel, A., Xia, T., Madler, L., Li, N., 2006. Toxic potential of materials at the nanolevel. Science 311, 622-627.

    Oberdorster, G., Oberdorster, E., Oberdorster, J., 2005. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect 113, 823-839.

    Powers, C.M., Slotkin, T.A., Seidler, F.J., Badireddy, A.R., Padilla, S., 2011. Silver nanoparticles alter zebrafish development and larval behavior: distinct roles for particle size, coating and composition. Neurotoxicol Teratol 33, 708-714.

    Schins, R.P., Knaapen, A.M., 2007. Genotoxicity of poorly soluble particles. Inhal Toxicol 19 Suppl 1, 189-198.

    Shaw, B.J., Al-Bairuty, G., Handy, R.D., 2012. Effects of waterborne copper nanoparticles and copper sulphate on rainbow trout, (Oncorhynchus mykiss): physiology and accumulation. Aquat Toxicol 116-117, 90-101.

    Shaw, B.J., Handy, R.D., 2011. Physiological effects of nanoparticles on fish: a comparison of nanometals versus metal ions. Environ Int 37, 1083-1097.

    Shih, T.H., Horng, J.L., Hwang, P.P., Lin, L.Y., 2008. Ammonia excretion by the skin of zebrafish (Danio rerio) larvae. Am J Physiol Cell Physiol 295, C1625-1632.

    Stawicki, T.M., Esterberg, R., Hailey, D.W., Raible, D.W., Rubel, E.W., 2015. Using the zebrafish lateral line to uncover novel mechanisms of action and prevention in drug-induced hair cell death. Front Cell Neurosci 9, 46.

    Wiesner, M.R., Lowry, G.V., Alvarez, P., Dionysiou, D., Biswas, P., 2006. Assessing the risks of manufactured nanomaterials. Environ Sci Technol 40, 4336-4345.

    Wood, C.M., 2012. Fish Physiology: Homeostasis and Toxicology of Essential Metals.

    Xin, Q., Rotchell, J.M., Cheng, J., Yi, J., Zhang, Q., 2015. Silver nanoparticles affect the neural development of zebrafish embryos. J Appl Toxicol 35, 1481-1492.

    Yan, J.J., Chou, M.Y., Kaneko, T., Hwang, P.P., 2007. Gene expression of Na+/H+ exchanger in zebrafish H+ -ATPase-rich cells during acclimation to low-Na+ and acidic environments. Am J Physiol Cell Physiol 293, C1814-1823.

    Zabirnyk, O., Yezhelyev, M., Seleverstov, O., 2007. Nanoparticles as a novel class of autophagy activators. Autophagy 3, 278-281.

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