簡易檢索 / 詳目顯示

研究生: 馮瑩茜
Phong, Ying-Chiann
論文名稱: N-乙醯半胱氨酸對斑馬魚仔魚側線毛細胞的影響
Effects of N-acetyl cysteine on Lateral Line Hair Cell of 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: 2019
畢業學年度: 107
語文別: 英文
論文頁數: 46
中文關鍵詞: N-乙醯半胱氨酸抗氧化劑新黴素斑馬魚側線毛細胞耳毒性胚胎毒性
英文關鍵詞: N-acetyl cysteine, antioxidant, neomycin, zebrafish, lateral line, hair cell, ototoxicity, embryotoxicity
DOI URL: http://doi.org/10.6345/THE.NTNU.SLS.005.2019.D01
論文種類: 學術論文
相關次數: 點閱:114下載:0
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報
  • N-乙醯半胱氨酸(N-acetyl cysteine,NAC)是一種作為營養食品的抗氧化劑。在水產養殖上使用N-乙醯半胱氨酸餵食吳郭魚亦可以降低微囊藻毒素和柱孢藻毒素所產生的氧化壓力。此研究的目的是想要探討抗氧化劑NAC的潛在毒性及致死劑量,以便未來在魚類上作為抗氧化劑的應用。此外測試NAC對於新黴素所造成耳毒性傷害是否有保護作用。我們將斑馬魚長時間及短時間曝露於NAC,並觀察NAC對斑馬魚的影響。我們的研究結果顯示NAC會對斑馬魚仔魚造成死亡的現象,並且其藥效與濃度成正相關。另外,NAC對各個發育的指標都會帶來負面的影響,如存活率、孵化率、斑馬魚仔魚的體長及心跳速率。另外,使用FM1-43這活體染劑,可得知NAC在急性或是慢性處理下都會減少斑馬魚仔魚的毛細胞。急性NAC處理後會增加毛細胞的氧化壓力。除此之外,NAC亦會減少斑馬魚仔魚卵黃囊上的離子細胞密度。從以上的結果可得知不管是急性或是慢性的NAC處理都會對斑馬魚造成毒性傷害。

    N-acetyl cysteine (NAC), an antioxidant used as a nutritional supplement, was used to protect tilapia from oxidative stress-induced by Microcystin and Cylindrospermopsin in the aquaculture sector. The aim of the present study was to investigate the potential toxicity and lethal dose of NAC before applying it to be an antioxidant in fish. Furthermore, we test for the protective effect of NAC against neomycin-induced ototoxicity. We investigate the embryonic toxicity of NAC on zebrafish larvae by acute or chronic exposure to NAC. Our results showed that NAC caused a lethal effect on zebrafish embryo in a dose-dependent manner. By the development assessments, survival rate, body length, and heart rate significantly decreased after the NAC treatment. By labeling hair cells with FM1-43, the hair cells number decreased in both acute and chronic NAC treatment groups. Oxidative stress of lateral line hair cell increased after acute NAC treatment. Moreover, NAC reduced the density of ionocyte on zebrafish yolk sac. Our findings demonstrate that NAC may cause toxicity to zebrafish in both acute and chronic treatment.

    Introduction 1 N-acetyl cysteine 1 Ototoxicity 2 Advantages of zebrafish lateral line 3 Zebrafish yolk sac ionocyte 4 Zebrafish model 5 Purpose 6 Experimental design 7 Materials and methods 8 Zebrafish husbandry 8 Drug administration 8 Embryos developmental assessments 9 Staining of hair cells within neuromasts 9 Rhodamine123 staining of ionocyte 9 ROS detection 10 The analysis of ion content in zebrafish larvae 10 Statistical analysis 11 Results 12 N-acetyl cysteine treatment affects zebrafish embryogenesis 12 Chronic treatment of N-acetyl cysteine affects the zebrafish larvae lateral line hair cells number 13 Chronic treatment of N-acetyl cysteine affects zebrafish larvae yolk sac ionocyte density 13 N-acetyl cysteine affects ion contents of zebrafish 14 Acute treatment of N-acetyl cysteine affects zebrafish lateral line hair cell number 14 Acute treatment of N-acetyl cysteine affects zebrafish larvae yolk sac ionocyte density 15 The oxidative stress level of zebrafish lateral line hair cell was increased after acute treatment of N-acetyl cysteine 15 Co-administration of N-acetyl cysteine does not have a protective effect against neomycin-induced hair cell damage 16 Pre-treatment with N-acetyl cysteine does not have a protective effect against neomycin-induced hair cell loss 16 Discussion 18 N-acetyl cysteine induces reductive stress on zebrafish larvae 18 Effect of N-acetyl cysteine on zebrafish larvae ion regulation 19 Effect of N-acetyl cysteine on zebrafish lateral line hair cell 21 Protective effect of NAC against neomycin-induced hair cell damage 22 Conclusion 24 References 25 Figures 31 Figure 1. Effect of N-acetyl cysteine treatment on zebrafish embryogenesis. 31 Figure 2. Effect of N-acetyl cysteine treatment on zebrafish lateral line hair cells. 32 Figure 3. Effect of N-acetyl cysteine treatment on zebrafish yolk sac ionocyte. 33 Figure 4. Effect of N-acetyl cysteine treatment on zebrafish ion content. 34 Figure 5. Effect of N-acetyl cysteine acute treatment on zebrafish lateral line hair cell. 36 Figure 6. Effect of N-acetyl cysteine acute treatment on zebrafish yolk sac ionocyte density. 37 Figure 7. Oxidative stress of N-acetyl cysteine treatment on zebrafish lateral line hair cell. 40 Figure 8. The protective effect of N-acetyl cysteine on neomycin-induced hair cell damage. 41 Figure 9. The protective effect of pre-treatment with N-acetyl cysteine on neomycin-induced hair cell damage. 43

    Aruoma, O.I., Halliwell, B., Hoey, B.M. & Butler, J. (1989) The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid. Free Radic Biol Med, 6, 593-597.
    Bavarsad Shahripour, R., Harrigan, M.R. & Alexandrov, A.V. (2014) N-acetylcysteine (NAC) in neurological disorders: mechanisms of action and therapeutic opportunities. Brain Behav, 4, 108-122.
    Bers, D.M. (2002) Cardiac excitation-contraction coupling. Nature, 415, 198-205.
    Bleckmann, H. & Zelick, R. (2009) Lateral line system of fish. Integr Zool, 4, 13-25.
    Chiu, L.L., Cunningham, L.L., Raible, D.W., Rubel, E.W. & Ou, H.C. (2008) Using the zebrafish lateral line to screen for ototoxicity. J Assoc Res Otolaryngol, 9, 178-190.
    Covarrubias, L., Hernandez-Garcia, D., Schnabel, D., Salas-Vidal, E. & Castro-Obregon, S. (2008) Function of reactive oxygen species during animal development: passive or active? Dev Biol, 320, 1-11.
    Crowell, C., Lyew, R.V., Givens, M. & Deering, S.H. (2008) Caring for the mother, concentrating on the fetus: intravenous N-acetylcysteine in pregnancy. Am J Emerg Med, 26, 735 e731-732.
    Eftekhari, P., Hajizadeh, S., Raoufy, M.R., Masjedi, M.R., Yang, M., Hansbro, N., Li, J.J. & Foster, P.S. (2013) Preventive effect of N-acetylcysteine in a mouse model of steroid resistant acute exacerbation of asthma. EXCLI J, 12, 184-192.
    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.
    Feghali, J.G., Liu, W. & Van De Water, T.R. (2001) L-n-acetyl-cysteine protection against cisplatin-induced auditory neuronal and hair cell toxicity. Laryngoscope, 111, 1147-1155.
    Fettiplace, R. (2009) Defining features of the hair cell mechanoelectrical transducer channel. Pflugers Arch, 458, 1115-1123.
    Giari, L., Dezfuli, B.S., Astolfi, L. & Martini, A. (2012) Ultrastructural effects of cisplatin on the inner ear and lateral line system of zebrafish (Danio rerio) larvae. J Appl Toxicol, 32, 293-299.
    Goss, G.G., Perry, S.F., Wood, C.M. & Laurent, P. (1992) Mechanisms of ion and acid-base regulation at the gills of freshwater fish. J Exp Zool, 263, 143-159.
    Green, J.L., Heard, K.J., Reynolds, K.M. & Albert, D. (2013) Oral and intravenous acetylcysteine for treatment of acetaminophen toxicity: A systematic review and meta-analysis. West J Emerg Med, 14, 218-226.
    Guh, Y.J., Lin, C.H. & Hwang, P.P. (2015) Osmoregulation in zebrafish: ion transport mechanisms and functional regulation. EXCLI J, 14, 627-659.
    Gutierrez-Praena, D., Puerto, M., Prieto, A.I., Jos, A., Pichardo, S., Vasconcelos, V. & Camean, A.M. (2012) Protective role of dietary N-acetylcysteine on the oxidative stress induced by cylindrospermopsin in tilapia (Oreochromis niloticus). Environ Toxicol Chem, 31, 1548-1555.
    Harris, J.A., Cheng, A.G., Cunningham, L.L., MacDonald, G., Raible, D.W. & Rubel, E.W. (2003) Neomycin-induced hair cell death and rapid regeneration in the lateral line of zebrafish (Danio rerio). J Assoc Res Otolaryngol, 4, 219-234.
    He, Z., Guo, L., Shu, Y., Fang, Q., Zhou, H., Liu, Y., Liu, D., Lu, L., Zhang, X., Ding, X., Liu, D., Tang, M., Kong, W., Sha, S., Li, H., Gao, X. & Chai, R. (2017) Autophagy protects auditory hair cells against neomycin-induced damage. Autophagy, 13, 1884-1904.
    Heard, K. & Schaeffer, T.H. (2011) Massive acetylcysteine overdose associated with cerebral edema and seizures. Clin Toxicol (Phila), 49, 423-425.
    Hobbie, S.N., Akshay, S., Kalapala, S.K., Bruell, C.M., Shcherbakov, D. & Bottger, E.C. (2008) Genetic analysis of interactions with eukaryotic rRNA identify the mitoribosome as target in aminoglycoside ototoxicity. Proc Natl Acad Sci U S A, 105, 20888-20893.
    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.
    Hwang, P.P., Lee, T.H. & Lin, L.Y. (2011) Ion regulation in fish gills: recent progress in the cellular and molecular mechanisms. Am J Physiol Regul Integr Comp Physiol, 301, R28-47.
    Hwang, P.P., Lee, T.H., Weng, C.F., Fang, M.J. & Cho, G.Y. (1999) Presence of Na-K-ATPase in mitochondria-rich cells in the yolk-sac epithelium of larvae of the teleost Oreochromis mossambicus. Physiol Biochem Zool, 72, 138-144.
    Imamura, S. & Adams, J.C. (2003) Distribution of gentamicin in the guinea pig inner ear after local or systemic application. J Assoc Res Otolaryngol, 4, 176-195.
    Kerksick, C. & Willoughby, D. (2005) The antioxidant role of glutathione and N-acetyl-cysteine supplements and exercise-induced oxidative stress. J Int Soc Sports Nutr, 2, 38-44.
    Kopke, R.D., Liu, W., Gabaizadeh, R., Jacono, A., Feghali, J., Spray, D., Garcia, P., Steinman, H., Malgrange, B., Ruben, R.J., Rybak, L. & Van de Water, T.R. (1997) Use of organotypic cultures of Corti's organ to study the protective effects of antioxidant molecules on cisplatin-induced damage of auditory hair cells. Am J Otol, 18, 559-571.
    Krishnan, V & Strauss, R. (2013) Is there a role for reactive oxygen species in zebrafish embryogenesis? Thesis Submitted in partial fulfillment of the requirements for the degree of Master of Science in Biology in the College of Science of Northeastern University May 6, 2013
    Lieschke, G.J. & Currie, P.D. (2007) Animal models of human disease: zebrafish swim into view. Nat Rev Genet, 8, 353-367.
    Lin, C.H. & Hwang, P.P. (2016) The Control of Calcium Metabolism in Zebrafish (Danio rerio). Int J Mol Sci, 17.
    MacRae, C.A. & Peterson, R.T. (2015) Zebrafish as tools for drug discovery. Nat Rev Drug Discov, 14, 721-731.
    Mahmoudi, G.A., Astaraki, P., Mohtashami, A.Z. & Ahadi, M. (2015) N-acetylcysteine overdose after acetaminophen poisoning. Int Med Case Rep J, 8, 65-69.
    Maniu, A., Perde-Schrepler, M. & Cosgarea, M. (2011) Protective effect of L-N-acetylcysteine against gentamycin ototoxicity in the organ cultures of the rat cochlea. Rom J Morphol Embryol, 52, 159-164.
    Martinez-Banaclocha, M.A. (2012) N-acetyl-cysteine in the treatment of Parkinson's disease. What are we waiting for? Med Hypotheses, 79, 8-12.
    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.
    Mokhtari, V., Afsharian, P., Shahhoseini, M., Kalantar, S.M. & Moini, A. (2017) A Review on Various Uses of N-Acetyl Cysteine. Cell J, 19, 11-17.
    Monroe, J.D., Rajadinakaran, G. & Smith, M.E. (2015) Sensory hair cell death and regeneration in fishes. Front Cell Neurosci, 9, 131.
    Moreira, P.I., Harris, P.L., Zhu, X., Santos, M.S., Oliveira, C.R., Smith, M.A. & Perry, G. (2007) Lipoic acid and N-acetyl cysteine decrease mitochondrial-related oxidative stress in Alzheimer disease patient fibroblasts. J Alzheimers Dis, 12, 195-206.
    Ou, H.C., Raible, D.W. & Rubel, E.W. (2007) Cisplatin-induced hair cell loss in zebrafish (Danio rerio) lateral line. Hear Res, 233, 46-53.
    Owens, K.N., Cunningham, D.E., MacDonald, G., Rubel, E.W., Raible, D.W. & Pujol, R. (2007) Ultrastructural analysis of aminoglycoside-induced hair cell death in the zebrafish lateral line reveals an early mitochondrial response. J Comp Neurol, 502, 522-543.
    Palmer, L.A., Doctor, A., Chhabra, P., Sheram, M.L., Laubach, V.E., Karlinsey, M.Z., Forbes, M.S., Macdonald, T. & Gaston, B. (2007) S-nitrosothiols signal hypoxia-mimetic vascular pathology. J Clin Invest, 117, 2592-2601.
    Parng, C., Seng, W.L., Semino, C. & McGrath, P. (2002) Zebrafish: a preclinical model for drug screening. Assay Drug Dev Technol, 1, 41-48.
    Perez-Torres, I., Guarner-Lans, V. & Rubio-Ruiz, M.E. (2017) Reductive Stress in Inflammation-Associated Diseases and the Pro-Oxidant Effect of Antioxidant Agents. Int J Mol Sci, 18.
    Pinto-Teixeira, F., Muzzopappa, M., Swoger, J., Mineo, A., Sharpe, J. & Lopez-Schier, H. (2013) Intravital imaging of hair-cell development and regeneration in the zebrafish. Front Neuroanat, 7, 33.
    Puerto, M., Prieto, A.I., Pichardo, S., Moreno, I., Jos, A., Moyano, R. & Camean, A.M. (2009) Effects of dietary N-acetylcysteine on the oxidative stress induced in tilapia (Oreochromis niloticus) exposed to a microcystin-producing cyanobacterial water bloom. Environ Toxicol Chem, 28, 1679-1686.
    Rushworth, G.F. & Megson, I.L. (2014) Existing and potential therapeutic uses for N-acetylcysteine: the need for conversion to intracellular glutathione for antioxidant benefits. Pharmacol Ther, 141, 150-159.
    Schacht, J., Talaska, A.E. & Rybak, L.P. (2012) Cisplatin and aminoglycoside antibiotics: hearing loss and its prevention. Anat Rec (Hoboken), 295, 1837-1850.
    Sheth, S., Mukherjea, D., Rybak, L.P. & Ramkumar, V. (2017) Mechanisms of Cisplatin-Induced Ototoxicity and Otoprotection. Front Cell Neurosci, 11, 338.
    Srinivasan, V., Corwin, D. & Verceles, A.C. (2015) An accidental overdose of N-acetylcysteine during treatment for acetaminophen toxicity. Clin Toxicol (Phila), 53, 500.
    Teves, J.M.Y., Bhargava, V., Kirwan, K.R., Corenblum, M.J., Justiniano, R., Wondrak, G.T., Anandhan, A., Flores, A.J., Schipper, D.A., Khalpey, Z., Sligh, J.E., Curiel-Lewandrowski, C., Sherman, S.J. & Madhavan, L. (2017) Parkinson's Disease Skin Fibroblasts Display Signature Alterations in Growth, Redox Homeostasis, Mitochondrial Function, and Autophagy. Front Neurosci, 11, 737.
    Timme-Laragy, A.R., Goldstone, J.V., Imhoff, B.R., Stegeman, J.J., Hahn, M.E. & Hansen, J.M. (2013) Glutathione redox dynamics and expression of glutathione-related genes in the developing embryo. Free Radic Biol Med, 65, 89-101.
    Ton, C. & Parng, C. (2005) The use of zebrafish for assessing ototoxic and otoprotective agents. Hear Res, 208, 79-88.
    Tran Ba Huy, P., Bernard, P. & Schacht, J. (1986) Kinetics of gentamicin uptake and release in the rat. Comparison of inner ear tissues and fluids with other organs. J Clin Invest, 77, 1492-1500.
    Tsai, J.C., Jain, M., Hsieh, C.M., Lee, W.S., Yoshizumi, M., Patterson, C., Perrella, M.A., Cooke, C., Wang, H., Haber, E., Schlegel, R. & Lee, M.E. (1996) Induction of apoptosis by pyrrolidinedithiocarbamate and N-acetylcysteine in vascular smooth muscle cells. J Biol Chem, 271, 3667-3670.
    Ufer, C., Wang, C.C., Borchert, A., Heydeck, D. & Kuhn, H. (2010) Redox control in mammalian embryo development. Antioxid Redox Signal, 13, 833-875.
    Wang, Q. & Steyger, P.S. (2009) Trafficking of systemic fluorescent gentamicin into the cochlea and hair cells. J Assoc Res Otolaryngol, 10, 205-219.
    Whitfield, T.T. (2002) Zebrafish as a model for hearing and deafness. J Neurobiol, 53, 157-171.
    Wu, W.J., Sha, S.H. & Schacht, J. (2002) Recent advances in understanding aminoglycoside ototoxicity and its prevention. Audiol Neurootol, 7, 171-174.
    Zhang, H., Limphong, P., Pieper, J., Liu, Q., Rodesch, C.K., Christians, E. & Benjamin, I.J. (2012) Glutathione-dependent reductive stress triggers mitochondrial oxidation and cytotoxicity. FASEB J, 26, 1442-1451.

    無法下載圖示 本全文未授權公開
    QR CODE