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研究生： 王俞方
Wang, Yu-Fang
論文名稱： 快速且高通量的斑馬魚毒性篩選平台之建立與應用
Establish and Application of a Rapid and High Throughput Toxicity Test Method with Zebrafish Animal Model
指導教授： 蘇五洲
Su, Wu-Chou
楊孔嘉
Young, Kung-Chia
傅子芳
Fu, Tzu-Fun
學位類別： 碩士
Master
系所名稱： 醫學院 - 食品安全衛生暨風險管理研究所
Department of Food Safety / Hygiene and Risk Management
論文出版年： 2021
畢業學年度： 109
語文別： 英文
論文頁數： 54
中文關鍵詞： 斑馬魚 、替代測試 、斑馬魚胚胎毒性試驗 、奈米石墨烯 、胚胎殼
外文關鍵詞： Zebrafish, AlternativeToxicology Tests, Zebrafish Embryo Acute Toxicity Test, Graphene Oxide Quantum Dots, Chorion
相關次數： 點閱：262  下載：0 
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    • 現在的世界是各種新興化學物質以及奈米材料發展越來越快速蓬勃發展以及合成的時代，新的物質不斷的產出，並且廣泛的應用在我們的生活中。然而這些新興化學物質對於環境、對於生物體、甚至對於人類的影響又評估之必要性。傳統利用囓齒類動物作為評估方式較為耗時，且耗費金錢。因此發展一個快速且方便的毒性評估方式的發展便非常的重要。且在3Rs (Replacement, Reduction, and Refinement) 越來越被重視的現在，推行替代方案並減少使用實驗動物，成為許多國家討論重點並努力發展的方向。
    　　斑馬魚胚胎實驗即為替代方案之一。以國際經濟合作暨發展組織(OECD)所訂定的急毒性試驗為藍本，每天觀察胚胎發育之存活率 (Survival Rate)、胚胎孵化率 (Hatching rate)、胚胎凝結數 (Coagulated embryos)、缺乏體節胚胎數 (Lack of somite formation)，尾端未分離胚胎數 (Non-detachment of the tail)、缺乏心跳數胚胎數 (Lack of heartbeat)。這些雖然可作為胚胎存活之判定以了解測試中物質對胚胎毒性，但仍希望可以更做細節的了解毒性所影響的器官或組織。因此本實驗利用食用色素建立並測試OECD毒性試驗以及新的試驗方式，以確認新的試驗方式所得之死亡率與OECD實驗方式一致，以判定新的實驗方式是可行的。並利用所建立的方法，測試氧化石墨烯奈米粒子的毒性。在新的實驗設計會針對胚胎受精後三天計算心跳數，拍照測量眼睛大小，以及身體長度。以外觀了解所測試物質對於胚胎心臟影響，眼睛影響，以及發育狀態。氧化石墨烯是應用相當廣泛的奈米物質，也容易以其為基礎衍生出各種不同的衍生物，相當適合做為比較各種不同衍生物對斑馬魚胚胎可能產生的影響。我們利用新的試驗方式了解氧化石墨烯對於斑馬魚胚胎所產生之毒性會影響整體發育包括心跳速率，眼睛大小，以及身體長度，也了解到卵殼對於毒性試驗影響甚鉅。在實驗中利用血管螢光魚也可清楚知道除了上述影響之外，對於氧化石墨烯也會影響血管發育。
    　我們的研究結果為測試藥物及毒物的安全性提供了一個可更快速評估毒性，且可觀察到更細微斑馬魚胚胎在物質暴露下所產生的變化，並利用不同種特定組織螢光基因轉殖斑馬魚更清楚探究物質對於胚胎的影響，以期能更了解物質對於斑馬魚胚胎的影響。未來在奈米材料的發展上，可利用此方法快速的比較評估新物質對於生物體的影響。

    In today’s modern world, new materials are constantly being developed, which we incorporate into our lives. However, the toxicity of these materials is not always clear and the impact to the environment, organisms, even humans is usually not evaluated. The traditional toxicity test method used involves a rodent animal model that can take a few weeks and needs sufficient financial support. Under this situation, developing a convenient and quick alternative method is necessary. Moreover, since the 3Rs policy, that is, replacement, reduction, and refinement, is gaining important, there are many countries focusing on alternative methods in animal experimentation.
    In compliance with the 3Rs principle, zebrafish embryos are considered as replacement or refinement alternatives. Based on the Organization for Economic Co-operation and Development (OECD) guidelines for the testing of chemicals and pesticides, Test No. 236: Fish Embryo Toxicity (FET) test (TG 236), we can observe survival rate, hatching rate, coagulated embryos, lack of somite formation, non-detachment of the tail, and lack of heartbeat of the embryo. Although those signatures can reflect the toxicity to zebrafish embryo, further information regarding these nanomaterials is needed.
    The project used the survival rate of zebrafish embryos exposed to food coloring to confirm whether the OECD TG 236 protocol is comparable to the modified protocol. A graphene oxide (GO) toxicity test to the zebrafish embryo using the modified protocol was established. The modified protocol not only observes the morphologies in TG 236 but also provides more morphologies, such as heartbeat rate, eye size, and body length. We used the GO base materials to reveal the impact on the zebrafish and the impact of the chorion on the toxicity test. The transgenic tissue-specific fluorescence zebrafish, such as the vessel fluorescence zebrafish embryo, helped examine the impact on the embryo vessel development with graphene oxide quantum dots (GOD).
    This protocol provides a faster toxicity evaluation method for nanomaterials that helps improve their developing progress.

    摘要　I ABSTRACT　II ACKNOWLEDGEMENTS　III TABLE OF CONTENTS　IV LIST OF FIGURES　VI LIST OF TABLES　VII LIST OF APPENDICES　VIII ABBREVIATIONS　IX I.　INTRODUCTION　1 II.　BACKGROUND　3 II.1.　Nanoparticles　3 II.2.　Zebrafish (Denio rerio)　3 II.3.　The role of zebrafish in 3R policy　4 II.4.　Fish Embryo Acute Toxicity (FET) Test　5 II.5.　Graphene Oxide Quantum Dots　5 II.6.　Toxicity of nanoparticles　5 III.　SPECIFIC AIMS　7 IV.　RATIONALE AND HYPOTHESIS　8 V.　MATERIALS AND METHODS　9 V.1.　Fish line and maintain　9 V.2.　Graphene Oxide　9 V.3.　Zebrafish embryo exposed method with OECD TG 236 protocol and modified protocol　9 V.4.　De-chorion method　10 V.5.　Survival rate and morphological change in zebrafish embryo　10 V.6.　Oxidative stress detection methods　10 a.　H2DCFDA　10 b.　Thiobarbituric acid-reactive substance (TBARS)　11 c.　CellROX　11 V.7.　Whole mount in situ hybridization　11 V.8.　Locomotor behaviors in zebrafish (Danio rerio) larvae　11 V.9.　Statistical analysis　12 VI.　RESULTS　13 VI.1.　Establish the toxicity test OECD TG 236 with zebrafish embryos using red food coloring　13 VI.1.1　Establishing the OECD TG 236 operating protocol with red food coloring　13 VI.1.2　Modified method to evaluate the toxicity using the zebrafish model　13 VI.2.　Evaluate the toxicity of GOD molecules with the established OECD TG 236 protocol　13 VI.2.1　GO and GOD toxicity test results with OECD TG 236 protocol and modified protocol　14 VI.2.2　The effect of the zebrafish chorion to the toxicity test　15 VI.2.3　GOD 5K-F of different synthesis batch have difference impact on the zebrafish embryos　15 a.　Survival rate 15 b.　Heart rate 15 c.　Eye size and body length 16 VI.2.4　Survival rate of zebrafish embryos exposed to GOD 3K-C and GOD 100K-C　 16 VI.3.　Potential impact of GOD on the development of zebrafish embryos with tissue-specific fluorescence　16 VI.3.1　Impact of GOD 3K-C on the zebrafish embryo　16 VI.3.2　Development-related defects　17 VII.　DISCUSSION　18 VII.1.　Chorion　18 VII.2.　Toxicity of Nanoparticles　19 VII.3.　The toxicity of the GOD 3K-C　20 VII.4.　Nanomaterial uptake in the zebrafish embryo　22 VIII.　CONCLUSION　23 REFERENCES　24 FIGURES　29 TABLES　48 APPENDICES　50

    1. Gupta, D.S.K., Study of Nanotechnology and Its Application. Journal of Physics & Optics Sciences. SRC/JPSOS/112 J Phy Opt Sci, 2020. 2(1): p. 2.
    2. Vance, M.E., et al., Nanotechnology in the real world: Redeveloping the nanomaterial consumer products inventory. Beilstein journal of nanotechnology, 2015. 6(1): p. 1769-1780.
    3. Schrand, A.M., et al., Toxicity testing of nanomaterials. New technologies for toxicity testing, 2012: p. 58-75.
    4. Chahardehi, A.M., H. Arsad, and V. Lim, Zebrafish as a successful animal model for screening toxicity of medicinal plants. Plants, 2020. 9(10): p. 1345.
    5. Cheng, S.H., et al., Cellular and molecular basis of cadmium‐induced deformities in zebrafish embryos. Environmental Toxicology and Chemistry: An International Journal, 2000. 19(12): p. 3024-3031.
    6. Belanger, S.E., J.M. Rawlings, and G.J. Carr, Use of fish embryo toxicity tests for the prediction of acute fish toxicity to chemicals. Environmental toxicology and chemistry, 2013. 32(8): p. 1768-1783.
    7. Feynman, R.P., There's plenty of room at the bottom. California Institute of Technology, Engineering and Science magazine, 1960.
    8. Khan, I., K. Saeed, and I. Khan, Nanoparticles: Properties, applications and toxicities. Arabian journal of chemistry, 2019. 12(7): p. 908-931.
    9. Salamanca-Buentello, F., et al., Nanotechnology and the developing world. PLoS Med, 2005. 2(5): p. e97.
    10. Khan, F.R. and S. Alhewairini, Zebrafish (Danio rerio) as a model organism. Current Trends in Cancer Management, 2018: p. 3-18.
    11. Engeszer, R.E., et al., Zebrafish in the wild: a review of natural history and new notes from the field. Zebrafish, 2007. 4(1): p. 21-40.
    12. Arunachalam, M., et al., Natural history of zebrafish (Danio rerio) in India. Zebrafish, 2013. 10(1): p. 1-14.
    13. Spence, R., et al., The distribution and habitat preferences of the zebrafish in Bangladesh. Journal of fish biology, 2006. 69(5): p. 1435-1448.
    14. Blackburn, P.R., et al., The CRISPR system—keeping zebrafish gene targeting fresh. Zebrafish, 2013. 10(1): p. 116-118.
    15. Tannenbaum, J. and B.T. Bennett, Russell and Burch's 3Rs then and now: the need for clarity in definition and purpose. Journal of the American Association for Laboratory Animal Science, 2015. 54(2): p. 120-132.
    16. Ema, M., et al., Historical control data on prenatal developmental toxicity studies in rabbits. Congenital anomalies, 2012. 52(3): p. 155-161.
    17. Ema, M., et al., Historical control data on developmental toxicity studies in rodents. Congenital anomalies, 2014. 54(3): p. 150-161.
    18. Kari, G., U. Rodeck, and A.P. Dicker, Zebrafish: an emerging model system for human disease and drug discovery. Clinical Pharmacology & Therapeutics, 2007. 82(1): p. 70-80.
    19. Dooley, K. and L.I. Zon, Zebrafish: a model system for the study of human disease. Current opinion in genetics & development, 2000. 10(3): p. 252-256.
    20. Teraoka, H., W. Dong, and T. Hiraga, Zebrafish as a novel experimental model for developmental toxicology. Congenital anomalies, 2003. 43(2): p. 123-132.
    21. Gandhirajan, R.K., N. Endlich, and S. Bekeschus, Zebrafish larvae as a toxicity model in plasma medicine. Plasma Processes and Polymers, 2020: p. e2000188.
    22. Selderslaghs, I.W., R. Blust, and H.E. Witters, Feasibility study of the zebrafish assay as an alternative method to screen for developmental toxicity and embryotoxicity using a training set of 27 compounds. Reproductive toxicology, 2012. 33(2): p. 142-154.
    23. Yang, L., et al., Zebrafish embryos as models for embryotoxic and teratological effects of chemicals. Reproductive Toxicology, 2009. 28(2): p. 245-253.
    24. Chakraborty, C., et al., Zebrafish: A complete animal model to enumerate the nanoparticle toxicity. Journal of nanobiotechnology, 2016. 14(1): p. 1-13.
    25. Geim, A.K. and K.S. Novoselov, The rise of graphene, in Nanoscience and technology: a collection of reviews from nature journals. 2010, World Scientific. p. 11-19.
    26. Neuberger, N., H. Adidharma, and M. Fan, Graphene: A review of applications in the petroleum industry. Journal of Petroleum Science and Engineering, 2018. 167: p. 152-159.
    27. Han, T.-H., et al., Graphene-based flexible electronic devices. Materials Science and Engineering: R: Reports, 2017. 118: p. 1-43.
    28. Tonelli, F.M., et al., Graphene-based nanomaterials: biological and medical applications and toxicity. Nanomedicine, 2015. 10(15): p. 2423-2450.
    29. Akhavan, O. and E. Ghaderi, Toxicity of graphene and graphene oxide nanowalls against bacteria. ACS nano, 2010. 4(10): p. 5731-5736.
    30. Yadav, S.K., et al., Mechanically Robust, Electrically Conductive Biocomposite Films Using Antimicrobial Chitosan‐Functionalized Graphenes. Particle & Particle Systems Characterization, 2013. 30(8): p. 721-727.
    31. Narayanan, K.B., G.T. Park, and S.S. Han, Antibacterial properties of starch-reduced graphene oxide–polyiodide nanocomposite. Food Chemistry, 2021. 342: p. 128385.
    32. Nel, A., et al., Toxic potential of materials at the nanolevel. science, 2006. 311(5761): p. 622-627.
    33. Gonzalez, L., D. Lison, and M. Kirsch-Volders, Genotoxicity of engineered nanomaterials: A critical review. Nanotoxicology, 2008. 2(4): p. 252-273.
    34. Vranic, S., et al., Live imaging of label-free graphene oxide reveals critical factors causing oxidative-stress-mediated cellular responses. ACS nano, 2017. 12(2): p. 1373-1389.
    35. Srikanth, K., et al., Graphene oxide induces cytotoxicity and oxidative stress in bluegill sunfish cells. Journal of Applied Toxicology, 2018. 38(4): p. 504-513.
    36. Nanda, S.S., S.S.A. An, and D.K. Yi, Oxidative stress and antibacterial properties of a graphene oxide-cystamine nanohybrid. International journal of nanomedicine, 2015. 10: p. 549.
    37. Fenoglio, I., et al., Reactivity of carbon nanotubes: free radical generation or scavenging activity? Free Radical Biology and Medicine, 2006. 40(7): p. 1227-1233.
    38. Westerfield, M., The zebrafish book: a guide for the laboratory use of zebrafish. http://zfin. org/zf_info/zfbook/zfbk. html, 2000.
    39. Hsieh, C.-T., et al., Electrochemical capacitors based on graphene oxide sheets using different aqueous electrolytes. The Journal of Physical Chemistry C, 2011. 115(25): p. 12367-12374.
    40. Zhou, X., et al., In situ synthesis of metal nanoparticles on single-layer graphene oxide and reduced graphene oxide surfaces. The Journal of Physical Chemistry C, 2009. 113(25): p. 10842-10846.
    41. Hummers Jr, W.S. and R.E. Offeman, Preparation of graphitic oxide. Journal of the american chemical society, 1958. 80(6): p. 1339-1339.
    42. Yeh, T.-F., et al., Elucidating quantum confinement in graphene oxide dots based on excitation-wavelength-independent photoluminescence. The journal of physical chemistry letters, 2016. 7(11): p. 2087-2092.
    43. Kimmel, C.B., et al., Stages of embryonic development of the zebrafish. Developmental dynamics, 1995. 203(3): p. 253-310.
    44. Chen, Y., et al., Specific nanotoxicity of graphene oxide during zebrafish embryogenesis. Nanotoxicology, 2016. 10(1): p. 42-52.
    45. Ahmad, F., et al., Zebrafish embryos and larvae in behavioural assays. Behaviour, 2012. 149(10): p. 1241.
    46. Vilcacundo, R., et al., Digestibility of quinoa (Chenopodium quinoa Willd.) protein concentrate and its potential to inhibit lipid peroxidation in the Zebrafish larvae model. Plant Foods for Human Nutrition, 2017. 72(3): p. 294-300.
    47. Rissone, A. and F. Candotti, Detection of Reactive Oxygen Species Using MitoSOX and CellROX Green in Zebrafish. 2016.
    48. Lee, G.-H., et al., Zebrafish larvae exposed to ginkgotoxin exhibit seizure-like behavior that is relieved by pyridoxal-5′-phosphate, GABA and anti-epileptic drugs. Disease models & mechanisms, 2012. 5(6): p. 785-795.
    49. Thisse, C., et al., Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos. Development, 1993. 119(4): p. 1203-1215.
    50. Wilson, C., Aspects of larval rearing. ILAR journal, 2012. 53(2): p. 169-178.
    51. Lee, K.J., et al., In vivo quantitative study of sized-dependent transport and toxicity of single silver nanoparticles using zebrafish embryos. Chemical research in toxicology, 2012. 25(5): p. 1029-1046.
    52. Liu, X.T., et al., Toxicity of multi-walled carbon nanotubes, graphene oxide, and reduced graphene oxide to zebrafish embryos. Biomedical and Environmental Sciences, 2014. 27(9): p. 676-683.
    53. Chen, Z.-Y., et al., The effect of the chorion on size-dependent acute toxicity and underlying mechanisms of amine-modified silver nanoparticles in zebrafish embryos. International journal of molecular sciences, 2020. 21(8): p. 2864.
    54. Lee, K.J., et al., In vivo imaging of transport and biocompatibility of single silver nanoparticles in early development of zebrafish embryos. ACS nano, 2007. 1(2): p. 133-143.
    55. Kim, D.-H., et al. Mechanical property characterization of the zebrafish embryo chorion. in The 26th Annual International Conference of the IEEE Engineering in Medicine and Biology Society. 2004. IEEE.
    56. Mandrell, D., et al., Automated zebrafish chorion removal and single embryo placement: optimizing throughput of zebrafish developmental toxicity screens. Journal of laboratory automation, 2012. 17(1): p. 66-74.
    57. M. Martinez-Sales1, J.P., S. Sadeghi1, J. Núñez2, C. Soler2, M.A. Silvestre2, Effect of a pronase treatment at mid-blastula transition stage on length of zebrafish (Danio rerio) embryos at 24 hours, in 9th Iberian and 6th Iberoamerican congress on Environmental contamination and toxology-CICTA 2013. 2013.
    58. Wesstrom, S., D. Korest, D. Hunter, AND S. Padilla. , Dechorionation of Zebrafish Embryos on Day 1 Post Fertilization Alters Response to an Acute Chemical Challenge at 6 Days Post Fertilization, EPA, Editor. April 25 - 27, 2018.
    59. Zoroddu, M.A., et al., Toxicity of nanoparticles. Curr Med Chem, 2014. 21(33): p. 3837-3853.
    60. Fu, P.P., et al., Mechanisms of nanotoxicity: generation of reactive oxygen species. Journal of food and drug analysis, 2014. 22(1): p. 64-75.
    61. Souza, J.P., et al., Toxicological effects of graphene oxide on adult zebrafish (Danio rerio). Aquatic toxicology, 2017. 186: p. 11-18.
    62. Tarabová, B., et al., Fluorescence measurements of peroxynitrite/peroxynitrous acid in cold air plasma treated aqueous solutions. Physical Chemistry Chemical Physics, 2019. 21(17): p. 8883-8896.
    63. Domingues, I. and C. Gravato, Oxidative stress assessment in zebrafish larvae, in Teratogenicity Testing. 2018, Springer. p. 477-486.
    64. Weinberg, E.S., et al., Developmental regulation of zebrafish MyoD in wild-type, no tail and spadetail embryos. Development, 1996. 122(1): p. 271-280.
    65. Mani, R., et al., Chronic exposure to copper oxide nanoparticles causes muscle toxicity in adult zebrafish. Environmental Science and Pollution Research, 2019: p. 1-12.
    66. Chaudhuri, B., Biopolymers-graphene oxide nanoplatelets composites with enhanced conductivity and biocompatibility suitable for tissue engineering applications, in Fullerens, Graphenes and Nanotubes. 2018, Elsevier. p. 457-544.
    67. Childs, S., et al., Patterning of angiogenesis in the zebrafish embryo. 2002.
    68. de Almeida, F.L.A., et al., Differential expression of myogenic regulatory factor MyoD in pacu skeletal muscle (Piaractus mesopotamicus Holmberg 1887: Serrasalminae, Characidae, Teleostei) during juvenile and adult growth phases. Micron, 2008. 39(8): p. 1306-1311.
    69. Fenaroli, F., et al., Nanoparticles as drug delivery system against tuberculosis in zebrafish embryos: direct visualization and treatment. ACS nano, 2014. 8(7): p. 7014-7026.
    70. Wang, Z.G., et al., Toxicity of graphene quantum dots in zebrafish embryo. Biomedical and Environmental Sciences, 2015. 28(5): p. 341-351.

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