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研究生: 林银月
Que, Ma. Katreena Danielle E.
論文名稱: 探討COOH-SWCNT奈米碳管對秀麗線蟲動物實驗之影響
In vivo testing of COOH-SWCNT on Caenorhabditis elegans
指導教授: 侯文哲
Hou, Wen-Che
學位類別: 碩士
Master
系所名稱: 工學院 - 環境工程學系
Department of Environmental Engineering
論文出版年: 2018
畢業學年度: 106
語文別: 英文
論文頁數: 106
中文關鍵詞: 動物實驗奈米碳管奈米毒性秀麗線蟲單壁奈米碳管(COOH-SWCNT)
外文關鍵詞: In vivo, carbon nanotubes, nanotoxicity, C. elegans, COOH-SWCNT
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  • 秀麗線蟲 (Caenorhabditis elegans ,C. elegans)為評估奈米毒性效應之理想動物實驗系統,該系統具有易於維護且價格便宜等優點。奈米材料工程(Engineered nanomaterials, ENMs)應用端非常廣泛,導致在過去幾年中產量大增,其在環境中潛在的毒性效應與對健康的不利影響逐漸受到重視。碳基之ENMs如單壁奈米碳管(single-walled carbon nanotubes, SWCNT)在含水介質中不易分散,為了將其有效應用於水環境中,將該材料加入可與水相溶之官能基(如:羧基(-COOH)),增加其溶解度是相當必要的。然而,這項改動也可能間接導致該材料的毒性改變。在本次研究中,將使用C. elegans評估carboxylated single-walled carbon nanotubes (COOH-SWCNT) ENMs (濃度為0.001-1000 μg/ L)對其之各種毒性效應。本研究結果顯示,儘管在急毒性測試方面並沒有發現C. elegans有致死之現象,但暴露COOH-SWCNT濃度高於1 μg/L之C. elegans在第8天觀察到壽命有顯著降低之現象。在神經與生殖毒性評估之結果顯示,當COOH-SWCNT暴露濃度高於1 μg/L時,其C. elegans運動反應及卵的大小數量均顯著的下降。此外,在COOH-SWCNT暴露濃度高於0.01 μg/L時,對C. elegans生長同樣發現相似之結果。在活性氧(reactive oxygen species, ROS)之研究結果也觀察到在第8天出現下降之趨勢,但並沒有發現顯著之影響。這可能歸因於前人的研究發現腺蟲體內檸檬酸循環(負責能量與ROS產生) 會抑制SWCNTs與線蟲暴露COOH-SWCNT 24小時後轉移至新鮮培養基之恢復能力所影響。Colocalization 實驗結果發現 COOH-SWCNT確實在線蟲體內具有生物累積效應,當暴露高濃度之COOH-SWCNT (10,000 μg/L)三小時後,沿著線蟲的頭部與中部區域COOH-SWCNT誘導了ROS產生,並可以穿透線蟲之腸道組織。總體而言,當COOH-SWCNT濃度高於1 μg/L時,即可導致C. elegans次級標靶器官退化與活性降低相關等生長延遲現象,進而導致壽命降低而非具有立即的毒性效應影響。

    The Caenorhabditis elegans (C. elegans) in vivo model is an ideal system for nanotoxicity assessment due to its many attractive characteristics such as easy maintenance, cheap cost, etc. Engineered nanomaterials (ENMs) have many useful applications and their production has increased rapidly over the past few years. Their potential existence in the environment has raised an important concern regarding their toxicity and adverse health effects. Carbon-based ENMs such as single-walled carbon nanotubes (SWCNTs) have poor dispersion in aqueous media that impedes water-based applications. Therefore their functionalization with water-compatible functionalities such as carboxyl groups (-COOH) is necessary for solubility improvement. However, functionalization with different functional groups lead to altered toxicities. In this study, the nanotoxicity of carboxylated single-walled carbon nanotubes (COOH-SWCNT) ENMs (0.001-1000 μg/L) was evaluated using the C. elegans system for different endpoints such as survival (lethality and lifespan), behavioral or locomotion (head thrash and body bend), development (growth), reproduction (brood size), and biochemical (reactive oxygen species (ROS) and colocalization). We found that while no lethality occurred for COOH-SWCNT treatment of C. elegans, a significant reduction in their lifespan was observed for concentrations >1 μg/L COOH-SWCNT at day 8 after 24-hour acute exposure. For consideration of neurological and reproductive toxicity (the secondary targeted organs), the tests of locomotion and brood size number were significantly decreased at concentrations >1 μg/L. Furthermore, a similar result was found on the growth of C. elegans which was retarded significantly at concentrations >0.01 μg/L. Oxidative stress did not seem to cause the significant toxic effects since the intracellular ROS showed a decreasing trend at day 8. This may be attributed to the ability of C. elegans to recover and reduce ROS toxicity. Colocalization experiments showed that the COOH-SWCNT was bioaccumulated in the worms, inducing ROS production mainly along the head and middle regions at 3-hrs treatment with high concentrations (10,000 μg/L). In addition, intestinal penetration of the COOH-SWCNTs was also observed. Overall, concentrations >1 μg/L can cause the toxic effects to C. elegans as observed from the deterioration of the secondary targeted organs and the retardation of their growth which correlated with the decreasing their lifespan while not necessarily causing immediate lethality.

    Contents Abstract iii ACKNOWLEDGEMENT v Contents vii List of Figures x List of Tables xii Symbols xiii 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 3 2.1 Nanoparticles (NPs) and Engineered Nanomaterials (ENMs) 3 2.2 Nanoparticles and Nanomaterials Classifications 4 2.3 Carbon-based nanomaterials 5 2.3.1 Single-walled carbon nanotubes (SWCNTs) and their properties 12 2.3.2 Carboxylic Acid Functionalized SWCNTs (COOH-SWCNTs) 16 2.4 CNT Nanotoxicity Factors 19 2.4.1 Bioaccumulation of COOH-SWCNT 22 2.4.2 In vitro-based nanotoxicity testing and studies on functionalized SWCNTs 22 2.4.3 In vivo-based nanotoxicity testing and studies on functionalized SWCNTs 24 2.5 C. elegans as an in vivo model 28 2.5.1 Brief background on C. elegans 28 2.5.2 Environmental Applications of C. elegans 32 2.5.3 Environmental toxicology of Engineered Nanomaterials (ENMs) such as carbon nanotubes and others on C. elegans 33 3 METHODOLOGY 35 3.1 Characterization of COOH-SWCNTs 35 3.2 C. elegans Maintenance and Culturing 35 3.2.1 Preparation of Nematode Growth Media (NGM) Plates 35 3.2.2 Seeding of OP50 E.coli Bacteria on NGM Plates 35 3.2.3 Transferring of on Fresh NGM Plates 36 3.2.4 Age Synchronization by Bleaching Method 36 3.2.5 Seeding Age-Synchronized Worms to NGM Plates for Experiment 37 3.3 Nanotoxicity Assays using C. elegans 42 3.3.1 COOH-SWCNTs Exposure Concentration Preparation 42 3.3.2 Acute Exposure Lethality Assay 42 3.3.3 Reproductive or Brood Size Assay 43 3.3.4 Growth Measurement Assay 43 3.3.5 Locomotion Assay (Head Thrash and Body Bend Assay) 43 3.3.6 Lifespan Assay (Ageing Assay) 44 3.3.7 Reactive Oxygen Species (ROS) Assay 44 3.3.8 Distribution, Translocation and Colocalization of COOH-SWCNTs in C. elegans 45 4 RESULTS AND DISCUSSION 47 4.1 Characterization of COOH-SWCNTs 47 4.2 Evaluation of the lethality effects of COOH-SWCNT on C. elegans 54 4.3 Locomotion Assay and Brood Size Assay to determine toxicity effects of COOH-SWCNT on secondary targeted organs 56 4.4 Decreased growth of due to treatment with COOH-SWCNTs 60 4.5 The effects of COOH-SWCNT on the lifespan and ageing of C. elegans 64 4.6 ROS production induced by COOH-SWCNTs 69 4.7 Translocation, and Colocalization of COOH-SWCNT and ROS. 73 5 CONCLUSION 80 6 SUGGESTIONS 81 REFERENCES 82

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