奈米二氧化鈦對斑馬魚及溼地蕨類的影響

The effects of titanium dioxide nanoparticles (TiO2-NPs) on zebrafish (Danio rerio) and wetland fern.

江仲民(Chung-Min Chung)

指導教授 ： 周映孜 ； 陳德豪

國立屏東科技大學/農學院/生物科技研究所/碩士(2015年)

https://doi.org/10.6346/NPUST.2015.00207

摘要

在近幾十年來隨著奈米工業的快速發展，導致空氣中的奈米金屬微粒含量逐漸增加，這些奈米金屬微粒對於環境中的生物而言可能是一種潛在的風險。奈米二氧化鈦被廣泛地運用在各種消費型產品當中，例如化妝品、保養品以及建築塗料。根據統計每一年約會製造出5000公噸的奈米二氧化鈦用於相關商品的製造。當奈米二氧化鈦吸收紫外線後具有光毒性會產生活性氧化物(ROS)。鱗蓋鳳尾蕨為在亞熱帶及台灣常見的一種蕨類植物，已知鱗蓋鳳尾蕨可以有效的吸收土壤中的重金屬砷，具有相當大的潛力可以用於環境上做為植物修復系統。在本篇實驗以斑馬魚及鱗蓋鳳尾蕨做為材料，探討奈米二氧化鈦對環境中生物的影響。
將斑馬魚曝露奈米二氧化鈦(10 mg/L)以一般日光燈管的光源及水族用太陽燈管模擬陽光照射，進行為期六天的短期急毒性測試，結果發現曝露了奈米二氧化鈦在兩種光源處理下都會影響到斑馬魚的行為，而經由雙因子分析(Two way ANOVA)分析後證明了太陽燈管和二氧化鈦會有交互作用產生，藉由AmpliteTM Colorimetric Acetylcholinesterase Assay kit排除了觀察到的行為異常並不是因為乙醯膽鹼酯(Acetylcholinesterase，AChE)濃度改變造成的神經毒性，模擬陽光照射後並沒有引起相關抗氧化酵素超氧化物岐化酶(SOD)及過氧化氫酶(Catalase)的濃度改變及造成脂質過氧化(MDA)的產生，但是在第六天的實驗經由吖啶橙(Acridine Orange，AO)進行染色，藉由螢光密度測量法測試細胞凋亡，經染色觀察後發現，曝露奈米二氧化鈦後一般日光管燈照射組減少了細胞凋亡產生而太陽燈管照射組則顯著提高了細胞凋亡程度。
將鱗蓋鳳尾蕨的原葉體曝露在不同濃度的奈米二氧化鈦(10、100、1000 mg/L)六天，每兩天進行一次觀察，在三次的觀察結果皆發現奈米二氧化鈦導致葉綠體型態改變、黃化及細胞酸化的現象，但是以Trypan blue測試後並沒有發現細胞的存活率受到影響，我們也以薄層色層分析法(TLC)測試奈米二氧化鈦對植物光合色素影響進行分析，發現在1000 mg/L時會導致葉綠素a含量降低。為了詳細了解奈米二氧化鈦對鱗蓋鳳尾蕨的影響，我們將曝露6天的鱗蓋鳳尾蕨以蛋白質體學比較蛋白質變化，將差異性蛋白質以液相串聯式質譜儀(LC-MS/MS)分析後在資料庫進行比對，發現在1000 mg/L會抑制砷還原酶(arsenate reductase)的表現，10、100 mg/L時會誘導假定的砷還原酶(putative arsenate reductase)，而在曝露了奈米二氧化鈦之後則會導致釋氧增強蛋白(Oxygen-evolving enhancer protein 1)，ATP轉運結合蛋白及ATP合成酶二型(ATP-binding cassette transporter and ATP synthase beta chain)在100 mg/L時被誘導，類似肌動蛋白的葉綠體移動相關蛋白(Kinesin-like protein for actin-based chloroplast movement 1)則只有在對照組中被發現。此外我們將鱗蓋鳳尾蕨的原葉體以固態培養的方式進行為期五個禮拜的生長實驗，在葉綠體型態、細胞存活率以及光合色素含量並沒有看到影響，只有觀察到細胞酸化的現象，經統計軟體分析後發現曝露在濃度10及1000 mg/L時會顯著抑制其生長，然而在濃度100 mg/L時卻看到了相反的結果，在蛋白質表現的部分，曝露奈米二氧化鈦後發現Protochlorophyllide 的氧化還原酶(protochlorophyllide oxidoreductase A, PORA)的表現受到了抑制，而核酮糖-1,5-二磷酸羧化酶/氧化酶大次單元(ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, partial, RUBL)的表現則被誘導。
此外我們對以鱗蓋鳳尾蕨作為植物修復工具，清除水體環境中的奈米二氧化鈦進行探討，結果發現其可以有效的清除奈米二氧化鈦以及改善奈米二氧化鈦對斑馬魚所造成的行為異常。

關鍵字

奈米二氧化鈦 ； 植物修復 ； 鱗蓋鳳尾蕨 ； 葉綠素a ； 光毒性

並列摘要

Over the decades, nanotechnology has become a rapidly developing industry, therefore nanoparticles concentration increased in the air, causing potential risk to organisms. TiO2-NPs have been widespread used in consumer products, such as cosmetics, sunscreens and building paints. According to statistics, manufacturing products containing TiO2-NPs stand at 5000 t/a for the current production. The phototoxicity of titanium dioxide nanoparticles (TiO2-NPs) can produce reactive oxygen species (ROS) when TiO2-NPs absorb ultraviolet (UV) radiation. Pteris vittata L can absorb arsenic in the soil, making it potential for phytoremediation. The study focuses on the effect of exposure to TiO2 nanoparticles in zebrafish and Pteris vittata L.
The zebrafish exposed to TiO2-NPs (concentration at 10 mg/L), under ambient laboratory light and simulated solar radiation (SSR) through a short-term of acute toxicity test for 6 days, shows that SSR have effect on the behavior of zebrafish. Using the analysis of two way ANOVA confirm that TiO2-NPs and SSR have interaction. However, using the AmpliteTM Colorimetric Acetylcholinesterase Assay kit has exclude factors of neurological injury, and also that behavioral effect was not based on the concentration change of Acetylcholinesterase (AChE). The TiO2 did not affect related antioxidant enzymes activities and production of lipid peroxidation (MDA). For 6 days, using a fluorescence method to analyze the relative levels of apoptosis based on densitometric techniques by AO (Acridine Orange) stain, found that exposed to TiO2-NPs will reduce apoptosis, in contrast exposed to TiO2-NPs with SSR which significantly increased the levels of apoptosis.
Prothallus of Pteris vittata L was exposed to TiO2-NPs (concentration at 10, 100, and 1000 mg/L) for toxicity test for six days; observation was done once every two days. It was found that TiO2-NPs induced prothallium chlorosis, chloroplast morphological changes and cellular acidification on Pteris vittata L following observed after three times, but did not affect the cell viability. We also analyzed the TiO2 effects on photosynthesis pigment of Pteris vittata L by Thin Layer Chromatography (TLC), it was found that exposed to TiO2-NPs will reduce chlorophyll a content on 1000 mg/L concentration. To learn more about TiO2-NPs effect on Pteris vittata L, comparative proteomic to protein variable were exposed to TiO2-NPs for six days. The proteins were identified by LC-MS/MS. TiO2-NPs 1000 mg/L concentration was found to cause arsenate reductase expression to diminished, whileTiO2-NPs 10、100 mg/L concentration induced putative arsenate reductase expression. Exposed TiO2-NPs induced Oxygen-evolving enhancer protein 1 protein expression, which ATP-binding cassette transporter and ATP synthase beta chain found at TiO2-NPs 100 mg/L. Kinesin-like protein for actin-based chloroplast movement 1 was found in control. We also culture the prothallus of Pteris vittata L on a solid medium for growth toxicity test for five weeks. Using the analysis of statistics software , it was found that TiO2-NPs can significantly inhibit Pteris vittata L growth, when exposed to TiO2-NPs at concentration 10、1000 mg /L. In contrast, Pteris vittata L exposed to TiO2-NPs at concentration of 100 mg/L cause growth. It was found that TiO2-NPs induced cellular acidification on Pteris vittata L, but did not affect the cell viability、chloroplast morphological、photosynthesis pigment. We used comparative proteomic to protein variable expose to TiO2-NPs for five weeks. Found that TiO2-NPs exposure can cause protochlorophyllide oxidoreductase A (PORA) expression diminished, however, on the ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit, partial (RUBL) expression was induced.
Our study also demonstrated phototoxicity of TiO2-NPs effect on zebrafish behavior by TiO2-NPs with SSR interaction. In addition, we investigate the suitability of Pteris vittata L for the phytoremediation of a wide range of aquatic environment with TiO2. We showed that Pteris vittata L could clean TiO2 of aquatic environment as a tool of phytoremediation, and improve to behavioral effects of TiO2 on zebrafish.

並列關鍵字

Titanium dioxide nanoparticles (TiO2-NPs) ； phytoremediation ； Pteris vittata L ； chlorophyll a ； phototoxicity

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