1. 奈米氧化鋅微粒毒性研究 2. 奈米碳球毒性研究

Abstract

Part Ι: 奈米氧化鋅用途廣泛，可以被應用在塗料、油漆、橡膠輪胎工業、化妝品等產品上，但相關的毒理研究並不多。因此我們利用全身性呼吸暴露的方式，將奈米氧化鋅微粒暴露於健康大鼠，觀察其肺部及系統性的毒性效應。 本實驗利用呼吸暴露的方式，於實驗動物進行奈米氧化鋅微粒的暴露，我們以奈米微粒產生系統產生粒徑約為30 nm的奈米氧化鋅微粒，產生微粒的同時以微粒粒徑濃度分佈自動監測儀（SMPS+CPC）進行微粒粒徑及濃度的監測，分別進行了低暴露組（5 hrs）以及高暴露組（10 hrs）的暴露，並以在相同暴露條件下，未填入鋅粉於爐管內空燒的實驗（暴露5hrs）為對照組，每組暴露6隻大鼠，共計18隻。暴露後24小時進行犧牲，並採集周邊血液樣本，測試血液中血球數目（CBC/DC）及細胞激素的濃度（IL-6、TNF-α），同時採集其肺泡灌洗液（BALF）進行肺部發炎及傷害指標的分析，包括總細胞數、血球分類計數、乳酸脫氫酵素（LDH）及總蛋白質（Total protein）。 實驗結果顯示奈米產生器可穩定產生奈米粒徑的氧化鋅微粒，低暴露組（5 hrs）所產生的奈米氧化鋅微粒，中數粒徑（count median diameter, CMD）為34.0 nm，數目濃度約為5.3 × 105 #/cm3；高暴露組（10 hrs）的中數粒徑為24.7 nm，數目濃度約為4.1 × 106 #/cm3。 動物實驗結果發現大鼠暴露於低暴露組及高暴露組的奈米氧化鋅微粒後，在肺泡灌洗液中的嗜中性球百分比（Neutrophils）及乳酸脫氫酵素（LDH）相較於對照組有顯著增加，而且呈現劑量反應關係（p＜0.05）。另外，暴露於高暴露組的奈米氧化鋅微粒後，還發現周邊血液中的白血球數目（WBC）、血小板數目（PLT）以及細胞激素（IL-6）相較於對照組有顯著增加（p＜0.05）。 本研究發現，奈米氧化鋅微粒會誘使健康大鼠發生急性肺部發炎與傷害反應，同時有系統性發炎產生，顯示奈米氧化鋅微粒可能具有急性毒性效應，值得進一步關注，不過真正機制需要未來研究加以探討。 Part Ⅱ: 奈米碳球（carbon nanocapsules, CNC）為新興材料，目前國內已成功開發可大量生產並純化奈米碳球的方法，但是其毒性並不清楚。一旦經由呼吸將微粒吸入可能會造成健康危害。所以我們將大鼠暴露於奈米碳球，觀察其肺部及系統性毒性。 本實驗利用氣管灌注的方式，對實驗動物進行奈米碳球的暴露，包括三種奈米碳球（水溶性：CNC-(OH)n、CNC-(COOH)n及非水溶性：純CNC），兩種劑量（1與5 mg/kg），並以生理食鹽水（PBS）為對照組，每一個組別各需要6隻大鼠。實驗動物於微粒灌注後24小時進行犧牲，並採集周邊血液樣本，測試血液中血球數目（CBC/DC）及氧化壓力（oxidative stress），同時採集其肺泡灌洗液（BALF）進行肺部發炎及傷害指標的分析，包括總細胞數、血球分類計數、乳酸脫氫酵素（lactate dehydrogenase, LDH）及總蛋白質（Total protein）。 實驗發現大鼠暴露於高劑量的水溶性奈米碳球微粒（CNC-(OH)n及CNC-(COOH)n）後，在肺泡灌洗液中的總細胞及嗜中性球數目相較於控制組有顯著增加（p＜0.05），但是總蛋白質及乳酸脫氫酵素（LDH），及周邊血液血球及分類計數和氧化壓力皆沒有明顯變化。至於純CNC暴露後，各項指標皆沒有顯著差異。 研究顯示經過表面處理的奈米碳球（水溶性），在較高劑量時可能造成肺部發炎反應，真正機轉有待進ㄧ步評估。

Part Ι: Zinc oxides (ZnO) have been used widely, including manufacturing of rubber, paint and cosmetics. However, its toxicity remains unclear. We conducted an animal study to determine its pulmonary and systemic toxicity. Healthy Sprague Dawley (SD) rats were exposed by inhalation to 30 nm of zinc oxides nanoparticles (ZnO) for 5hr (low-exposure group) and 10hr (high-exposure group). Scanning Mobility Particle Sizer (SMPS+CPC) were used to monitor the size and concentration of particles. Each experimental group has six animals. The same number of rats was exposed to filtered-air as controls. Total cells, differential counts, total protein and lactate dehydrogenase activity (LDH) were determined in bronchoalveolar lavage fluid. Complete blood counts and differential counts (CBC/DC) and cytokines levels (IL-6 and TNF-α) in peripheral blood were also determined. Our results showed that the nanoparticles generator can stably produce about 30 nm zinc oxides nanoparticles. The count median diameter (CMD) of zinc oxides nanoparticles for low-exposure group was 34.0 nm, and the number concentration was 5.3 × 105 #/cm3. The CMD for high-exposure group was 24.7 nm, and the number concentration was 4.1 × 106 #/cm3. In both low-exposure group and high-exposure group, the percentage of neutrophils and LDH increased significantly as compared to the controls (P<0.05). There was also a dose-response relationship. Further, WBC, platelet and IL-6 in peripheral blood were also increased in high-exposure group (P<0.05). Our preliminary study has demonstrated that ZnO nanoparticles may cause lung and systemic toxicity. However, factors that determine the toxicity of nanoparticles need further investigation. PartⅡ: Carbon nanocapsules (CNC) have been developed and have a great potential to be applied in many fields. However, its toxicity remains unclear. We conducted animal study to determine its pulmonary and systemic toxicity. Healthy Sprague Dawley (SD) rats were intratracheally administered with the carbon nanocapsules including pure CNC (hydrophobic) and CNC-(OH)n and CNC-(COOH)n (hydrophilic) obtained from Industrial Technology Research Institute at doses of 1 mg/kg and 5 mg/kg suspended in phosphate-buffered saline (PBS). The animal numbers of each experimental group were six. The same number of rats was exposed to PBS as controls. Total cells and differential counts and total protein and lactate dehydrogenase activity (LDH) were determined in bronchoalveolar lavage fluid. Complete blood counts and differential counts (CBC/DC), and oxidative stress (the contents of H2O2 and O2- free radicals determined by Multi Luminescence Spectrometer) in peripheral blood were also determined. Our results showed that after exposure to the high dose (5 mg/kg) of CNC-(OH)n and CNC-(COOH)n (hydrophilic), the counts of total cells and neutrophils increased significantly as compared to the controls (P＜0.05). But the levels of total protein, LDH, CBC/DC and oxidative stress (the contents of H2O2 and O2- free radicals) were not significantly different. In the case of exposing to pure CNC (hydrophobic), the above markers were not different. Our study indicates that the high dose (5 mg/kg) of the carbon nanocapsules with surface modification may induce pulmonary inflammation. However, real mechanism needs to be further assessed.