奈米氧化鋅微粒呼吸暴露誘發實驗動物肺部毒性之量度單位研究

Abstract

奈米材料已被廣泛的運用在許多領域中，其中奈米氧化鋅在催化、光學、磁學、力學等方面展現出許多特殊功能，使其在陶瓷、化工、電子、光學、生物、醫藥等許多領域有重要的應用價值。然而奈米氧化鋅相關的毒理研究並不多，需要進一步的研究探討。 另一方面，許多研究指出奈米微粒健康效應與傳統使用的質量濃度相關較小，而與顆粒濃度或表面積濃度相關較大，但是目前研究多以氣管灌注模式暴露實驗動物。氣管灌注方式暴露動物，無法掌握奈米微粒粒徑及濃度，所以我們開發微粒產生及暴露系統，利用全身性呼吸暴露的方式，將不同粒徑以及濃度之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。除此之外我們也探討毒性效應與量度單位(dose metric)之關係，並且進一步利用基準劑量模式(bench mark dose)推估氧化鋅之基準計量。 我們以微粒產生系統產生粒徑約為35 nm及250 nm的氧化鋅微粒並以全身性暴露腔進行大鼠暴露，產生微粒的同時以掃瞄式電移動微粒分析儀自動監測儀（SMPS+C）進行微粒粒徑及濃度的監測。分別進行35nm粒徑:低、中、高濃度以及250nm:低、中、高濃度的暴露，每組暴露6隻大鼠，並以在相同暴露條件下，微粒通過高效能過濾器(HEPA)之空氣為對照組(4隻/組)，共計60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），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）。 實驗結果顯示微粒產生器可穩定產生粒徑約為35 nm 以及250 nm的氧化鋅微粒，35nm組低、中、高濃度所產生的奈米氧化鋅微粒，數目濃度分別約為1.5×106、2.1×106、7.9×106 #/cm3；250nm組低、中、高濃度之數目濃度分別約為6.2×104、1.5×105、4.5×105 #/cm3。 動物實驗結果發現，不論是在35 nm或250 nm粒徑下，大鼠暴露於低、中、高濃度的氧化鋅微粒後，在肺泡灌洗液中的嗜中性球百分比（Neutrophils）及總細胞數相較於對照組有顯著增加，並且呈現劑量反應關係（p＜0.05）。同時，暴露於高濃度組別之乳酸脫氫酵素也顯著高於控制組（p＜0.05）。另外，在周邊血液方面，暴露於兩個粒徑之高濃度組的白血球數目（WBC）相較於對照組有顯著增加（p＜0.05）。 毒性效應與量度單位的結果顯示，相較於重量濃度與顆粒濃度，表面積濃度與肺部毒性效應之劑量曲線在兩種粒徑最為類似。進一步利用基準劑量模式推估得到表面積濃度之基準劑量下限為1.1×104 mm2/m3(以35nm、100nm、250nm氧化鋅換算之重量濃度分別為1.14、3.24、8.11mg/m3)。本研究發現，健康大鼠在暴露低於目前氧化鋅燻煙之容許濃度標準(PEL, 5 mg/m3)之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以及系統性發炎反應，而目前法規是否能妥善維護勞工之安全，仍有待進一步研究。

Nano-sized zinc oxide (ZnO) was widely applied in industrial field. However, there are literatures indicating that nano-size particles may induce inflammation, thrombosis and cardiovascular diseases, or even penetrate into systemic circulation. Previous studies have shown that total surface area of nanoparticles (NPs) is the best dose metric for particle toxicity. But these studies were conducted with intratracheal instillation, in which the surface area may not be correctly estimated because of particle agglomeration. In this study, I used inhalation exposure to compare relationship of different metrics with lung inflammation induced by zinc oxide particles. I further calculated the benchmark dose to determine the reference dose of zinc oxide. ZnO NPs were produced in a furnace system and SMPS was used to monitor particle size and number. Healthy SD rats were exposed to 35nm NPs at 1.5×106, 2.1×106, 7.9×106 particles/cm3 and filtered air for 6 hrs. Similarly, rats were exposed to 250nm ZnO at 6.2×104, 1.5×105, 4.5×105 particles/cm3 and filtered air. The total number of cells and proportion of neutrophils in bronchoalveolar lavage (BAL) were determined. The best fit model for lung inflammation with number, mass and surface area concentrations were compared. We also used the Benchmark dose software to calculate the reference dose for ZnO NPs on this effect. The results showed that in both 35-nm and 250-nm ZnO exposed group, percentage of neutrophils, total cells and total protein in BAL fluid increased with the ZnO particles level (p<0.05, test for trend). Further, LDH and WBC in high-dose group increased significantly compared to the controls (P<0.05). As we investigated the relationship between inflammatory markers and the particle parameters, we could find that surface area concentration had the best correlation with the inflammation markers when compared with the mass and number concentration. Significant dose-response curves were also observed in BALF total cells and percentage of neutrophils. These results indicate that total surface area of particles may play an important role in ultrafine particles related toxicity. According to our results, we choose the surface area concentration as dose to present the toxicity. The BMDL of surface area-based dose of ZnO NPs was estimated at 1.1×104 mm2/m3, which is equal to a mass-based dose of 1.14 mg/m3 for 35 nm, 3.24 mg/m3 for 100 nm, and 8.11 mg/m3 for 250 nm ZnO particles. Our results suggest that current US OSHA standard for ZnO fume (PEL, 5 mg/m3) may not be sufficient to protect workers exposed to ZnO NPs against lung inflammation.