應用核磁共振與質譜技術共同探討急性呼吸暴露奈米與微米粒徑之氧化鋅顆粒對大鼠呼吸系統之影響

Abstract

急性呼吸暴露氧化鋅粒子雖已被證實會引發人類與實驗嚙齒類動物的諸多不良健康效應，但氧化鋅粒子造成的呼吸系統損傷的分子機制尚未能一虧全貌。有鑑於此，本研究結合核磁共振技術為基礎的代謝體學和質譜技術為基礎的脂質體學來量測大鼠呼吸暴露氧化鋅粒子後所誘發之呼吸系統的代謝擾動。 氧化鋅呼吸暴露的動物模式的建立以不同粒徑：奈米（35 nm）或微米（250 nm）、不同粒子濃度的氧化鋅粒子暴露於SD品系的雄性大鼠，並以呼吸過濾後空氣的同品系大鼠作為控制組。暴露實驗後二十四小時犧牲動物並收集肺沖洗液和肺部組織再分別以氫核磁共振光譜或質譜儀進行代謝體或脂質體的分析。肺沖洗液和肺部水溶性代謝物的核磁共振光譜中主成分分析和偏最小平方法的結果顯示暴露微粒粒徑的氧化鋅粒子的組別其代謝物擾動的程度有劑量反應關係，然暴露不同濃度奈米粒徑氧化鋅粒子組別的代謝物組成差異則較不明顯。微米粒徑暴露組別中特定代謝物量的改變：如肺沖洗液中牛磺酸（taurine）、甲酸鹽（formate）的下降以及肺組織中葡萄糖的下降或含磷酸膽鹼官能基脂質（phosphorylcholine-containing lipids）的增加等可能與抗氧化、能量代謝、DNA損傷以及細胞膜結構之穩定有關。 另一方面，透過統合一系列多變量與單變量統計方法不但可以探索因氧化鋅粒子暴露所引發之脂質擾動外，也可篩選出潛在的氧化鋅暴露的生物指標。肺部的質譜光譜中主成分分析和偏最小平方法的結果顯示微米粒徑各組與奈米粒徑高劑量暴露組的脂質組成與控制組、奈米粒徑的中低濃度的暴露組不甚相同。不同脂質的差異可能藉由抗氧化、細胞膜結構的改變以及訊息傳遞等機制來應對氧化鋅暴露所引發的氧化壓力與發炎反應。此外，此研究也篩選出兩個能反映出氧化鋅粒子暴露的脂質：PC(18:0/18:1)和PC(16:0/15:0)。綜合上述結果可知利用代謝體學和脂質體學的研究策略不但可以探索氧化鋅粒子所引發的分子毒理機制，也可以篩選出潛在的氧化鋅粒子暴露的生物指標。此外，此平台也可應用於研究其他肺部毒性反應或健康效應。

Zinc oxide (ZnO) particles induce acute occupational inhalation illness in humans and rats. However, the possible molecular mechanisms of ZnO particles on the respiratory system remain unclear. In this study, metabolic responses of the respiratory system of rats inhaled ZnO particles were investigated by a nuclear magnetic resonance (NMR)-based metabolomic approach coupling with a liquid chromatography-mass spectrometry (LC-MS)-based lipidomic approach. Male Sprague–Dawley rats were treated with a series of doses of nano-sized (35 nm) or fine-sized (250 nm) ZnO particles. The corresponding control groups inhaled filtered air. After 24h, bronchoalveolar lavage fluid (BALF) and lung tissues were collected, extracted and prepared for 1H and J-resolved NMR analysis as well as LC-MS analysis, followed by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). PCA and PLSDA models from analysis of BALF and hydrophilic lung NMR spectra demonstrated that dose response trends were restricted to the 250 nm ZnO particle exposure group and were not observed in the 35 nm ZnO particle exposure group. Increased isoleucine and valine, as well as decreased acetate, trimethylamine n-oxide, taurine, glycine, formate, ascorbate and glycerophosphocholine, were recorded in the BALF of rats treated with moderate and high dose 250 nm ZnO exposures. Decreases in taurine and glucose, as well as an increase of phosphorylcholine-containing lipids and fatty acyl chains, were detected in the lung tissues from 250 nm ZnO-treated rats. These metabolic changes may be associated with cell anti-oxidation, energy metabolism, DNA damage and membrane stability. On the other hand, PCA, PLS-DA, and the Mann-Whitney U test with false discovery rate control were conducted to explore the perturbed lipid species and discriminate a potential pulmonary biomarker signature after ZnO particle exposure. The PCA and PLS-DA models revealed that the fine-sized ZnO particle-treated groups and the high-dose nano-sized group were separated from the control groups as well as the low and moderate nano-sized groups. Furthermore, our results suggested that lipids involved in anti-oxidation, membrane conformation, and cellular signal transduction were altered in response to ZnO-induced oxidative stress and inflammation. Two lipids, PC(18:0/18:1) and PC(16:0/15:0), exhibited good performance (AUC> 0.8) of discriminative ability in distinguishing ZnO particle exposure from the control. These findings not only provide a foundation for the exploration of possible ZnO particle-mediated mechanisms but also suggest a lipid biomarker for ZnO particle exposure. In addition, using this integrated metabolomic and lipidomic approaches can be applied to study other pulmonary toxicity or diseases.