應用核磁共振的代謝體學探討萘對小鼠呼吸系統的致毒反應

Abstract

肺部疾病已達其跨界域之流行性，並對人類造成重大的影響。Clara cells為易感受的氣管支氣管表皮細胞，當許多環境的異質物進入此表皮細胞後被細胞內的酵素CYP450代謝，過程中生成的活性中間產物造成細胞的傷害。Naphthalene為多環芳香烴中最廣存於周遭空氣中的一員，並已從動物研究中證實其可能的致癌性。 代謝體學與組織病理學方法被應用於探究易感受的小鼠受到腹腔注射的方式暴露於Naphthalene之毒性反應。代謝體學方法聯結了一維與二維核磁共振氫譜實驗與多變量統計分析，可用來解釋小鼠的肺泡灌洗液及肺臟組織代謝物變化情形，並探究Naphthalene毒性的劑量與時間效應對小鼠造成代謝物的變化。小鼠的肺臟組織經由組織病理的固定與包埋後，於光學與電子顯微鏡下檢視其形態上的改變。 無監督的主成分分析結果顯示，肺泡灌洗液及肺臟組織代謝物變化情形與Naphthalene毒性之劑量與時間效應呈現顯著相關。在高劑量暴露下，肺泡灌洗液中乙烷和丙酮的含量顯著增加，顯示Naphthalene引起的脂質過氧化傷害與細胞膜完整性缺失有關。高劑量的實驗結果發現磷脂醯膽鹼類顯著減少，使其無法繼續做為細胞膜磷脂類的前驅物以及維持滲透壓調節的角色，最終造成表皮細胞腫脹與空泡化。從肺臟組織的結果發現，鞘磷脂的量在200 mg/kg Naphthalene暴露後顯著上升，顯示與自我保護機制下產生的細胞凋零有關。從肺部的脂相分析中可知丙酮含量在暴露12小時候達顯著增加，顯示脂質過氧化傷害的發生早於12小時。由肺泡灌洗液結果顯示，暴露4、12與24小時的丙酮含量無太大的變化，推斷其在組織的脂肪層中大量累積。 此研究設計，可用於探討疾病模式及開發其相對應的生物指標，非侵入性的檢驗方法更可以應用於人體上，由於流行病學的資料與傳統的組織病理實驗結果無法具體提供生化分子層面之變異，因此藉由代謝體學的實驗來找出其對應的生化反應機制，一旦機制被解開後，對於此疾病的預防、治療或是新藥的開發都是一突破性的里程碑。

Pulmonary diseases have reached their transboundary properties and pose significant impact to human. Clara cell, a susceptible tracheobronchial epithelial cell type, is injured via CYP450-dependent metabolic activation when reactive intermediates of various environmental xenobiotics are produced. Naphthalene is a major polycyclic aromatic hydrocarbon in ambient polluted air, and has been demonstrated possible carcinogenic from animal investigations. Metabolomics and histopathology approaches are applied to examine naphthalene toxicity in a susceptible species, mouse, using ip administration. Dose response and time course experiments were carried out in male ICR mice whose bronchoalveolar lavage fluid (BALF) and lung were then taken for metabolic analysis. BALF and the lung metabolomes were analyzed by using 1D 1H and 2D JRES NMR following principal component analysis (PCA). Mouse lung was embedded and examined by light and electron microscopes on morphologic changes. Unsupervised PCA results of BALF and lung metabolomes noted a clear trend of naphthalene dose and time effects. Elevated ethane and acetone from BALF suggested a correlation between naphthalene-induced lipid peroxidation and loss of membrane integrity. Decreased glycerophosphocholine, result of higher dose effects, failed to serve as a pro-phospholipid source and an osmoregulator for cell membranes which eventually results in swollen and vacuolated Clara cells. Sphingomyeline in lung were found increased in 200 mg/kg dose and may be related to programmed cell death as a self-protection strategy. The amount of acetone in lung increased at 12 h post-dose has indicated the early initiation of lipid peroxidation before 12 h. It is postulated to accumulate in lung lipophilic layer in view of no huge difference from BALF investigation. This study design can be used in investigating pathogenesis of diseases and related biomarker development. Non-invasive screening method can be further applied in human. Due to inadequate molecular information provided by epidemiology and histopathology, metabolomics hereby excavates correlated biochemical mechanisms. The underlying mechanisms will be very informative to disease prevention, therapy, or new drug design. Such breakthrough is as a milestone.