應用質譜分析小鼠肺部灌洗液的脂質體研究萘引發的呼吸系統毒性

Mass Spectrometry-Based Lipidomics to Characterize Naphthalene-Induced Respiratory Toxicity on Bronchoalveolar Lavage Fluid of Mice

林尚廷(Shang-Ting Lin)

指導教授 ： 林靖愉

國立臺灣大學/公共衛生學院/環境衛生研究所/碩士(2018年)

https://doi.org/10.6342/NTU201803516

摘要

萘廣泛存在於環境中，被美國環保署訂為人類可能性致癌物。先前的研究發現萘的急性暴露會導致小鼠呼吸系統的毒性，對克氏細胞造成囊泡化和腫脹等情形，同時改變細胞膜的通透性，並具有劑量效應性。肺部灌洗液（Bronchoalveolar lavage fluid）中除含有免疫細胞外，還有表面活性劑（surfactant）相關的脂質、磷脂質和蛋白質。因此肺部灌洗液中的分子組成可以提供有價值的資訊，反應肺部的的生化改變。然而因為技術上的考量，肺部灌洗液的脂質研究數量仍然相當稀少。因此本研究希望透過脂質體學的方法探討肺部灌洗液中脂質體的改變來了解萘引發的肺部毒性。
本研究使用七週大的雄性ICR小鼠，並分為三組，控制組是將小鼠利用腹腔內注射施予橄欖油，低劑量與高劑量組則分別使用腹腔內注射施予使用含有萘劑量100或200 mg/kg body weight的橄欖油，暴露後24小時後麻醉犧牲，並抽取肺部灌洗液。經脂質萃取等前處理後，利用極致高效液相層析串聯式質譜儀分析含有磷酸膽鹼的脂質，包含磷脂醯膽鹼（phosphatidylcholine）和神經磷脂（sphingomyelin）。得到的圖譜數據經過前處理之後，再利用偏最小平方判別等方法進行多變量統計分析和單變量統計分析來評估萘所引發的脂質擾動。
偏最小平方判別法中的分數圖顯示控制組、低劑量、高劑量三組的脂質體散布在不同的區域，並且有明顯的分離。PC(16:0/16:0)和PC(16:0/14:0)兩種脂質在高劑量組有下降的趨勢，推測和表面活性劑（surfactant）的功能有關，多種磷脂醯膽鹼(diacyl-phosphatidylcholines, plasmanylcholines 和 plasmenylcholines)在高劑量組有上升趨勢，則推測和抗氧化作用等保護作用有關。因此，利用質譜儀為基礎的脂質體學研究，可以發現到小鼠暴露到萘之後，肺部灌洗液中的脂質變化，幫助我們了解呼吸系統萘的毒性機制，並可以進一步應用在生物指標的開發上。

關鍵字

脂質體學 ； 萘 ； 質譜儀 ； 肺部灌洗液 ； 毒性 ； 磷脂醯膽鹼 ； 神經磷脂

並列摘要

Naphthalene, as a suspected carcinogen listed by the US Environmental Protection Agency, widely exists in the environment. Previous studies indicated that naphthalene caused acute respiratory toxicity in mice. Acute exposure to naphthalene induced swelling and vacuolation of Clara cells and altered membrane integrity with dose-response effects. Lipidomic analysis of respiratory system may help us realize naphthalene-induced toxicity.
Bronchoalveolar lavage fluid (BALF) consists of inflammatory cells and acellular components such as surfactant-like lipids, phospholipids and proteins. The molecular composition of the fluid provides valuable information of biochemical alterations in the lungs. However, limited numbers of studies examined the lipid constituent of BALF due to technical limitation. In the present study, we aim to study the respiratory toxicity of naphthalene by measuring lipid components in BALF.
7-week male ICR mice were randomly divided into 3 groups (n=6), treated with olive oil (control) or naphthalene (100, 200 mg/kg body weight in olive oil) by intraperitoneal injection. After 24 hr., mice were anesthetized and BALF were collected for further analysis. Lipidomics, a platform able to examine considerable lipids at the same time, can provide integrated information of biological changes induced by environmental stimuli. In this study, phosphorylcholine-containing lipids, such as phosphatidylcholine and sphingomyelin, were profiled by ultra-performance liquid chromatography tandem mass spectrometer (UPLC-MS/MS). The MS spectra were further processed and analyzed by partial least squares discriminant analysis (PLS-DA).
The PLS-DA score plot showed that lipidome of control, low-dose, and high-dose groups were separated clearly. Moreover, the responses of the low-dose and high-dose groups are different; possibly indicated that different mechanisms were involved. Decreasing level of PC(16:0/16:0) and PC(16:0/14:0) after mice treated with high-dose naphthalene may be related with function of surfactant. Besides, increasing level of diacyl-phosphatidylcholines, plasmanylcholines, and plasmenylcholines after mice treated with high-dose naphthalene may be associated with anti-oxidation and protective mechanisms.
We concluded that MS-based lipidomics is a powerful tool to exhibit lipid perturbation in BALF, helping us to understand the toxic mode of actions of naphthalene in the respiratory system and develop biomarkers.

並列關鍵字

Lipidomics ； Phosphorylcholine-containing lipids ； Mass spectrometry ； Naphthalene ； Bronchoalveolar lavage fluid ； Toxicity

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