脂質體學探討萘對小鼠不同器官脂質影響

Lipidomics to study lipid effects of naphthalene on various organs of mice

陳泳宏(Yung-Hung Chen)

指導教授 ： 林靖愉

國立臺灣大學/公共衛生學院/環境與職業健康科學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202003394

摘要

萘為常見的環境汙染物，也是最簡單的多環芳香烴，主要是由不完全燃燒所產生。萘的毒性具有器官以及物種專一性，小鼠暴露萘後，肺上皮細胞的非纖毛細胞(克氏細胞)對於萘的攻擊最為敏感，從細胞型態變化上能觀察與劑量相關，過去的動物實驗發現在暴露100 mg/kg的萘24小時後，可以觀察到細胞的腫脹以及空泡化，而在200 mg/kg 的萘暴露下，觀察到細胞的脫落甚至壞死，細胞膜上的變化表示萘對於細胞膜的重要成分—脂質有重大的影響。脂質為細胞膜的重要成分，同時也是能量代謝來源以及協助細胞分子間訊號的傳遞。過去研究觀察到小鼠在萘暴露後對於肺臟以及血液內含磷酸膽鹼的脂質擾動。本研究利用質譜儀為基礎的脂質體學探討萘對於小鼠目標器官(肺)以及非目標器官(肝、腎)中脂質體的影響，嘗試分析更多種脂質種類以了解萘的毒理機制。
本研究中，七週大的雄性ICR小鼠隨機分派成控制組、低暴露組 (100 mg/kg) 以及高暴露組 (200 mg/kg)，暴露24小時後，針對肺臟、肝臟以及腎臟三種器官萃取脂質並進行極致高效能液相層析搭配四極柱串聯飛行時間質譜儀分析，接著圖譜處理及統計分析。在偏最小平方判別法結果中顯示三組之間有明顯的分群，再以Kruskal-Wallis test 以及 Dunnett’s test 的事後檢定，找出顯著改變的重要脂質。最後，在肺部有209個顯著改變脂質，其中甘油磷脂占最大部分，變化最大出現在磷脂絲氨酸。除了甘油磷脂，本研究也觀察到醯基肉鹼、神經醯胺、心磷脂等多種脂質受到萘擾動，其擾動程度以及變化方向，可能與萘造成的代謝擾動、細胞損傷以及發炎反應有關。低劑量組與高劑量組存在著劑量關係，兩組之脂質變化趨勢相同且高劑量組之變化較大。然而，在暴露萘之後，在小鼠的肝與腎臟內沒有顯著的脂質改變。
此研究透過更全面性的脂質體學分析平台研究萘對於小鼠器官的脂質效應，能夠更完整的了解萘可能的毒理機制，並且提供公共衛生上具潛力的生物標記的開發以及更多元的肺部傷害研究方向。

關鍵字

萘 ； 肺 ； 脂質體學 ； 質譜儀 ； 毒理學 ； 生物標記

並列摘要

Naphthalene is a common air pollutant in the environment, and the simplest polycyclic aromatic hydrocarbon. Previous studies showed that naphthalene induced respiratory toxicity in a mouse model. Naphthalene led to the non-ciliated, bronchiolar epithelial cells which called Clara cells injury in the airways of mice. After 24-hour exposure of 100 mg/kg naphthalene, Clara cells became swelling and vacuolation; while under 200 mg/kg naphthalene exposure, Clara cells became exfoliating or even necrosis. The morphology study indicated that naphthalene changed the cell morphology and likely altered the composition of cell membrane in the mouse lung. Lipids are important component of cell membrane. Lipids are also sources of metabolic energy and able to assist in transmission of cellular signals. In previous studies, we observed that naphthalene disrupted phosphocholine-containing lipids in the lung and serum of mice. In this study, we applied mass spectrometry (MS) based lipidomic platform to study naphthalene-induced lipid effects in target organ (lungs) and non-target organs (liver and kidneys) in a mouse model. By using high-through-put and high-resolution analysis platform, we intend to obtain changes of more lipid classes to explain naphthalene-induced toxicity more completely.
In this study, 7-week-old male ICR mice were treated with naphthalene (100, 200 mg/kg, ip) or only olive oil (control). After 24h, mouse lungs, liver, and kidneys were collected, and further lipids extracted for ultra-high-performance liquid chromatography quadrupole-time of flight tandem MS analysis, followed by spectral pretreatment and statistical analysis. The results of partial least squares discriminant analysis showed obvious separation among three groups. Then, the Kruskal-Wallis test and Dunnett’s post hoc test were applied to identify the important lipids with significant changes. Finally, 209 lipids with significant changes were identified in the lungs. Among them, glycerophospholipids were the most abundant changed lipids, and the most dramatically changed lipid was phosphatidylserines. In addition to glycerophospholipids, we also observed that numerous lipids, such as acylcarnitines, ceramides, and cardiolipins, etc, were disturbed by naphthalene. The different degree and direction of the disturbance in lipids may be correlated to metabolic disorder, cell membrane damage, and inflammation caused by naphthalene. Dose responses were also observed. The trends of lipid changes in two groups were similar, but the changes in the high dose group were greater. However, no significant changes of lipids were found in the liver and kidneys.
This study applied a more comprehensive lipidomic analysis platform to study the lipid effects of naphthalene on mouse organs. It can provide more complete information of the molecular events of naphthalene on the mouse lungs, and promote the development of potential biomarkers and diverse directions for future studies in the field of respiratory toxicology.

並列關鍵字

naphthalene ； lungs ； lipidomics ； mass spectrometry ； toxicology ； biomarkers

參考文獻

Adkins, B., Jr., Van Stee, E. W., Simmons, J. E., Eustis, S. L. 1986. 'Oncogenic response of strain A/J mice to inhaled chemicals'. Journal of Toxicology and Environmental Health, 17(2-3): 311-322.

Andrieu-Abadie, N., Gouaze, V., Salvayre, R., Levade, T. 2001. 'Ceramide in apoptosis signaling: Relationship with oxidative stress'. Free Radical Biology and Medicine, 31(6): 717-728.

Agency for Toxic Substances and Disease Registry (ATSDR). (1995). Toxicological Profile for Naphthalene, 1-Methylnaphthalene and 2-Methylnaphthalene. Atlanta, GA, Department of Health and Human Services.

Barbosa, B. S., Martins, L. G., Costa, T., Cruz, G., Tasic, L. 2018. 'Qualitative and Quantitative NMR Approaches in Blood Serum Lipidomics'. Methods in Molecular Biology, 1735: 365-379.

Bartke, N., Hannun, Y. A. 2009. 'Bioactive sphingolipids: metabolism and function'. Journal of Lipid Research, 50: S91-S96.