Mass spectrometry-based lipidomics to explore the naphthalene toxicity in a tolerant mouse model

萘是個微溶於水並且可於許多環境中接觸到的多環芳香烴，也是一種具有器官及物種專一性的毒化物質。呼吸道遠端支氣管上的非纖毛細胞(Clara cell)對萘毒性具有易感受性。萘會引起小鼠呼吸道的代謝體擾動，包括脂質過氧化、細胞膜的破壞、能量供給失衡等代謝機制。 本實驗室稍早的研究利用ICR品系的雄性小鼠進行萘耐受性動物實驗，分別分為三組:單一劑量組、重複劑量暴露組及對照組，單一劑量暴露組連續七天注射橄欖油，再於第八天注射300 mg/kg萘；重複劑量暴露組連續七天注射200 mg/kg萘，再於第八天注射300 mg/kg萘；對照組連續八天注射橄欖油。24小時後觀察各器官的形態及代謝體擾動，單一劑量組所發現的呼吸道細胞膜上的外觀型態損壞和能量代謝擾動有關，然而在重複劑量暴露組會誘發Glutathione抗氧化機制，使小鼠呼吸道對對萘毒性產生耐受性。結果也顯示不只在主要標的器官-肺臟會受到萘重複暴露的影響，肝臟及腎臟組織也都會觀察到代謝體擾動的現象。 甘油磷脂類是生物膜的主要組成成分。脂質具有許多的生物意義，如細胞膜組成、細胞間訊息傳遞、活化酶等。因此本研究主要是要探討萘引起細胞傷害分子機制，注重於細胞膜主要成分磷醘膽鹼類(phosphorylcholine-containing lipids)，包括脂質甘油磷酸膽鹼(phosphatidylcholine)和鞘磷脂(sphingomyelin)在耐受性小鼠受萘暴露後所造成的改變，以期更了解萘的制毒機制。 我們使用脂質體學方法探討萘傷害及萘耐受性老鼠的分子機制並將結果與組織病理學比對，透過超高效液相層析質譜儀(UPLC-MS/MS)和多變量分析，觀察肺臟、肝臟、腎臟的脂質體變化，探討不同萘暴露方式對小鼠的影響。由肺部非纖毛細胞組織病理學研究中顯示，在單一劑量組的小鼠肺臟組織會有空泡化跟腫脹的非纖毛細胞產生。而重複劑量暴露組與控制組間沒有顯著差異。偏最小平方判別分析(Partial Least Squares Discriminant Analysis, PLS-DA) 磷醘膽鹼類脂質結果發現單一劑量組與重複劑量暴露組有明顯分群趨勢。 本研究目標是要瞭解萘耐受性小鼠所引起的保護機制，藉此知道萘的制毒機制。本研究發現二醯基甘油磷酸膽鹼(Diacyl PCs)、含多元不飽和脂肪酸鏈的甘油磷酸膽鹼(PC with polyunsaturated fatty acyl (PUFA) chains)和醛磷脂醯膽鹼(plasmenylcholine)在重複劑量暴露組中有升高趨勢，可能與萘耐受性現象相關。Naphthalene, a slightly water soluble volatile aromatic hydrocarbon, is present in both groundwater and air emissions from a variety of sources. Naphthalene is a site- and species- selective cytotoxicant. The cell injury is found in mouse distal airways where the nonciliated or Clara cell is particularly susceptible to naphthalene toxicity. Naphthalene induced airway injury is related to lipid peroxidation, disruptions of membrane components, and imbalanced energy supply based on NMR-based metabolomics studies. Previously, our lab has investigated the mechanisms of naphthalene toxicity in various mouse tissues among injured, tolerant, and the control mice using 1H NMR-based metabolomics. Male ICR mice were administered seven repeated injections (ip) of naphthalene (0, 200 mg/kg/day) in olive oil and gave a challenge dose (300 mg/kg/day) at the eighth day. From the metabolomic results, the single exposure effects of naphthalene on the respiratory system are associated with cellular membrane damages and energy metabolism disturbance. However, the repeated exposure induced the antioxidation mechanism associated with glutathione in the airway; therefore, mice become tolerant to naphthalene toxicity. Furthermore, there is no airway injury in this model. Glycerophospholipids are key components of biological membranes. They also have a variety of biological functions, such as cellular messengers, enzyme activators and etc. In this study, we especially focus on the most abundant phosphorylcholine-containing lipids, including phosphatidylcholine (PC) and sphingomyelin (SM) and associate the changes of them with protective effects in a naphthalene tolerant mouse model. Moreover, critical toxic mechanisms of naphthalene will be revealed in this study. In this study, we intend to understand the mechanism of naphthalene-induced cell injury or tolerance by profiling changes of phosphorylcholine-containing lipids including PCs and SM by using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) following multivariate statistical analysis. Phosphorylcholine-containing lipid profiling from naphthalene tolerant mice and injury mice will be compared and related with naphthalene toxicity.. From the results of the histopathology, we can easily recognize the single naphthalene dose group had vacuolated and swollen Clara cell in the airways. The lung of the repeated naphthalene dose group appeared to be similar as that in the control which administered with the vehicle (olive oil). Partial least-square-discriminate analysis (PLS-DA) model shows differences between the phosphorylcholine-containing lipid profiling from the analysis of the lung, liver, and kidney from the mice received single dose or the repeated dose of naphthalene. Diacyl PCs, PC with polyunsaturated fatty acyl (PUFA) chains and plasmenylcholine are elevated in the repeated dose group than those in the single dose group, which may be associated with the tolerant phenomenon. Our goal attempts to reveal the mechanisms of tolerance in a naphthalene tolerant mouse model and further understand naphthalene toxicity.摘要 iii Abstract v List of figures iii List of tables iv 1. Introduction 1 1.1 Background 1 1.2 Naphthalene acute toxicity from single dose treatment in mouse 2 1.3 Tolerance to multiple doses of naphthalene in mouse 5 1.4 Metabolomics 6 1.5 Lipidomics 8 2. Specific aims 11 3. Materials and methods 12 3.1 Experiment flow chart 12 3.2 Animal treatments 13 3.3 Histopathology 13 3.4 Sample preparation for lipid analysis 14 3.5 Phosphorylcholine-containing lipids profiling by UPLC/MS/MS 15 3.6 Structural identification 16 3.7 Spectral processing 18 3.8 Multivariate data analysis 18 3.9 Statistical analysis 20 4. Results 21 4.1 Histopathology 21 4.2 LC-MS/MS profiling results among lung, liver, and kidney 21 4.3 Metabolic responses of naphthalene in the mouse lung 22 4.4 Metabolic responses of naphthalene in the mouse liver 25 4.5 Metabolic responses of naphthalene in the mouse kidney 26 5. Discussion 29 5.1 Protection against oxidative stress in the lung of tolerant mice 30 5.1.1 Lysophosphatidylcholine 31 5.1.2 Diacylphosphatidylcholine 33 5.1.3 Phosphatidylcholine with polyunsaturated fatty acyl (PUFA) chains 35 5.1.4 Surfactant phosphatidylcholine 37 5.1.5 Plasmanylcholine (O- PC) and Plasmenylcholine (P- PC) 38 5.2 Less PCs involved in the liver of tolerant mice 40 5.3 Kidney 41 6. Conclusion 44 References 45 Figures 54 Tables 6