自工業革命至今，空氣汙染一直是人類關注的健康議題。過去的流行病學研究及動物實驗發現微粒空氣汙染和對呼吸系統及心血管系統的不良健康效應相關，如肺間質細胞發炎、氣喘、慢性阻塞性肺病 (COPD)、動脈硬化、缺血性心臟病、心律不整、心臟衰竭等。前人研究多鎖定特定細胞激素與組織，預期微粒空氣汙染暴露會造成這些細胞激素的上升以及組織的損傷。以核磁共振儀為基礎的代謝體學由另一個方向切入，以驗證已知生化指標、釐清可能的毒理機制、及尋找新的指標為目標，建立及比較生物體體內內生性代謝物的資訊。我們設計了一個亞慢性大氣微粒全身性呼吸暴露動物實驗，將32隻Sprague-Dawley大鼠隨機分為微粒空氣暴露組及乾淨空氣暴露組(控制組) 進行一天24小時，一週七天，為期十二週的微粒空氣汙染暴露。為校正個體差異，本研究將暴露前兩天設為基準點(baseline)，並於暴露後五天、七天、四週及十二週針對同一批老鼠(N=8/group/time-point)以連續採樣的方式採集血液及尿液進行以核磁共振儀為基礎的代謝體學研究，並於暴露後七天、四週及十二週犧牲(N=3/group/time-point)，進行血液學及肺泡灌洗液發炎反應分析。本研究採用主成份分析法(PCA)、部分最小平方判別分析法(PLS-DA)以及學生T檢定(Student’s t-test)進行代謝物辨認、資料分群檢測及半定量統計分析，以利後續針對PM可能的毒理機制的建構。
實驗過程中，PM2.5於暴露滿五天、七天、四週及十二週的平均質量濃度分別為20.86μg/m3、20.87μg/m3、19.30μg/m3、及13.73μg/m3。暴露後七天、四週及十二週實驗動物的血液學檢查、細胞計數及肺泡灌洗液發炎指標於兩組間均未呈現明顯差異。血清代謝體的PCA分析發現，四週及十二週的微粒空氣暴露組及控制組呈現分群之趨勢，在代謝物半定量分析的部分，若以暴露組和控制組相較，暴露七天微粒空氣在兩組中代謝物含量無顯著差異；五天時微粒組的血清在citrate, choline及creatine的量呈上升趨勢，四週時creatine、acetate與胺基酸valine、methionine、tyrosine呈上升趨勢，飽和、不飽和脂肪鏈、低密度脂蛋白及極低密
度脂蛋白成下降趨勢；而暴露十二週時choline、phosphocholine、胺基酸valine、alanine、glutamine、methionine、tyrosine、能量代謝相關的acetoacetate、pyruvate、citrate、creatine、glucose成下降趨勢，飽和脂肪鏈、formate、低密度脂蛋白及極低密度脂蛋白則呈上升趨勢。以上PCA及半定量分析結果顯示亞慢性PM2.5暴露和生物體內代謝物表現之擾動有相關，而這些代謝物的擾動表現於能量代謝、脂質代謝及細胞膜恆定等層面。
本研究藉由核磁共振譜的代謝體學，分析健康SD大鼠暴露微粒空氣汙染後體內代謝物組成的改變，藉以評估微粒空氣汙染對生物體造成的危害及可能機制，以幫助未來生物標記之發展。

Both epidemiological and animal studies had indicated clear association between adverse health effects of the pulmonary and cardiovascular system with particulate matter (PM) air pollution. These studies approached the outcomes specifically, suggesting exposure of PM lead to elevation of cytokines and tissue lesions. The use of NMR-based metabolomics provided a global profiling of the endogenous metabolites in our body, aiming at the verification of known biomarkers, clarification of possible toxicological mechanisms, and finding of novel markers.
A 12-week whole body ambient PM inhalation exposure design was conducted with 16 male SD rats randomly divided into the ambient air exposure group and the filtered-air group for a 24 hr/day, 7 days/week exposure. Serial sampling of serum was held for adjustment of individual variation at time points of -2 days(baseline), 7 days, 4 weeks, and 12 weeks of exposure for NMR-based metabolomic analysis. Another 16 male SD rats in the same age were also randomly divided into the two groups and were sacrificed for hematological analysis, cell differential and inflammatory biomarker analysis of bronchial alveolar lavage fluid (BALF) at time points of 5 days, 7 days, 4 weeks, and 12 weeks of exposure (N=3/group/time-points). Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and Student’s t-test were employed to identifying critical metabolites for the clarification of possible
mechanisms of toxicological effects of PM in the body.
The estimated average mass concentration of PM2.5 at 7 days, 4 weeks, and 12 weeks were 20.86μg/m3, 20.87μg/m3, 19.30μg/m3, 13.73μg/m3, respectively. PCA scores plot from the analysis of NMR spectra of serum showed a trend separation and clustering according to exposure and control groups at both 4 and 12 weeks of exposure. Compared to the control group, decrease levels of the -CH2 of LDL and VLDL and increase level of creatine, citrate and choline were found at 5 days of exposure; saturated and unsaturated fatty acid chains, and increase levels of valine, acetate, methionine, tyrosine and creatine were found at 4 weeks of exposure, while valine, alanine, acetoacetate, pyruvate, glutamine, citrate, methionine, choline, phosphocholine, creatine, glucose and tyrosine were found decreased and increase level of –CH2 of LDL and VLDL, saturated fatty acid chain and formate were found at 12 week of exposure.
Our findings suggested that sub-chronic inhalation of PM at ambient level might have an association with the perturbation of TCA cycle, energy metabolism, amino acid and lipid metabolism, and disruption of energy production and the increase of ROS production.

口試委員會審定書…………………………………………………….…..I
誌謝……………………………………………………………………. …II
中文摘要……………………………………………………………….....III
Abstract ……………………………………………………………………V
Contents …………………………………………………………………VII
List of Tables..…………………………………………………………….IX
List of Figures……………………………………………………………...X

Chapter 1 Introduction
1.1 Research Background………………………………………………1
1.2 Objectives…………………………………………………………..4

Chapter 2 Literature review
2.1 Particulate matter and Respiratory Diseases……………………….5
2.2 Particulate Matter and Cardiovascular Diseases…………………...6
2.3 Metabolomics………………………………………………………8
2.3.1 NMR spectroscopy as a tool of reaching Metabolomics……..10
2.3.2 Metabolomics and particulate matter-related diseases………. 11
2.4 Exposure Routes…………………………………………………..12

Chapter 3 Materials and Methods
3.1 Animals and Animal Care…………………………………………15
3.2 Exposures to Ambient Particulate Matter…………………………15
3.2.1 Study Design…………………………………………………15
3.2.2 The TAPES…………………………………………………...17
3.2.3 Exposure Monitoring…………………………………………17
3.3 Metabolomic Parameters………………………………………….18
3.3.1 Sample Collection and Preparation…………………………..18
3.3.2 1H NMR Spectral Acquisition………………………………..19
3.3.3 Spectral Processing and Spectral Data Analysis…………….. 19
3.3.4 Metabolite Identification……………………………………..21
3.4 Measurement of biomarkers………………………………………21
3.4.1 Collection and preparation of Samples……………………… 21
3.4.2 Measurement of Inflammatory Factors and Blood Analysis…22
3.5 Statistical Analysis……………………………………………….. 22

Chapter 4 Results
4.1 Exposure Data……………………………………………………. 23
4.2 Body weight change of experimental animals…………………….23
4.3 Hematological analysis……………………………………………24
4.4 Inflammatory Factors……………………………………………..24
4.5 1H NMR Spectroscopy of Rat Serum……………………………..24
4.5.1 Metabolic Response to Acute PM Inhalation Exposure……...26
4.5.2 Metabolic Response to Chronic PM Inhalation Exposure…...26

Chapter 5 Discussion…………………………………………………..28
Chapter 6 Conclusion………………………………………………….35
Reference…………………………………………………………………36

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