粉塵暴露引起肺部細胞損傷之揮發性代謝組學研究

Volatile metabolomics study on lung cell injury caused by dust exposure

彭馨頤(Hsin-Yi Peng)

指導教授 ： 楊孝友

國立臺灣大學/公共衛生學院/職業醫學與工業衛生研究所/碩士(2019年)

https://doi.org/10.6342/NTU201900702

摘要

石英粉塵可以增加氧化壓力，脂質過氧化，並導致肺部纖維化之損傷。在之前我們的研究中對塵肺症勞工吐氣中的揮發性有機化合物進行分析，用於檢測塵肺症勞工的吐氣代謝變化。但塵肺症的內源性代謝揮發性生物標誌物仍然須被鑑定。本研究的目的是在確認石英的細胞毒性作用後，分析損傷肺泡上皮細胞頂空中的代謝物來探討可能的生物標誌物。我們在石英處理的人肺泡上皮細胞中進行了體外細胞培養實驗。本研究中使用的石英首先通過能量色散光譜掃描式電子顯微鏡確認其物理和化學性質。將A549人肺泡上皮細胞暴露於50, 100, 200, 500和1000 μg/mL的石英24, 48, 72小時，並通過LDH測量細胞膜損傷，8-isoprostane測定氧化壓力，以及透過IL-6測定發炎反應。當存在劑量反應關係時，我們確定了細胞毒性的存在並使用氣相層析質譜儀分析細胞頂空中的揮發性有機化合物。實驗結果顯示，檢測100顆石英的平均直徑為2.3μm。當暴露劑量大於或等於200 μg/mL以及暴露時間大於24小時，毒性作用顯著增加。基於細胞毒性研究的結果，我們分析了當細胞暴露200, 500和1000 μg/mL的石英24小時後的頂空氣體。損傷細胞產生辛烷、3,3-二甲基辛烷、2-甲基2-丙醇和2,3-二甲基庚烷四種有劑量反應關係的揮發性有機化合物，可能是塵肺症的潛在內源性的生物偵測指標。未來仍有必要進行動物實驗或流行病學前瞻性研究，以驗證生物標誌物可以在臨床應用上將塵肺症患者與健康人區分開來。

關鍵字

石英 ； 揮發性有機化合物 ； 氧化壓力 ； 體外細胞研究 ； 氣相層析質譜儀

並列摘要

Quartz dust can increase oxidative stress, lipid peroxidation, and cause pulmonary fibrosis. Analysis of volatile organic compounds (VOCs) in breath had been used to detect the metabolomics changes in pneumoconiosis patients. Potential metabolic VOCs biomarkers in pneumoconiosis are waiting to be identified. The objective of this study was to analyse endogenous VOCs in the headspace of injury cells after confirmed the cytotoxic effects of quartz. We conducted an in vitro study in quartz-treated human alveolar epithelial cells. Quartz used in this study was first examined by scanning electron microscopy with energy dispersive spectroscopy to confirm the physical and chemical properties. We exposed human alveolar A549 cell lines to 50, 100, 200, 500 and 1000 μg/mL of quartz for 24, 48, 72 hours, and measured the cell membrane damage by LDH assay, generation of reactive oxidative stress by 8-isoprostane assay, and inflammatory reaction by the interleukin-6 assay. We determined the existence of cytotoxicity when a dose-response relationship existed. When the cytotoxic effects were confirmed, we then analyzed the VOCs in the headspace of cells using gas chromatography-mass spectrometry (GC-MS). In our results, the mean diameter of quartz was 2.3 μm. The toxicity effects were significantly increased when the exposure dose was greater or equal to 200, 500 and 1000 μg/ mL and 24 hours. Based on the results of the cytotoxicity study, we analyzed the VOCs when cells were exposed to quartz at the concentration of 200 μg/mL for 24 hours. The injury cells generated octane, octane, 3,3-dimethyl-, 2-propanol, 2-methyl- and heptane, 2,3-dimethyl- of VOCs that might be potential volatile metabolites of pneumoconiosis. An in vivo study and prospective independent epidemiology study is warranted to validate the biomarkers could distinguish pneumoconiosis patients from healthy before clinical application.

並列關鍵字

quartz ； volatile organic compounds (VOCs) ； oxidative stress ； in vitro study ； gas chromatography-mass spectrometry (GC-MS)

參考文獻

1. Wade, W.A., et al., Severe Occupational Pneumoconiosis Among West Virginian Coal Miners One Hundred Thirty-eight Cases of Progressive Massive Fibrosis Compensated Between 2000 and 2009. Chest, 2011. 139(6): p. 1458-1462.

2. Cohen, R.A., Resurgent coal mine dust lung disease: wave of the future or a relic of the past? Occup Environ Med, 2016. 73(11): p. 715-716.

3. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans: Silica, Some Silicates, Coal Dust and Para-Aramid Fibrils. Lyon, 15-22 October 1996. IARC monographs on the evaluation of carcinogenic risks to humans, 1997. 68: p. 1-475.

4. Greenberg, M.I., J. Waksman, and J. Curtis, Silicosis: a review. Dis Mon, 2007. 53(8): p. 394-416.

5. Cox, C.W., C.S. Rose, and D.A. Lynch, State of the art: Imaging of occupational lung disease. Radiology, 2014. 270(3): p. 681-96.