利用體外試驗研究探討短期臭氧暴露之揮發性有機物質變化

An in vitro Study of short-term Ozone Exposure Induced Volatile Organic Compounds

10.6288/TJPH2011-30-01-09

胡佩儀(Pei-Yei Hu)；馬一中(Yee-Chung Ma)

臭氧暴露 ； 人類肺部細胞 ； 揮發性有機物 ； 氣相層析質譜儀 ； Ozone exposure ； MRC-5 ； VOCs ； GC/MS

台灣公共衛生雜誌

30卷1期（2011 / 02 / 15）

82 - 94

繁體中文

目標：探討人類肺部細胞MRC-5在不同濃度、時間暴露之臭氧衝擊式系統下，藉由產生揮發性有機物質(Volatile organic compounds, VOCs)之種類及含量變化，間接從細胞脂質過氧化了解氧化壓力程度。為確認VOCs為臭氧與細胞膜上不飽和脂肪酸PUFA (Polyunsaturated fatty acids)所反應物質，再進行臭氧暴露至PUFA (Arachidonic Acid, AA; Linoleic Acid, LA)。方法：在細胞部分，暴露時間為30分鐘，臭氧濃度範圍在0.1至2ppm，暴露後觀察時間為30，60，120分鐘。10ppm PUFA暴露至1ppm臭氧30分鐘後，以採樣管進行收集，再以氣相層析質譜儀分析。結果：比較臭氧與空氣暴露之收集氣體並分析後，有18種物質在兩組間達統計上顯著差異。其中以methyl cyclopentane物質為最，不論是在高或低濃度臭氧暴露皆達顯著差異。3-methyl-butanal在暴露30分鐘於高濃度臭氧暴露下有顯著；carbon disulfide及dimethyl disulfide兩者暴露臭氧1.5ppm 30分鐘後亦有顯著。此外，暴露空氣與臭氧至PUFA （AA及LA），結果顯示暴露至AA有顯著差異為2-methyl-1-propene，而LA則為pentane。結論：利用體外試驗之臭氧暴露系統所得到有顯著差異之VOCs物質，可做為臭氧暴露後氧化壓力之生物標誌。

Objectives: The aim of this study was to evaluate the impinger in vitro exposure system used to investigate ozone exposure induced oxidative stress. Comparisons of Volatile Organic Compounds (VOCs) levels in collected gas and MRC-5 cell membrane integrity were made after ozone exposure. In order to validate the source of the VOCs, the reactions of ozone with two polyunsaturated fatty acids (PUFA); Arachidonic Acid (AA) and Linoleic Acid (LA) were also determined. Methods: MRC-5 cells were treated with air and ozone concentrations ranging from 0.1 to 2 ppm for 30 min, and VOCs were collected during and after ozone exposure at 30, 60, 90, and 120 min intervals. PUFAs 10 ppm were exposed to 1 ppm ozone for 30 min. Sampling tubes were used to collect the reacting gas after ozone exposure on human lung cells or PUFAs and these were then analyzed by GC/MS. Results: When the VOCs from air and ozone exposure in MRC-5 cells were compared at different time points, 18 compounds were found to be significantly different. The methyl cyclopentane level increased acutely after oxidative stress to lung tissue. 3-methyl butanal was significantly associated with a high ozone level; carbon disulfide and dimethyl disulfide increased immediately following ozone exposure at 1.5 ppm for 30 min. 2-butanal and methyl cyclopentane were significant different after exposure to ozone 2 ppm as measured by the Tukey HSD test. 2-methyl-1-propene and pentane were also significantly different in the reactive gases of AA and LA after ozone exposure. Conclusions: VOCs may be significant markers of oxidative stress after the human lung cell is exposed to ozone in the impinger exposure system.

1. Aufderheide, M.(2008).An efficient approach to study the toxicological effects of complex mixtures.Exp Toxicol Pathol,60,163-80.
2. Bosson, J.,Pourazar, J.,Forsberg, B.,Adelroth, E.,Sandström, T.,Blomberg, A.(2007).Ozone enhances the airway inflammation initiated by diesel exhaust.Respir Med,101,1140-6.
3. Buszewski, B.,Kesy, M.,Ligor, T.,Amann, A.(2007).Human exhaled air analytics: biomarkers of diseases.Biomed Chromatogr,21,553-66.
4. Buszewski, B.,Ulanowska, A.,Ligor, T.,Denderz, N.,Amann, A.(2009).Analysis of exhaled breath from smokers, passive smokers and non-smokers by solid-phase microextraction gas chromatography/mass spectrometry.Biomed Chromatogr,23,551-6.
5. Chen, X.,Xu, F.,Wang, Y.(2007).A study of the volatile organic compounds exhaled by lung cancer cells in vitro for breath diagnosis.Cancer,110,835-44.
6. Corradi, M.,Alinovi, R.,Goldoni, M.(2002).Biomarkers of oxidative stress after controlled human exposure to ozone.Toxicol Lett,134,219-25.
7. de Zwart, L.L.,Meerman, J.H.,Commandeur, J.N.,Vermeulen, N.P.(1999).Biomarkers of free radical damage applications in experimental animals and in humans.Free Radic Biol Med,26,202-6.
8. Foucaud, L.,Bennasroune, A.,Klestadt, D.,Laval-Gilly, P.,Falla, J.(2006).Oxidative stress induction by short time exposure to ozone on THP-1 cells.Toxicolo In Vitro,20,101-8.
9. Frampton, M.W.,Pryor, W.A.,Cueto, R.,Cox, C.,Morrow, P.E.,Utell, M.J.(1999).Ozone exposure increases aldehydes in epithelial lining fluid in human lung.Am J Respir Crit Care Med,159,1134-7.
10. Janic, B.,Umstead, T.M.,Phelps, D.S.,Floros, J.(2003).An in vitro cell model system for the study of the effects of ozone and other gaseous agents on phagocytic cells.J Immunol Methods,272,125-34.
11. Lestari, F.,Markovic, B.,Green, A.R.,Chattopadhyay, G.,Hayes, A.J.(2006).Comparative assessment of three in vitro exposure methods for combustion toxicity.J Appl Toxicol,26,99-114.
12. Meadows, J.,Smith, R.C.,Reeves, J.(1986).Uric acid protects membranes and linolenic acid from ozone-induced oxidation.Biochem Biophys Res Commun,137,536-41.
13. Miekisch, W.,Schubert, J.K.,Noeldge-Schomburg, G.F.(2004).Diagnostic potential of breath analysis-focus on volatile organic compounds.Clin Chim Acta,347,25-39.
14. Mohler, E.R. 3rd,,Reaven, P.,Stegner, J.E.,Fineberg, N.S.,Hathaway, D.R.(1996).Gas chromatographic method using photoionization detection for the determination of breath pentane.J Chromatogr B Biomed Appl,685,201-9.
15. Phillips, M.(2003).United States 6540691United States 6540691,未出版
16. Phillips, M.(2001).United States Patent 6254547United States Patent 6254547,未出版
17. Phillips, M.,Cataneo, R.N.,Greenberg, J.,Munawar, M.,Nachnani, S.,Samtani, S.(2005).Pilot study of a breath test for volatile organic compounds associated with oral malodor: evidence for the role of oxidative stress.Oral Dis,11,32-4.
18. Pleil, J.D.,Hubbard, H.F.,Sobus, J.R.,Sawyer, K.,Madden, M.C.(2008).Volatile polar metabolites in exhaled breath condensate (EBC): collection and analysis.J Breath Res,2,026001.
19. Pyo, J.S.,Ju, H.K.,Park, J.H.,Kwon, S.W.(2008).Determination of volatile biomarkers for apoptosis and necrosis by solid-phase microextraction-gas chromatography/mass spectrometry: a pharmacometabolomic approach to cisplatin's cytotoxicity to human lung cancer cell lines.J Chromatogr B Analyt Technol Biomed Life Sci,876,170-4.
20. 陳孟筠(2006)。台北=Taipei，國立台灣大學環境衛生研究所=Institute of Environmental Health, National Taiwan University。