利用體外試驗研究探討短期臭氧暴露之揮發性有機氣體變化

Abstract

本研究之目的為探討人類肺部纖維細胞MRC-5受到臭氧衝擊式暴露系統下之氧化壓力情形，主要利用採樣管收集細胞反應後產生之氣體及分析當中揮發性有機物質(volatile organic compounds, VOCs)，並觀察細胞膜完整程度以了解所受到的氧化性壓力程度。為了更進一步確認分析出來之VOCs為臭氧暴露至細胞膜上不飽和脂肪酸所反應物質，再利用臭氧衝擊式系統暴露至不飽和脂肪酸(arachidonic acid, AA; linoleic acid, LA)後以採樣管收集反應氣體並分析之。 本實驗為體外試驗研究-細胞部分，暴露時間為30分鐘，暴露濃度為0.1, 0.5, 1, 1.5, 2 ppm，暴露後觀察所經過時間為30, 30-60, 60-120 分鐘，另外不飽和脂肪酸濃度為10 ppm受到臭氧1ppm暴露30分鐘後，以採樣管收集暴露後所產生VOCs，再以熱脫附系統搭配低溫濃縮捕集以及氣相層析質譜儀進行分析。 實驗結果顯示比較臭氧與空氣暴露後各不同時間點其收集氣體發現有18種物質在兩組間達統計上顯著差異。其中直鏈及支鏈碳氫化合物以methylcyclopentane物質為最，不論是在高或低濃度臭氧暴露皆有顯著差異。醛類物質3-methylbutanal在暴露高濃度2 ppm臭氧30分鐘及暴露停止後經過30及60分鐘有顯著差異;另外在含硫物質carbon disulfide及 dimethyldisulfide兩者皆於暴露臭氧1.5 ppm 30分鐘停止後及再經過120分鐘有統計上顯著差異。空氣或臭氧暴露前後各時間點進行比較皆無顯著差異。比較臭氧濃度與生成化合物之半定量間共有4種化合物達統計上顯著意義，有2-butenal、Methylcyclopentane、2-methylhexane、eicosane等物質，且進行Tukey HSD事後檢定有2-butenal與methylcyclopentane在高濃度2 ppm臭氧暴露有顯著差異。此外，暴露空氣與臭氧至不飽和脂肪酸AA及LA並比較暴露30分鐘後之情況，結果顯示暴露至AA有顯著差異為2-methyl-1-propene ，而暴露至LA 後產生之pentane有顯著差異。 最後利用體外試驗之臭氧暴露系統所得到有顯著差異之VOCs物質並依據物質生成特性分成四類: 高或低臭氧暴露濃度下能分析出該物質、高臭氧暴露濃度下能分析出該物質、暴露30時即可分析出該物質及暴露經過120分鐘方能分析出該物質，期盼未來可做為臭氧暴露後相關研究導致氧化壓力之生物標誌。

The main aim of this study was to evaluate the impinger in vitro exposure system used for investigating ozone exposure induced oxidative stress. Comparison of VOCs levels of collecting gas and membrane integrity of MRC-5 cell line after ozone exposure were observed. In order to validate the VOCs source, the reaction of ozone with two polyunsaturated fatty acids (PUFA; arachidonic acid, AA; linoleic acid, LA) was performed. MRC-5 cells were treated to 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, 30-60, 60-120 min. PUFA 10 ppm were exposed to 1 ppm ozone for 30 min. Using sampling tubes to collect the reacting gas after ozone exposure on human lung cells or PUFA and analyze by the thermal desorption GC/MS. As the result, ozone induced a significant reduction in cell survival rate as determined using the pre- and post- exposure time and concentration of ozone. Comparing the VOCs from air and ozone exposure in MRC-5 cells at different time points, 18 compounds were significantly different. Methylcyclopentane level was acutely increased in lung cells oxidative stress. 3-methylbutanal product was significantly associated with high ozone level. carbon disulfide and dimethyldisulfide were immediately increased following ozone 1.5 ppm exposure for 30 min. There were four compounds significant associated with ozone concentrations. 2-butenal, methylcyclopentane were significant different from ozone 2 ppm by Tukey HSD test. 2-methyl-1-propene and pentane were identified and changing significantly different in the collected gas of AA and LA by ozone exposure. The significant VOCs would be as markers of oxidative stress after ozone exposure on the human lung cell in the impinger exposure system.