臭氧及其氧化產物對細胞毒性之探討-以氣、液介面細胞株為測試對象

Biological response of ozone and its oxidative products―Air Liquid Interface (ALI) in vitro study

陳思寧(Ssu-Ning Chen)

指導教授 ： 吳佩芝

長榮大學/健康科學學院/職業安全與衛生學系(所)/碩士(2011年)

摘要

近年來全球暖化加上各種臭氧前驅物排放增加下，臭氧在各大城市均有攀升的趨勢。這也使得臭氧及其氧化產物對健康及環境生態之影響日益受到重視。室內臭氧濃度隨著室外濃度增加的情況下，更容易與許多室內常見的單帖類(Monoterpenes)化學物質，這形成各類二次反應產物。但相關臭氧暴露及二次反應之產物及其對生物機制之影響，所知仍有限。傳統上體外細胞毒性測試受限於細胞培養方式，?H予細胞之暴露方式會改變污染物物化特性，並與實際暴露狀態有所差距。因此，本研究希望透過小型暴露艙室搭配氣、液介面(Air Liquid Interface，ALI)細胞培養技術，使人類呼吸道細胞能直接暴露於臭氧及其氧化產物，探討臭氧及其氧化產物長時間暴露對細胞存活率之影響，並確認暴露及生物反應之間的劑量效應之關係。
本研究利用暴露艙(體積9.24×10-3 m3）控制系統模擬6小時長時間不同室內臭氧濃度(30、60、120 ppb)及其與β-pinene 氧化反應暴露狀態，進而探討不同臭氧及氧化產物對ALI 細胞株(A549)細胞存活率及氧化性傷害之變化情形。透過微粒電移動度掃描分徑器(SMPS)持續 6小時監測暴露艙內反應生成之二次有機氣膠濃度及粒徑分佈，並使用Trypan Blue細胞染色法來計算培養及暴露後細胞株的細胞存活率。本研究亦使用2’,7’-dichlorofluorescin diacetate（DCFH-DA） probe與螢光酵素免疫分析儀量化暴露細胞液中的reactive oxygen species（ROS）含量。
研究結果顯示，ALI細胞培養技術能長時間(6小時)維持細胞生長，當ALI細胞培養系統分別暴露臭氧6小時後，細胞存活率相較於對照組均有顯著下降趨勢，呈現一劑量效應關係，暴露濃度愈高時，細胞存活率則愈低；進一步將細胞暴露於臭氧/β-pinene反應產物後，相較於單純暴露臭氧時，細胞存活率下降趨勢更為顯著，此趨勢也存在一劑量關係。但ALI細胞在暴露過臭氧/β-pinene反應產物後，ROS濃度卻沒比單純臭氧暴露來的低，可能是β-pinene與臭氧反應，降低了氧化物的濃度，因而細胞暴露到的氧化傷害減小。也顯示二次反應產物對細胞的傷害，除了氧化性傷害外，亦可能有其他機制，需進一步驗證之。
本研究所建構之A549 ALI細胞直接暴露系統，可應用於各類氣、粒狀污染物之細胞毒性研究探討上。研究結果顯示，混合的二次衍生化產物會對細胞造成更明顯的毒性傷害，未來可利用此暴露系統進一步探討驗證並從機制面了解不同混合物或反應產物對細胞毒性的影響。

關鍵字

臭氧 ； 氧化產物 ； 氣、液介面細胞株 ； 細胞存活率 ； 氧化壓力

並列摘要

Global warming and increasing emissions of various precursors, elevated ozone levels in major cities of the world have been predicted in the near future. More and more indoor secondary reaction products will derive from increasing ozone and a variety of indoor emissions monoterpenes from nature plants and consuming products. This addresses a health concern of increasing ozone and its oxidative products exposure in general population. Yet, only limit studies have examined the effects and its biological mechanisms are still unclear. Conventional in vitro assays are limited by the requirement that cells are in the cell growth medium, and gas and particles should be collected and resuspended for cell exposure. The process is likely to change the gas and surface chemistry, morphology, band size of particles. Our study therefore aim to develop an air-liquid interface (ALI) cell exposure system to be adapted for assessing various air pollutants in the future. ALI system with human lung epithelial cell line was used to exame the cell toxicity of exposing to different levels of ozone and it secondary reaction products.
In this study, a small scale chamber (9.24×10-3 m3) injecting different ozone levels by using ozone generator/monitor (dasibi, USA) to simulate different indoor ozone concentrations (30,60,120 ppb). β-pinene was then injected consistently for producing secondary reaction for A549 cell ALI exposure. The changes of cell viability and ROS were measured in different exposure scenario. Use Scanning Mobility Particle Sizer (SMPS) was used to monitor the concentration and aerodynamic diameters for the reactive secondary organic aerosols (SOA). Cell viability was measured using Trypan Blue staining method and reactive oxygen species (ROS) in culture media was measured by using 2’,7’-dichlorofluorescin diacetate (DCFH-DA) probe and fluorescent enzyme immunoassay analyzer.
The viability by using conventional cell culture (87.75%) and ALI with collagen transwell (89.3%) were similar, showing the suitable for supporting cell viability and the use for exposure test. Our chamber could simulate a consistent exposure scenario for ozone and β-pinene reaction with higher number concentrations of SOA in higher ozone levels. The compositions of SOA are mainly ultrafine particles (96.7%-98.5%) with diameters less than 100 nm. Significantly decrease in cell viability was found when exposing to higher ozone levels with a clear dose-response relationship. Comparing to the results of ozone exposure, a significant drop of cell viability with dose-dependent was also found when the cell exposed to ozone/β-pinene. The ROS levels also have been found to significantly increase with ozone concentrations in chamber. However, the productions of ROS tend to less when exposing to ozone and β-pinene reactive products comparing to those exposing to ozone. This phenomenon suggests the need of further examining the mechanism of cell toxicity in future study.
Our study shows that, ALI cell exposure system could be used for assessing prolonged exposure of gas/particles mixtures or reactive products on cell toxicity. Comparing to ozone exposure, dramatically reduction of cell viability could caused by ozone/β-pinene secondary products, which addressing a rising health concerns due to more indoor chemical reactions in the future.

並列關鍵字

Ozone ； Oxidative products ； Air Liquid Interface (ALI) ； cell viability ； Reactive Oxygen Species (ROS)

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