산화적 스트레스가 대기분진 (PM2.5)의 유전독성에 미치는 영향 = Role of free radicals in the genotoxicity of airborne fine particulate matter (PM2.5)

국문 초록 (Abstract)

미세먼지 (PM2.5)는 PAHs와 그 유도체 등을 포함한 다양한 화합물이 혼합되어 있으며, 인체 건강에 영향을 미치고, 유전독성을 유발한다고 알려져 있다.
본 연구에서는 도시대기환경의 미세먼지가 호흡기계의 유전독성 유발에 미치는 영향을 조사하고, 원인물질을 추정하였다. 이를 위해 교통 혼잡지역인 수원역 부근의 대기 중 미세먼지(PM2.5)를 채취하여 유기용매로 추출한 유기물질을 추출 분획을 시료로 유전독성을 평가하고 원인 물질군을 확인하였으며, 유전독성을 유발하는 기전 중 산화적 스트레스의 관련성에 대해 확인하였다.
Dichloromethane으로 PM2.5 시료를 추출하여 crude 추출물을 얻었고, 이를 산-염기 추출법에 의해 염기성, 산성, 중성 유기추출분획을 분리하였다. 유기용매층의 중성 추출물은 silica gel column chromatography에 의해 극성에 따라 각각 5개(aliphatic and aromatic, slightly polar, moderately polar 및 highly polar fraction) 분획으로 추출하였다. 이렇게 얻어진 각 시료를 대상으로 Comet assay와 MN assay 등의 in vitro bioassay로 유전독성을 평가하였다.
MN assay와 Comet assay를 이용한 실험 결과 in vitro 시험계에서 교통혼잡지역 미세먼지에서 유전 독성이 유발되었다. 또한 유발된 유전독성은 다이옥신류, PAHs 및 그 유도체 등이 포함되어 있는 추출 분획에서 높게 나타남을 알 수 있었다. 또한 FPG와 ENDOIII를 이용한 변형된 Comet assay및 free radical scavenger의 병용투여 실험에서 이들 분획의 유전독성이 활성산소에 의한 DNA의 산화적 손상에 의해 유도됨을 확인할 수 있었다. 또한 주요 원인 물질군으로 다이옥신류, PAHs 및 그 유도체류일 것으로 추정되었다.

다국어 초록 (Multilingual Abstract)

The airborne particulate matters (PM2.5) collected from traffic area of Suwon city was smaller than 2.5?m in aerodynamic diameter (PM2.5), indicating that these particles may be able to reach the pulmonary alveoli. Generally, it has been reported that PM2.5 is associated with a range of health effects including lung cancer. Therefore, we investigated genotoxic effects of PM2.5 and the role of oxidative stress. Because PM2.5 is a complex mixture comprised of thousands of chemical compounds (U.S. EPA, 2004), we used bioassay-directed fractionation to identify the causative chemicals for genotoxic effect of PM2.5. The samples were extracted with dichloromethane (crude extract, CE) and the CE were fractionated by acid-base partitioning into the organic base (F1), acid (F2) and five neutral fractions (F3-F7). The genotoxic activity of the organic extracts was determined using CBMN assay and Comet assay in human lung epithelial cell (BEAS-2B). In addition, to determine whether the genotoxic effect of fraction was directed or indirected, BEAS-2B cells were treated with combination of PM2.5 samples and endonuclease enzyme or free radical scavenging agents in Comet assay and MN assay. Our results showed that CE significantly stimulated DNA breakage and micronucleus formation in a dose-dependent manner (from 1 mg/ml to 50 mg/ml). Fraction of PM2.5 showed that F3, F4 and F5 significantly stimulated DNA breakage and micronucleus formation. And in modified Comet assay using endonucleases, CE and its fraction samples (F3, F4 and F5) increased DNA breakage compared with control groups, these results indicate that CE and fraction samples of PM2.5 induced oxidative damage. In addition, DNA breakage of PM2.5 was significantly decreased by scavenger agents, in comparison with independently treated fraction. These results clearly confirmed that PM2.5 collected in Suwon traffic area has genotoxic effects and oxidative stress may play a distinct role in genotoxic effects. Therefore, we suggested that PM2.5 collected in Suwon area contains genotoxic chemicals, and aliphatic/chlorinated hydrocarbons, PAH/their alkylderivatives, and nitro-PAH/ketones/quinones may be considered as important causative agents of genotoxic effects.

목차 (Table of Contents)

• I. INTRODUCTION = 1
• II. MATERIALS AND METHODS = 5
• 1. Materials = 5
• 1.1. Reagents = 5
• 1.2. Instruments = 8
• 1.3. PM2.5 Sampling & preparation = 9
• 1.4. Cell culture = 10
• 2. Methods = 11
• 2.1. In vitro single cell gel electrophoresis (In vitro Comet Assay) = 11
• 2.2. In vitro cytokinesis-block micronucleus (CBMN) Assay = 12
• 2.3. Data analysis = 14
• III. RESULTS = 15
• 1. Genotoxic effect of crude extract (CE) of ambient air PM2.5 = 15
• 2. Identification of biological active fraction = 16
• 3. Oxidative DNA damage by major fraction of PM2.5 = 21
• 4. Role of free radicals on the genotoxicity of major fraction of PM2.5 = 23
• IV. DISCUSSION = 26
• V. REFERENCES = 29
• ABSTRACT = 37
• 국문요약 = 39