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  • 學位論文

評估AhR受體報導基因快速篩檢法檢測臺灣環境與生物樣品中之戴奧辛濃度

Evaluation of AhR reporter gene assay in Taiwanese environmental and biological samples

指導教授 : 趙浩然

摘要


以往高解析氣相層析/高解析質譜儀(HRGC/HRMS)檢測戴奧辛與類戴奧辛化合物濃度,常有耗時與分析費用高的缺點。然而,歐美等先進國家已開發出生物偵測法-化學活化冷光酵素基因表現法(CALUX®),可快速大量篩檢受戴奧辛污染的環境與生物或食品樣品。化學活化冷光酵素基因表現法是使用離體冷光基因表現,藉由戴奧辛與類戴奧辛物質與AhR (aryl hydrocarbon receptor)的高親合度來偵測戴奧辛與類戴奧辛化合物的濃度。本論文針對兩種臺灣本土細胞株系統和BioDetection Systems公司的CALUX®系統對戴奧辛標準品、法定管制之17種戴奧辛組成環境標準品及環境或生物真實樣品,進行研究與討論。本研究與國家衛生研究院鄒粹軍研究員合作並取得兩株本土細胞株: (1)Huh7-DRE-Luc,類似CALUX®系統,(2)腺病毒轉殖細胞株AdEasy-4#westeur024#DRE-Luc/H4IIE (攜帶4倍戴奧辛反應單元),進行土壤、底泥、魚體、螃蟹中的戴奧辛類化合物濃度調查及學術應用。本研究發現Huh7-DRE-Luc與HRGC/HRMS在土壤、底泥的檢測上有良好顯著的線性關係,並可經由2,3,7,8-TCDD與1,2,3,7,8-PeCDD兩種戴奧辛同構物預測Huh7-DRE-Luc的反應濃度(決定係數=0.962,顯著p值小於0.001)。隨後,本研究調降Huh7-DRE-Luc暴露樣品時的培養箱溫度 (由37°C降為35°C)且添加癌細胞誘發藥物PMA (phorbol-12-myristate-13-acetate,佛波酯),提升Huh7-DRE-Luc檢測土壤、底泥、魚體樣品中戴奧辛類化合物的敏感度與穩定性。另外,本研究將AdEasy-4#westeur024#DRE-Luc/H4IIE與環保署環境檢驗所第三組開發的快速索式萃取淨化濃縮技術結合,檢測魚類與螃蟹等戴奧辛濃度較低的樣品,仍具有良好的快篩能力。最後,本研究分析臺灣土壤戴奧辛背景值調查計畫所建置的統計資料庫(由環檢所第五組操作CALUX®進行土壤檢測)發現,800組樣品中的平均戴奧辛毒性當量濃度為36.0 pg-BEQs/g d.w.。其中採樣地點、土壤利用情況、土壤顏色、土壤濕度可能與戴奧辛類化合物濃度高低有相關性。此外,本研究發現八德市附近的工業區土壤與顏色較深的土壤,可能含有較高的戴奧辛類化合物濃度。

並列摘要


Recent techniques for detection and quantification of dioxins and dioxin-like compounds involve mainly the costly and time-consuming high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS). To ensure environmental safety, it is extremely crucial to develop high-throughput assays for screening potential contamination of dioxin-like chemicals in various field samples from environments and foods. The Chemical Activated LUciferase eXpression (CALUX®) bioassay is an in vitro luciferase-reporter-gene assay for detecting the Toxic Equivalents (TEQ) levels of dioxins and dioxin-like compounds based on their high affinity with aryl hydrocarbon receptor (AhR). As compared with HRGC/HRMS, the CALUX® bioassay is a fast and low-cost method for high-throughput screening of those highly dioxin-contaminated samples from environments and foods. The technique relies on selection of stable dioxin-responsive cell lines that can maintain their responsiveness to dioxins and dioxin-like compounds. This manuscript describes that two stable cell lines, Huh7-DRE-Luc and AdEasy-4#westeur024#DRE-Luc/H4IIE established by Dr. Tsou in NHRI of Taiwan, and CALUX® were used to screen the dioxin concentrations in soil, sediment, fish, and crab. Significant correlations were found between BEQs (Bioanalytical EQuivalents) (by AdEasy-4#westeur024#DRE-Luc/H4IIE) and PCDD/F WHO-TEQs (by HRGC/HRMS). BEQs is significantly correlated with WHO-TEQs of 2,3,7,8-TCDD and 1,2,3,7,8-PeCDD using a multiple stepwise linear regression model (adjusted R2=0.962, p<0.001). Huh7-DRE-Luc was modified by reducing the incubation temperature of the cell culture from 37°C to 35°C and by adding phorbol-12-myristate-13-acetate (PMA), and the modified bioassay was used to examine samples from soil, sediment, and fish. The results of this bioassay were shown to be significantly related to those of the HRGC/HRMS assay of dioxins. A new and easy fast-screening test (AdEasy-4#westeur024#DRE-Luc/H4IIE) for dioxins in biological samples from highly dioxin-contaminated areas was developed. A good correlation was found between the results of the HRGC/HRMS assay and those of the Ad-DR bioassay. Environmental Analysis Laboratory (EAL) in Taiwan’s EPA determined dioxin levels in 800 soil samples collected from Taiwan by the CALUX® bioassay. An in vitro CALUX® bioassay was carried out with the help of an automated Soxhlet system and fast cleanup column. The mean dioxin level of 800 soil samples was 36.0 pg-bioanalytical equivalents (BEQs)/g dry weight (d.w.). After all, we found that four variables, including soil sampling location, land use, soil brightness, and soil moisture, may be related to soil-dioxin contamination. Soil samples collected in northern Taiwan, and especially in Bade City, soils near industrial areas, and soils with darker color may contain higher dioxin-BEQ levels.

參考文獻


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被引用紀錄


曾迺婷(2017)。從性騷擾防治法(再)申訴過程探討防治網絡協同合作圖像〔碩士論文,國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342%2fNTU201700147

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