環保專案成果報告查詢系統

專案計畫編號	EPA-99-1624-02-01
經費年度	099
計畫經費	1,960 千元
專案開始日期	2010/07/13
專案結束日期	2010/12/31
專案主持人	鄒粹軍副研究員
主辦單位	環檢所
承辦人	陳元武
執行單位	財團法人國家衛生研究院
專案分類	環境監測及檢測
中文關鍵字	類戴奧辛化合物、化學活化冷光酵素基因表現法;類戴奧辛化合物、化學活化冷光酵素基因表現法
英文關鍵字	Dioxin-like compounds, CALUX
協同主持人	趙浩然;王雅玢副教授
共同主持人
計畫聯絡人	鄒粹軍副研究員
計畫聯絡電話	037-246166#36521
計畫聯絡信箱	tctsou@nhri.org.tw

中文摘要	目前國內環境與生物檢體的類戴奧辛化合物濃度檢測，大多利用高解析氣相層析/高解析質譜儀(HRGC/HRMS)進行檢測，該檢測技術已相當成熟且準確，但過程耗費時間且分析成本偏高。在歐、美、日等已開發國家，均已開發出化學活化冷光酵素基因表現法(CALUX)，並設定該技術為戴奧辛先期篩檢方法，以降低社會所付出的戴奧辛檢驗成本。延續98年度環檢所計畫，以人類肝腫瘤Huh7-DRE-Luc細胞株之研究成果，本團隊嘗試建構腺病毒(Ad-DRE-Luc)為冷光基因載體，並利用大鼠肝腫瘤細胞株（H4IIE）為感染宿主，開發出以CALUX原理為基礎之DRE-driven Luciferase assay，本計畫的研究成果如下：(1)引進環檢所開發出之快速前處理標準方法程序（包含索氏萃取、硫酸矽膠管柱淨化與CAPE管淨化），已建構完成；(2) 完成細胞分析方法標準程序（期末報告3-2-2節），並搭配QA/QC指標（期末報告3-3節及附錄5）；(3)以Hill equation公式配適2,3,7,8-TCDD檢量線的R2均可達到0.995以上，且液態標準品(EDF)測試件次達12件次，當EDF液態標準品稀釋倍數為10倍及30倍時，DRE-driven Luciferase測值僅以1~2倍比值高於毒性當量實際值；(4)環境基質CRM參考標準品共檢測23件次，DRE-driven Luciferase測值在400~736 ng-ADL-TEQ/kg d.w.之間，較Huh7-DRE-Luc測值(2108~4141 ng-DL-TEQ /kg d.w.)要更接近GC-HRMS測值(466 ng-WHO1998 TEQ /kg d.w.) ；(5)土壤底泥樣品已由49個樣本中完成99件次(包含98、99年度樣品)，雖然99年度樣品比值(平均比值:土99=12.1，底99=9.25)不比98年度樣品(平均比值:土98=4.24，底98=3.14)好，但若將99年度之GC-HRMS及DRE-driven Luciferase測值取對數值(Log)計算R2分別為土壤=0.815、底泥=0.84，仍皆屬於高度相關；(6)生物體樣品共分析40個樣品40件次，雖然其DRE-driven Luciferase與GC-HRMS測值之平均比值為8.6倍，高於先前液態標準品、CRM樣品、土壤與底泥樣本之任一平均比值，但若以「篩檢樣品」的定位來看，DRE-driven Luciferase仍可準確評估樣品中類戴奧辛化合物之濃度高低排序。
中文關鍵字	類戴奧辛化合物、化學活化冷光酵素基因表現法;類戴奧辛化合物、化學活化冷光酵素基因表現法

中文摘要

目前國內環境與生物檢體的類戴奧辛化合物濃度檢測，大多利用高解析氣相層析/高解析質譜儀(HRGC/HRMS)進行檢測，該檢測技術已相當成熟且準確，但過程耗費時間且分析成本偏高。在歐、美、日等已開發國家，均已開發出化學活化冷光酵素基因表現法(CALUX)，並設定該技術為戴奧辛先期篩檢方法，以降低社會所付出的戴奧辛檢驗成本。延續98年度環檢所計畫，以人類肝腫瘤Huh7-DRE-Luc細胞株之研究成果，本團隊嘗試建構腺病毒(Ad-DRE-Luc)為冷光基因載體，並利用大鼠肝腫瘤細胞株（H4IIE）為感染宿主，開發出以CALUX原理為基礎之DRE-driven Luciferase assay，本計畫的研究成果如下：(1)引進環檢所開發出之快速前處理標準方法程序（包含索氏萃取、硫酸矽膠管柱淨化與CAPE管淨化），已建構完成；(2) 完成細胞分析方法標準程序（期末報告3-2-2節），並搭配QA/QC指標（期末報告3-3節及附錄5）；(3)以Hill equation公式配適2,3,7,8-TCDD檢量線的R2均可達到0.995以上，且液態標準品(EDF)測試件次達12件次，當EDF液態標準品稀釋倍數為10倍及30倍時，DRE-driven Luciferase測值僅以1~2倍比值高於毒性當量實際值；(4)環境基質CRM參考標準品共檢測23件次，DRE-driven Luciferase測值在400~736 ng-ADL-TEQ/kg d.w.之間，較Huh7-DRE-Luc測值(2108~4141 ng-DL-TEQ /kg d.w.)要更接近GC-HRMS測值(466 ng-WHO1998 TEQ /kg d.w.) ；(5)土壤底泥樣品已由49個樣本中完成99件次(包含98、99年度樣品)，雖然99年度樣品比值(平均比值:土99=12.1，底99=9.25)不比98年度樣品(平均比值:土98=4.24，底98=3.14)好，但若將99年度之GC-HRMS及DRE-driven Luciferase測值取對數值(Log)計算R2分別為土壤=0.815、底泥=0.84，仍皆屬於高度相關；(6)生物體樣品共分析40個樣品40件次，雖然其DRE-driven Luciferase與GC-HRMS測值之平均比值為8.6倍，高於先前液態標準品、CRM樣品、土壤與底泥樣本之任一平均比值，但若以「篩檢樣品」的定位來看，DRE-driven Luciferase仍可準確評估樣品中類戴奧辛化合物之濃度高低排序。

中文關鍵字

類戴奧辛化合物、化學活化冷光酵素基因表現法;類戴奧辛化合物、化學活化冷光酵素基因表現法

類型	檔名	檔案大小	說明	最新版公開日期
期末報告	污染場址本土戴奧辛生物快速篩選檢測技術開發計畫定稿本.pdf	7MB	污染場址本土戴奧辛生物快速篩選檢測技術開發計畫結案報告	2011/2/1 上午 12:00:00

摘要	Recent techniques for 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 samples from various matrices. 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, CALUX bioassay is a fast and low-cost method for high-throughput screening of those highly dioxin-contaminated samples from environment and foods. We have established a recombinant adenovirus carrying a DRE-driven luciferase gene (Ad-DRE-Luc) and use a rat hepatoma cell line H4IIE as the host. On the basis of this DRE-driven luciferase assay system, this project aims to develop a high-throughput screening bioassay for PCDD/Fs measurement in samples from various matrices. In this study, we have established a fast sample cleanup system and the standard cell culture procedure for the assay. After fitted into the Hill equation, the regression curve of 2,3,7,8-TCDD was shown with R2 ≥ 0.995. The DRE-driven luciferase assay system was tested with liquid standards (EDF-5416 and EDF-5417), a certified reference material (CRM) (DX-1), samples from soil and sediment, and a variety of bio-samples. Our results showed that the sensitivity of Ad-DRE-Luc/H4IIE system in dioxin measurement is significantly higher than that of the previous Huh7-DRE-Luc system. The Ad-DRE-Luc/H4IIE system has been demonstrated to be a powerful technique for pre-screening dioxin samples from different sources, including organisms.
英文關鍵字	Dioxin-like compounds, CALUX

摘要

Recent techniques for 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 samples from various matrices. 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, CALUX bioassay is a fast and low-cost method for high-throughput screening of those highly dioxin-contaminated samples from environment and foods. We have established a recombinant adenovirus carrying a DRE-driven luciferase gene (Ad-DRE-Luc) and use a rat hepatoma cell line H4IIE as the host. On the basis of this DRE-driven luciferase assay system, this project aims to develop a high-throughput screening bioassay for PCDD/Fs measurement in samples from various matrices. In this study, we have established a fast sample cleanup system and the standard cell culture procedure for the assay. After fitted into the Hill equation, the regression curve of 2,3,7,8-TCDD was shown with R2 ≥ 0.995. The DRE-driven luciferase assay system was tested with liquid standards (EDF-5416 and EDF-5417), a certified reference material (CRM) (DX-1), samples from soil and sediment, and a variety of bio-samples. Our results showed that the sensitivity of Ad-DRE-Luc/H4IIE system in dioxin measurement is significantly higher than that of the previous Huh7-DRE-Luc system. The Ad-DRE-Luc/H4IIE system has been demonstrated to be a powerful technique for pre-screening dioxin samples from different sources, including organisms.

英文關鍵字

Dioxin-like compounds, CALUX