食品加工過程中胺類經亞硝化反應可產生亞硝胺(N-nitrosamines)，其已被證實為食品中重要 的致癌物質。目前台灣尚未訂定食品亞硝胺的管制標準，同時國際上食品亞硝胺的快速篩檢 方法也沒有進展。本計畫將建立各種類型食品樣本的前處理方法，同時發展兩類型的快速分 析方法，分別是「子計畫一：酵素免疫分析法及快速奈米金免疫檢測試紙」及「子計畫二： 連線固相萃取-液相層析串聯質譜儀搭配同位素稀釋法」。未來將以”液相層析串聯質譜儀”分 析9 種致癌性亞硝胺所得到的精準定量結果作為”酵素免疫分析法及快速奈米金免疫檢測試 紙”的基準(Gold standard)，以協助”酵素免疫分析法及快速奈米金免疫檢測試紙”的開發與分析 品質確認。方法開發完成後，本計畫將採集台灣市面上民眾常接觸各類含亞硝胺的食品，初 步進行含量調查及國人的暴露量評估。 飲食暴露到硝酸鹽(NO3 -)或亞硝酸鹽(NO2 -)，進入人體後也可與體內胺類生成亞硝胺，因 此抑制亞硝胺的生成及其在體內作用機制成為重要研究課題。「子計畫三：亞硝胺前驅物在 動物體內反應生成、毒性機制與解毒模式之探討」將在質譜技術的支援下，領先採用穩定同 位素15N 標示的亞硝酸鹽/硝酸鹽(15N-NO2 -及15N-NO3 -)施予小鼠，探討各種內生性亞硝胺的 生成及提出解毒方案(抑制生成或減緩代謝物的作用)。此外，量測人體體液中的亞硝胺，所 代表的是外在暴露及體內生成亞硝胺的總合。本計畫將建立非侵入性的尿液分析法來評估亞 硝胺在體內的劑量，對於亞硝胺的暴露及健康風險評估有很大的幫助。藉此生物偵測工具可 逐年觀察國人體內亞硝胺濃度的改變，作為政府單位在管制食品亞硝胺或其前驅物成效的直 接證據。 亞硝胺在體內代謝活化後能與體內大分子鍵結，造成 DNA 或蛋白質的化學修飾。有幾 種亞硝胺所造成的DNA 特定鍵結(adducts)曾被探討，然而亞硝胺在生物體內可形成的DNA adducts 種類眾多，至今仍不斷有大量新的DNA adducts 被報導出來，但都無法完整且同一時 間描述基因損傷的全貌。「子計畫四：運用新穎基因鍵結體學全面評估亞硝胺所造成的DNA 鹼基修飾」，期以鍵結體地圖(adductome map)的疊圖比對探究各種亞硝胺暴露造成DNA 鹼 基修飾的重要特徵，並進行修飾鹼基的結構鑑定。目前亞硝胺造成蛋白質修飾的研究很少， 「子計畫五：運用蛋白質體學評估亞硝胺所造成的蛋白質修飾」將透過蛋白質體學技術與質 譜分析，找出亞硝胺在人類肝細胞及血清中蛋白質的作用標的。本計劃鑑定出各種亞硝胺造 成DNA 鹼基及蛋白質的特徵修飾產物，將有利於亞硝胺毒性作用機制的深入探討，並可作 為人類亞硝胺暴露及效應的新指標。
The name N-nitrosamine is related to compounds that present the NNO characteristic group. N-nitrosamines are formed in food as a result of natural chemical interactions, but mainly through food processing activity. Many volatile N-nitrosamines are potent carcinogens, and their rapid detection and adverse health effects assessment are of great importance. This proposal contains five projects. In order to characterize/quantify the nitrosamines in food, we are going to develop high-throughput screening assays (Project 1 and 2) for 9 common N-nitrosamines, namely N-nitrosodimethylamine (NDMA), N-nitrosomethylethylamine (NMEA), N-nitrosodiethylamine (NDEA), N-nitrosopyrrolidine (NPyr), N-nitrosomorpholine (NMor), N-nitrosodi-n-propylamine (NDPA), N-nitrosopiperidine (NPip), N-nitrosodi-n-butylamine (NDBA) and N-nitrosodiphenylamine (NDPhA). Project 1 aims to produce specific monoclonal antibodies for immunoassays and develop rapid gold nanoparticle immune chromatographic strips for N-nitrosamines assay. Project 2 aims to develop isotope-dilution LC-MS/MS with automated solid-phase extraction (as the current gold standard) for high-throughput analysis of N-nitrosamines and will be further used to help in the validation of newly developed immunoassays (Project 1). Furthermore, both methods (Project 1 and 2) will be applied to determine the N-nitrosamines levels in various foods that are frequently consumed by Taiwanese people to characterize the N-nitrosamines distribution in food, and also to determine the urinary N-nitrosamines levels of residents in Taiwan to investigate the body's internal exposure level of N-nitrosamines. Dietary exposure to nitrates/nitrites can react with amines in vivo to produce N-nitrosamines. Its underlying mechanism and the possible suppression of endogenous N-nitrosamine formation are of interest. Project 3 aims to investigate the formation and toxicity and of the N-nitrosamines from precursors by treating the mice with stable isotope labeled nitrate/nitrites (15N-NO2 - or 15N-NO3 -) and determining endogenously formed 15N-nitrosamine using LC-MS/MS method (Project 1). Possible approaches to suppress the endogenous N-nitrosamine formation are also investigated. N-nitrosamines are able to cause damage to cellular constituents including DNA and proteins. However, there is limited information available in the literature. Project 4 aims to adopt a novel DNA adductomics method using LC-MS/MS to explore/characterize putative DNA adducts that induced by different N-nitrosamine and to compare profiles of putative DNA adducts that are formed by the external exposure to N-nitrosamine. Project 5 aims to investigate the mechanism of N-nitrosamine-induced protein modification (nitration or alkylation) in albumin, human liver cells and human blood. Specific modified proteins induced by different nitrosamines will be characterized/identified by immuno-dot blot or proteinomic and mass spectrometry analysis. Overall, the results obtained from this proposal may help our government to establish an initiative/general standard for N-nitrosamines in food and to conduct the risk assessment of N-nitrosamine.
