| Abstract: | 氮氧化物(NOx)為普遍存在於環境的空氣汙染物，燃燒過程中空氣中大量的2 N 可氧化產生 NOx。因此除了吸煙及交通工具外，高溫燃燒的鑄造、熔接及煉焦爐等工作環境皆會長時間暴露 高量的NOx。NOx 在體內最終會轉換形成硝酸鹽類(NO2 -及NO3 -)，因此體液中NO2 -及NO3 -濃度 的量測常用作環境NOx 的暴露指標。暴露NOx 會使體內活性含氮物質(reactive nitrogen species, RNS)增高進而造成硝化傷害，其中過氧亞硝酸根陰離子(peroxynitrite, PN)可與DNA 作用形成 8-nitroguanine (8-NO2-G) 或與蛋白質反應生成3-nitrotyrosine (3-NTYR) 及 3-nitro-4-hydroxyphenylacetic acid (NHPA)。PN 也能釋出活性含氧物質(reactive oxygen species, ROS) 造成核酸(DNA 及RNA) 氧化傷害。8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) 及 8-oxoguanosine (8-oxoGuo) 分別為DNA 及 RNA 主要氧化傷害產物。研究也證實暴露NOx 不僅 使體內產生大量的 ROS/RNS，NO可在體內自行氧化成強亞硝化物質 2 3 N O 並與二級胺衍生形成亞 硝胺(N-nitrosamines)。亞硝胺經代謝活化後可產生烷基偶氮離子，進而造成DNA 烷基化傷害，如 alkylguanines、alkyladenines及 alkylthymines；同時亞硝胺代謝亦會產生ROS 造成氧化傷害。 2 3 N O 也可直接攻擊DNA 導致去胺作用(deamination)，當CpG 島上的5-methylcytosine 鹼基經去胺化及 氧化作用可形成thymidine glycol (dTg) 、5-hydroxymethyl-2'-deoxyuridine (5-hmdU) 和 5-hydroxymethyl-2'-deoxycytidine (5-hmdC)，將干擾基因的表觀遺傳調控機制(epigenetic mechanism)， 這與許多慢性疾病及癌症有關。 本計畫預計執行四年，將利用液相層析串聯質譜儀(LC-MS/MS)搭配連線固相萃取(on-line solid phase extraction, on-line SPE)及同位素稀釋法(isotope dilution)建立多項NOx 暴露的生物偵測/ 效應指標，用以評估NOx 暴露及其可能造成的健康危害。逐年目標如下，第一年：運用on-line SPE LC-MS/MS 搭配isotope dilution 開發尿液中亞硝酸鹽/硝酸鹽(NO2 -及NO3 -)、尿液中10 種常見亞硝 胺及DNA 甲基鍵結損傷(O4-methylthymine)的分析方法;第二年：運用on-line SPE LC-MS/MS 搭配 isotope dilution 開發尿液中DNA/蛋白質硝化傷害指標(8-NO2-G、3-NTYR 及NHPA)及尿液/細胞中 DNA 表觀遺傳物質5-mdC 的氧化/去胺化指標(5-hmdC、dTg 及5-hmdU)分析方法;第三年：運用 on-line SPE LC-MS/MS 搭配isotope dilution 開發尿液及細胞中RNA 氧化傷害指標(8-oxoGuo)及動 物試驗驗證指標適用性：將小鼠置於暴露腔中，探討NO 及NO2 (或其15N 同位素)暴露後，測試動 物體內各種NOx 生物指標的形成及其特異性;第四年：選擇煉焦爐廠(NOx 高暴露職業)進行空氣採 樣分析、血液與尿液收樣及問卷調查。將各項開發出之生物偵測方法應用於煉焦工人族群分析， 並探討NOx 外在暴露與各項生物指標間的相關性。
Nitrogen oxides (NOx) is a common air pollutant that is formed when nitrogen reacts with oxygen. In addition to cigarette smoke and vehicle emission, high levels of NOx emissions can be easily found in manufacturing environment of high temperature, including coke oven, welding and foundry work. In vivo, NOx is rapidly oxidized to nitrite and nitrate, and thus the levels of nitrite and nitrate in body fluids have been used as indicators of NOx exposure. NOx exposure could increase reactive nitrogen species (RNS) in vivo that are able to cause nitrative damage to cellular constituents (i.e., DNA and proteins). RNS (e.g., peroxynitrite, PN) can react with DNA to form nitrative lesion 8-nitroguanine (8-NO2-G) or with protein to form 3-nitrotyrosine (3-NTYR) and 3-nitro-4-hydroxyphenylacetic acid (NHPA). RNS can also release reactive oxygen species (ROS) and cause oxidative damage to nucleic acids (DNA and RNA). 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 8-oxoguanosine (8-oxoGuo) are the most used biomarkers for oxidative lesion of DNA and RNA, respectively. Meanwhile, RNS (e.g., N2O3) can react with secondary amines to form N-nitrosamines, that are capable of further alkylating nucleobase to form mutagenic lesions (e.g., alkylguanines, alkyladenines and alkylthymines). Furthermore, N2O3 has been shown to promote the nitrosative deamination of purines and pyrimidines. Particularly, deamination/oxidation of 5-methylcytosine in CpG dinucleotides that results in the formation of thymidine glycol (dTg), 5-hydroxymethyl-2'-deoxyuridine (5-hmdU) and 5-hydroxymethyl-2'-deoxycytidine (5-hmdC), can affect genetic and epigenetic processes. This is a four-year project, which aims to develop serial “on-line SPE LC-MS/MS” methods to quantitate various biomarkers for assessing the potential adverse health effect of NOx exposure. Subaims of each year are stated as follows: 1st year: To develop isotope dilution LC-MS/MS with on-line SPE methods for determining nitrite/nitrate, N-nitrosamines and alkylated DNA adducts; 2nd year: To develop isotope dilution LC-MS/MS with on-line SPE methods for determining 8-NO2-G, 3-NTYR/NHPA and 5-hmdC/dTg/5-hmdU; 3rd year: To develop isotope dilution LC-MS/MS with on-line SPE methods for determining 8-oxoGuo. Moreover, these newly developed methods will be firstly validated in a mouse model of NOx (or 15N-NOx) exposure; 4th year: Newly developed methods will be comprehensively applied in a total of 150 blood and urine samples of coke oven workers to assess the possible health effects of occupational exposure to NOx. |
