質譜法建立氮氧化物之生物暴露與效應指標---職業暴露危害研究

中山醫學大學機構典藏 CSMUIR > 健康管理學院 > 公共衛生學系暨碩士班 > 研究計劃 > Item 310902500/10350

Please use this identifier to cite or link to this item: https://ir.csmu.edu.tw:8080/ir/handle/310902500/10350

Title:	質譜法建立氮氧化物之生物暴露與效應指標---職業暴露危害研究 Development and Application of Mass Spectrometry-Based Biomarkers for Assessing Occupational Exposure to Nitrogen Oxides
Authors:	胡瓊文;潘致弘;趙木榮;巖正傑
Contributors:	公共衛生學系
Keywords:	氮氧化物,硝酸鹽類,亞硝胺,核酸氧化損傷;基因烷基化損傷;硝化損傷;基因甲基化;基因去胺化;動物模式;煉焦爐工人 Nitrogen oxides;nitrite/nitrate;N-nitrosamines;oxidative DNA/RNA lesions;alkylated DNA lesions;nitrative lesions;DNA methylation;DNA deamination;mice;coke oven workers
Date:	2014
Issue Date:	2015-02-25T09:19:00Z (UTC)
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 2N 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 32ON 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。 32ON 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.
URI:	https://ir.csmu.edu.tw:8080/ir/handle/310902500/10350
Appears in Collections:	[公共衛生學系暨碩士班] 研究計劃

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