質譜法發展新穎的基因鍵結體學(DNA Adductomics)：全面評估檳榔及香菸所造成的DNA鹼基修飾

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Title:	質譜法發展新穎的基因鍵結體學(DNA Adductomics)：全面評估檳榔及香菸所造成的DNA鹼基修飾 Development and Application of Novel Dna Adductomics for Assessing Human Exposure to Areca Nuts and Cigarette Smoke
Authors:	趙木榮
Contributors:	中山醫學大學：職業安全與衛生系
Date:	2012
Issue Date:	2012-06-25T08:13:43Z (UTC)
Abstract:	質譜法發展新穎的基因鍵結體學(DNA Adductomics)：全面評估檳榔及香菸所造成的 DNA 鹼基修飾趙木榮、?正傑、張耀仁細胞內 DNA 鹼基?與外在及內生性??所衍生的親電性分子反應產生DNA 鍵結物 (DNA adducts)且種?眾多，DNA adducts 的形成為細胞癌化的關鍵起始步驟。先前的研究?只以少?幾種DNA adducts ?作為特定化合物??或疾病的效應指標，但?無法完整且同一時間描述混合毒物??或疾病所造成的DNA 鹼基修飾全貌。Kanaly et al. 2006 ?首先提出運用LC-MS/MS 於同一時間分析基因組上所有DNA adducts 的構想，係將水解後的DNA 分子以中性丟失2’-deoxyribose 作為adducted 2’-deoxynucleosides 的分析特徵。運用質譜技術進?大範圍的中性丟失掃瞄，可同時間偵測所有adducted 2’-deoxynucleosides (已知及未知)，以描繪出樣本中各種DNA adducts 的修飾鍵結體地圖(adductome map)；此概?最近統稱為基因鍵結體學(DNA adductomics)。DNA adductomics 擁有極佳的優勢?探究人群環境中??多重混合毒物所造成的DNA adducts 全貌(profiles)，且可篩選出未知的DNA adducts。但目前此技術尚處萌芽階段，同時遭遇?各種分析問題如敏感?較低、結構資訊?足及層析區段的基質效應?一等。本研究目的在於運用 LC-MS/MS 搭配?線固相萃取(on-line solid phase extraction, SPE)建?新穎的DNA adductomics 研究方法，並克服DNA adductomics 目前的分析難題，接著運用於探討檳榔及香菸??所造成的DNA 鹼基修飾。期以adductome map 的疊圖比對探究檳榔及香菸??造成DNA 鹼基修飾特徵，並試圖以修飾鹼基的結構鑑定，追朔人?尚未發現的重要致癌成分。本計畫預計執?時間為四?：第一?將運用LC-MS/MS 搭配on-line SPE 開發具高敏感?及高選擇性的DNA adductomics 分析方法，同時建?層析質譜訊號輸出系統以快速進?adductome map 的疊圖比對。第二?將引入多段式on-line SPE 方法改?DNA adductomics 技術以首?運用於?液樣本分析，並將檳榔及香菸煙霧萃取液直接與calf thymus DNA 混合或施予小鼠，以探討所形成的adductome map 特徵並進?修飾鹼基的結構推測。第三?將展開抽菸及嚼食檳榔族群的血液及?液收樣，將所開發出的DNA adductomics 技術運用白血球 DNA 及?液分析，以探討所形成的adductome map 特徵並進?修飾鹼基的結構推測。第四?將選出對檳榔及香菸??具關鍵性或特?性的修飾鹼基並以市售或自?合成標準品?完全確認修飾鹼基的分子結構，未?可作為檳榔及香菸??及致癌效應的新指標。此外，我們也將由新發現的未知修飾鹼基，試圖追朔在檳榔及香菸中尚未發現的重要致基因毒物。 Development and application of novel DNA adductomics for assessing human exposure to areca nuts and cigarette smoke Mu-Rong Chao, Cheng-Chieh Yen, Yan-Zin Chang Cellular DNA is highly vulnerable to modification by exposure to electrophilic molecules that originate from both exogenous and endogenous sources, either directly or following metabolic activation. These modifications result in the formation of DNA adducts. If DNA adducts are not repaired properly, they can lead to mutations in critical genes such as those involved in the regulation of cellular growth and subsequent development of cancer. Recently, Kanaly et al. (2006) suggested a novel technique to detect multiple known or unknown DNA adducts simultaneously by using LC-MS/MS. The strategy, named the DNA adductome approach, is based on chromatographic separation of a nucleoside mixture combined with the detection of the constant neutral loss (CNL) of 2’-deoxyribose from positively ionized 2’-deoxynucleoside adducts over a certain range of transitions. This has led to the concept of producing an adductome map of all DNA adducts in a sample. This approach holds great potential for gaining further insight into DNA adduct profile differences between human populations exposed to mixtures of environmental genotoxins, but until now has been constrained by a variety of factors such as lower sensitivity, limited structural information and regional differences in matrix effects along the chromatogram. In this study, we are going to develop a novel DNA adductomics method, which overcome the drawbacks of previous works, for assessing external exposure to areca nuts and cigarette smoke. This project will last four years with several objects as follows: 1st year: To develop a highly sensitive and specific DNA adductomics method by LC-MS/MS coupled with on-line solid phase extraction (SPE). This method would allow putative adducts to be structurally characterized in the same analytical run by the combination of CNL with the acquisition of product ion mass spectra, and to be rapidly organized into an adductome map. 2nd year: To fully investigate 2’-deoxynucleoside adducts formed following the treatment of calf thymus DNA and mice with areca nut and cigarette smoke extracts. A multi-step on-line SPE procedure will be firstly applied to overcome issues of matrix effect often observed in urine analysis. 3rd year: To fully investigate 2’-deoxynucleoside adducts formed in lymphocyte DNA and urine of areca nut chewers and/or smokers. 4th year: To identify the important and specific putative DNA adducts on the adductome maps via comparison with their correspondent authentic standards and stable isotope analogues followed by LC-MS/MS analyses. Additionally, based on the structural information of newly identified adducts, we will attempt to backtrack the genotoxicants (either parent compound or putative reactive metabolites), which result from exposure to areca nuts and cigarette smoke.
URI:	https://ir.csmu.edu.tw:8080/ir/handle/310902500/4274
Relation:	公共衛生學
Appears in Collections:	[School of Occupational Safety and Health] Research Project Report

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