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

中山醫學大學機構典藏 CSMUIR > 健康管理學院 > 職業安全衛生學系暨碩士班 > 研究計劃 > Item 310902500/10339

Please use this identifier to cite or link to this item: https://ir.csmu.edu.tw:8080/ir/handle/310902500/10339

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;巖正傑;張耀仁
Keywords:	基因鍵結體學;DNA鹼基修飾;液相層析串聯質譜儀;檳榔;香菸 DNA adducts,DNA damage,adductomics,LC-MS/MS,areca nuts,cigarette smoke 基因鍵結體學;DNA鹼基修飾;液相層析串聯質譜儀;檳榔;香菸 DNA adducts;DNA damage;adductomics;LC-MS/MS;areca nuts;cigarette smoke
Date:	2014
Issue Date:	2015-02-25T09:18:41Z (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、巌正傑、張耀仁細胞內 DNA 鹼基易與外在及內生性暴露所衍生的親電性分子反應產生DNA 鍵結物 (DNA adducts)且種類眾多，DNA adducts 的形成為細胞癌化的關鍵起始步驟。先前的研究都只以少數幾種DNA adducts 來作為特定化合物暴露或疾病的效應指標，但都無法完整且同一時間描述混合毒物暴露或疾病所造成的DNA 鹼基修飾全貌。Kanaly et al. 2006 年首先提出運用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。運用質譜技術進行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)；此概念最近統稱為基因鍵結體學(DNA adductomics)。DNA adductomics 擁有極佳的優勢來探究人群環境中暴露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，同時遭遇了各種分析問題如敏感度較低、結構資訊不足及層析區段的基質效應不一等。本研究目的在於運用 LC-MS/MS 搭配連線固相萃取(on-line solid phase extraction, SPE)建立新穎的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暴露所造成的DNA 鹼基修飾。期以adductome map 的疊圖比對探究檳榔及香菸暴露造成DNA 鹼基修飾特徵，並試圖以修飾鹼基的結構鑑定，追朔人類尚未發現的重要致癌成分。本計畫預計執行時間為四年：第一年將運用LC-MS/MS 搭配on-line SPE 開發具高敏感度及高選擇性的DNA adductomics 分析方法，同時建立層析質譜訊號輸出系統以快速進行adductome map 的疊圖比對。第二年將引入多段式on-line SPE 方法改良DNA adductomics 技術以首度運用於尿液樣本分析，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 特徵並進行修飾鹼基的結構推測。第三年將展開抽菸及嚼食檳榔族群的血液及尿液收樣，將所開發出的DNA adductomics 技術運用白血球 DNA 及尿液分析，以探討所形成的adductome map 特徵並進行修飾鹼基的結構推測。第四年將選出對檳榔及香菸暴露具關鍵性或特異性的修飾鹼基並以市售或自行合成標準品來完全確認修飾鹼基的分子結構，未來可作為檳榔及香菸暴露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。 Vitamin B-6 may directly scavenge free radical or indirectly through glutathione antioxidant systeme to act as an antioxidant. However, the effect of different vitamin B-6 status before and after the occurrence of oxidative stress on antioxidant defense capacities in animal models, and the association between vitamin B-6 status, glutathione status and antioxidant enzyme activities in human models has not been fully studied. This is a 3-y study and the specific aims are: 1) to determine whether low or high levels of vitamin B-6 before and after the occurrence of oxidative stress have direct or indirect effects on glutathione status and antioxidant defense capacities in animal models; 2) to investigate whether vitamin B-6 status is associated with plasma and erythrocyte glutathione concentration and glutathione-dependent enzyme activities in human subjects under normal condition or persistent oxidative stress; 3) to study whether the supplementation of vitamin B-6 has a beneficial effect on antioxidant defense capacity in subjects under persistent oxidative stress; 4) to investigate the effect of vitamin B-6 status on oxidative stress, glutathione status and antioxidant capacities in patients with colorectal cancer and matched healthy controls. In an animal study, 5 week old male BALB/c mouse (n = 170) will be weight and evenly divided into one of three dietary treatment groups feed either a control diet (n = 70), a vitamin B-6 deficient diet (n = 50) or vitamin B-6 supplemental diet (7 mg/kg diet of pyridoxine-HCl is supplemented to the control diet) (n = 50) for 28 days. At the 35th d and 49th d, 10 mice of each group will be sacrificed to get blood and tissue samples. The rest of mice (n = 60) in control group will then be stratified again by weight and evenly divided into control (n = 20) (as a positive control group), vitamin B-6 deficient (n = 20) or vitamin B-6 supplement (n = 20) diet for another 28 d. The rest of mice in vitamin B-6 group (n = 40) and vitamin B-6 supplement group (n = 40) will also be stratified again by weight and evenly divided into either vitamin B-6 deficient (n = 20) or vitamin B-6 supplemented diet (n = 20) for another 28 d. During this period (28 d), mice will be given homocysteine thiolactone adding in the diet (50 mg/kg body weight/d) to induce oxidative stress. In human observational cross-sectional study, 70 industrial workers will be recruited as the exposure group, and office employees will be as the reference group. In a double-blind 2 × 2 factorial intervention and follow-up study, industrial workers of the exposure group will be asked to participate this intervention study if they have plasma pyridoxal 5’-phosphate (PLP) concentration < 30 nmol/L. Participants will be randomized into 4 groups - group A: vitamin C (100 mg/d) plus vitamin B-6 (50 mg/d); group B: vitamin C; group C: vitamin B-6 or group D: placebo for 12 weeks. Participants will discontinue the intervention after blood drawn at the 12th week, and will be asked to go back to their normal diet without taking any nutritional supplements for another 8 weeks. In a case-control study, 140 healthy volunteers will be recruited as the control group, and 70 colorectal cancer patients will be as the case group. Collecting samples will be measured hematological values, plasma homocysteine, plasma and erythrocyte PLP, pyridoxal and glutathione, antioxidant enzyme activities, lipid peroxidation and total antioxidant capacity levels and urinary 8-hydroxydeoxyguanosine concentration. Hopefully, the results of this study could provide more pictures on vitamin B-6, oxidative stress and antioxidant activities in either animal or human models. Additionally, the effects of pyridoxine supplementation on the occurrence of oxidative stress will be understood.
URI:	https://ir.csmu.edu.tw:8080/ir/handle/310902500/10339
Appears in Collections:	[職業安全衛生學系暨碩士班] 研究計劃

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