五、第三年研究目標與成果
4. 檳榔嚼食者唾液中基因鍵結體全貌與 ASNAs 量測 (對應分項目標 4) (1) 檳榔嚼食者唾液之基因鍵結體全貌
本研究將HRMS-based DNA adductomics 技術應用於檳榔嚼食者的唾液分析，以探討檳榔嚼 食者口腔細胞的DNA 修復產物(2'-dN)排至唾液中的變化。本研究從收集的檳榔嚼食者唾液中挑 選3 個嚼食白灰檳榔的男性，年齡介於 31 至 59 歲，BMI 範圍為 22-30 kg/m2；此外，也另外採 集 3 個非檳榔嚼食者唾液作為控制組，其性別、年齡與 BMI 皆與嚼食者相近。圖二十八為唾液 樣本經蛋白質沈澱、濃縮5 倍後的基因鍵結體分析結果。檳榔嚼食者唾液中可測得 18 個離子訊 號(圖二十八(B))，比控制組(圖二十八(A))多出 1 個，此訊號為 m/z 341.0656 (13.05 分鐘, Ion A)。
圖二十八、(A)非檳榔嚼食者(控制組)與(B)檳榔嚼食者唾液的 adductome map；(C)具訊號強度表 現顯著差異的特徵離子box plot (Ion 1-2)
我們也進一步利用 PCA 分析法尋找控制組與嚼食者唾液間訊號強度上有差異的離子。各 3 位的檳榔嚼食者與非嚼食者，其數據結果經 PCA 分析後呈現顯著的分群，表示組間具有明顯差 異。透過PCA loadings plot 可找到 2 個離子訊號造成兩族群間的差異。此 2 個訊號分別為：Ion 1 (8.14 min, m/z 241.1547)與 Ion 2 (22.70 min, m/z 288.1443)，這些訊號於嚼食者唾液中強度皆大於 非嚼食者如圖二十八(C)。而原先由 map (原始數據)上發現的新生成基因鍵結物 Ion A，後續的數 據處理流程研判可能為 ESI 游離化過程產生的離子鍵結型態，進一步查證發現此訊號與 dT (m/z 243.0975) 的時間一樣，推測此訊號為 dT 接合上硫酸基團[M+ H2SO4+H]+。
(2) 檳榔(白灰)暴露產生的基因鍵結體相關性探討 (CT-DNA、細胞與唾液)
本研究為探討檳榔暴露後的基因鍵結體全貌，實驗設計從基礎的 CT-DNA 模型、TW2.6 口 腔頰黏膜細胞到人類的唾液，以觀察暴露於檳榔特有毒物(arecoline 及 MNPN)與複雜成分的檳榔 汁液(檳榔子及白灰檳榔)下的基因鍵結體變化。表十五綜合 CT-DNA 及 TW2.6 細胞暴露於白灰 檳榔汁液及白灰檳榔嚼食者唾液的分析結果。以DD-NL-MS3可測得的2'-dN 訊號量排序：TW2.6 細胞 (32 ions) > CT-DNA (27 ions) > 唾液(18 ions)。在 CT-DNA、細胞或著唾液實驗中，相較各 自的控制組雖無發現有新生成的基因鍵結物，但藉由統計方式 PCA，可找出共 8 個 2'-dN 訊號 (表十五)在暴露組中的強度大於控制組：CT-DNA (3 個)；TW2.6 細胞(4 個)；唾液(2 個)。此 8 個 2'-dN 的離子訊號，透過標準品的確認，其中 2 個可鑑定出為 5-MedC 與 N6-MedA，皆與表觀遺 傳的調控有關[Hu et al. 2013; Huang et al. 2015]。而另外 6 個 2'-dN 訊號因缺乏典型的鹼基離子碎 片尚待進一步的鑑定。
表十五、CT-DNA 及 TW2.6 細胞暴露於白灰檳榔汁液及白灰檳榔嚼食者唾液的 2'-dN 綜合比較
22.70 288.1443 253.1062 (100%)
271.1175 [M-NH3+H]+ (96.5%)
22.73 223.1540 165.0546 (100%) 224.1274 (78.07%) 123.0664 [M-dR-HCN+H]+ (14.89%) 135.0539 [Ade]+ (2.46%)
148.0618 [Ade+C+H]+ (1.68%)
N6-MedA
v
37.85 327.1412 197.0786 (100%) 164.0807 (43.58%)
40.38 278.1750 260.1644 (100%) 164.1068 (80.79%) N6-MedA: N6-methyl-2'-deoxyadenosine
(3) 檳榔嚼食者唾液中 ASNAs 的量測結果
針對唾液中ASNAs 的量測，我們挑選 5 位檳榔嚼食者(年齡：35-64；BMI：20.9-28.0 kg/m2)進 行分析以初步探討台灣檳榔嚼食者族群唾液中ASNAs 的分佈。唾液經分析後，5 種 ASNAs 只測得 2 種(MNPN 與 NGCO)且集中於其中 2 位檳榔嚼食者。首先，MNPN 於 2 位嚼食者唾液中測得的濃 度分別為0.89 和 0.86 ng/mL；而 NGCO 則是在此 2 位中的其一被測得，其濃度為 4.04 ng/mL。
針對唾液中的ASNAs 量測，過去文獻報導可測得 MNPN、NGCO 與 NGCI 等 3 種[IARC 2004]，
相較下本研究只測得其中 2 種，推測可能先前研究皆使用 GC-TEA 分析法其特異性較低，可能受
唾液基質干擾而產生假陽性；抑或唾液中的 ASANs 雖存在但濃度低於本研究方法的偵測極限
(LOD) ： MNPN (0.34 ng/mL) 、 MNPA (0.48 ng/mL) 、 NGCI(0.45 ng/mL) 、 NNIP(0.05 ng/mL) 與 NGCO(0.17 ng/mL)。本研究測得的 MNPN 與 NGCO 的濃度與過去文獻發表的濃度範圍相符。對於 檳榔嚼食者唾液中ASNAs 檢出率偏低(5 位只測得 2 位)的原因，可能原因有：(1) 台灣食用未成熟 檳榔子，而過去文獻報導僅限於印度族群食用成熟檳榔種子，檳榔種類及添加物造成差異；(2)個體 間唾液基質不同而造成ASAs 亞硝化形成 ASNAs 的差異。未來本研究將再最佳化前處理流程，將 唾液蛋白質沉澱後以手動固相萃取(manual SPE)進一步純化，以並擴大分析樣本數以探討嚼食檳榔 者唾液中ASNAs 的含量。
六、成果自評
本研究成功以 LIT-Orbitrap MS 建立 DNA adductomics 分析法，並予以最佳化；建立資料依靠 (data-dependent, DD) 模 式 於 HRMS-based DNA adductomics 分 析 系 統 及 HRMS-based DNA adductomics 的通用訊號處理平台，可將離子層析資訊整理輸出並進行比對。所開發方法進一步運 用驗證於DNA 暴露直接甲基化劑及小鼠暴露於各種亞硝胺試驗的基因鍵結體學研究，並獲得 DNA adducts 的修飾基分子結構訊息。更重要的，我們運用 HRMS-based DNA adductomics 研究方法，以 高準確質量測定繪製adductome map，探討檳榔成分造成口腔黏膜細胞的 DNA adducts 全貌。經由 adductome map 的疊圖比對探究檳榔暴露造成 DNA 鹼基修飾的重要特徵，重新檢視檳榔嚼塊中潛 在的化學致癌因子。
在科技部3 年度的計劃經費支持下發表如下，SCI 期刊論文 6 篇，另有 1 篇論文投稿中：
1. Mu-Rong Chao, Marcus S. Cooke, Chung-Yih Kuo, Chih-Hong Pan, Hung-Hsin Liu, Hao-Jan Yang, Szu-Chieh Chen, Yi-Chen Chiang and Chiung-Wen Hu. Children are particularly vulnerable to environmental tobacco smoke exposure: Evidence from biomarkers of tobacco-specific nitrosamines, and oxidative stress. Environment International, 2018, 120, 238-245. (SCI; Impact factor = 7.297, Ranking in Environmental Sciences = 7/242 = 2.9%).
2. Marcus S. Cooke, Chiung-Wen Hu, Yuan-Jhe Chang and Mu-Rong Chao* (Corresponding Author).
Urinary DNA adductomics – A novel approach for exposomics. Environment International, 2018, 121, 1033-1038. (SCI; Impact factor = 7.577, Ranking in Environmental Sciences = 18/265 = 6.8%).
3. Chiung-Wen Hu, Marcus S. Cooke, Yuan-Jhe Chang and Mu-Rong Chao* (Corresponding Author).
Direct-acting DNA ethylating agents associated with tobacco use primarily originate from the tobacco itself, not combustion. Journal of Hazardous Materials, 2018, 358, 397-404. (SCI; Impact factor = 9.038; Ranking in Environmental Sciences = 8/265 = 3.0%).
4. Yuan-Jhe Chang, Marcus S. Cooke, Chiung-Wen Hu* and Mu-Rong Chao* (Co-corresponding Author). Novel approach to integrated DNA adductomics for the assessment of in vitro and in vivo environmental exposures. Archives of Toxicology, 2018, 92, 2665-2680. (SCI; Impact factor = 5.059, Ranking in Toxicology = 9/92 = 9.8%).
5. Tsu-Shing Wang, Cheng-Ping Lin, Yu-Pong Chen, Mu-Rong Chao, Chien-Chun Li and Kai-Li Liu.
CYP450-mediated mitochon drial ROS production involved in arecoline N-oxide-induced oxidative damage in liver cell lines. Environmental Toxicology, 2018, 33, 1029-1038. (SCI; Impact factor = 3.118; Ranking in Water Resources = 21/94 = 22.3%).
6. Chiung-Wen Hu, Yuan-Jhe Chang, Marcus S. Cooke and Mu-Rong Chao* (Corresponding Author).
DNA crosslinkomics: A tool for the comprehensive assessment of interstrand crosslinks using high resolution mass spectrometry. Analytical Chemistry, 2019, 91, 15193-15203. (SCI; Impact factor = 6.785, Ranking in Chemistry, analytical = 7/86 = 8.1%).
7. Yuan-Jhe Chang, Marcus S. Cooke, Yet-Ran Chen, Shun-Fa Yang, Pei-Shan Li, Chiung-Wen Hu* and Mu-Rong Chao* (Co-corresponding Author). Is High Resolution a Strict Requirement for Mass Spectrometry-Based Cellular DNA Adductomics? Journal of Hazardous Materials, 2020, Submitted.
研討會論文8 篇：
1. Pin-Hsuan Wang, Pei-Shan Li, Yuan-Jhe Chang, Marcus S. Cooke, Chiung-Wen Hu and Mu-Rong Chao*. Simultaneous analysis of urinary 8-oxo-7,8-dihydro-2’-deoxyadenosine and 2-hydroxy-2’-deoxyadenosine by LC-MS/MS. 7th Asia Oceania Mass Spectrometry Conference, pp. 128, December 11-13, 2017, Biopolis, Singapore.
2. Wan-Zhen Xiao, Chi-Syuan Liao, Chiung-Wen Hu, Mu-Rong Chao*. Analysis of areca-nut derived nitrosamines by LC-MS/MS with on-line solid-phase extraction. 7th Asia Oceania Mass Spectrometry Conference, pp. 210, December 11-13, 2017, Biopolis, Singapore.
3. Yi-Jhen Wang, Chi-Syuan Liao, Yuan-Jhe Chang, Chiung-Wen Hu, Mu-Rong Chao*. Simultaneous analysis of five areca nut-specific nitrosamines by isotope-dilution liquid chromatography-tandem mass spectrometry with on-line solid-phase extraction. The 67th Annual Conference on Mass Spectrometry, 1P-29, May 15-17, 2019, Tsukuba, Japan.
4. Pei-Shan Li, Yuan-Jhe Chang, Chiung-Wen Hu, Mu-Rong Chao*. Comprehensive characterization and identification of DNA adducts induced by formaldehyde with high-resolution mass spectrometry-based DNA adductomics approach. The 67th Annual Conference on Mass Spectrometry, 1P-32, May 15-17, 2019, Tsukuba, Japan.
5. Jia-Wei Jian, Yuan-Jhe Chang, Pei-Shan Li, Chiung-Wen Hu, Mu-Rong Chao*. Application of DNA adductomics approach for non-targeted screening formaldehyde-derived DNA adducts by LC-QqQ-MS/MS with constant neutral loss scanning. The 67th Annual Conference on Mass Spectrometry, 1P-33, May 15-17, 2019, Tsukuba, Japan.
6. Yuan-Jhe Chang, Marcus S. Cooke, Chiung-Wen Hu*, Mu-Rong Chao*. “Measurement of the totality of DNA adducts by mass-spectrometry-based omics approach”，2020 年第十七屆台灣質 譜學會年會暨學術研討會，pp. 73，September 1-3，嘉義。(Invited Lecture)
7. 杜姍容，張元哲，李佩珊，簡嘉葳，胡瓊文*，趙木榮*，“比較液相層析串聯質譜儀「恆定中
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