NDMA (mM) for 24 hr
4.4 小鼠模式下 NDMA 對蛋白質甲基化及解毒酵素蛋白表現之影響
NDMA 處理人類細胞血管內皮 EA.hy926 細胞及 WRL-68 肝細胞，均可造成細胞內明顯 ROS 產生量大幅增加的氧化壓力，並且造成細胞死亡，為進一步了解 NDMA 處理對動物體內 解毒代謝及抗氧化酵素系統可能造成之改變，本計畫利用 BALB/c 小鼠進行探討。以四週齡 BALB/c 小鼠腹腔注射 NDMA，每週 3 次連續 5 週，結果發現肝臟組織蛋白總甲基化程度，在 注射濃度 10 mg/kg 組蛋白質甲基化程度相較於對照組顯著增加約 28%(圖十八)，NDMA 處理 在小鼠肝臟造成三個甲基化標的蛋白質(圖十九)。
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除蛋白甲基化的改變之外，NDMA 處理也對小鼠肝臟中多種蛋白質表現照成明顯的影響，
表現增加的蛋白質包括：(1)伴護蛋白 GRP75 (75 kDa Glucose regulated protein)、(2)MnSOD (Manganese-dependent superoxide dismutase)、(3)CAT (Catalase)、(4)GCLC (Glutamate–cysteine ligase catalytic subunit) 、 (5) GCLM (Glutamate–cysteine ligase modifier subunit) 、 (6)GSTp (Glutathione S-transferase pi)，表現下降的蛋白質包括(7)GPX1 (Glutathione peroxidase 1)、
(8)HO-1 (Heme oxygenase 1)，(9)GR (Glutathione reductase)的表現程度則不受影響(圖二十)
圖十八、二甲基亞硝胺造成小鼠肝臟蛋白質甲基化之免疫圓點轉漬分析。四週齡小鼠經 5 mg/kg 或 10 mg/kg NDMA 注射，每週 3 次連續 5 週，於第 6 週結束詴驗，犧牲小鼠摘取肝臟製備蛋 白質溶解液，進行免疫西方轉漬分析，測量蛋白質的甲基化之程度。
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圖十九、二甲基亞硝胺對小鼠肝臟蛋白質甲基化之影響。四週齡小鼠經 5 mg/kg 或 10 mg/kg NDMA 注射，每週 3 次連續 5 週，於第 6 週結束詴驗，犧牲小鼠摘取肝臟製備蛋白質溶解液，
進行免疫西方轉漬分析，測量蛋白質的甲基化圖譜。
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圖二十、二甲基亞硝胺對小鼠肝臟伴護蛋白解毒酵素及抗氧化酵素表現的影響。四週齡小鼠經 5 mg/kg 或 10 mg/kg NDMA 注射，每週 3 次連續 5 週，於第 6 週結束詴驗，犧牲小鼠摘取肝 臟製備蛋白質溶解液，進行免疫西方轉漬分析，測量個別蛋白質的表現程度。
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(A) protein methylation
(B) GRP75 methylation
(C) MnSOD 量。數據為三次詴驗帄均值。(A)protein methylation, (B)GRP75/75 kDa glucose regulated protein, (C)MnSOD/Mn-dependent superoxide dismutase, (D)CAT/Catalase, (E)GPX1/Glutathionn peroxidase 1, (F)HO-1/Heme oxygenase 1, (G)GCLC/Glutamate–cysteine ligase catalytic subunit、
(H) GCLM/Glutamate–cysteine ligase modifier subunit 、 (I)GR/Glutathion reductase, (J) GSTp/Glutathione S-transferase pi。
100 et al., 2012; Kang et al., 2007; Yamazaki et al., 1992)，某些文獻則又指出單胺氧化酶(monoamine oxidase/MAO)及黃素單胺氧化酶(flavin monooxygenase/FMO)也都扮演重要角色(Lake et al., 1982; Phillips et al., 1982)。本計畫結果發現 CYP450、FMO 及 MAO 的選擇性抑制劑，都能有 效地抑制 NDMA 在 WRL-68 肝細胞造成 ROS 產生量增加，然而，根據抑制劑可能作用的重疊 性判斷，CYP450 酵素及單胺氧化酶(MAO)對 NDMA 代謝產生 ROS 的角色是同等重要的，FMO 抑制劑因同時具有強烈抑制 CYP450 的活性，角色重疊重要性較低。更有趣且重要的發現是 NDMA 誘發的 ROS 可被粒線體專一性抗氧化劑(Mito-tempo)完全抑制，這顯示 NDMA 誘發的 ROS 產量增加與粒線體的關係密切，綜合這些發現，我們提出一個新且重要可能分子機制， 白質進出粒線體扮演重要的伴護(chaperon)功能(Liu et al., 2015; Horst et al., 1997)，該蛋白質也 是串聯內質網與粒線體並控制鈣離子恆定的主角(Szabadkai et al., 2006)。據此我們認為 GRP75 可能與 CYP450 2E1 的粒線體轉運有關，NDMA 處理透過 GRP75 協助 CYP450 2E1 進入粒線 體並代謝 NDMA，造成 ROS 產量增加，同時其代謝產物(如甲基重氮烷陽離子/methyldiazonium ion)也讓 GRP75 蛋白發生甲基化並降低其原有蛋白伴護的能力，進而誘發該蛋白質的表現程度 (圖二十一 A & B)，這項發現也再一次說明粒線體極可能是 NDMA 在哺乳動物細胞作用的標 的。
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圖二十二、二甲基亞硝安在哺乳動物細胞可能之代謝途徑 (Fournier et al., 2006)。
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圖二十三、二甲基亞硝胺透過粒線體中代謝酵素產生 ROS 造成細胞氧化傷害可能之作用機 制。
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(五) 成果自評
1. 利用二維西方轉漬及質譜分析技術，以及免疫沉澱技術成功定義二甲基亞硝胺 NDMA 在人 類臍靜脈内皮 EA.hy926 细胞主要的甲基化蛋白標的：GRP75 蛋白，這是首次且重要的發 現，進一步研究發現 GRP75 蛋白的甲基化現象，也同樣出現在人類 WRL-68 肝細胞，除此 monoamine oxidase/MAO、flavin-containing monooxidase/FMO 以及粒線體選擇性抗氧化劑，
成功建立粒線體可能是 NDMA 主要的作用胞器，粒線體外膜 MAO 及基質內 CYP450(因逆 境壓力誘導轉入粒線體基質)聯合代謝 NDMA 造成細胞 ROS 產量增加，這項發現首次提出 粒線體氧化傷害在 NDMA 致病機制上的角色，也提出因逆境壓力誘導轉入粒線體基質的 CYP450，尤其是 2E1 型可能在 NDMA 的代謝致病機制上扮演重要角色，這些發現對發展 NDMA 的致病危害防治將提供新的思考方向。
1 發表的論文有 2 篇，分別為 Tobacco-specific nitrosamine NNK and its metabolite NNAL in patients with chronic obstructive pulmonary disease 及 Urinary levels of oxidative stress biomarkers in patients with mechanical ventilation。我們除了張貼壁報論文外，公衛系專題生廖啟軒同學及蔡依虹專任助理也 在 English Poster Discussion 各自進行 5 分鐘的專題報告並回答現場學者提問。
二、與會心得
肺部疾病發炎可造成呼吸器官硝化壓力(nitrosative stress)升高。此次會議有不少學者討論呼吸 器官中產生 Reactive nitrogen species (RNS)造成肺部分子損傷，並且開發出各式生物偵測指標(如 3-nitrotyrosine)來監測 nitrosative stress。RNS 與體內胺類反應也可生成亞硝胺，但目前仍未有質譜 分析技術。在我們的「食品中致癌性亞硝胺之快速篩檢與暴露危害評估」研究計畫中，我們已能 成功分析尿液中的各式亞硝胺，該技術未來將嘗試朝向運用於肺積水分析，以協助探討肺部發炎 所產生致癌物。