Protein Adducts as Biomarkers of Environmental Exposure to Naphthalene

本研究係利用生物性指標分析方法，針對萘之醌類化物與蛋白質所形成之共價鍵結產物-胼合物(protein adduct)，作為推估萘醌類化物於標的器官之組織計量。運用鹼性烷化分析方法(alkaline permethylation)，以碘甲烷為衍生劑在強鹼環境下催化，將萘之醌類化物與蛋白質上的cysteine之硫原子鍵結之胼合物自結構中移除，並經由溶劑萃取濃縮，利用氣相層析電子撞擊離子源(GC/EIMS)進行分析。本研究除了建立1,2-naphthoquinone (1,2-NPQ)與1,4-naphthoquinone (1,4-NPQ)對cysteine、glutathione (GSH)、bovine serum albumin (BSA)和人類血清白蛋白形成之胼合物之定性分析，也建立了運用高效能液相層析儀純化1,2-naphthoquinone-4-cysteine (1,2-NPQ-Cys.)，作為定量所需檢量線之標準品。同時對鹼性烷化在萘醌胼合物上之應用，也完成了其最佳化的反應條件測試，推估鹼性烷化之偵測極限在此研究中約為1 nmol。運用此一鹼性烷化分析法量測BSA於生理條件下與1,2-NPQ反應所生成protein adduct總量與添加1,2-NPQ濃度之關係為13.3μmol/g per μM。本研究中亦針對1,2-NPQ與1,4-NPQ於活體外與BSA和人類血清反應之一次移除速率常數(First-Order Elimination Rate constants)Ke進行探討，推估其值約略介於0.05-1.2 (h-1)，而其中1,2-NPQ與BSA及人類血清白蛋白質(serum albumin)之二次反應速率常數(Second-Order Reaction Rate conatants)之推估值，約介於3-60 (Lg-1h-1)。此外人類T47-D腫瘤細胞之實驗結果顯示，添加1,2-naphthalene catechol之人體乳癌細胞，以鹼性烷化法分析胞內蛋白質，可以偵測到1,2-NPQ-protein adduct，且1,2-NPQ -protein adduct之生成量與添加濃度與反應時間相關。而添加Fe(Ⅱ)之螯合劑dipyridyl(DPD)及Lipophilic Cu (Ⅰ)之螯合劑Neocuprion(NC)等抑制誘發氧化損害之試劑，結果顯示添加DPD會降低約57%的 NPQ-protein adducts之生成，但添加Cu (Ⅰ)則似乎不會影響NPQ-protein adducts之生成。The objective of this research is to develop a biochemical assay using protein adducts as biomarker of exposure to assess the cumulative body burden of naphthoquinones in target organs. The alkaline permethylation procedure using methyl iodine and sodium hydroxide as catalysts was applied to cleave cysteinyl adducts of naphthoquinones. The cleaved adducts are recovered by organic solvent extractions and analyzed by gas chromatography/electron-impacted mass spectrometer (GC-EIMS). Cysteinyl adducts of 1,2-naphthoquinone (1,2-NPQ) and 1,4-naphthoquinone (1,4-NPQ) on cysteines (Cys.), glutathione (GSH), bovine serum albumins (BSA), and human serum albumins are characterized by the alkaline permethylation procedure. Additionally, the synthetic cysteinyl adduct of 1,2-NPQ was further purified by HPLC-UV and was used as standards to quantify modified proteins. Results from the optimization of the alkaline permethylation procedure reveal that the optimal condition for adduct cleavage is when cysteine adducts of 1,2-NPQ react with 4N NaOH and 0.8 mL of CH3I at 80℃ for 4 hours. The limit of detection is estimated to be 1 nmol on column. Regression analysis of the concentration-adduct levels of indicates that the production of 1,2-NPQ-modified BSA adducts was estimated to be 13.3μmol/g per μM. Results from the reaction rate constants analyses indicated that for the elimination of naphthoquinones (1,2-NPQ and 1,4-NPQ) with proteins (First-Order Elimination Rate constants;Ke), values of ke were estimated to be between 0.05-1.2 (h-1). Additionally, kr (Second-Order Reaction Rate constants) were estimated to be between 3-60 (Lg-1h-1). When human T47-D breast cancer cells were incubated with 1,2-naphthalene catechol (1,2-NACT) (0 — 2.5 mM) at 37C for 1.5 h, formation of 1,2-NPQ-protein adducts was detected in cytoplasmic proteins of cells exposed to 1,2-NACT. Results also confirmed that 1,2-NACT was converted to 1,2-NPQ which subsequently binds to proteins. Regression analysis of the concentration-adduct relationship indicates that the production of protein adducts in T47D cells exposed to NCAT increases proportionally with increased concentration of NCAT (0－2.5 mM) (r2 =0.999 ).第一章 前言 1 第二章 文獻回顧 2 2-1 多環芳香族碳氫化合物 2 2-1-1 多環芳香族碳氫化合物的特性 2 2-1-2 多環芳香族碳氫化合物的產生 6 2-1-3 多環芳香族碳氫化合物在環境中的降解 7 2-1-4 多環芳香族碳氫化合物的代謝與毒性關係 10 2-2 萘之代謝途徑及致癌機制 14 2-2-1 萘之物化特性與健康危害 14 2-2-2 萘之代謝途徑與影響代謝之因子 16 2-2-3 萘醌類化物之致癌機制 18 2-3 蛋白質胼合物(Protein Adduct)作為生物性指標(Biomarker)之應用 20 2-3-1 生物指標之定義與分類 20 2-3-2 萘醌類化物之生物指標研究 22 2-4 運用蛋白質胼合物(Protein Adduct)作為生物指標之動力學模式 24 2-4-1 蛋白質胼合物(Protein Adduct)作為生物指標 24 2-4-2 蛋白質胼合物(Protein Adduct)的毒理學動力模式 26 第三章 實驗材料與方法 28 3-1 實驗材料 29 3-2 Naphthalene Quinone之Cysteine / Glutathione adduct 之合成 30 3-2-1 1,2-NPQ-4-Cysteine Adduct之製備 31 3-2-2 1,2-NPQ-4-Glutathione Adduct之製備 32 3-2-3 1,2-NPQ-4-BSA Adduct之製備 32 3-2-4 1,4-NPQ-2-Cysteine Adduct之製備 33 3-2-5 1,4-NPQ-2-Glutathione Adduct之製備 33 3-2-6 1,4-NPQ-2-BSA Adduct之製備 33 3-3 鹼性烷化之分析方法(Alkaline Permethylation) 34 3-4 氣相層析質譜儀(Gas Chromatography/Mass Spectrometer -Electron Impact)之分析條件 35 3-5 HPLC純化1,2-NPQ-Cysteine Adduct之條件設定 36 3-6 鹼性烷化條件之最佳化 37 3-7 鹼性烷化偵測之運用 38 3-7-1 Ke及Kr之動力學實驗方法 38 3-7-2 人類乳癌細胞株T47D之實驗方法 39 第四章 結果 40 4-1 萘之醌類衍生物與Cysteine、GSH和BSA生成腁合物(Adduct)之定性質譜圖 40 4-1-1 1,2-NPQ Thioether Adduct 40 (1)1,2-NPQ-cysteine adduct 40 (2)1,2-NPQ-GSH adduct 42 (3)1,2-NPQ-BSA adduct 44 4-1-2 1,4-NPQ Thioether Adduct 45 (1) 1,4-NPQ-cysteine adduct 45 (2) 1,4-NPQ-GSH adduct 47 (3) 1,4-NPQ-BSA adduct 48 4-2 運用HPLC純化標準品並製作檢量線 50 4-3 1,2-NPQ與BSA之劑量-效應關係 51 4-4 鹼性烷化衍生對1,2-NPQ-cysteine反應條件最佳化之探討 52 4-4-1 反應時間與溫度的影響 52 4-4-2 氫氧化鈉當量濃度的影響 53 4-4-3 碘甲烷用量的影響 54 4-5 1,2-NPQ與人類血清白蛋白(albumin)反應之結果 55 4-6 1,2-NPQ與BSA及人類血清白蛋白中之反應速率常數(Ke和Kr)之推估結果 56 4-7 1,2-NCAT 於T47-D乳癌細胞之實驗結果 57 4-7-1 1,2-NCAT的添加濃度於人體T47-D乳癌細胞形成NPQ-protein adducts之關係 57 4-7-2 1,2-NCAT與人體T47-D乳癌細胞反應時間形成NPQ-protein adducts之關係 58 4-7-3 添加Cu (Ⅰ)及Fe(Ⅱ)抑制劑於人體T47-D乳癌細胞對形成NPQ-protein adducts之影響 59 第五章 討論 61 第六章 結論與建議 66 第七章 參考文獻 6