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標題: 以蛋白質胼合物作為生物性指標以推估五氯酚醌類代謝物在標的器官組織劑量之研究
Application of Protein Adducts as Biomarkers to Investigate the Dosimetry of Quinonoid Derivative of Pentachlorophenol
作者: 林蔚宗
Lin, wel-jung
關鍵字: 五氯酚;PCP;蛋白質胼合物;protein adducts
出版社: 環境工程學系
本研究係利用生物性指標分析方法,針對五氯酚之醌類化合物與蛋白質所形成之共價鍵結產物-胼合物(protein adducts),作為推估五氯酚醌類化物於標的器官之組織劑量。本研究中建立一運用鹼性烷化分析方法(alkaline permethylation),以碘甲烷(CH3I)為衍生劑在強鹼環境下催化,將五氯酚之醌類化物與蛋白質上之cysteine之硫原子鍵結產生之胼合物,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與化學離子源質譜儀(GC-EI-MS/GC-NICI-MS),進行定性及定量分析。本研究除了建立Tetrachloro-1,2-benzoquinone(Cl4-1,2-BQ)與Tetrachloro-1,4-benzoquinone(Cl4-1,4-BQ)對N-Acetyl-cysteine (NAC)、glutathione (GSH)、bovine serum albumin (BSA)、和人類乳癌細胞MCF-7之細胞質內蛋白質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Cl3-1,2-BQ-Y(NAC)與Cl3-1,4-BQ-Y(NAC),作為定量所需檢量線之標準品。同時完成對以鹼性烷化衍生法於五氯酚之醌類蛋白質胼合物上之應用,其最佳化之反應條件測試,並推估鹼性烷化衍生法對五氯酚之偵測極限在此研究中約為1 pmole。運用此一鹼性烷化分析法量測於生理條件下,BSA與Cl4-1,2-BQ及Cl4-1,4-BQ反應所生成protein adduct總量與添加Cl4-1,2-BQ、Cl4-1,4-BQ濃度關係,分別為32.3 pmol/g per pM與38.3 pmol/g per pM。在PCP於人類乳癌細胞實驗中,其細胞蛋白質可偵測到Cl3-1,2-BQ-Y、Cl3-1,4-BQ-Y、Cl4-1,2-SQ-Y及Cl4-1,4-SQ-Y之胼合物。本研究中亦針對Cl4-1,2-BQ 與Cl4-1,4-BQ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0.282-36.4 (h-1),而其與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 constants)之推估值,約介於0.067-3.028 (Lg-1h-1)。

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 pentachlorophenol (PCP)-derived benzoquinones and benzosemiquinones in target organs. The alkaline permethylation procedure using methyl iodine and sodium hydroxide as catalysts was applied to cleave cysteinyl adducts of pentachlorophenol-derived quinonoids. The cleaved adducts are recovered by organic solvent extractions and analyzed by gas chromatography-electron-impact/negative-ion-chemical-ionization-mass spectrometer (GC-EI-MS/GC-NICI-MS). Cysteinyl adducts of tetrachloro-1,2-benzoquinone(Cl4-1,2-BQ)and tetrachloro-1,4-benzoquinone(Cl4-1,4-BQ) on n-acetyl-cysteine (NAC), glutathione (GSH), bovine serum albumins (BSA), and cytoplasmic proteins derived from human MCF-7 breast cancer are characterized by the alkaline permethylation procedure. N-acetyl-cysteinyl adducts of Cl4-1,2-BQ and Cl4-1,4-BQ were synthesized and purified by HPLC-UV and were used as standards to quantify modified proteins. Results from the optimization of the alkaline permethylation procedure reveal that the optimal condition for adducts cleavage is when cysteinyl adducts of PCP-derived quinones react with NAOH/CH3I at 80℃ for 4 hours. The limit of detection is estimated to be 1 pmole on column. Regression analyses of the concentration-adduct levels indicated that the production of Cl4-1,2-BQ and Cl4-1,4-BQ-modified BSA adducts were estimated to be 32.3 pmol/g per pM and 38.3 pmol/g per pM, respectively. Results from the reaction rate constants analyses indicated that for the elimination of pentachlorophenol-derived quinones (Cl4-1,2-BQ and Cl4-1,4-BQ) with proteins (first-order elimination rate constants;ke), values of ke were estimated to be between 0.282-36.4 (h-1) whereas the second-order reaction rate constant, kr) were estimated to be between 0.067-3.028 (Lg-1h-1).
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