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標題: 探討環境中多環芳香烴碳氫化合物及戴奧辛之暴露對人體細胞及血清中萘及雌性激素醌纇代謝物蛋白質腁合物之影響
Investigation of the effects of environmental exposure to PAHs and dioxin on the production of naphthalene and estrogen derived protein adducts in human cultured cell and in human serum derived from blood donors in taiwan
作者: 林政佑
Lin, Cheng-yu
關鍵字: naphthalene;雌性激素;estrogen;protein adducts;蛋白質腁;合物
出版社: 環境工程學系所
The objective of this research is to develop a biochemical assay using protein adducts as biomarker of environmental exposure to polycyclic aromatic hydrocarbons (PAHs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). This methodology ultilizes trifluoroacetic acid (TFAA) and methanesulfonic acid (MSA) as catalysts to cleave cysteinyl adducts of naphthalene- and estrogen -derived quinones on proteins. The cleaved adducts are recovered by organic solvent extractions and analyzed by gas chromatography- negative-ion- chemical -ionization-mass spectrometer (GC-NICI-MS). The isomeric forms of cysteinyl adducts of naphthoquinones, including 1,2-naphthoquinone (1,2-NPQ) and 1,4-naphthoquinone (1,4-NPQ) as well as estrogen quinones, including estrogen-2,3-quinones (2-OHE2) and estrogen-3,4-quinones (4-OHE2), are characterized by using a modified procedure after adducts cleavage by the TFAA/MSA. Additionally, we refined the original protocol to purify synthetic cysteinyl adducts of estrogen and naphthalene quinones by HPLC-UV and were used as standards to quantify the modified proteins. The first part of this study was to refine the modified MT assay to analyze background level of naphthalene quinine-derived in human serum albumin and to measure the cysteinlyl adducts of naphthalene quinones in cellular proteins derived from human breast cancer cells (MCF-7) exposed to naphthalene-derived quinonoids. The second part of this study was to apply the modified MT assay to analyze the background levels of estrogen quinine-derived in human serum albumin. The third part of this study was to to develop a method for the simultaneous analysis of naphthalene and estrogen quinone-derived protein adducts in human serum albumin. Overall, we conclude that the methodology developed in this study may be applied to epidemiological study to serve as biomarkers of environmental exposure to PAHs and modulators of estrogen homeostasis.
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