Development of a chemical assay to analyze the protein adducts of estrone (E1) quinone adducts on human blood proteins

雌酮-2,3-醌類代謝物 (E1-2,3-Q) 和雌酮-3,4-醌類代謝物 (E1-3,4-Q) 為雌性激素之活性代謝產物，並且雌性激素認為會誘發遺傳毒性。本研究為建立一化學分析方法，將雌性激素之雌酮 (estrone, E1) 醌類化合物，作為環境暴露累積組織劑量之生物指標。本研究以TFAA (trifluoroacetic anhydride) 作為衍生劑，在強酸環境下催化，將雌性激素之醌類化物與蛋白質上之Cysteine之硫原子鍵結產生之胼合物，自蛋白質結構中切除，並經由溶劑萃取濃縮，再利用氣相層析電子撞擊與負離子化學離子化質譜儀 (GC/EI/MS及GC/NICI/MS)，進行定性與定量分析。運用此分析方法，在時間效應實驗結果顯示， E1-3,4-Q-2-S-Alb和E1-2,3-Q-4-S-Alb之蛋白質胼合物都會迅速在五分鐘即達到最大鍵結量，而隨著時間加長並沒有顯著之差異。運用此分析方法，對小牛血清白蛋白和人類血清白蛋白分析其背景值。實驗結果顯示，人類血清白蛋白 (n=5) 之E1-3,4-Q-2-S-Alb和E1-2,3-Q-4-S-Alb蛋白質胼合物平均值分別為71.5及265 pmol/g，而小牛血清白蛋白 (n=5) 之E1-3,4-Q-2-S-Alb以及E1-2,3-Q-4-S-Alb 蛋白質胼合物平均值46.4及118 pmol/g。將人類乳癌細胞株MCF-7以TCDD預處理(10 nM, 72小時)後，添加雌性激素及共同添加COMT抑制劑。實驗結果顯示，共同添加COMT抑制劑相較於單獨暴露於雌性激素之實驗組，其結果發現前者比後者增加3倍之雌性激素醌類蛋白質胼合物。進一步的分析台灣女性乳癌患者 (n=10) 與健康婦女 (n=10)　血清白蛋白胼合物之背景值。研究結果顯示乳癌患者相較於健康人控制組之背景值約高出2倍左右。本研究預期此一蛋白質胼合物分析法，將能作為一高效率之生物指標，提供流行病學中評估雌性激素代謝活化之干擾物質長期暴露族群之研究。Both estrone-2,3-quinone (E1-2,3-Q) and estrone-3,4-quinone (E1-3,4-Q) are reactive metabolites of estrogen that are thought to be responsible for the estrogen-induced genotoxicity. The objective of this research is to develop a biochemical assay using estrone (estrone, E1) quinone adducts as biomarker of exposure to assess the cumulative body burden of estrogen-derived quinones in target organs. This method ultilizes trifluoroacetic anhydride (TFAA) as catalysts to cleave cysteinyl adducts of estrogen-derived quinones on proteins. The cleaved adducts are recovered by organic solvent extractions and analyzed by gas chromatography-electron-impact/negative-ion-chemical-ionization-massspectrometer (GC-EI-MS/GC-NICI-MS). Time-course experiments suggested that both E1-2,3-Q-4-S-Alb and E1-3,4-Q-2-S-Alb rapidly reached maximum values at 5 min mark and remained constant thereafter. We applied this methodology to analyze the background levels of estrone quinone-derived adducts on bovine serum albumin (BSA) and human serum albumin (HSA). Results showed that cysteinyl adducts of E1-2,3-Q-4-S-Alb and E1-3,4-Q-2-S-Alb were detected in HSA (n=5) with mean levels at 71.5 and 265 (pmol/g), respectively. Similarily, we detected these adducts in BSA (n=5) with mean levels at 46.4 and 118 pmol/g for E1-2,3-Q-4-S-Alb and E1-3,4-Q-2-S-Alb, respectively. In human MCF-7 breast cancer cells, with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) pretreatment (10 nM for 72 h), production of estrogen quinone-derived protein adducts was detected in cells exposed to E1. Co-treatment of a catechol-o-methyl transferase (COMT) inhibitor further enhanced the production of estrogen quinone-derived adducts (~3 fold). Further investigation indicated that the background levels of these adducts in human albumin (Alb) derived from female breast cancer patients (n=10) were ~2 fold greater than those of healthy controls (n=10). Overall, we concluded that the methodology developed in this study may be applied to epidemiological study to serve as biomarkers of environmental exposure to modulators of estrogen homeostasis.目錄 摘要 i Abstract ii 縮寫表 (List of Abbreviation) iv 目錄 vi 表目錄 x 圖目錄 xii 第一章 前言 1 1-1 研究起源 1 1-2 研究目的 2 第二章 文獻回顧 3 2-1 雌性激素 3 2-1-1 雌性激素環境流布及影響 6 2-1-2 雌性激素代謝機制 9 2-1-3 雌性激素去活化 13 2-1-4 雌性激素代謝物毒性 16 2-1-5 雌性激素與乳癌之相關性 18 2-2 戴奧辛 20 2-2-1 戴奧辛之性質與環境流布 20 2-2-2 戴奧辛之毒性與健康傷害 21 2-2-3 戴奧辛之致癌機制 25 2-2-4 戴奧辛之暴露效應 27 2-2-5 戴奧辛與雌性激素之關係 28 2-3 蛋白質胼合物(Protein adduct) 30 2-3-1蛋白質胼合物種類 30 2-3-2 胼合物於分子流行病學之應用 32 2-4 生物指標(Biomarker) 34 2-4-1 生物指標之定義與應用 34 2-4-2 利用蛋白質胼合物作為生物指標 35 第三章 實驗架構與設計 37 第四章 實驗材料及方法 40 4-1 實驗材料 40 4-1-1 水 40 4-1-2 化學品及耗材 40 4-1-3 實驗設備 42 4-1-4 細胞株來源 42 4-2 實驗方法 43 4-2-1 2/4-OH E1-N-acetyl-L-Cysteine Adducts之合成 43 4-2-2 E1 /E2-N-acetyl-L-Cysteine Adducts製備 44 4-2-3 E1 /E2-HSA Adducts製備 45 4-2-4 E1-d4雌性激素同位素蛋白質胼合物內標準品製備 46 4-2-5 TFAA derivatization分析方法 48 4-3氣相層析質譜儀分析條件 49 4-4 HPLC純化E1-NAC Adducts之條件 52 4-5透析同位素內標準品 54 4-5-1 血清白蛋白透析 54 4-5-3 同位素內標準品透析 55 4-6 雌性激素醌類蛋白質胼合物之時間效應 56 4-7 COMT對雌性激素醌類蛋白質胼合物影響之細胞實驗 57 4-7-1 試藥處理 57 4-7-2 實驗步驟 57 4-9分析驗證之程序 57 4-9-1檢量線 57 4-9-2偵測極限 58 4-10 統計分析 58 4-10苗栗大千醫院健康婦女之背景資料 59 4-11彰化基督教醫院乳癌婦女之背景資料 59 第五章 實驗結果 60 5-1 雌性激素醌類化物與蛋白質胼合物之定性圖譜 61 5-1-1 E1-NAC adduct 之定性圖譜 61 5-1-2 E1-HSA adduct 之定性圖譜 62 5-1-3 E1-d4-HSA adduct 同位素內標準品之定性圖譜 63 5-2 運用HPLC純化雌性激素醌類化物與蛋白質胼合物製作檢量線及定量分析 73 5-2-1 TFAA/MSA分析之最低偵測極限 73 5-3 分析由Sigma公司所購置血清白蛋白之雌性激素雌酮醌類蛋白質胼合物之背景值 79 5-4 雌性激素雌酮醌類蛋白質胼合物之時間效應實驗 83 5-5 雌性激素雌酮醌類蛋白質胼合物對乳癌細胞株生成之影響 85 5-6 苗栗大千醫院健康婦女之血清白蛋白雌性激素醌類蛋白質胼合物背景值 89 5-7 彰化基督教醫院乳癌婦女之血清白蛋白雌性激素醌類蛋白質胼合物背景值 91 第六章 討論 93 6-1 定性用離子之評估與選擇 93 6-2 雌酮醌類adduct同分異構物之判定 94 6-3 雌性激素雌酮醌類之血清白蛋白胼合物之化學性質 98 6-4 戴奧辛環境曝露及COMT酵素對於雌性激素雌酮醌類蛋白質胼合物生成之影響 100 6-5 苗栗大千醫院健康婦女與彰化基督教醫院乳癌病人雌性激素醌類蛋白質胼合物之背景值比較 102 第七章 結論與建議 108 7-1 結論 108 7-1-1 雌性激素雌酮E1醌類蛋白質胼合物之化學特性 108 7-1-2 人類乳癌細胞株MCF-7之細胞實驗 108 7-1-3 醫院婦女之雌性激素醌類蛋白質胼合物 109 7-2 未來研究方向與建議 110 參考文獻 11

Hemoglobin adducts as biomarkers of estrogen homeostasis: Elevation of estrogenquinones as a risk factor for developing breast cancer in Taiwanese Women

The aim of this study was to establish a methodology to analyze estrogen quinone-derived adducts, including 17β-estradiol-2,3-quinone (E2-2,3-Q) and 17β-estradiol-3,4-quinone (E2-3,4-Q), in human hemoglobin (Hb). The methodology was then used to measure the levels of these adducts in Hb derived from female breast cancer patients (n=143) as well as controls (n=147) in Taiwan. Our result confirmed that both E2-2,3-Q- and E2-3,4-Q-derived adducts, including E2-2,3-Q-4-S-Hb and E2-3,4-Q-2-S-Hb, were detected in all breast cancer patients with median levels at 434 (215-1472) and 913 (559-2384) (pmol/g), respectively. Levels of E2-2,3-Q-4-S-Hb correlated significantly with those of E2-3,4-Q-2-S-Hb (r=0.622-0.628, p<0.001). By contrast, median levels of these same estrogen quinone-derived adducts in healthy controls were 71.8 (35.7-292) and 139 (69.1-453) (pmol/g). This translated to ~6-fold increase in mean values of E2-2,3-Q-4-S-Hb and E2-3,4-Q-2-S-Hb in breast cancer patients compared to those in the controls (p<0.001). Our findings add further support to the theme that cumulative body burden of estrogen quinones is an important indicator of breast cancer risk. We hypothesize that combination of genetic events and environmental factors may modulate estrogen homeostasis and enhance the production of estrogen quinones which lead to subsequent generation of pro-mutagenic DNA lesions in breast cancer patients

Hemoglobin adducts as biomarkers of estrogen homeostasis:Elevation of estrogenquinones as a risk factor for developingbreast cancer in Taiwanese Women

The aim of this study was to establish a methodology to analyze estrogen quinone-derived adducts,including 17 -estradiol-2,3-quinone (E2-2,3-Q) and 17 -estradiol-3,4-quinone (E2-3,4-Q), in humanhemoglobin (Hb). The methodology was then used to measure the levels of these adducts in Hb derivedfrom female breast cancer patients (n = 143) as well as controls (n = 147) in Taiwan. Our result confirmedthat both E2-2,3-Q- and E2-3,4-Q-derived adducts, including E2-2,3-Q-4-S-Hb and E2-3,4-Q-2-S-Hb,were detected in all breast cancer patients with median levels at 434 (215–1472) and 913 (559–2384)(pmol/g), respectively. Levels of E2-2,3-Q-4-S-Hb correlated significantly with those of E2-3,4-Q-2-S-Hb(r = 0.622–0.628, p < 0.001). By contrast, median levels of these same estrogen quinone-derived adducts inhealthy controls were 71.8 (35.7–292) and 139 (69.1–453) (pmol/g). This translated to ∼6-fold increasein mean values of E2-2,3-Q-4-S-Hb and E2-3,4-Q-2-S-Hb in breast cancer patients compared to those inthe controls (p < 0.001). Our findings add further support to the theme that cumulative body burden of estrogen quinones is an important indicator of breast cancer risk. We hypothesize that combination ofgenetic events and environmental factors may modulate estrogen homeostasis and enhance the produc-tion of estrogen quinones which lead to subsequent generation of pro-mutagenic DNA lesions in breastcancer patients