EPA-93-E3S5-02-02

093

1,260 千元

2005/03/16

2005/12/31

陳福安

環檢所

藥學系 陳福安

環境監測及檢測

塑化劑、雌激素效應、MVLN細胞

陳福安

08-7624002 ext. 328

fachen@ccsun.tajen.edu.tw

本計畫以美國環保署公告方法4425為品管要求進行MVLN雌激素專一性轉錄分析法之建立與標準化，其評估項目包括冷光酵素檢量線建立、檢體溶劑選擇與用量影響評估、檢體暴露時間影響評估、雌激素標準品檢量線建立、檢測感度與精密度。冷光酵素標準曲線線性方程式為y＝2274.6x－1.6944（0.01 ~ 0.16 pg/ml），相關係數（r2）達0.9985，有很好的線性關係，精密度RSD ％ 範圍1.40 ~ 14.43 ％，冷光酵素偵測極限可達0.01 pg/ml。DMSO、Methylene chloride（MECL）、Hexane（HX）、Methanol（MEOH）、Isooctane（ISOT）與Acetone（ACT）六種溶劑對MVLN之細胞毒性大小順序為HX＞DMSO＞ACT MEOH＞MECL ISOT，要讓MVLN細胞存活率低於80 ％之溶劑濃度分別為HX 0.5 ％、DMSO 2 ％、ACT 8 ％、MEOH 8 ％、MECL＞8 ％、ISOT＞8 ％。MVLN細胞暴露於雌激素(E2)下之冷光值會隨著E2的濃度而上升，具濃度效應，其標準曲線均呈Sigmoidal curve，約1 nM已達最大效應。MVLN細胞暴露於雌激素 1、2、3天之濃度-相對雌激活性圖樣與RSD ％並無太大差異，整體實驗重覆數3~6次之RSD ％範圍為0.18 ~ 15.23 ％。另MVLN細胞暴露於雌激素 1、2、3天之細胞檢測感度分別為1.59 pM、0.29 pM及2.54 pM。本計畫完成方法確立後進行42種塑化劑之雌激素活性檢測與四種塑化劑間的交互作用評估，結果發現42種塑化劑中有五種塑化劑Butyl benzyl phthalate（ BBP，10 uM）、Bis（4-methyl-2-pentyl）phthalate (BMPP，10 uM)、Diethoxyethyl phthalate（DEEP，1,10 uM）、Hexyl-2-ethylhexyl phthalate (HEHP，10 uM) 及Monobenzyl phthalate（MBP，10 uM）呈現顯著雌激素效應（p<0.01）。這五種的對雌激素的相對力價分別為BBP（1/2.44×106）、BMPP（1/1.42×106）、DEEP（1/4.50×105）、HEHP（1/3.76×106）及MBP（1/1.81×106），此結果顯示這五種塑化劑是屬弱雌激性化學物質。在交互作用方面，結果初步推論BBP、BMPP、DEEP、HEHP可能沒有發生物種間與對雌激素間的交互作用。

塑化劑、雌激素效應、MVLN細胞

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[期末報告]

2005/3/1 上午 12:00:00

This plan established and standardized the MVLN estrogen specific transcription assay according to QC/QA criteria of USEPA method 4425. The evaluated items included establishment of luciferase standard curve, evaluation of solvent selection for sample and used quantity, evaluation of exposure time of sample, establishment of 17b-estradiol (E2) standard curve, and detection sensitivity and precision. The luciferase standard curve gave the equation of y = 2274.6X -1.6944 after linear regression（0.01 ~ 0.16 pg/ml）. The correlation coefficient of the linear regression analysis achieved 0.9985. The precision and detection limit were 1.40 ~ 14.43 %（RSD） and 0.01 pg/ml, respectively. The cytotoxicity of MVLN to DMSO, methylene chloride（MECL）, hexane（HX）, methanol（MEOH）, isooctane（ISOT）and acetone（ACT）decreased in the order of HX＞DMSO＞ACT  MEOH＞MECL  ISOT. The luciferase induction was increased as the concentration of E2 increased. The standard curve showed sigmoidal pattern having Cmax at 1 nM. Exposure time of MVLN cells to E2 (1~3 days) did not affect significantly their concentration-estrogenicity plots. The precision of E2 in 3 to 6 replicates were 0.18 ~ 15.23 %. The detection limit for E2 exposure time of 1, 2 and 3 days were 1.59 pM, 0.29 pM and 2.54 pM, respectively. The method was further applied to estimate the estrogenicity of 42 plasticizers and interactions between 4 plasticizers. The results revealed that 5 plasticizers as butyl benzyl phthalate（BBP，10 uM）、bis（4-methyl-2-pentyl）phthalate (BMPP，10 uM)、diethoxyethyl phthalate（DEEP，1,10 uM）、hexyl-2-ethylhexyl phthalate (HEHP，10 uM) and monobenzyl phthalate（MBP，10 uM）possessed significant estrogenicity (p<0.01). In case of interaction study, the results implied that BBP, BMPP, DEEP, HEHP and E2 have possibly no interaction each other.