本實驗所建立的實驗方法，如酵母菌轉殖株篩選實驗，土鯽魚活體實驗，
皆可敏感有效進行環境荷爾蒙的偵測，並可推廣到其他魚類的應用。酵母菌轉 殖株篩選實驗除可用於疑似環境荷爾蒙的大量快速篩選，尚可仿效國外發表的 文獻加以改良用於野外水體的快速測定；而在活體實驗方面，卵黃前質測定方 法的建立可用於野外土鯽魚的長期監控，判別所採的環境樣品是否遭到環境荷 爾蒙污染之依據，尚可與本實驗的結果進行比較分析而更瞭解環境荷爾蒙所造 成的影響。
另 外 ， 苦 花 的 雌 性 激 素 受 體 基 因 亦 在 NCBI (http://www.ebi.ac.uk/submission/webin.html) 網站註冊，11 種污染物對酵母菌轉 殖株的轉錄活化分析亦在 IWA 的國際會議上[21]進行口頭報告，除了酵母菌轉 殖株實驗結果，土鯽魚活體混合浸泡實驗兩兩混合皆為加成效應與 Arukwe et al.及 Thorpe et al.等實驗結果不同，將進行分析探討並投稿至國際期刊，綜上可 知，本研究之成果基本上有達成預期目標，研究內容與計畫亦符合，研究成果 亦有相當高的應用價值。
參考文獻
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65:143-150.
11. Kime, D.E. 1999. A strategy for assessing the effects of xenobiotics on fish reproduction. Sci. Total Environ.225:3-11.
12. Knudsen, F.R., Arukwe, A. and Pottinger, T.G. 1998. The in vivo effect of
combinations of octylphenol, butylbenzylphthalate and estradiol on liver estradiol receptor modulation and induction of zona radiata proteins in rainbow trout: no evidence of synergy. Environ. Pollut. 103:75-80.
13. Tully, D.B., Cox, V. T., Mumtaz, M.M., Davis, V.L. and Chapin, R.E. 2000. Six high-priority organochlorine pesticides, either singly or in combination, are nonestrogenic in transfected HeLa cells. Reprod. Toxicol. 14: 95-102.
14. Arukwe, A., Celius, T., Walther, B.T. and Goksoyr, A. 2000. Effects of xenoestrogen treatment on zona radiata protein and vitellogenin expression in Atlantic salmon (Salmo Salar). Aquat. Toxicol. 49: 159-170.
15. Stelzer, A. and Chan, H.M. 1999. The relative estrogenic activity of technical toxaphene mixture and two individual congeners. Toxicology. 138: 69-80.
16. Graumann, K., Breithofer, A. and Jungbauer, A. 1999. Monitoring of estrogen mimics by a recombinant yeast assay: synergy between natural and synthetic compounds? Sci. Total Environ. 225:69-79.
17. Rehmann K., Schramm K.W. and Kettrup A.A. 1999. Applicability of a yeast oestrogen screen for the detection of oestrogen-like activities in environmental samples. Chemosphere. 38(14): 3303-3312.
18. Chang, C. F., Lau, E. L., Lin, B. Y. and Jeng, S. R. 1996. Characterization of vitellogenin induced by estradiol-17β in protandrous black porgy, Acanthopagrus
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丁望賢、吳建誼，周瓊瑤，王正雄， 2000 第一屆環境荷爾蒙與持久性有機 污染物研討會：pp 153-155. 台大，臺北
20. Thorpe, K.L., Hutchinson, T.H., Hetheridge, M.J., Scholze, M., Sumpter, J.P.
and Tyler, C.R. 2001. Assessing the biological potency of binary mixtures of environmental estrogens using vitellogenin induction in juvenile rainbow trout (Oncorhynchus mykiss). Environ Sci Technol. 35(12): 2476-81.
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圖 1、環境荷爾蒙的分子作用機制。內分泌干擾物（endocrine disrupting chemicals）會模擬雌性激素（estrogen）與雌性激素受體結合（ER），進而誘發 不正常的基因表現
圖 2 雌性激素受體基因全長之選殖流程圖
圖 3、苦花(Varicorhinus barbatulus) 雌性激素受體 alpha (ERα) 的核酸序列 及轉譯出蛋白質
圖 4、苦花(Varicorhinus barbatulus) 雌性激素受體 beta (ERβ) 的核酸序列 及轉譯出蛋白質
0
solvent control
0 500 1000 1500 2000
control-01 control-02
Vitellogenin (ug/ml)
圖 7、土鯽魚活體實驗溶劑控制組（solvent control）之卵黃前質生成量長條圖
E2
0 5000 10000 15000
(1nM) (5nM) (10nM) (20nM)
concentration
Vitellogenin (ug/ml)
圖 8、土鯽魚活體實驗 E2 之卵黃前質生成量長條圖
BPA
0 5000 10000 15000 20000
(0.5uM) (1uM) (5uM) (10nM)
concentration
Vitellogenin (ug/ml)
圖 9、土鯽魚活體實驗 BPA 之卵黃前質生成量長條圖
4NP
0 2000 4000 6000 8000 10000 12000
(0.1uM) (0.5uM) (1uM) (5uM)
concentration
Vitellogenin (ug/ml)
圖 10、土鯽魚活體實驗 4NP 之卵黃前質生成量長條圖
E2+BPA
(0.5nM+0.25uM) (2.5nM+0.5uM) (5nM+2.5uM) (10nM+5uM)
concentration
(0.5nM+0.05uM) (2.5nM+0.25uM) (5nM+0.5uM) (10nM+2.5uM)
concentration
Vitellogenin (ug/ml)
圖 12、土鯽魚活體實驗 E2+4NP 之卵黃前質生成量長條圖
BPA+4NP
0 2000 4000 6000 8000 10000 12000 14000 16000
(0.25uM+0.05uM) (0.5uM+0.25uM) (0.25uM+0.5uM) (5uM+2.5uM) concentration
Vitellogenin (ug/ml)
圖 13、土鯽魚活體實驗 BPA+4NP 之卵黃前質生成量長條圖
表 1、日本環境廳公佈之疑似內分泌干擾物質一覽表
表 1、日本環境廳公佈之疑似內分泌干擾物質一覽表（接續）
表 2、環境荷爾蒙混合效應相關文獻
表 3、E2， 4-NP， 4-t-OP ，BPA 對不同酵母菌轉殖株參數(ligand potency and ligand efficiency)整理
Yeast contained ERα Yeast contained ERβ Chemical
Potency (M) Efficiency
(Miller units) Potency (M) Efficiency (Miller units) E2 4.1×10-9 160 2.9×10-9 112 4-NP 6.4×10-7 90 6.3×10-7 26 4-t-OP 4.1×10-7 73 7.5×10-7 32 BPA 1.0×10-5 75 9.1×10-5 35
表 4、土鯽魚活體實驗溶劑控制組卵黃前質生成平均值、標準差、變異係數 控制組 solvent control 控制組 01 控制組 02
卵黃前質平均值 (ug/ml) 867 1192
標準差 513.160 366.217
變異係數(%) 59 31
表 5、土鯽魚活體實驗浸泡 E2、BPA、4NP 兩星期後卵黃前質生成平均值、標 準差、變異係數
E2 (浸泡濃度) 1nM 5nM 10nM 20nM
卵黃前質平均值 (ug/ml) 1233 4088 7650 12192 標準差 563.656 1887.459 2031.971 1885.733
變異係數(%) 46 46 27 15
BPA (浸泡濃度) 0.5uM 1uM 5uM 10nM
卵黃前質平均值 (ug/ml) 1983 3421 8046 14296 標準差 910.729 1125.579 1404.976 2913.019
變異係數(%) 46 33 17 20
E2+BPA 0.5nM+0.25uM 2.5nM+0.5uM 5nM+2.5uM 10nM+5uM 卵黃前質平均值 (ug/ml) 1967 5000 10317 20108 標準差 1245.325 1166.905 1389.544 3098.219
變異係數(%) 63 23 13 15
E2+4NP 0.5nM+0.05uM 2.5nM+0.25uM 5nM+0.5uM 10nM+2.5uM
卵黃前質平均值 (ug/ml) 1817 6100 9283 11367
標準差 1027.538 1561.249 1778.576 2081.065
變異係數(%) 57 26 19 18
BPA+4NP 0.25uM+0.05uM 0.5uM+0.25uM 0.25uM+0.5uM 5uM+2.5uM
卵黃前質平均值 (ug/ml) 600 1267 9300 11767
標準差 556.776 1051.586 1302.881 1576.653
變異係數(%) 93 83 14 13
表 7、土鯽魚活體實驗混合浸泡實驗(E2+BPA、E2+4NP、BPA+4NP)預測加成 反應卵黃前質生成量
E2+BPA 0.5nM+0.25uM 2.5nM+0.5uM 5nM+2.5uM 10nM+5uM
卵黃前質平均值 (ug/ml) 1608 3755 7848 13244
E2+4NP 0.5nM+0.05uM 2.5nM+0.25uM 5nM+0.5uM 10nM+2.5uM
卵黃前質平均值 (ug/ml) 1608 3746 5879 10744
BPA+4NP 0.25uM+0.05uM 0.5uM+0.25uM 0.25uM+0.5uM 5uM+2.5uM
卵黃前質平均值 (ug/ml) 2181 3005 6077 11796