臺灣博碩士論文加值系統

在魚類血漿中，雖然雌雄魚皆可發現卵黃前質（Vitellogenin），但正常雄魚體內之濃度遠較雌魚為低，惟若暴露在疑似雌性激素干擾物下，雄魚血漿中卵黃前質將會異常提升，呈現高於正常雄魚的雌性化現象。烷基酚類化合物（Alkylphenol polyethoxylates, APEs ）包括壬基苯酚（4-Nonylphenol, NP）和辛基苯酚（4-tert-Octylphenol, OP）被懷疑為雌性激素干擾物－環境荷爾蒙的一群人造化合物。本研究主要目的是要了解魚體在不同的暴露途徑下，觀測烷基酚類化合物是否為國內本土雄性鯉魚（Cyprinus carpio, Carp）之雌性激素化學物質，並進而探討其影響的劑量反應關係。
本研究利用雄性鯉魚為實驗對象，以肌肉注射、餵食和水中暴露等作為暴露途徑，每組至少有八條魚，雌二醇（17α-Ethynylestradiol, EE2）是天然之雌性激素，在試驗中為暴露雌性激素之環境荷爾蒙的對照試劑，使用酵素免疫分析法（ELISA）分析血漿中卵黃前質，試驗時使用一次抗體稀釋倍數為10000倍，二次抗體稀釋倍數為5000倍。
研究結果顯示，無論以注射（4mg/1.4kg）、餵食（2.85μg/day）和水中暴露（40μg/L.day）三暴露途徑，均證明雌二醇、壬基苯酚和辛基苯酚確實會使雄魚雌性化，且此效應在兩週後即表現出來，但是比起雌二醇，壬基苯酚和辛基苯酚兩者為弱的雌性激素，注射雌二醇、壬基苯酚、辛基苯酚與同期對照組分別為883.81（n=8）、8.21（n=8）、2.18（n=7）及1.45μg/mL（n=8）；餵食雌二醇、壬基苯酚、辛基苯酚與同期對照組分別為7568.66（n=4）、12.22（n=4）、49.39（n=2）及1.45μg/mL（n=8）；水中暴露雌二醇、壬基苯酚和辛基苯酚與同期對照組分別為6910.62（n=8）、10.89（n=5）、6.40（n=5）及1.45μg/mL（n=8）。而在劑量-反應實驗部份，本研究的最低劑量（4μg/L.day）下，水中暴露壬基苯酚和辛基苯酚及雌二醇均使雄性鯉魚造成雌性化效應，最遲四週即可使血漿中卵黃前質顯著上升，但其產生的劑量-反應曲線變化複雜，不一定為直線，又本研究的壬基苯酚最低劑量組（4μg/L.day），出現卵黃前質濃度大量上升的效應，因此應再進一步降低劑量濃度，探討更低劑量的反應。本研究對於辛基苯酚（OP）和壬基苯酚（NP）何者對鯉魚雌性化影響的效應較強，尚無明確定論。

Vitellogenin, a yolk-precursor protein normally found only in the blood plasma of sexually mature female teleosts and other egg-laying vertebrates, was used as an indicator of exposure of male carp（Cyprinus carpio）to exogenous estrogens. Alkylphenol polyethoxylates（e.g., 4-Nonylphenol（NP）and 4-tert-Octylphenol （OP））are probable estrogenic. The purposes of this study were twofolds：to examine the estrogenic effect of different alkyphenolic compounds to male carp, and to clarify the dose-response relationship.
In this experiment, groups of adult male carp（n=8-10）held in grass tanks（110L）were maintained in clean water, either in the absence of chemical, or in the presece of various alkylphenolic chemicals, for periods of 2-6 weeks. The carp was treated through three exposure routes, including injection（4mg/1.4kg）, feeding（2.85μg/day）, and water exposure（40μg/L.day）. 17α-Ethynylestradiol（EE2）served as a positive control. Blood plasma was analyzed for vitellogenin content using an enzyme-linked immunosorbent assay（ELISA）. A titer of primary antibody of 1:10,000 with a second antibody conjugate of 1:5,000 was chosen for the routine assay.
This study demonstrated that all of the alkylphenolic chemicals were weakly estrogenic and could cause very significant elevations in the concentrations of vitellogenin measured in the plasma of exposed fish. The vitellogenin concentratons of male carp exposed to EE2, NP, OP（through injection）, and control groups（no treatment）were 883.81μg/mL（n=8）, 8.21μg/mL（n=8）, 2.18μg/mL（n=7）and 1.45μg/mL（n=8）respectively; their vitellogenin concentratons were 7568.66μg/mL（n=4）, 12.22μg/mL（n=4）, 49.39μg/mL（n=2）and 1.45μg/mL（n=8）through feeding; and the concentratons were 6910.62μg/mL（n=8）, 10.89μg/mL（n=5）, 6.40μg/mL（n=5）and 1.45μg/mL（n=8）through water exposure. Dose-response study showed a complicated response. The response curve was not linear. The estrogenic effect appeared in four weeks for both NP and OP. Furthermore, the lowest treated concentration of NP（4μg/L.day）had significantly elevated the plasma vitellogenin levels of the male carp. Therefore, the estrogenic effect of NP and OP for lower concentrations should be studied further.

目 錄 i
表目錄 iii
圖目錄 iv
摘 要 v
Abstract vii
簡寫一覽表 ix
第一章 前言 1
第一節 研究緣起 1
第二節 研究目的 3
第二章 文獻探討 4
第一節 環境荷爾蒙的危機 4
第二節 環境荷爾蒙物質-烷基酚類化合物之研究 10
第三節 烷基酚類化合物影響之相關研究 12
第四節 環境荷爾蒙物質檢測的方法 15
第五節 雌性化指標-卵黃前質 16
第三章、材料與方法 17
第一節 烷基酚類化合物（NP、OP）與雌二醇（EE2）三者，在不同暴露途徑下之雌性化效應 17
第二節 烷基酚類化合物與雌二醇對雄性鯉魚雌性化影響的劑量-反應關係 24
第三節 血漿卵黃素前質濃度之分析 26
第四節 統計分析 31
第四章、結果 32
第一節 酵素免疫分析測試結果 32
第二節 烷基酚類化合物（NP、OP）與雌二醇（EE2）三者，在不同暴露途徑下之雌性化效應 32
第三節 烷基酚類化合物與雌二醇對雄性鯉魚雌性化影響的劑量-反應關係 37
第五章 討論 41
參考文獻 45

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