- 學位論文
氣管灌注B[a]P對大鼠肝臟與肺臟內雌激素氫氧化活性之比較
Intratracheal Instillation of Benzo[a]pyrene Differentially Affects Estrogen Hydroxylation in the Liver and Lung of Rats
摘要
目前已知雌激素與其代謝物與多種癌症之形成有關，而benzo[a]pyrene (B[a]P)所活化的cytochrome P4501 (CYP1)酵素會促進17β-estradiol hydroxylation (E2 hydroxylation)，其中2-hydroxyestradiol (2-OHE2)及4-hydroxyestradiol (4-OHE2)具有基因毒性。在動物實驗B[a]P會促進母鼠的肺臟腫瘤形成，為了探討B[a]P是否在肝與肺增加E2 hydroxylase酵素活性，因此本研究首先建立E2及其代謝物(2-OHE2、4-OHE2)之氣相層析質譜儀(GC/EI-MS)分析方法，用於偵測經4 mg/kg B[a]P氣管灌注處理後24小時的Sprague-Dawley (SD) 大鼠，其肝臟與肺臟內E2 2-hydroxylase與E2 4-hydroxylase之酵素活性是否上升。我們建立的分析方法的偵測極限依序E2為8 pg；2-OHE2和4-OHE2皆為4 pg。而在酵素反應實驗，我們偵測到在肝臟中，公鼠與母鼠對照組部分E2 2-hydroxylase活性分別為107.5與35.0 pmol/min/mg而E2 4-hydroxylase之酵素活性分別為1.3與0.4 pmol/min/mg；經過B[a]P處理過後E2 2-hydroxylase酵素活性分別為67.1與24.5 pmol/min/mg而E2 4-hydroxylase之酵素活性分別為1.0與0.3 pmol/min/mg，結果顯示，公大鼠之E2 hydroxylase酵素活性較母鼠高約3倍，另外經B[a]P處理過後公、母大鼠肝臟之E2 hydroxylation活性皆呈現下降的現象。肺臟部分，公鼠與母鼠對照組部分E2 2-hydroxylase酵素活性分別為0.24與0.22 pmol/min/mg，而E2 4-hydroxylase之酵素活性分別為0.03與0.05 pmol/min/mg；經過B[a]P處理過後E2 2-hydroxylase之酵素活性分別增加為0.51與0.46 pmol/min/mg；E2 4-hydroxylase之酵素活性分別增加為0.06與0.073 pmol/min/mg。與肝臟結果不同的是，公母大鼠於肺臟內之酵素活性相似，另一方面經B[a]P處理後2- and 4-E2 hydroxylase活性皆明顯地上升，這顯示B[a]P對於肺臟的腫瘤形成機轉，或許與增加E2 hydroxylation活性的酵素有關。 由於GC/EI-MS靈敏度不足以進行肝、肺與血液中內生性雌激素與其代謝物之分析，因此為了探討經B[a]P氣管灌注處理24小時後是否造成肝、肺與血液中之內生性estrone (E1)、E2與其相關代謝物濃度產生變化，我們在此利用API3000建立經dansyl chloride衍生化之液相層析串聯式質譜儀 (LC/MS/MS)分析方法，以期望經衍生化後能更進一步增加儀器偵測靈敏度，結果顯示經衍生化後E2與其代謝物偵測極限可達5 fg/μL以下，先前文獻所測得之停經期婦女血液內生性E2濃度約為20 fg/μL，因此使用此方法應足以進行分析，有繼續開發下去的價值。
並列摘要
Many studies have pointed out that estrogen and its metabolites (2- and 4-hydroxylated estrogen) involve in the formation of many cancers. Benzo[a]pyrene (B[a]P), an animal carcinogen, is known to increase cytochrome P4501 (CYP1) enzymes activities, which catalyze 17β-estradiol (E2) hydroxylation. In animal studies, B[a]P induced liver and lung tumors formation in female mice. The objective of this thesis was to compare effects of intratracheal instillation of B[a]P on activities of E2 hydroxylation in the liver and the lung of rats. First, we established a gas chromatographic /electron impact-mass spectrometry (GC/EI-MS) analysis method for quantification of E2 and its metabolites (2-OHE2, 4-OHE2). In our analysis method, detection limit for E2 is 8 pg; 2-OHE2 and 4-OHE2 are 4 pg. Rats were intratracheally instilled with 4 mg/kg B[a]P. At 24 weeks later, we utilized E2 as the substrate to quantify activities of E2 2- and 4-hydroxylation in the liver and lung. In the liver, E2 2-hydroxylase activity was higher in male (107.5 pmol/min/mg) than in (35.0 pmol/min/mg) female controls. Similarly, E2 4-hydroxylase activity was higher in male (1.3 pmol/min/mg) than in female (0.4 pmol/min/mg) controls. After B[a]P treatment for 24 hr, E2 2-hydroxylase activities respectively were reduced to 67.1 and 24.5 pmol/min/mg in the livers of male and female rats. Similarly, E2 4-hydroxylase activities were respectively reduced to 1.0 and 0.3 pmol/min/mg in the livers of male and female rats. These results showed that E2 hydroxylase enzyme activity of the male rat were approximately 3 times higher than the female rats. Moreover, treatment with B[a]P reduced E2 hydroxylation activity in the liver. In the lung, there was no gender difference in E2 2- and 4-hydroxylase activities. After B[a]P treatment, both E2 2 and 4-hydroxylase activities were significantly increased in the lung of male and female rats. It implied that B[a]P induced E2 hydroxylase activities might a role in lung carcinogenesis in females. However, the sensitivity of GC/EI-MS is insufficient to quantify the endogenous estrogen and its metabolites in the liver, the lung and the blood. Later, we established a liquid chromatography-mass spectrometry/mass spectrometry (LC/MS/MS) analysis method. E2 and its metabolites were derived by dansyl chloride before analysis. The detection limit was lowered to 5 fg/μL. It is more sensitive than reported in literatures - 20 fg/μL. Therefore, this method should be used for quantifying endogenous estrogen and its metabolites in the future.