臺灣博碩士論文加值系統

摘 要
土震素A ( territrem A; TRA ) 為Aspergillus terreus 23-1菌株經米培養，以氯仿萃取分離的震顫性黴菌毒素之一。本實驗室先前的研究指出，土震A在成熟六週大的老鼠以phenobarbital前處理的肝臟微粒體中有MA1與MA2兩種代謝物產生。因此，王 (1995) 認為細胞色素P450同功CYP2C11、2B1/2和3A1/2參與土震素A的代謝。然而，其真正的代謝過程與何種P450酵素參與仍然尚未釐清。本研究之目的在於確認大鼠肝臟微粒體中參與土震素A代謝的P450酵素與其代謝途徑。
為了證實這個問題，我們以互相比較的研究方式來探討土震素A與testosterone在相同組別的大鼠肝臟微粒體中之代謝，而testosterone在P450的代謝方式則在過去的文獻中已被探討。所使用的方法有：(1) 肝臟微粒體為兩週大 (雄性) 與六週大 (雄性與雌性) 的Wistar大鼠及經由phenobarbital (PB) 或dexamethasone (DEX) 處理 (2) 代謝受質為土震A、MA1與testosterone，經由與肝臟微粒體及NADPH的反應後以HPLC定量其代謝產物 (3) 以化學抑制劑cimetidine、orphenadrine與SKF-525A及免疫抑制以CYP2C11、2B1/2和3A1/2的抗血清來研究其代謝受質抑制作用。
研究結果指出 (1) 無論前處理PB或DEX的雌性或雄性大鼠其肝臟微粒體中均能夠增加其testosterone 6b-, 16a-, 16b-與2a-hydroxylase的活性，如此推測CYP2C11、2B1/2和3A1/2酵素活性可被PB或DEX誘導及增強 (2) 兩週大的雄性Wistar大鼠肝臟微粒體中只有testosterone 6b-hydroxylase活性表現 (3) 由免疫染色分析結果可知經PB與DEX處理的雌性或雄性大鼠其肝臟微粒體中CYP2B和3A蛋白都有增加 (4) 在以土震A與MA1為受質的肝臟微粒體代謝研究發現，在雄性大鼠前處理PB與DEX其代謝物MA1、MI與MA2都增加，於雌性大鼠則只有MA1產生 (5) 由化學抑制劑cimetidine、orphenadrine與SKF-525A及免疫抑制血清CYP2C11、2B1/2和3A1/2 antiserums的研究分析，在成熟的雄性大鼠肝臟微粒體中testosterone 6b-, 16a-, 16b-與2a-hydroxylase的活性都受到抑制，但於testosterone 6b-hydroxylase活性受抑制時其土震素A之代謝物MA1、MI與MA2才受抑制。
綜合以上研究結果，我們證實 (1) 雄性大鼠肝臟微粒體土震素A的代謝首先經水解作用 (hydroxylation) 產生MA1，而後經氧化作用 (oxidation) 產生MI，最後再經脫醛作用 (decarbonylation) 產生MA2。而雌性大鼠肝臟微粒體土震素A的代謝僅能行水解作用產生MA1，而無法再經氧化及脫醛作用產生MI及MA2 (2) 而由兩週與六週大的雄性大鼠之化學與免疫抑制研究推論，CYP3A1/2為主要參與土震素A代謝P450酵素。由此推斷，於雄性Wistar成熟大鼠肝臟微粒體土震素A的代謝過程中，CYP3A2佔了95%以上的角色，而CYP3A1只參與在土震素A至MA1的代謝步驟。

Abstract
Territrem A (TRA) is a tremogenic mycotoxin isolated from the chloroform extracts of the sub-merged rice culture of Aspergillus terreus 23-1. The previous studies from our laboratory have indicated that two metabolites designated as MA1 and MA2 were found from TRA by liver microsomes of phenobarbital-pretreated adult male of 6 weeks old Wistar rats. It was then suggested by Wang (1995) that cytochrome P450 isoforms such as CYP2C11, 2B1/2 and 3A1/2 may be involved in TRA metabolism. However, its real patterns of metabolism and of which kind of P450 involved are still not clarified. The aim of the present investigation is to elucidate the P450 isoforms and define its metabolic pathway involved in the metabolism of TRA by rat liver microsomes.
In order to solve the problems, the comparative study of metabolizing TRA and testosterone were carried out with the same rat liver microsomes, since the metabolic pathway and P450 involved in testosterone in rat liver microsomes were well documented in the past literature. The methods used in the present investigation are following: (1) Liver microsomal fractions used, are obtained from 2 weeks (male) and 6 weeks (both adult male and female) Wistar rats. (2) Substrates used are TRA, MA1 and testosterone. Their metabolites are quantified by HPLC after incubation of the reaction mixture including the above described substrates, microsomes and NADPH. (3) For inhibition study, chemicals such as cimetidine, orphenadrine and SKF-525A, and polyclonal antiserums for CYP2B1, CYP3A2 and CYP2C11 are used.
In these studies, we have observed the following results. (1) Pre-treatment of phenobarbital (PB) and dexamethasone (DEX) of both adult male and female Wistar rats could enhance the activities of testosterone 6b-, 16a-, 16b- and 2a-hydroxylase in their liver microsomes, that is interpreted as the increase of the activities of CYP3A1/2, 2B1/2 and 2C11. (2) By immunoblotting method, it is confirmed that the proteins of CYP3A and CYP2B increase in liver microsomes of both adult male and female rats after PB and DEX treatment. However, increase of CYP2C11 protein was only observed in liver microsomes of PB-pretreated adult male rats but not in those of adult female rats. (3) With cimetidine, orphenadrine and SKF-525A and polyclonal antiserums for CYP2B1, CYP3A2 and CYP2C11, the all activities of testosterone 6b-, 16a-, 16b- and 2a-hydroxylase in liver microsomes of adult male Wistar rats are inhibited. (4) In microsomes of 2 weeks old male Wistar rats, only testosterone 6b-hydroxylase is observed. (5) When TRA is used as the only substrate in the experiments with liver microsomes of PB and DEX pre-treated adult Wistar rats, it is shown that the products MA1, MA2 and the new compound designated as MI, increase in male ones but only MA1 produced in female ones. However, when MA1 is the substrate in the same experimental conditions, the products MI and MA2 increased in male ones but they are not found in female ones. (6) With the analysis of LC-MS of MI fraction, the molecule weight of MI is 524. Moreover, when MI used as the substrate, it is shown that MA2 is produced. Therefore, we conclude that MI is the intermediate product between MA1 and MA2. (7) When TRA is used as the substrate with liver microsomes of 2 weeks old male Wistar rats, MA1, MI and MA2 are formed as the products. (8) With cimetidine and polyclonal antiserums, the metabolism of TRA and MA1 in 2 weeks old male rat liver microsomes, are completely inhibited.
Therefore, it is concluded that (1) TRA transforms to MA1 via hydroxylation, then to MI via oxidation and finally to MA2 via decarbonylation in the adult male rat liver microsomes. However, in adult female rat liver microsomes, TRA transforms only to the stage of MA1 via hydroxylation without further metabolism. (2) From chemical and immuno inhibition study, we propose that CYP3A1/2 may be the major P450 isoenzyme of TRA metabolism in liver microsomes of 2 weeks and adult Wistar male rats, since that both of their testosterone 6b-hydroxylase activity and TRA and MA1 metabolism are also inhibited. We also suggest that CYP3A2 has major role (probably up to 95%) in metabolic pathway from TRA to MA2 as described above in male adult Wistar rats and CYP3A1 may be involved in the metabolic pathway from TRA to MA1.

目 錄
中文摘要…………………………………………………….. I
英文摘要………………………………………………….....III
縮寫表………………………………………………………..V
第一章 誘導劑誘發大鼠肝臟微粒體之細胞色素P450同功
…………………………………………………….1
第二章 土震素A及其代謝產物之分析…………………...19
第三章 土震素A在大鼠肝臟微粒體中的代謝反應及其酵素動力學之研究………………………………………30
第四章 不同細胞色素P450同功參與土震素A代謝之研究……………………………………………………39
第五章 總結…………………………………………………53
第六章 參考文獻……………………………………………58
圖表…………………………………………………………..66

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