- 學位論文
人體肝臟細胞色素P450同功酶參與土震素B和土震素C代謝作用之研究
Role of Human Hepatic Cytochrome P450s in Territrem B and C Metabolism
摘要
土震素為土麴菌編號23-1 (Aspergillus terreus 23-1) 經米培養，由氯仿萃取分離純化出土震素A、B、C (TRA、TRB、TRC) 三種震顫性黴菌毒素，過去研究指出土震素A可經由成熟雄性大鼠肝臟微粒體代謝生成MA1、MAX以及MA2，而雌性大鼠僅能代謝至MA1；土震素B於兩性大鼠肝臟微粒體中均可被代謝成MB1、MB2、MB3、MB4 (同土震素C)，TRC再經由氫氧化作用 (hydroxylation) 產生MC (同MB1)，但其代謝物產量則有年齡及性別上的差異性。然後由不同細胞色素P450同功酶之化學、抗體抑制劑以及supersomes確定CYP3A1/2為主要參與TRA、TRB及TRC代謝的P450酵素，其中CYP3A1只參與TRA至MA1的步驟。 土震素為早期在台灣穀倉黴米中篩選出的黴菌Aspergillus terreus 23-1菌株在米培養產生的毒素，目前已知土震素A可經由人肝臟微粒體代謝生成MA1，且證實CYP3A4為主要參與TRA代謝作用的P450酵素。爲了探討在人體肝臟中細胞色素P450同功酶參與TRB與TRC代謝作用之情形，因此本實驗採用人肝臟微粒體及以基因工程轉殖並表現人類細胞色素P450 3A4的中國倉鼠肺臟纖維母細胞 (Chinese hamster lung fibroblast) V79衍生之V79MZh3A4細胞株，作為探討對於土震素B及土震素C代謝研究的生物體外模式。 研究結果顯示：（1）人肝臟對於TRB之代謝作用，可經由4β-C hydroxylation生成MB2及O-demethylation生成MB4，且MB2之產量均大於MB4；對於TRC之代謝作用，則經由4β-C hydroxylation代謝生成MC。人類肝臟對於TRB或是TRC的代謝作用，和人類之性別及年齡均無顯著相關性。（2）藉由化學抑制劑、抗體抑制劑、V79MZh3A4細胞株及以不同細胞色素P450同功酶之cDNA利用baculovirus轉植入insect cell (BTI-TN-5B1-4) 之supersomes的實驗進一步證實CYP3A4/5為主要協助人體對於TRB與TRC進行代謝作用的P450同功酶。（3）在V79MZh3A4細胞株對TRB、TRC和testosterone相互競爭抑制之作用探討中得知，testosterone對TRB代謝生成MB2過程的影響為競爭性抑制作用 (competitive inhibition)，testosterone對TRB代謝生成MB4過程的影響為未競爭抑制作用 (uncompetitive inhibition)；相對TRB對testosterone代謝生成6β-hydroxytestosterone過程的影響為mixed inhibition。Testosterone與TRC之間則互相為混合型抑制作用 (mixed inhibition)。（4）在探討人肝臟微粒體中CYP3A4/5對於TRB 4β-C hydroxylation及TRB O-demethylation之參與情形的實驗結果得知，由基因重組系統及人肝臟微粒體所求得的CLCYP3A4及CLCYP3A5值，可更加證實了在人肝臟微粒體中，CYP3A4是趨向參與TRB 4β-C hydroxylation生成MB2過程之P450酵素，而CYP3A5是趨向參與TRB O-demethylation生成MB4過程之P450酵素。
並列摘要
Territrem A, B and C, the structure related tremogenic mycotoxins isolated from the chloroform extracts of rice culture of Aspergillus terreus 23-1. The previous study on metabolism of Territrem A (TRA) by liver microsome from Wistar rats showed that three metabolites, MA1, MAX and MA2 were formed in male rats, but only one metabolite, MA1 in female rats. The studies were further carried with specific chemical and antibody inhibitors for CYP isoforms and the sypersomes expressed with specific type of CYP isoforms. The results indicated that CYP3A1, which is dominated in female rat liver, plays a mean role in metabolic pathway from TRA to MA1 and that CYP3A2, which is dominated in male rat liver, plays the mean role in metabolic pathway of TRA to MA1, MAX and MA2. On the other hand, four metabolites, such as MB1, MB2, MB3 and MB4 (which structure is the same as Territrem C (TRC) ) were formed from Territrem B (TRB) and one metabolite, MC (which structure is the same as MB4) from Territrem C respectively by liver microsome from each of male and female rats. In order to elucidate the role of human hepatic CYP isoforms in TRB and TRC, the experiments were performed with several enzyme sources such as from different age and sex, V79MZh3A4 cell line derived from Chinese hamster, in which human CYP3A4 were expressed, and several supersomes having enzyme or different tyoe of CYP isoforms. The following are the resultes obtained： (1) In human liver microsome, MB2, and MB4 were main products from TRB and MC from TRC. There were no age and gender difference in ability to metabolize TRB and TRC. (2) CYP3A4/5 had the major role in metabolism of TRB and TRC. (3) The rate of production of MB2 from TRB or MC from TRC were competitively inhibited by the present of testosterone. However the rate of the production of 6β-hydroxytestosterone from testosterone were inhibited by the present of TRB or TRC in mixed type of inhibition (non competitive and non uncompetitive inhibition). The rate of the production of MB4 from TRB was also inhibited by the present of testosterone in mixed type of inhibition. (4) It suggested that CYP3A4 had the major role in 4β-C hydroxylation of TRB, and CYP3A5 had the major role in O-demethylation of TRB.