| Summary: | 碩士 === 國立臺灣大學 === 毒理學研究所 === 90 === Territrem (territrem A.B.C) is a tremogenic mycotoxin isolated from the chloroform extracts of the sub-merged rice culture of Aspergillus terreus 23-1. The previous studies indicated that three metabolites, designated as MA1, MAX MA2, were obtained when TRA was the substrate in liver microsomes of adult male Wistar rats. However, only MA1 was formed from TRA in liver microsomes of adult female rats. From chemical inhibition, immuno inhibition and CYP3A1/2 supersomes studies, suggested that CYP3A1/2 mainely involved in TRA metabolism. When TRB was the substrate, four metabolities, designated as MB1, MB2, MB3 and MB4, were formed in liver microsomes of adult male Wistar rats.
Territrems were producted by Aspergillus terreus, which contamined in the stored unhalled rice in Taiwan. Therefore, the metabolism of territrems in human liver microsomes may have same as in liver microsomes of rat. CYP3A4 amino acid sequences was 73% homologous with CYP3A1. Therefore, the studies were undertaken using V79MZh3A4 cell lines, in which human cytochrome P450 3A4 were expressed. The methods used in the present investigation are following: (1) the cytotoxicity of TRA, TRB or testosterone (2) the metabolism of TRA, TRB or testosterone (3) the enzyme kinetic study for TRA, TRB or testosterone (4) the inhibition study between TRA, TRB and testosterone.
In these studies, I have obtained the following results. (1) 5μM TRA, 5μM TRB or 100 μM testosterone had no cytotoxicity. (2) The immunobloting assay showed that CYP3A4 protein was detected in V79MZh3A4 cells, but not in V79MZ cells. (3) V79MZh3A4 cells could expressed the activities of testosterone 6β-hydroxylase. Otherwise, the testosterone 6β-hydroxylase was not determined in V79MZ cells. (4) V79MZh3A4 cells metabolized TRA to MA1, and also metabolized TRB to MB2 and MB4, but not in V79MZ cells. (5) In the enzyme kinetics study, V79MZh3A4 cells could metabolize TRA judged from the value of Vmax/Km of MA1 calculatedly activity, and TRB judged from the value of Vmax/Km of MB2 more than MB4 calculatedly activity. (6) In the inhibition study, the rate of production of MB2 that is formed by TRA to TRB is a noncompetitive inhibitor reaction; the rate of production of MB4 is a uncompetitive inhibitor reaction. However, testosterone and TRA form MA1 or testosterone and TRB form MB2 and MB4 all have inhibition. And yet, the relationship of inhibition still needs more experiments to prove. Therefore, it is concluded that the different metabolic reaction performed due to different substitution of chemical structure of territrems. For instances, cytochrome P450 3A4 is responsible for 4βC-hydroxylation of TRA ,and 4βC-hydroxylation, O-demethylation of TRB.
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