| Summary: | 碩士 === 國立臺灣大學 === 毒理學研究所 === 87 === 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.
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