The metabolism of territrem A (TRA) was investigated in liver microsomes of male Wistar rats. The results indicated that three metabolites were produced from TRA and these metabolic reactions were inhibited by metyrapone, an inhibitor of cytochrome P-450. Based on analysis by high-performance liquid chromatography (HPLC), mass, and nuclear magnetic resonance (NMR) spectroscopic techniques, the structure of these metabolites were identified as 4beta-hydroxymethyl-4beta-demethylterritrem A (MA1), 4beta-oxo-4beta-demethylterritrem A (MAX), and 2-dihydro-4beta-demethylterritrem A (MA2). It was proposed that reactions proceeded by three sequential oxidative reactions in the pyran moiety of TRA: first, hydroxylation at the 4beta-C methyl group of TRA to form MA1; second, oxidation at the 4beta hydroxyl group of MA, to form MAX; and third, decarbonylation at the 4beta-C oxo group of MAX to form MA2.
The cytochrome P-450 isoforms involved in territrem A (TRA) metabolism in liver microsomes of male Wistar rats have been characterized. Pretreatment with phenobarbital (PB) or dexamethasone (DEX) resulted in a similar significant increase in TRA metabolic activity. Although PB treatment resulted in a significant elevation in CYP2B, CYP2C11, and CYP3A levels, only CYP3A levels were significantly increased by DEX treatment. Cimetidine markedly reduced the formation of the TRA metabolites 4beta-hydroxymethyl-4beta-demethylterritrem A (MA(1)), 4beta-oxo-4beta-demethylterritrem A (MAX) and 2-dihydro-4beta-demethylterritrem A (MA(2)) in liver microsomes from 2-wk-old rats (mainly containing CYP3A2) and 7-wk-old rats (containing CYP2B, CYP2C11, and CYP3A2). SKF 525A, which inhibits CYP2B, CYP2C11, and CYP3A2, and orphenadrine, which inhibits CYP2B, also decreased MA(2) formation in liver microsomes from 7-wk-old phenobarbital-pretreated rats. The formation of MA(1) and MAX was not affected. Furthermore, an immunoinhibition study demonstrated that anti-CYP3A2 antibody reduced MA(1), MAX, and MA(2) formation to nondetectable levels in liver microsomes from 2- and 7-wk-old rats, whereas anti-CYP2C11 or anti-CYP2B antibody, respectively, had no marked effect on MA(1), MAX, and MA(2) formation in liver microsomes from 7-wk-old untreated or PB-treated rats. These results suggest that the CYP3A isoform is mainly responsible for MA(1), MAX, and MA(2) formation in liver microsomes in male Wistar rats.
Liver microsomal territrem A (TRA) metabolism was studied in 7-wk-old female Wistar rats. Pretreatment with phenobarbital (PB) or dexamethasone (DEX) resulted in a significant increase in 4 beta-hydroxymethyl-4 beta-demethylterritrem A (MA1) production. SKF 525A (0.025 and 0.05 mM), a general cytochrome P-450 (CYP450) inhibitor, blocked MA1 formation in liver microsomes from PB-pretreated female rats. Anti-CYP2B antibody had no marked effect on MA1 formation, although orphenadrine (0.5 mM), which inhibits CYP2B, blocked MA1 formation in liver microsomes from PB-treated female rats. An immunoinhibition study showed that anti-CYP3A2 antibody reduced MA1 formation to nondetectable levels in liver microsomes from PB-treated female rats. Furthermore, immunoblotting showed that CYP3A1 protein was expressed in 7-wk-old female rat and only MA1 was formed from TRA using supersomes from CYP3A1-expressing baculovirus-infected insect cells. Further, Western blot analysis indicated that CYP3A2 protein was expressed in 2-wk-old rats of both sexes and 7-wk-old male rats, and 3 metabolites of TRA, such as MA1, MAX, and MA2, were formed using supersomes from CYP3A2-expressing baculovirus-infected insect cells. These results suggest that MA1 formation in liver microsomes of 7-wk-old female Wistar rats is mediated by CYP3A1.
The aim of the present study was to assess the effects of sex difference on metabolism of territrem A (TRA) by liver microsomes from 7-wk-old Wistar rats. Metabolism of TRA to 2-dihydro-4beta-demethylterritrem A (MA2) through 4beta-hydroxymethyl-4beta-demethylterritrem A (MA1) and 4beta-oxo-4beta-demethylterritrem A (MAX) was observed in intact male rats. However, in intact female rats only MA1 was formed, although the amount of MA, formed in females was much less than in males. Phenobarbital pretreatment enhanced this step and was not affected by gonadectomy. In the gonadectomized rats of both sexes, MA2 was formed from TRA when the animals were further treated by testosterone and was significantly enhanced by treatment with phenobarbital. However, estradiol treatment or estradiol in combination with phenobarbital treatment did not affect MA2 formation from TRA in gonadectomized rats.
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