br Acknowledgements br Introduction Three structurally relat
Introduction Three structurally related fungal metabolites have been isolated from chloroform extracts of submerged rice cultures of Aspergillus terreus 23-1 from stored unhulled rice in Taiwan; these were named territrems A–C to indicate their biological origin (A. terreus) and their tremorgenic activity (Ling et al., 1979, Ling et al., 1984, Ling, 1994). These mycotoxins also possess irreversible anti-acetylcholinesterase activity (Chen et al., 1999). The irreversible inhibition of acetylcholinesterase (EC 22.214.171.124.) by territrem B (TRB) is mediated by tight noncovalent binding of TRB to the enzyme (Chen et al., 1999). Metabolism of TRB to 4β-hydroxylmethyl-4β-demethylterritrem B (MB2), O-demethylation of the methoxy group of the aromatic moiety of TRB to form MB4 (same structure as TRC), and metabolism of TRC to 4β-hydroxylmethyl-4β-demethylterritrem C (MC) were observed in liver microsomes from 14-week-old male and female Wistar rats; however, the amounts of MB2, MB4, and MC formed in females were much lower than in males (Peng et al., 2005a). Experiments using supersomes or various inhibitors of, or tryptophan hydroxylase against, isoforms of cytochrome P450 (CYP450) demonstrated that the formation of MB2, MB4, and MC is mediated by both CYP3A1 and CYP3A2 (Peng et al., 2005a). Further experiments (Peng et al., 2005b) showed an age-related decline in TRB and TRC metabolism in both genders, which was more marked in males and was associated with changes in CYP3A1 and CYP3A2 mRNA and protein levels. Significant gender differences in TRB and TRC metabolism during the juvenile to senescent stage were also seen, with male rats exhibiting greater activities than females. Four isoforms of the CYP3A subfamily, CYP3A4, CYP3A5, CYP3A7, and CYP3A43, are expressed at different levels in the human liver, kidney, and gastrointestinal tract (Thummel and Wilkinson, 1998). CYP3A4 is the dominant CYP3A isoform in the human liver and small intestine (Domanski et al., 2000), but CYP3A5 is also found in the liver, intestinal mucosa (Wrighton et al., 1990, Paine et al., 1997), and other extrahepatic tissues, including the kidney (Haehner et al., 1996), lung (Kivistö et al., 1996), and prostate gland (Yamakoshi et al., 1999). CYP3A7 is primarily a fetal enzyme (Kitada and Kamataki, 1994, Schuetz et al., 1994). More recently, human CYP3A43 has been identified and cloned (Domanski et al., 2001), although its contribution to hepatic or extrahepatic CYP3A-dependent drug clearance is thought to be negligible (Westlind et al., 2001). TRA metabolism in the human liver has been studied (Peng et al., 2003). The only metabolite generated was found to be 6β-hydroxymethyl-6β-demethylterritrem A (MA1) and experiments using various cytochrome P450 (CYP450) inhibitors, antibodies, or supersomes demonstrated that CYP3A4 is the major enzyme responsible for TRA metabolism by human liver microsomes, although CYP2C9 and CYP2D6 play a minor role. However, the roles of human liver cytochrome P450s in TRB and TRC metabolism have not been investigated. The aim of the present study was therefore to determine which CYP450 isoforms play a major role in TRB and TRC metabolism.
Materials and methods
Discussion The metabolism of TRB and TRC in liver microsomes from Wistar rats of both genders and in supersomes from baculovirus-transformed insect cells expressing different human CYP450 isoforms was characterized in a previous study (Peng et al., 2005a). In 14-week-old male and female rats, CYP3A1 and CYP3A2 are the main CYP450 isoenzymes responsible for MB2, MB4, and TRC formation. The aim of the present study was to identify the human CYP450 isoforms responsible for the metabolism of TRB to MB2 and MB4 and of TRC to MC using isoform-specific chemical inhibitors and antibodies, supersomes from baculovirus-transformed insect cells expressing different human CYP450 isoforms, and CYP3A4-expressing V79MZh3A4 cells. All the data suggested that, in humans, CYP3A4 and CYP3A5 play the major role in the metabolism of TRB and TRC.