Elsevier

Toxicology

Volume 218, Issues 2–3, 1 February 2006, Pages 172-185
Toxicology

Territrems B and C metabolism in human liver microsomes: Major role of CYP3A4 and CYP3A5

https://doi.org/10.1016/j.tox.2005.10.010Get rights and content

Abstract

Human liver microsomes, supersomes from baculovirus-transformed insect cells expressing different human CYP450 isoforms, and control and CYP3A4 cDNA-transfected V79 Chinese hamster cells were tested for their ability to metabolize territrem B (TRB) and territrem C (TRC). Two TRB metabolites, designated MB2 and MB4, and one TRC metabolite, designated MC, were formed by all of these preparations. Of the nine supersomes from baculovirus-transformed insect cells expressing different human CYP450 isoforms (1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5), only those expressing CYP3A4 or CYP3A5 metabolized TRB and TRC. MB2, MB4, and MC were formed by CYP3A4 cDNA-transfected V79MZ Chinese hamster cells, but not by non-transfected cells. In order to investigate which CYP450 isoforms were responsible for MB2, MB4 and MC formation in human liver microsomal preparations, six isoform-specific chemical inhibitors (furafylline, sulfaphenazole, omeprazole, quinidine, ketoconazole, and diethyldithiocarbamate) and antibodies against CYP3A4 were used. MB2, MB4, and MC formation was markedly inhibited by ketoconazole, but less affected by quinidine and sulfaphenazole. Anti-CYP3A4 antibody markedly inhibited MB2, MB4, and MC formation and also 6β-hydroxytestosterone formation from testosterone. The CYP3A-dependent reaction of testosterone 6β-hydroxylation showed a high correlation with 4β-C hydroxylation of TRB (r2 = 0.97, P < 0.0001), O-demethylation of TRB (r2 = 0.95, P < 0.0001), and 4β-C hydroxylation of TRC (r2 = 0.99, P < 0.0001). Immunoblotting and RT-PCR showed that CYP3A4 and CYP3A5 were expressed in all four human liver microsomal preparations tested (HLM1–HLM4). The amount of MB2, MB4, and MC formed using different HLM preparations was related to the 6β-testosterone hydroxylase activity of the preparations. However, the extent of MB2, MB4, and MC formation was not related to the age or gender of the person from whom the microsomal sample was prepared. It was therefore suggest that CYP3A4 and CYP3A5 are the major enzymes responsible for TRB and TRC metabolism by human liver microsomes.

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 3.1.1.7.) 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 antibodies 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.

Section snippets

Chemicals

Diethyldithiocarbamate, furafylline, ketoconazole, NADPH, omeprazole, quinidine, sulfaphenazole, and testosterone propionate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 6β-Hydroxytestosterone was purchased from Steroids Inc. (Newport, RI, USA). Polyclonal goat antibodies against human CYP2C, CYP2D6, or CYP3A4 were purchased from Gentest Corp. (Woburn, MA, USA). Specific antibodies to CYP3A4 and CYP3A5 raised in rabbits by immunization with synthetic peptides were provided by

Results

When TRB or TRC was incubated with human liver microsomes, supersomes from baculovirus-transformed insect cells expressing different human CYP450 isoforms, or CYP3A4 cDNA-transfected V79 Chinese Hamster cells, the only TRB metabolites detected were MB2 and MB4 and the only TRC metabolite was MC (Table 1). These results suggested that CYP3A4 and CYP3A5 were involved in the 4β-C hydroxylation of TRB and TRC and the O-demethylation of TRB.

The initial velocities of 4β-C hydroxylation of TRB, O

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

Acknowledgments

This work was supported by grant no. NSC 93-2320-B-002-131 from the National Science Council, Taiwan. The authors are indebted to Drs. Kuo-Huang Ling and Thomas Barkas for reading the manuscript and for critical suggestions.

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