Substrate specificity for rat cytochrome P450 (CYP) isoforms: screening with cDNA-expressed systems of the rat

doi:10.1016/S0006-2952(01)00843-7

Biochemical Pharmacology

Volume 63, Issue 5, 1 March 2002, Pages 889-896

https://doi.org/10.1016/S0006-2952(01)00843-7 Get rights and content

Abstract

In this study, we performed a screening of the specificities of rat cytochrome P450 (CYP) isoforms for metabolic reactions known as the specific probes of human CYP isoforms, using 13 rat CYP isoforms expressed in baculovirus-infected insect cells or B-lymphoblastoid cells. Among the metabolic reactions studied, diclofenac 4-hydroxylation (DFH), dextromethorphan O-demethylation (DMOD) and midazolam 4-hydroxylation were specifically catalyzed by CYP2C6, CYP2D2 and CYP3A1/3A2, respectively. These results suggest that diclofenac 4-hydroxylation, dextromethorphan O-demethylation and midazolam 4-hydroxylation are useful as catalytic markers of CYP2C6, CYP2D2 and CYP3A1/3A2, respectively. On the other hand, phenacetin O-deethylation and 7-ethoxyresorufin O-deethylation were catalyzed both by CYP1A2 and by CYP2C6. Benzyloxyresorufin O-dealkylation and pentoxyresorufin O-dealkylation were also catalyzed by CYP1A2 in addition to CYP2B1. Bufuralol 1′-hydroxylation was extensively catalyzed by CYP2D2 but also by CYP2C6 and CYP2C11. p-Nitrophenol 2-hydroxylation and chlorzoxazone 6-hydroxylation were extensively catalyzed by CYP2E1 but also by CYP1A2 and CYP3A1. Therefore, it is necessary to conduct further study to clarify whether these activities in rat liver microsomes are useful as probes of rat CYP isoforms. In contrast, coumarin 7-hydroxylation and S- and R-mephenytoin 4′-hydroxylation did not show selectivity toward any isoforms of rat CYP studied. Therefore, activities of coumarin 7-hydroxylation and S- and R-mephenytoin 4′-hydroxylation are not able to be used as catalytic probes of CYP isoforms in rat liver microsomes. These results may provide useful information regarding catalytic probes of rat CYPs for studies using rat liver microsomal samples.

Introduction

CYP enzymes comprise a large family of hemoprotein [1], and enzymes from three families (CYP1, CYP2, and CYP3) are mainly involved in the biotransformation of xenobiotics in both humans and rats [2]. However, considerable differences exist in the expression and catalytic activities between human and rat CYP orthologues [2]. Although CYP enzymes make xenobiotics less toxic, the reactions frequently involve the formation of reactive intermediates or allow the leakage of free radicals capable of causing toxicity [3], [4]. Therefore, differences in the levels of individual CYP isoforms and indeed the expression of distinct isoforms significantly contribute to interspecies differences in the toxicity of chemicals and the susceptibility to chemically induced cancer [2]. Nevertheless, drug safety assessment studies, including pharmacological and toxicological studies, have been performed using rodents although they are not necessarily excellent surrogate models for man. To assess the effects and toxicities of xenobiotics (i.e. developmental drugs and environmental chemicals), it is necessary to identify CYP isoforms responsible for the metabolism of drugs in each species.

Currently, many different strategies are employed for the unambiguous identification of CYP isoforms responsible for the biotransformation of therapeutic agents in humans. These include the use of CYP isoform-selective inhibitors, immunoinhibitory antibodies, studies with purified and heterogeneously expressed CYP protein, and correlation analyses of metabolic rates of drugs with immunoquantified CYP levels or metabolic rates of specific substrates for each isoform. The use of cDNA-expressed human CYPs has greatly facilitated the evaluation of the metabolic specificity of probe substrates and the identification of individual CYP isoforms involved in metabolism of drugs. On the other hand, criteria and approaches for unambiguous identification of individual CYP isoforms responsible for the metabolism of drugs in rats have not been established. This is because the specificities of metabolic reactions used as probes of rat CYP isoforms have not been thoroughly evaluated.

In the present study, therefore, the metabolism of model substrates for human CYP-isoforms was examined using 13 rat CYP isoforms expressed in baculovirus-infected insect cells or human B-lymphoblastoid cells.

Section snippets

Chemicals

Bufuralol hydrochloride, 1′-hydroxybufuralol and 4-hydroxydiclofenac were purchased from Gentest. Midazolam, 1′- and 4-hydroxymidazolam were gifts from F. Hoffmann-La Roche. Zaltoprofen was a gift from Zeria Pharmaceutical. Dextrorphan, R- and S-mephenytoin, 4-hydroxymephenytoin, 6-hydroxychlorzoxazone, 6β-, 7α- and 16α-hydroxytestosterone were purchased from Ultrafine Chemicals. 7-Benzyloxyresorufin, chlorzoxazone, dextromethorphan hydrobromide, 7-ethoxyresorufin, 7-pentoxyresorufin and

Metabolism of diclofenac, dextromethorphan and midazolam

Fig. 1 shows the activities of DFH (A), DMOD (B), MD1H and MD4H (C) in cDNA-expressed rat CYPs. DFH activity, a catalytic marker of human CYP2C9 [6], was selectively catalyzed by CYP2C6 (15.4 pmol/min/pmol CYP). DMOD activity, a catalytic marker of human CYP2D6 [7], was extensively mediated by CYP2D2 (3.4 pmol/min/pmol CYP). MD1H and MD4H activities, which are known to be catalyzed by human CYP3A4/3A5 [8], were predominantly catalyzed by CYP3A1/3A2. CYP3A2 showed higher MD4H activity than did

Discussion

Since the 1990s, the use of cDNA-expressed human CYPs has greatly facilitated the evaluation of metabolic specificities of probe substrates and the identification of individual CYPs involved in the metabolism of many drugs in humans. On the other hand, the specificities of metabolic reactions used as probes of CYP isoforms have not been thoroughly evaluated by the study with cDNA-expressed rat CYP isoforms. Therefore, in the present study, the isoform-specificities of metabolic reactions known

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Substrate specificity for rat cytochrome P450 (CYP) isoforms: screening with cDNA-expressed systems of the rat

Abstract

Introduction

Section snippets

Chemicals

Metabolism of diclofenac, dextromethorphan and midazolam

Discussion

TIPS Rev.

Life Sci.

Biochem. Pharmacol.

Biochem. Pharmacol.

Biochem. Pharmacol.

Biochem. Pharmacol.

J. Chromatogr. B

J. Biol. Chem.

J. Biol. Chem.

Biochem. Pharmacol.

Biochem. Pharmacol.

Biochem. Biophys. Res. Commun.

P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature

Pharmacogenetics

P450 in the rat and man: methods of investigation, substrate specificities and relevance to cancer

Xenobiotica

Catalytic selectivity of human cytochrome P450 enzymes: relevance to drug metabolism and toxicity

Toxicol. Lett.

Role of human cytochrome P4503A4 in metabolism of medroxyprogesterone acetate

Clin. Cancer Res.

The role of CYP2D6 in primary and secondary oxidative metabolism of dextromethorphan: in vitro studies using human liver microsomes

Br. J. Clin. Pharmacol.

Oxidation of midazolam and triazolam by human liver cytochrome P450IIIA4

Mol. Pharmacol.

Human cytochrome P-450IA2 (P4501A2), the phenacetin O-deethylase, is primarily responsible for the hepatic 3-demethylation of caffeine and N-oxidation of carcinogenic arylamines

Proc. Natl. Acad. Sci. U.S.A.