Human cytochrome P450s involved in the metabolism of 9-cis- and 13-cis-retinoic acids

doi:10.1016/S0006-2952(01)00925-X

Biochemical Pharmacology

Volume 63, Issue 5, 1 March 2002, Pages 933-943

https://doi.org/10.1016/S0006-2952(01)00925-X Get rights and content

Abstract

The purpose of this work was to identify the principal human cytochrome P450s (CYPs) involved in the metabolism of the retinoic acid (RA) isomers, 9-cis- and 13-cis-RA, by using a combination of techniques including human liver microsomes (correlation of activity and inhibition), and lymphoblast microsomes expressing a single CYP. Concerning the 9-cis-RA, 4-OH- and 4-oxo-9-cis-RA were formed with human liver microsomes, and their formation correlated with activities linked to CYPs 3A4/5, 2B6, 2C8, 2A6, and 2C9. The use of lymphoblast microsomes expressing a single human CYP identified CYPs 2C9>2C8>3A7 as the most active in the formation of 4-OH-9-cis-RA. With regard to 13-cis-RA, specific P450 activities linked to CYPs 2B6, 2C8, 3A4/5, and 2A6 were correlated with the formation of 4-OH- and 4-oxo-13-cis-RA. Microsomes expressing a single CYP identified CYPs 3A7, 2C8, 4A11, 1B1, 2B6, 2C9, 2C19, 3A4 (decreasing activity) in the formation of 4-OH-13-cis-RA. The use of CYP-specific inhibitors in human liver microsomes disclosed that the formation of the 4-OH-9-cis-RA was best inhibited by sulfaphenazole (72%) and quercetin (66%), whereas ketoconazole and troleandomycin inhibited its formation by 55 and 38%, respectively; the formation of 4-OH-13-cis-RA was best inhibited by troleandomycin (54%) and ketoconazole (46%), whereas quercetin was a weak inhibitor (14%). In conclusion, adult human CYPs 2C9, 2C8, 3A4 have been identified as active in the 9-cis-RA metabolism, whereas CYPs 3A4 and 2C8 were active in 13-cis-RA metabolism. The fetal form CYP3A7 was also identified as very active in either 9-cis- or 13-cis-RA metabolism. The role of these human CYPs in the biological response or resistance to RA isomers remains to be determined.

Introduction

In addition to the importance of retinoids (Vitamin A and its derivatives) in embryogenesis, vertebrate development, differentiation, and homeostasis [1], these compounds are under investigation in the prevention and treatment of a variety of cancers (reviewed in [2], [3]). Retinoids are obtained in the diet either as preformed retinoids or as carotenoids (provitamin A). After several metabolic reactions in the intestines, retinol (Vitamin A) becomes the major retinoid absorbed and is stored in esterified form in the liver. After ester hydrolysis, retinol is transported by a plasma protein to the tissues. The conversion of retinol to retinal by retinol dehydrogenases and several CYPs is considered to be rate-limiting in the biosynthesis of RA [4]. The metabolism of retinals to retinoic acids is mediated by human CYPs 1A1, 1A2, 1B1 and 3A4 for the formation of all-trans-retinoic acid (atRA), and CYP1A2 for the formation of 9-cis-RA [5].

RA crosses the plasma membrane passively and is translocated by cellular retinoic acid binding proteins (CRABP I–II) to the nucleus where it can bind to nuclear receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs), each composed of three subtypes (α, β, γ). The RARs are activated by both atRA and 9-cis-RA and function as ligand-inducible transcriptional regulators when heterodimerized with RXR [6]. The RXRs can also homodimerize, and act as transcriptional regulators under certain conditions, and are only activated by 9-cis-RA [6], [7]. Biological responses to retinoids are therefore, modulated by the availability of a specific ligand, and also by the type of nuclear receptors available. In addition to the nuclear receptor-mediated responses to retinoids, it has also been suggested that retinoylation, or covalent binding of the retinoid to specific proteins, may also play a role in the cell response to atRA [8].

The metabolism of atRA is not only a simple catabolic process, as some oxidized metabolites display biological activity in the modulation of genes expressed in apoptosis, cellular growth and differentiation, embryonic development, and in the growth inhibition of several normal and neoplastic cells in vitro[9], [10], [11], [12], [13], [14], [15]. It has also been proposed that the metabolism of atRA may be linked to its growth inhibitory effects, as the most sensitive cell lines are intriguingly those that can metabolize atRA the most efficiently [16], [17].

Although atRA metabolism appears to play a central role in its molecular mechanism of action, either in terms of sensitivity or resistance, the human enzymes involved in its metabolism have only recently been identified. In humans, in addition to the already known CYP2C8 [18], [19] and CYP26 [20], [21] several other CYPs have been identified in the metabolism of atRA, i.e., CYPs 3A7, 3A5, 2C18, 3A4, 2C9 and 1A1 [22], [23], [24].

The atRA can isomerize in vitro and in vivo to its stereoisomers 9-cis- and 13-cis-RA, which possess different nuclear receptor binding properties [7] and pharmacological activities. The clinical pharmacology of RA isomers is also markedly different, e.g., the 13-cis-RA pharmacokinetics are stable over time with a half-life in the range of 13–22 hr [25], whereas atRA and 9-cis-RA pharmacokinetics are variable over time with a decrease in plasma concentrations after repeated dosage [26], [27].

Because metabolism plays an important role in the response to retinoids, and also because little information is presently available concerning the identity of the human CYPs involved in the metabolism of the principal atRA isomers (9-cis- and 13-cis-RA), the purpose of this study was to identify the principal human CYPs involved in their metabolism, to identify the metabolites, and also to compare to the CYPs already identified in the metabolism of atRA.

Section snippets

Chemicals

atRA, 9-, 13-cis-RA, quercetin, sulfaphenazole, troleandomycin, and NADPH were purchased from Sigma–Aldrich, and ketoconazole was purchased from ICN-Biochemicals. 4-Oxo-9- and 4-oxo-13-cis-RA, were kindly provided by Eva-Maria Gutknecht and Pierre Weber (Hoffmann-La Roche, Ltd., Basel, Switzerland). The 4-OH-13- and 4-OH-9-cis-RA were obtained by reduction of the corresponding 4-oxo standard metabolite using a molar excess of sodium borohydride (1 mg/mL). Stock solutions of retinoids (10⁻² M)

9-Cis- and 13-cis-RA metabolites separation and identification

Fig. 1 presents the separation of 9-cis-RA metabolites by reversed-phase HPLC. Two metabolite peaks were detected at retention times of 11.2 and 13.6 min which were identified as the 4-oxo-9- and the 4-OH-9-cis-RA, respectively, by co-elution with authentic standards and UV spectra. Fig. 2 presents the separation of the two major metabolites of 13-cis-RA detected at retention times of 10.9 and 13 min that were identified as the 4-oxo- and the 4-OH-13-cis-RA, respectively, by co-elution with

Discussion

The aims of this study were to identify the human CYPs involved in the metabolism of the retinoic acid isomers 9-cis- and 13-cis-RA, to determine their major metabolites, and to compare the identified CYPs with those already known to be involved in the metabolism of atRA [18], [19], [20], [21], [22], [23], [24].

In the first part of this work, phenotyped human liver microsomes were used to identify the CYPs most likely to be involved in the metabolism of 9-cis- and 13-cis-RA. It was found that

Acknowledgements

This investigation was supported in part by the Institut National de la Santé et de la Recherche Médicale (INSERM), the Centre National de la Recherche Scientifique (CNRS), the Association pour la Recherche sur le Cancer (ARC, Villejuif, France), and the Ligue Nationale Contre le Cancer. J.M. was supported by a studentship from the Association pour la Recherche sur le Cancer (ARC), C.C.C. by a studentship from the Fondation pour la Recherche Médicale (Paris, France), and N.I. by a studentship

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^☆: Part of this work was presented in abstract form: Proc Am Assoc Cancer Res 2001;42, abstract 1001.

¹: Both authors contributed equally to this, and are both considered as first author.

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Human cytochrome P450s involved in the metabolism of 9-cis- and 13-cis-retinoic acids☆

Abstract

Introduction

Section snippets

Chemicals

9-Cis- and 13-cis-RA metabolites separation and identification

Discussion

Acknowledgements

Cell

Cell

J. Biol. Chem.

Biol. Cell.

J. Biol. Chem.

Arch. Biochem. Biophys.

Biochem. Pharmacol.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Pharmacol.

Biochem. Pharmacol.

Biochem. Pharmacol.

J. Invest. Dermatol.

Retinoids in chemoprevention and differentiation therapy

Carcinogenesis

Biosynthesis of all-trans-retinoic acid from all-trans-retinol: catalysis of all-trans-retinol oxidation by human P-450 cytochromes

Drug Metab. Dispos.

Human cytochrome P-450 metabolism of retinals to retinoic acids

Drug Metab. Dispos.

Retinoids and their receptors in differentiation, embryogenesis, and neoplasia

FASEB J.

Retinoid metabolism and all-trans retinoic acid-induced growth inhibition in head and neck squamous cell carcinoma cell lines

Br. J. Cancer

Effects of retinoic acid on cell differentiation and reversion toward normal in human endometrial adenocarcinoma (RL95-2) cells

Anticancer Res.

Retinoic acid metabolites exhibit biological activity in human keratinocytes, mouse melanoma cells and hairless mouse skin in vivo

J. Pharmacol. Exp. Ther.