Human cytochrome P450s involved in the metabolism of 9-cis- and 13-cis-retinoic acids☆
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.
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Both authors contributed equally to this, and are both considered as first author.