Diversity of selective environmental substrates for human cytochrome P450 2A6: alkoxyethers, nicotine, coumarin, N-nitrosodiethylamine, and N-nitrosobenzylmethylamine
Introduction
Cytochrome P450 (CYP) enzymes mediate the oxidative metabolism of exogenous and endogenous compounds (Gonzalez, 1992). Individual CYP forms have been shown to catalyze the metabolic activation and inactivation of numerous diverse xenobiotics, either precarcinogens or promutagens (Guengerich, 1993). CYP2A is one of the 10 CYP2 subfamilies known to be present in humans (Maurice et al., 1991, Fernandez-Salguero and Gonzalez, 1995). This P450 subfamily contains three members. CYP2A6 constitutes 5–10% of the total CYP in human liver (Yamano et al., 1990, Shimada et al., 1996), while CYP2A7 is not functional (Ding et al., 1995) and CYP2A13 is present mainly in nasal mucosa (Su et al., 2000). Although coumarin is known to be the prototypic substrate of CYP2A6 (Yamano et al., 1990, Fernandez-Salguero et al., 1995, Pelkonen et al., 2000), it also catalyzes the metabolism of other toxic agents as diverse as nicotine (Nakajima et al., 1996), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (Yamazaki et al., 1992), N-nitrosodiethylamine (NDEA) (Yamazaki et al., 1992, Camus et al., 1993), and alkoxyethers (Hong et al., 1999, Le Gal et al., 2001). However, it is still unclear whether or not other P450 enzymes are significantly involved in the oxidation of these diverse toxic agents. The diversity and bulk of the chemical structure of these substrates (Fig. 1) raise the question of their specificities.
The aim of the present study is, therefore, to examine the kinetic parameters of five substrates of CYP2A6, namely coumarin, nicotine, N-nitrosobenzylmethylamine (NBzMA), NDEA, and methyl tert-butyl ether (MTBE), by human liver microsomes and heterologously expressed CYPs. In order to reassess the relative selectivity of these diverse substrates, their interindividual variation, intercorrelation, and inhibition by different chemicals were examined in two panels of human liver microsomes.
Section snippets
Materials and methods
Caution: NBzMA is an hazardous compound and a carcinogen in rodents and should be handled carefully according to recommendations of NCI Chemical Carcinogen Repository.
Kinetic parameters
The kinetic parameters of metabolism of five substrates for CYP2A6 were measured (Table 1). In preliminary experiments, the conditions of these measurements were optimized.
The products of NBzMA metabolism were analyzed by reverse-phase HPLC as semicarbazone derivatives with UV detection. Based on previous studies with various long-chain alkylnitrosamines, it was estimated that a nitrosamine substrate concentration of 0.25 mM would be near saturation in human liver microsomes. Semicarbazide was
Discussion
This study investigated the selectivity of five substrates that have been described for human CYP2A6. This P450 isoform plays a crucial role in the bioactivation of several drugs, such as coumarin and nicotine, and a number of environmental precarcinogens such as tobacco-related N-nitrosamines (Patten et al., 1998).
7-Hydroxylation of coumarin is known to be a characteristic reaction catalyzed by liver microsomal CYP2A6 (Pelkonen et al., 2000). Coumarin appears to be the most selective probe
Acknowledgements
Annabelle Le Gal is a fellowship student supported by a grant from the Conseil Régional de Bretagne. This work was financially supported by the European Community (INCO-DC Programme of the European Commission, grant No. ERB-IC18-CT98-0341).
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