In vitro investigation of cytochrome P450-mediated metabolism of dietary flavonoids

Abstract

Human and mouse liver microsomes and membranes isolated from Escherichia coli, which expressed cytochrome P450 (CYP) 1A2, 3A4, 2C9 or 2D6, were used to investigate CYP-mediated metabolism of five selected dietary flavonoids. In human and mouse liver microsomes kaempferol, apigenin and naringenin were hydroxylated at the 3′-position to yield their corresponding analogs quercetin, luteolin and eriodictyol, whereas hesperetin and tamarixetin were demethylated at the 4′-position to yield eriodictyol and quercetin, respectively. Microsomal flavonoid metabolism was potently inhibited by the CYP1A2 inhibitors, fluvoxamine and α-naphthoflavone. Recombinant CYP1A2 was capable of metabolizing all five investigated flavonoids. CYP3A4 recombinant protein did not catalyze hesperetin demethylation, but showed similar metabolic profiles for the remaining compounds, as did human microsomes and recombinant CYP1A2, although the reaction rates in general were lower as compared to CYP1A2. CYP2C9 catalyzed the 4′-demethylation of tamarixetin, whereas CYP2D6 did not seem to play any role in the metabolism of the selected flavonoids. The major involvement in flavonoid metabolism of human CYP1A2, which mediates the formation of metabolites with different biochemical properties as compared to the parent compound and furthermore is known to be expressed very differently among individuals, raises the important question of whether individual differences in the CYP enzyme activity might affect the beneficial outcome of dietary flavonoids, rendering some individuals more or less refractory to the health-promoting potential of dietary flavonoids.

Introduction

Flavonoids belong to the class of low molecular weight phenols that are widely distributed throughout the plant kingdom with more than 4000 different flavonoids identified thus far. Their basic chemical structure consists of two benzene rings that are linked by a heterocyclic pyrane or pyrone ring. This structure allows multiple patterns and substitutions that give rise to various subclasses such as isoflavonoids, flavones, catechins and anthocyanins. Despite the great similarity in overall structure between subgroups and within members of the subgroups, the biochemical and biological properties vary considerably with only minor modifications of the flavonoid structure. For instance, the number and specific position of hydroxyl groups on the three-ring structure can thus determine whether the compound exhibit estrogenic activity or not (Breinholt and Larsen, 1998) or function as an in vivo or an in vitro antioxidant (Breinholt et al., 1999, Vinson, 1998). The cytotoxic (Breinholt and Dragsted, 1998), mutagenic (Rueff et al., 1995, Jurado et al., 1991) or antimutagenic (Edenharder et al., 1997) potential of the flavonoids also varies with the substitution pattern. The increasing awareness that only minor alterations of the flavonoid structure can impact on the associated biological properties has increased the need for detailed studies on the biotransformation of flavonoids and the potential properties of the resulting metabolites. Recent studies from this laboratory suggest that flavonoids, which are believed to be important dietary protective factors against cancer and atherosclerosis, are extensively metabolized by cytochrome P450 (CYP), giving rise to metabolites with associated biological activities distinctly different from those of the parent compound (Breinholt et al., 1999, 2000; Nielsen et al., 1998). It could thus be speculated that some of the flavonoid metabolites rather than the parent compound might mediate the biological response. As the expression pattern of CYP isoforms differs greatly between individuals and some CYP isoforms are polymorphic, it could be speculated that individual differences in the ability to biotransform flavonoids may render some individuals more or less refractory to dietary cancer intervention. On this basis it could be speculated that individual differences in cancer susceptibility may not be solely due to inter-individual differences in metabolic activation and detoxification of carcinogens, but may also in part result from differences in the metabolism of anticarcinogens, giving rise to metabolites with biological activities that differ from the parent compound. The objectives of this study were firstly to determine whether human and mouse liver microsomes were able to metabolize a selected series of flavonoids, and secondly to determine which human hepatic CYP enzymes were responsible for flavonoid biotransformation, by the use of recombinant human CYP proteins and specific CYP inhibitors.