Oxidation of Benzo[a]pyrene by Recombinant Human Cytochrome P450 Enzymes

Abstract

The oxidation of benzo[a]pyrene (B[a]P) was examined using reconstituted systems prepared with recombinant human cytochrome P450 (P450) enzymes 1A1, 1A2, 2C8, 2C10, 2E1, and 3A4 and with microsomes prepared from Saccharomyces cerevisiae expressing recombinant human P450s 2C8, 2C9, and 2C18. Products measured by HPLC included the 3- and 9-phenols, the 4,5-, 7,8-, and 9,10-dihydrodiols (detected in the presence of epoxide hydrolase), and products in the polar fraction eluting immediately after the void volume. The most active enzyme in all reactions was P450 1A1. P450 3A4 and P450 1A2 formed appreciable amounts of several of the products, including the 3-phenol. P450 2C enzymes and P450 2E1 formed relatively low amounts of all B[a]P products. Consideration of these patterns along with knowledge of levels of expression of the P450s in human tissues and previous results with microsomes leads to the conclusion that P450 1A1 should dominate the oxidation of B[a]P in tissues where it is present and inducible. In human liver the level of P450 1A1 is low and P450 3A4, P450 2C subfamily enzymes, and P450 1A2 probably all contribute. Of the human P450s considered here, P450 1A2 was the most active hepatic enzyme forming the 7,8-dihydrodiol. 7,8-Benzoflavone stimulated the oxidation of B[a]P by P450 3A4 and inhibited the oxidations catalyzed by P450 1A2. The extent of inhibition of P450 1A1 was less (than with P450 1A2), probably due to the rapid oxidation of 7,8-benzoflavone by P450 1A1. The major 7,8-benzoflavone product appears to be the 5,6-oxide.

Differential Mechanisms of Cytochrome P450 Inhibition and Activation by α-Naphthoflavone

“…The progress curve was identical to (data not shown) and the kinetic parameters were indistinguishable (p Ͼ 0.05) from those obtained with ANF alone (Table I). The observation that BP had no effect on the CO binding kinetics when ANF was present, in conjunction with the known metabolism of ANF by this P450 (25), indicates that ANF competitively inhibits BP binding.…”

“…P450 3A4 is a major liver P450 that also metabolizes many important drugs (19), whereas P450 1A1 is normally undetectable in human liver but present in lung (20), where it is induced by cigarette smoking and is associated with lung cancer (19,21). ANF inhibits P450 1A1-mediated metabolism of BP by rat liver microsomal P450s (22,23) and human P450 1A1 (24,25), yet stimulates BP metabolism by both human liver microsomal P450s (26 -29) and P450 3A4 (14,25,29,30). Classical mechanisms such as competitive inhibition or noncompetitive modulation of substrate binding have usually been invoked to explain such inhibitory or stimulatory effects (14,15).…”

Cooperativity in Oxidation Reactions Catalyzed by Cytochrome P450 1A2

“…10, B and C) suggested that two benzo-[a]pyrene molecules could readily fit into the P450 1A2 active site. An assay of benzo[a]pyrene hydroxylation, using a fluorimetric assay (which detects mainly 3-hydroxylation (44,45,78)) showed highly cooperative behavior (Fig. 11).…”

The relationship between DNA adduct formation by benzo[a]pyrene and expression of its activation enzyme cytochrome P450 1A1 in rat

“…BaP is, however, oxidized also to other metabolites such as the other dihydrodiols, BaPdiones and hydroxylated metabolites (Bauer et al, 1995;Chun et al, 1996;Kim et al, 1998;Jiang et al, 2001;Zhu et al, 2008). Even though most of these metabolites are detoxification products, BaP-9-ol is a precursor of 9-hydroxy-BaP-4,5-epoxide which can form another adduct with deoxyguanosine ( Fig.…”

“…Furthermore, beside BaP itself, additional AHR ligands that induce CYP1A1 can modulate BaP activation. Therefore, here we investigated the effect of the flavonoid β-naphthoflavone (BNF), an inducer of this enzyme, and another member of the CYP1A subfamily, CYP1A2, which can to some degree also participate in BaP activation (Bauer et al, 1995), on BaP-derived DNA adduct formation in rat liver and small intestine.…”