Abstract
Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after activation by cytochrome P450 (P450). Here, we investigated whether NADH:cytochrome b5 reductase (CBR) in the presence of cytochrome b5 can act as sole electron donor to human P450 1A1 during BaP oxidation and replace the canonical NADPH:cytochrome P450 reductase (POR) system. We also studied the efficiencies of the coenzymes of these reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of CBR, to mediate BaP oxidation. Two systems containing human P450 1A1 were utilized: human recombinant P450 1A1 expressed with POR, CBR, epoxide hydrolase, and cytochrome b5 in Supersomes and human recombinant P450 1A1 reconstituted with POR and/or with CBR and cytochrome b5 in liposomes. BaP-9,10-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol, BaP-3-ol, a metabolite of unknown structure, and two BaP-DNA adducts were generated by the P450 1A1-Supersomes system, both in the presence of NADPH and in the presence of NADH. The major BaP-DNA adduct detected by 32P-postlabeling was characterized as 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP (assigned adduct 1), while the minor adduct is probably a guanine adduct derived from 9-hydroxy-BaP-4,5-epoxide (assigned adduct 2). BaP-3-ol as the major metabolite, BaP-9-ol, BaP-1,6-dione, BaP-3,6-dione, an unknown metabolite, and adduct 2 were observed in the system using P450 1A1 reconstituted with POR plus NADPH. When P450 1A1 was reconstituted with CBR and cytochrome b5 plus NADH, BaP-3-ol was the predominant metabolite too, and an adduct 2 was also generated. Our results demonstrate that the NADH/cytochrome b5/CBR system can act as the sole electron donor both for the first and second reduction of P450 1A1 during the oxidation of BaP in vitro. They suggest that NADH-dependent CBR can replace NADPH-dependent POR in the P450 1A1-catalyzed metabolism of BaP.
Highlights
Benzo[a]pyrene (BaP) (Figure 1) is a polycyclic aromatic hydrocarbon (PAH) that has been classified as a human carcinogen (Group 1) by the International Agency for Research on Cancer.[1]
We investigated whether NADH:cytochrome b5 reductase (CBR) in the presence of cytochrome b5 can act as sole electron donor to human P450 1A1 during BaP oxidation and replace the canonical NADPH:cytochrome P450 reductase (POR) system
The metabolism of BaP has been extensively studied over the past decades,[2] and various studies have examined the role of P450 enzymes, P450 1A1 of several species, to metabolize this carcinogen.[2,6−8,15,16,19,28,44] the mechanism of the reaction cycle of BaP oxidation catalyzed by P450 1A1, the roles of P450 oxidoreductase (POR) and CBR as electron donors to P450 1A1, has not yet been fully resolved.[7,8,15,19]
Summary
Benzo[a]pyrene (BaP) (Figure 1) is a polycyclic aromatic hydrocarbon (PAH) that has been classified as a human carcinogen (Group 1) by the International Agency for Research on Cancer.[1]. Further bioactivation by P450 1A1 leads to the ultimate reactive species, BaP-7,8-dihydrodiol-9,10-epoxide (BPDE), that can react with DNA, forming adducts preferentially at guanine residues (Figure 1). The 10-(deoxyguanosin-N2-yl)7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-N2BPDE) adduct is the major product of the reaction of BPDE with DNA in vitro and in vivo.[4−10] BaP is oxidized to other metabolites such as other dihydrodiols, BaPdiones, and further hydroxylated metabolites.[2,6,11−16]
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