title: NADH:Cytochrome b5 Reductase and Cytochrome b5 Can Act as Sole Electron Donors to Human Cytochrome P450 1A1-Mediated Oxidation and DNA Adduct Formation by Benzo[a]pyrene.
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 (32)P-postlabeling was characterized as 10-(deoxyguanosin-N(2)-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.
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University , Albertov 2030, 128 40, Prague 2, Czech Republic.
authors.forename: Marie
authors.initials: M
authors.lastname: Stiborová
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University , Albertov 2030, 128 40, Prague 2, Czech Republic.
authors.forename: Radek
authors.initials: R
authors.lastname: Indra
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University , Albertov 2030, 128 40, Prague 2, Czech Republic.
authors.forename: Michaela
authors.initials: M
authors.lastname: Moserová
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University , Albertov 2030, 128 40, Prague 2, Czech Republic.
authors.forename: Eva
authors.initials: E
authors.lastname: Frei
authors.affiliation: Division of Radiopharmaceutical Chemistry, German Cancer Research Center (DKFZ) , Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
authors.forename: Heinz H
authors.initials: HH
authors.lastname: Schmeiser
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University , Albertov 2030, 128 40, Prague 2, Czech Republic.
authors.forename: Klaus
authors.initials: K
authors.lastname: Kopka
authors.affiliation: Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment and Health, King's College London , Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, United Kingdom.|NIHR Health Protection Research Unit in Health Impact of Environmental Hazards at King's College London in Partnership with Public Health England , Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, United Kingdom.
authors.forename: David H
authors.initials: DH
authors.lastname: Phillips
authors.affiliation: Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment and Health, King's College London , Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, United Kingdom.|NIHR Health Protection Research Unit in Health Impact of Environmental Hazards at King's College London in Partnership with Public Health England , Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, United Kingdom.
authors.forename: Volker M
authors.initials: VM
authors.lastname: Arlt
mesh_terms: D001564:Benzo(a)pyrene / Q000633:toxicity*; D042966:Cytochrome-B(5) Reductase / Q000378:metabolism*; D018736:DNA Adducts / Q000378:metabolism*; D006801:Humans; D010084:Oxidation-Reduction
chemical_list: D018736:DNA Adducts; D001564:Benzo(a)pyrene; D042966:Cytochrome-B(5) Reductase