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.

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

Department of Biochemistry Faculty of Science Charles University Albertov 2030 128 40 Prague 2 Czech Republic

Division of Radiopharmaceutical Chemistry German Cancer Research Center Im Neuenheimer Feld 280 69120 Heidelberg Germany

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

Erratum v

Chem Res Toxicol. 2016 Sep 19;29(9):1571. doi: 10.1021/acs.chemrestox.6b00283. PubMed

Zobrazit více v PubMed

IARC (International Agency for Research on Cancer) (2010) Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some Related Exposures, in IARC Monogr. Eval. Carcinog. Risks Hum., Vol. 92, pp 1–853, IARC, Lyon, France. PubMed PMC

Baird W. M.; Hooven L. A.; Mahadevan B. (2005) Carcinogenic polycyclic aromatic hydrocarbon-DNA adducts and mechanism of action. Environ. Mol. Mutagen. 45, 106–114. 10.1002/em.20095. PubMed DOI

Wohak L. E.; Krais A. M.; Kucab J. E.; Stertmann J.; Øvrebø S.; Seidel A.; Phillips D. H.; Arlt V. M. (2016) Carcinogenic polycyclic aromatic hydrocarbons induce CYP1A1 in human cells via a p53-dependent mechanism. Arch. Toxicol. 90, 291–304. 10.1007/s00204-014-1409-1. PubMed DOI PMC

Sims P.; Grover P. L.; Swaisland A.; Pal K.; Hewer A. (1974) Metabolic activation of benzo(a)pyrene proceeds by a diol-epoxide. Nature 252, 326–328. 10.1038/252326a0. PubMed DOI

Wood A. W.; Levin W.; Lu A. Y.; Yagi H.; Hernandez O.; Jerina D. M.; Conney A. H. (1976) Metabolism of benzo(a)pyrene and benzo(a)pyrene derivatives to mutagenic products by highly purified hepatic microsomal enzymes. J. Biol. Chem. 251, 4882–4890. PubMed

Bauer E.; Guo Z.; Ueng Y. F.; Bell L. C.; Zeldin D.; Guengerich F. P. (1995) Oxidation of benzo[a]pyrene by recombinant human cytochrome P450 enzymes. Chem. Res. Toxicol. 8, 136–142. 10.1021/tx00043a018. PubMed DOI

Arlt V. M.; Stiborova M.; Henderson C. J.; Thiemann M.; Frei E.; Aimova D.; Singhs R.; Costa; da G. G.; Schmitz O. J.; Farmer P. D.; Wolf C. R.; Philips D. H. (2008) Metabolic activation of benzo[a]pyrene in vitro by hepatic cytochrome P450 contrasts with detoxification in vivo: experiments with hepatic cytochrome P450 reductase null mice. Carcinogenesis 29, 656–665. 10.1093/carcin/bgn002. PubMed DOI

Arlt V. M.; Poirier M. C.; Sykes S. E.; John K.; Moserova M.; Stiborova M.; Wolf C. R.; Henderson C. J.; Phillips D. H. (2012) Exposure to benzo[a]pyrene of Hepatic Cytochrome P450 Reductase Null (HRN) and P450 Reductase Conditional Null (RCN) mice: Detection of benzo[a]pyrene diol epoxide-DNA adducts by immunohistochemistry and 32P-postlabelling. Toxicol. Lett. 213, 160–166. 10.1016/j.toxlet.2012.06.016. PubMed DOI PMC

Arlt V. M.; Krais A. M.; Godschalk R. W.; Riffo-Vasquez Y.; Mrizova I.; Roufosse C. A.; Corbin C.; Shi Q.; Frei E.; Stiborova M.; van Schooten F. J.; Phillips D. H.; Spina D. (2015) Pulmonary inflammation impacts on CYP1A1-mediated respiratory tract DNA damage induced by the carcinogenic air pollutant benzo[a]pyrene. Toxicol. Sci. 146, 213–225. 10.1093/toxsci/kfv086. PubMed DOI PMC

Krais A. M.; Speksnijder E. N.; Melis J. P.; Indra R.; Moserova M.; Godschalk R. W.; van Schooten F. J.; Seidel A.; Kopka K.; Schmeiser H. H.; Stiborova M.; Phillips D. H.; Luijten M.; Arlt V. M. (2016) The impact of p53 on DNA damage and metabolic activation of the environmental carcinogen benzo[a]pyrene: effects in Trp53(+/+), Trp53(±) and Trp53(−/−) mice. Arch. Toxicol. 90, 839–851. 10.1007/s00204-015-1531-8. PubMed DOI PMC

Chun Y. J.; Shimada T.; Guengerich F. P. (1996) Construction of a human cytochrome P450 1A1: rat NADPH-cytochrome P450 reductase fusion protein cDNA and expression in Escherichia coli, purification, and catalytic properties of the enzyme in bacterial cells and after purification. Arch. Biochem. Biophys. 330, 48–58. 10.1006/abbi.1996.0224. PubMed DOI

Kim J. H.; Stansbury K. H.; Walker N. J.; Trush M. A.; Strickland P. T.; Sutter T. R. (1998) Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-diol by human cytochrome P450 1B1. Carcinogenesis 19, 1847–1853. 10.1093/carcin/19.10.1847. PubMed DOI

Jiang H.; Gelhaus S. L.; Mangal D.; Harvey R. G.; Blair I. A.; Penning T. M. (2007) Metabolism of benzo[a]pyrene in human bronchoalveolar H358 cells using liquid chromatography-mass spectrometry. Chem. Res. Toxicol. 20, 1331–1341. 10.1021/tx700107z. PubMed DOI PMC

Zhu S.; Li L.; Thornton C.; Carvalho P.; Avery B. A.; Willett K. L. (2008) Simultaneous determination of benzo[a]pyrene and eight of its metabolites in Fundulus heteroclitus bile using ultra-performance liquid chromatography with mass spectrometry. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 863, 141–149. 10.1016/j.jchromb.2008.01.018. PubMed DOI PMC

Stiborová M.; Indra R.; Moserová M.; Šulc M.; Hodek P.; Frei E.; Schmeiser H. H.; Arlt V. M. (2016) NADPH- and NADH-dependent metabolism of and DNA adduct formation by benzo[a]pyrene catalyzed with rat hepatic microsomes and cytochrome P450 1A1. Monatsh. Chem. 147, 847–855. 10.1007/s00706-016-1713-y. PubMed DOI PMC

Šulc M.; Indra R.; Moserová M.; Schmeiser H. H.; Frei E.; Arlt V. M.; Stiborová M. (2016) The impact of individual cytochrome P450 enzymes on oxidative metabolism of benzo[a]pyrene in human livers. Environ. Mol. Mutagen. 57, 229–235. 10.1002/em.22001. PubMed DOI PMC

King H. W.; Thompson M. H.; Brookes P. (1976) The role of 9-hydroxybenzo(a)pyrene in the microsome mediated binding of benzo(a)pyrene to DNA. Int. J. Cancer 18, 339–344. 10.1002/ijc.2910180311. PubMed DOI

Fang A. H.; Smith W. A.; Vouros P.; Gupta R. C. (2001) Identification and characterization of a novel benzo[a]pyrene-derived DNA adduct. Biochem. Biophys. Res. Commun. 281, 383–389. 10.1006/bbrc.2000.4161. PubMed DOI

Stiborova M.; Moserova M.; Cerna V.; Indra R.; Dracinsky M.; Šulc M.; Henderson C. J.; Wolf C. R.; Schmeiser H. H.; Phillips D. H.; Frei E.; Arlt V. M. (2014) Cytochrome b5 and epoxide hydrolase contribute to benzo[a]pyrene-DNA adduct formation catalyzed by cytochrome P450 1A1 under low NADPH:P450 oxidoreductase conditions. Toxicology 318, 1–12. 10.1016/j.tox.2014.02.002. PubMed DOI

Guengerich F. P. (2008) Cytochrome P450 and chemical toxicology. Chem. Res. Toxicol. 21, 70–83. 10.1021/tx700079z. PubMed DOI

Porter T. D. (2002) The roles of cytochrome b5 in cytochrome P450 reactions. J. Biochem. Mol. Toxicol. 16, 311–316. 10.1002/jbt.10052. PubMed DOI

Schenkman J. B.; Jansson I. (2003) The many roles of cytochrome b5. Pharmacol. Ther. 97, 139–152. 10.1016/S0163-7258(02)00327-3. PubMed DOI

Guengerich F. P. (2005) Reduction of cytochrome b5 by NADPH-cytochrome P450 reductase. Arch. Biochem. Biophys. 440, 204–211. 10.1016/j.abb.2005.06.019. PubMed DOI

McLaughlin L. A.; Ronseaux S.; Finn R. D.; Henderson C. J.; Wolf C. R. (2010) Deletion of microsomal cytochrome b5 profoundly affects hepatic and extrahepatic drug metabolism. Mol. Pharmacol. 78, 269–278. 10.1124/mol.110.064246. PubMed DOI

Kotrbova V.; Mrazova M.; Moserova M.; Martinek V.; Hodek P.; Hudecek J.; Frei E.; Stiborova M. (2011) Cytochrome b5 shifts oxidation of the anticancer drug ellipticine by cytochromes P450 1A1 and 1A2 from its detoxication to activation, thereby modulating its pharmacological efficacy. Biochem. Pharmacol. 82, 669–680. 10.1016/j.bcp.2011.06.003. PubMed DOI

Stiborova M.; Indra R.; Moserova M.; Cerná V.; Rupertová M.; Martínek V.; Eckschlager T.; Kizek R.; Frei E. (2012) Cytochrome b5 increases cytochrome P450 3A4-mediated activation of anticancer drug ellipticine to 13-hydroxyellipticine whose covalent binding to DNA is elevated by sulfotransferases and N,O-acetyltransferases. Chem. Res. Toxicol. 25, 1075–1085. 10.1021/tx3000335. PubMed DOI

Henderson C. J.; McLaughlin L. A.; Wolf C. R. (2013) Evidence that cytochrome b5 and cytochrome b5 reductase can act as sole electron donors to the hepatic cytochrome P450 system. Mol. Pharmacol. 83, 1209–1217. 10.1124/mol.112.084616. PubMed DOI

Indra R.; Moserova M.; Sulc M.; Frei E.; Stiborova M. (2013) Oxidation of carcinogenic benzo[a]pyrene by human and rat cytochrome P450 1A1 and its influencing by cytochrome b5 – a comparative study. Toxicol. Lett. 221, S72.10.1016/j.toxlet.2013.05.057. PubMed DOI

Steinberg P.; Schlemper B.; Molitor E.; Platt K. L.; Seidel A.; Oesch F. (1990) Rat liver endothelial and Kupffer cell-mediated mutagenicity of polycyclic aromatic hydrocarbons and aflatoxin B1. Environ. Health Perspect. 88, 71–76. 10.1289/ehp.908871. PubMed DOI PMC

Marnett L. J.; Bienkowski M. J. (1980) Hydroperoxide-dependent oxygenation of trans-7,8-dihydroxy-7,8-dihydro benzo[a]pyrene by ram seminal vesicle microsomes, Source of the oxygen. Biochem. Biophys. Res. Commun. 96, 639–647. 10.1016/0006-291X(80)91403-5. PubMed DOI

Marnett L. J. (1990) Prostaglandin synthase-mediated metabolism of carcinogens and a potential role for peroxyl radicals as reactive intermediates. Environ. Health Perspect. 88, 5–12. 10.1289/ehp.90885. PubMed DOI PMC

West S. B.; Levin W.; Ryan D.; Vore M.; Lu A. Y. (1974) Liver microsomal electron transport systems. II. The involvement of cytochrome b5 in the NADH-dependent hydroxylation of 3,4-benzpyrene by a reconstituted cytochrome P-448-containing system. Biochem. Biophys. Res. Commun. 58, 516–522. 10.1016/0006-291X(74)90395-7. PubMed DOI

Lu A. Y.; West S. B.; Vore M.; Ryan D.; Levin W. (1974) Role of cytochrome b5 in hydroxylation by a reconstituted cytochrome P-450-containing system. J. Biol. Chem. 249, 6701–6709. PubMed

Finn R. D.; McLaughlin L. A.; Ronseaux S.; Rosewell I.; Houston J. B.; Henderson C. J.; Wolf C. R. (2008) Defining the in vivo role for cytochrome b5 in cytochrome P450 function through the conditional hepatic deletion of microsomal cytochrome b5. J. Biol. Chem. 283, 31385–31393. 10.1074/jbc.M803496200. PubMed DOI PMC

Henderson C. J.; McLaughlin L. A.; Scheer N.; Stanley L. A.; Wolf C. R. (2015) Cytochrome b5 is a major determinant of human cytochrome P450 CYP2D6 and CYP3A4 activity in vivo. Mol. Pharmacol. 87, 733–739. 10.1124/mol.114.097394. PubMed DOI

Milichovský J.; Bárta F.; Schmeiser H. H.; Arlt V. M.; Frei E.; Stiborová M.; Martínek V. (2016) Active site mutations as a suitable tool contributing to explain a mechanism of aristolochic acid I nitroreduction by cytochromes P450 1A1, 1A2 and 1B1. Int. J. Mol. Sci. 17, 213.10.3390/ijms17020213. PubMed DOI PMC

Stiborová M.; Martínek V.; Rýdlová H.; Hodek P.; Frei E. (2002) Sudan I is a potential carcinogen for humans: Evidence for its metabolic activation and detoxication by human recombinant cytochrome P450 1A1 and liver microsomes. Cancer Res. 62, 5678–5684. PubMed

Perkins D. M.; Duncan J. R. (1987) Improved isolation of rat microsomal cytochrome b5 reductase. J. Chromatogr. 405, 319–325. 10.1016/S0021-9673(01)81773-8. PubMed DOI

Roos P. H. (1996) Chromatographic separation and behavior of microsomal cytochrome P450 and cytochrome b5. J. Chromatogr., Biomed. Appl. 684, 107–131. 10.1016/0378-4347(96)00018-7. PubMed DOI

Omura T.; Sato R. (1964) The carbon monoxide-binding pigment of liver microsomes: I Evidence for its hemoprotein nature. J. Biol. Chem. 239, 2370–2378. PubMed

Wiechelman K. J.; Braun R. D.; Fitzpatrick J. D. (1988) Investigation of the bicinchoninic acid protein assay: Identification of the groups responsible for color formation. Anal. Biochem. 175, 231–237. 10.1016/0003-2697(88)90383-1. PubMed DOI

Stiborová M.; Sejbal J.; Bořek-Dohalská L.; Aimová D.; Poljaková J.; Forsterová K.; Rupertová M.; Wiesner J.; Hudeček J.; Wiessler M.; Frei E. (2004) The anticancer drug ellipticine forms covalent DNA adducts, mediated by human cytochromes P450, through metabolism to 13-hydroxyellipticine and ellipticine N2-oxide. Cancer Res. 64, 8374–8380. 10.1158/0008-5472.CAN-04-2202. PubMed DOI

Kotrbová V.; Aimová D.; Březinová A.; Janouchová K.; Poljaková J.; Hodek P.; Frei E.; Stiborová M. (2006) Cytochromes P450 reconstituted with NADPH:P450 reductase mimic the activating and detoxicating metabolism of the anticancer drug ellipticine in microsomes. Neuro Endocrinol. Lett. 27 (Suppl. 2), 18–20. PubMed

Indra R.; Moserova M.; Kroftova N.; Sulc M.; Martinkova M.; Adam V.; Eckschlager T.; Kizek R.; Arlt V. M.; Stiborova M. (2014) Modulation of human cytochrome P450 1A1-mediated oxidation of benzo[a]pyrene by NADPH:cytochrome P450 oxidoreductase and cytochrome b5. Neuro Endocrinol. Lett. 35 (Suppl. 2), 105–113. PubMed

Moserová M.; Kotrbová V.; Aimová D.; Šulc M.; Frei E.; Stiborová M. (2009) Analysis of benzo[a]pyrene metabolites formed by rat hepatic microsomes using high pressure liquid chromatography: optimization of the method. Interdiscip. Toxicol. 2, 239–244. 10.2478/v10102-009-0024-0. PubMed DOI PMC

Hodek P.; Koblihová J.; Kizek R.; Frei E.; Arlt V. M.; Stiborová M. (2013) The relationship between DNA adduct formation by benzo[a]pyrene and expression of its activation enzyme cytochrome P450 1A1 in rat. Environ. Toxicol. Pharmacol. 36, 989–996. 10.1016/j.etap.2013.09.004. PubMed DOI

Schmeiser H. H.; Stiborova M.; Arlt V. M. (2013) 32P-postlabeling analysis of DNA adducts. Methods Mol. Biol. 1044, 389–401. 10.1007/978-1-62703-529-3_21. PubMed DOI

Sligar S. G.; Cinti D. L.; Gibson G. G.; Schenkman J. B. (1979) Spin state control of the hepatic cytochrome P450 redox potential. Biochem. Biophys. Res. Commun. 90, 925–932. 10.1016/0006-291X(79)91916-8. PubMed DOI

Lewis D. F.; Hlavica P. (2000) Interactions between redox partners in various cytochrome P450 systems: functional and structural aspects. Biochim. Biophys. Acta, Bioenerg. 1460, 353–374. 10.1016/S0005-2728(00)00202-4. PubMed DOI

Hlavica P.; Schulze J.; Lewis D. F. (2003) Functional interaction of cytochrome P450 with its redox partners: a critical assessment and update of the topology of predicted contact regions. J. Inorg. Biochem. 96, 279–297. 10.1016/S0162-0134(03)00152-1. PubMed DOI

Meunier B.; de Visser S. P.; Shaik S. (2004) Mechanism of oxidation reactions catalyzed by cytochrome P450 enzymes. Chem. Rev. 104, 3947–3980. 10.1021/cr020443g. PubMed DOI

Berka K.; Hendrychová T.; Anzenbacher P.; Otyepka M. (2011) Membrane position of ibuprofen agrees with suggested access path entrance to cytochrome P450 2C9 active site. J. Phys. Chem. A 115, 11248–11255. 10.1021/jp204488j. PubMed DOI PMC

Lewis D. F. V.; Hlavica P. (2000) Interactions between redox partners in various cytochrome P450 systems: functional and structural aspects. Biochim. Biophys. Acta, Bioenerg. 1460, 353–374. 10.1016/S0005-2728(00)00202-4. PubMed DOI

Aono T.; Sakamoto Y.; Miura M.; Takeuchi F.; Hori H.; Tsubaki M. (2010) Direct electrochemical analyses of human cytochromes b5 with a mutated heme pocket showed a good correlation between their midpoint and half wave potentials. J. Biomed. Sci. 17, 90.10.1186/1423-0127-17-90. PubMed DOI PMC

Im S. C.; Waskell L. (2011) The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase and cytochrome b5. Arch. Biochem. Biophys. 507, 144–153. 10.1016/j.abb.2010.10.023. PubMed DOI PMC

Iyanagi T. (1977) Redox properties of microsomal reduced nicotinamide adenine dinucleotide-cytochrome bs reductase and cytochrome b5. Biochemistry 16, 2725–2730. 10.1021/bi00631a021. PubMed DOI

Yamazaki H.; Johnson W. W.; Ueng Y. F.; Shimada T.; Guengerich F. P. (1996) Lack of electron transfer from cytochrome b5 in stimulation of catalytic activities of cytochrome P450 3A4. Characterization of a reconstituted cytochrome P450 3A4/NADPH-cytochrome P450 reductase system and studies with apo-cytochrome b5. J. Biol. Chem. 271, 27438–27444. 10.1074/jbc.271.44.27438. PubMed DOI

Benzo[a]pyrene-Induced Genotoxicity in Rats Is Affected by Co-Exposure to Sudan I by Altering the Expression of Biotransformation Enzymes

Identification of Enzymes Oxidizing the Tyrosine Kinase Inhibitor Cabozantinib: Cabozantinib Is Predominantly Oxidized by CYP3A4 and Its Oxidation Is Stimulated by cyt b5 Activity

Identification of Human Enzymes Oxidizing the Anti-Thyroid-Cancer Drug Vandetanib and Explanation of the High Efficiency of Cytochrome P450 3A4 in its Oxidation

Exposure to endocrine disruptors 17alpha-ethinylestradiol and estradiol influences cytochrome P450 1A1-mediated genotoxicity of benzo[a]pyrene and expression of this enzyme in rats

The impact of chemotherapeutic drugs on the CYP1A1-catalysed metabolism of the environmental carcinogen benzo[a]pyrene: Effects in human colorectal HCT116 TP53(+/+), TP53(+/-) and TP53(-/-) cells

Cytochrome b 5 impacts on cytochrome P450-mediated metabolism of benzo[a]pyrene and its DNA adduct formation: studies in hepatic cytochrome b 5 /P450 reductase null (HBRN) mice

Comparison of human cytochrome P450 1A1-catalysed oxidation of benzo[a]pyrene in prokaryotic and eukaryotic expression systems

Cytochrome b5 plays a dual role in the reaction cycle of cytochrome P450 3A4 during oxidation of the anticancer drug ellipticine