title: Cytochrome b5 and epoxide hydrolase contribute to benzo[a]pyrene-DNA adduct formation catalyzed by cytochrome P450 1A1 under low NADPH:P450 oxidoreductase conditions.
abstract: In previous studies we had administered benzo[a]pyrene (BaP) to genetically engineered mice (HRN) which do not express NADPH:cytochrome P450 oxidoreductase (POR) in hepatocytes and observed higher DNA adduct levels in livers of these mice than in wild-type mice. To elucidate the reason for this unexpected finding we have used two different settings for in vitro incubations; hepatic microsomes from control and BaP-pretreated HRN mice and reconstituted systems with cytochrome P450 1A1 (CYP1A1), POR, cytochrome b5, and epoxide hydrolase (mEH) in different ratios. In microsomes from BaP-pretreated mice, in which Cyp1a1 was induced, higher levels of BaP metabolites were formed, mainly of BaP-7,8-dihydrodiol. At a low POR:CYP1A1 ratio of 0.05:1 in the reconstituted system, the amounts of BaP diones and BaP-9-ol formed were essentially the same as at an equimolar ratio, but formation of BaP-3-ol was ∼ 1.6-fold higher. Only after addition of mEH were BaP dihydrodiols found. Two BaP-DNA adducts were formed in the presence of mEH, but only one when CYP1A1 and POR were present alone. At a ratio of POR:CYP1A1 of 0.05:1, addition of cytochrome b5 increased CYP1A1-mediated BaP oxidation to most of its metabolites indicating that cytochrome b5 participates in the electron transfer from NADPH to CYP1A1 required for enzyme activity of this CYP. BaP-9-ol was formed even by CYP1A1 reconstituted with cytochrome b5 without POR. Our results suggest that in livers of HRN mice Cyp1a1, cytochrome b5 and mEH can effectively activate BaP to DNA binding species, even in the presence of very low amounts of POR.
authors: Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2, Czech Republic. Electronic address: stiborov@natur.cuni.cz.
authors: Marie
authors: M
authors: Stiborová
authors: Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2, Czech Republic.
authors: Michaela
authors: M
authors: Moserová
authors: Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2, Czech Republic.
authors: Věra
authors: V
authors: Černá
authors: Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2, Czech Republic.
authors: Radek
authors: R
authors: Indra
authors: Institute of Organic Chemistry and Biochemistry, v.v.i. Academy of Sciences, Flemingovo n. 2, 166 10 Prague 6, Czech Republic.
authors: Martin
authors: M
authors: Dračínský
authors: Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2, Czech Republic.
authors: Miroslav
authors: M
authors: Šulc
authors: Division of Cancer Research, Medical Research Institute, Jacqui Wood Cancer Centre, University of Dundee, Dundee DD1 9SY, United Kingdom.
authors: Colin J
authors: CJ
authors: Henderson
authors: Division of Cancer Research, Medical Research Institute, Jacqui Wood Cancer Centre, University of Dundee, Dundee DD1 9SY, United Kingdom.
authors: C Roland
authors: CR
authors: Wolf
authors: Research Group Genetic Alterations in Carcinogenesis, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
authors: Heinz H
authors: HH
authors: Schmeiser
authors: Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment and Health, King's College London, 150 Stamford Street, London SE1 9NH, United Kingdom.
authors: David H
authors: DH
authors: Phillips
authors: Division of Preventive Oncology, National Center for Tumour Diseases, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
authors: Eva
authors: E
authors: Frei
authors: Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment and Health, King's College London, 150 Stamford Street, London SE1 9NH, United Kingdom.
authors: Volker M
authors: VM
authors: Arlt
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001564:Benzo(a)pyrene / Q000378:metabolism* / Q000633:toxicity; D002273:Carcinogens / Q000378:metabolism* / Q000633:toxicity; D002478:Cells, Cultured / Q000378:metabolism; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism*; D015786:Cytochromes b5 / Q000378:metabolism*; D018736:DNA Adducts / Q000378:metabolism*; D004851:Epoxide Hydrolases / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*
chemical_list: D002273:Carcinogens; D018736:DNA Adducts; C056612:benzo(a)pyrene-DNA adduct; D001564:Benzo(a)pyrene; D015786:Cytochromes b5; D019363:Cytochrome P-450 CYP1A1; D009251:NADPH-Ferrihemoprotein Reductase; D004851:Epoxide Hydrolases
keywords: Benzo[a]pyrene; Cytochrome P450 1A1; Cytochrome b(5); Epoxide hydrolase; NADPH:cytochrome P450 oxidoreductase