Cytochrome b5 and epoxide hydrolase contribute to benzo[a]pyrene-DNA adduct formation catalyzed by cytochrome P450 1A1 under low NADPH:P450 oxidoreductase conditions

Marie Stiborová (Lead / Corresponding author), Michaela Moserová, Vera Černá, Radek Indra, Martin Dračínský, Miroslav Šulc, Colin J. Henderson, C. Roland Wolf, Heinz H. Schmeiser, David H. Phillips, Eva Frei, Volker M. Arlt

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    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 b, 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 b increased CYP1A1-mediated BaP oxidation to most of its metabolites indicating that cytochrome b 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 b without POR. Our results suggest that in livers of HRN mice Cyp1a1, cytochrome b and mEH can effectively activate BaP to DNA binding species, even in the presence of very low amounts of POR.
    Original languageEnglish
    Pages (from-to)1-12
    Number of pages12
    JournalToxicology
    Volume318
    Issue number1
    DOIs
    Publication statusPublished - 6 Apr 2014

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    Epoxide Hydrolases
    Cytochromes b5
    NADP
    Cytochrome P-450 Enzyme System
    Cytochromes b
    Oxidoreductases
    Metabolites
    Microsomes
    Liver
    NADPH-Ferrihemoprotein Reductase
    DNA Adducts
    Benzo(a)pyrene
    Enzyme activity
    benzo(a)pyrene-DNA adduct
    Hepatocytes
    Electrons
    Oxidation
    DNA
    Enzymes

    Cite this

    Stiborová, Marie ; Moserová, Michaela ; Černá, Vera ; Indra, Radek ; Dračínský, Martin ; Šulc, Miroslav ; Henderson, Colin J. ; Wolf, C. Roland ; Schmeiser, Heinz H. ; Phillips, David H. ; Frei, Eva ; Arlt, Volker M. / Cytochrome b5 and epoxide hydrolase contribute to benzo[a]pyrene-DNA adduct formation catalyzed by cytochrome P450 1A1 under low NADPH:P450 oxidoreductase conditions. In: Toxicology. 2014 ; Vol. 318, No. 1. pp. 1-12.
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    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 b, 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 b increased CYP1A1-mediated BaP oxidation to most of its metabolites indicating that cytochrome b 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 b without POR. Our results suggest that in livers of HRN mice Cyp1a1, cytochrome b and mEH can effectively activate BaP to DNA binding species, even in the presence of very low amounts of POR.",
    author = "Marie Stiborov{\'a} and Michaela Moserov{\'a} and Vera Čern{\'a} and Radek Indra and Martin Drač{\'i}nsk{\'y} and Miroslav Šulc and Henderson, {Colin J.} and Wolf, {C. Roland} and Schmeiser, {Heinz H.} and Phillips, {David H.} and Eva Frei and Arlt, {Volker M.}",
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    Stiborová, M, Moserová, M, Černá, V, Indra, R, Dračínský, M, Šulc, M, Henderson, CJ, Wolf, CR, Schmeiser, HH, Phillips, DH, Frei, E & Arlt, VM 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, vol. 318, no. 1, pp. 1-12. https://doi.org/10.1016/j.tox.2014.02.002

    Cytochrome b5 and epoxide hydrolase contribute to benzo[a]pyrene-DNA adduct formation catalyzed by cytochrome P450 1A1 under low NADPH:P450 oxidoreductase conditions. / Stiborová, Marie (Lead / Corresponding author); Moserová, Michaela; Černá, Vera; Indra, Radek; Dračínský, Martin; Šulc, Miroslav; Henderson, Colin J.; Wolf, C. Roland; Schmeiser, Heinz H.; Phillips, David H.; Frei, Eva; Arlt, Volker M.

    In: Toxicology, Vol. 318, No. 1, 06.04.2014, p. 1-12.

    Research output: Contribution to journalArticle

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    T1 - Cytochrome b5 and epoxide hydrolase contribute to benzo[a]pyrene-DNA adduct formation catalyzed by cytochrome P450 1A1 under low NADPH:P450 oxidoreductase conditions

    AU - Stiborová, Marie

    AU - Moserová, Michaela

    AU - Černá, Vera

    AU - Indra, Radek

    AU - Dračínský, Martin

    AU - Šulc, Miroslav

    AU - Henderson, Colin J.

    AU - Wolf, C. Roland

    AU - Schmeiser, Heinz H.

    AU - Phillips, David H.

    AU - Frei, Eva

    AU - Arlt, Volker M.

    PY - 2014/4/6

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    N2 - 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 b, 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 b increased CYP1A1-mediated BaP oxidation to most of its metabolites indicating that cytochrome b 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 b without POR. Our results suggest that in livers of HRN mice Cyp1a1, cytochrome b and mEH can effectively activate BaP to DNA binding species, even in the presence of very low amounts of POR.

    AB - 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 b, 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 b increased CYP1A1-mediated BaP oxidation to most of its metabolites indicating that cytochrome b 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 b without POR. Our results suggest that in livers of HRN mice Cyp1a1, cytochrome b and mEH can effectively activate BaP to DNA binding species, even in the presence of very low amounts of POR.

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