Cytochrome b5 impacts on cytochrome P450-mediated metabolism of benzo[a]pyrene and its DNA adduct formation: studies in hepatic cytochrome b5 /P450 reductase null (HBRN) mice

Lindsay Reed, Iveta Mrizova, Frantisek Barta, Radek Indra, Michaela Moserova, Klaus Kopka, Heinz H. Schmeiser, C. Roland Wolf, Colin J. Henderson, Marie Stiborova, David H. Phillips, Volker M. Arlt (Lead / Corresponding author)

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    Abstract

    Benzo[a]pyrene (BaP) is an environmental pollutant that, based on evidence largely from in vitro studies, exerts its genotoxic effects after metabolic activation by cytochrome P450s. In the present study, Hepatic Reductase Null (HRN) and Hepatic Cytochrome b 5 /P450 Reductase Null (HBRN) mice have been used to study the role of P450s in the metabolic activation of BaP in vivo. In HRN mice, cytochrome P450 oxidoreductase (POR), the electron donor to P450, is deleted specifically in hepatocytes. In HBRN mice the microsomal haemoprotein cytochrome b 5 , which can also act as an electron donor from cytochrome b 5 reductase to P450s, is also deleted in the liver. Wild-type (WT), HRN and HBRN mice were treated by i.p. injection with 125 mg/kg body weight BaP for 24 h. Hepatic microsomal fractions were isolated from BaP-treated and untreated mice. In vitro incubations carried out with BaP-pretreated microsomal fractions, BaP and DNA resulted in significantly higher BaP-DNA adduct formation with WT microsomal fractions compared to those from HRN or HBRN mice. Adduct formation (i.e. 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP [dG-N2-BPDE]) correlated with observed CYP1A activity and metabolite formation (i.e. BaP-7,8-dihydrodiol) when NADPH or NADH was used as enzymatic cofactors. BaP-DNA adduct levels (i.e. dG-N2-BPDE) in vivo were significantly higher (~ sevenfold) in liver of HRN mice than WT mice while no significant difference in adduct formation was observed in liver between HBRN and WT mice. Our results demonstrate that POR and cytochrome b 5 both modulate P450-mediated activation of BaP in vitro. However, hepatic P450 enzymes in vivo appear to be more important for BaP detoxification than its activation.

    Original languageEnglish
    Pages (from-to)1625-1638
    Number of pages14
    JournalArchives of Toxicology
    Volume92
    Issue number4
    Early online date24 Jan 2018
    DOIs
    Publication statusPublished - Apr 2018

    Fingerprint

    Cytochromes b5
    NADPH-Ferrihemoprotein Reductase
    Metabolism
    Cytochrome P-450 Enzyme System
    Oxidoreductases
    Liver
    Chemical activation
    benzo(a)pyrene-DNA adduct
    Environmental Pollutants
    Detoxification
    Electrons
    Benzo(a)pyrene
    Cytochromes
    Metabolites
    NADP
    NAD

    Cite this

    Reed, Lindsay ; Mrizova, Iveta ; Barta, Frantisek ; Indra, Radek ; Moserova, Michaela ; Kopka, Klaus ; Schmeiser, Heinz H. ; Wolf, C. Roland ; Henderson, Colin J. ; Stiborova, Marie ; Phillips, David H. ; Arlt, Volker M. / Cytochrome b5 impacts on cytochrome P450-mediated metabolism of benzo[a]pyrene and its DNA adduct formation : studies in hepatic cytochrome b5 /P450 reductase null (HBRN) mice. In: Archives of Toxicology. 2018 ; Vol. 92, No. 4. pp. 1625-1638.
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    abstract = "Benzo[a]pyrene (BaP) is an environmental pollutant that, based on evidence largely from in vitro studies, exerts its genotoxic effects after metabolic activation by cytochrome P450s. In the present study, Hepatic Reductase Null (HRN) and Hepatic Cytochrome b 5 /P450 Reductase Null (HBRN) mice have been used to study the role of P450s in the metabolic activation of BaP in vivo. In HRN mice, cytochrome P450 oxidoreductase (POR), the electron donor to P450, is deleted specifically in hepatocytes. In HBRN mice the microsomal haemoprotein cytochrome b 5 , which can also act as an electron donor from cytochrome b 5 reductase to P450s, is also deleted in the liver. Wild-type (WT), HRN and HBRN mice were treated by i.p. injection with 125 mg/kg body weight BaP for 24 h. Hepatic microsomal fractions were isolated from BaP-treated and untreated mice. In vitro incubations carried out with BaP-pretreated microsomal fractions, BaP and DNA resulted in significantly higher BaP-DNA adduct formation with WT microsomal fractions compared to those from HRN or HBRN mice. Adduct formation (i.e. 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP [dG-N2-BPDE]) correlated with observed CYP1A activity and metabolite formation (i.e. BaP-7,8-dihydrodiol) when NADPH or NADH was used as enzymatic cofactors. BaP-DNA adduct levels (i.e. dG-N2-BPDE) in vivo were significantly higher (~ sevenfold) in liver of HRN mice than WT mice while no significant difference in adduct formation was observed in liver between HBRN and WT mice. Our results demonstrate that POR and cytochrome b 5 both modulate P450-mediated activation of BaP in vitro. However, hepatic P450 enzymes in vivo appear to be more important for BaP detoxification than its activation.",
    author = "Lindsay Reed and Iveta Mrizova and Frantisek Barta and Radek Indra and Michaela Moserova and Klaus Kopka and Schmeiser, {Heinz H.} and Wolf, {C. Roland} and Henderson, {Colin J.} and Marie Stiborova and Phillips, {David H.} and Arlt, {Volker M.}",
    note = "Lindsay Reed is supported by a King’s College London Health Faculty PhD Studentship funded by the Medical Research Council (Grant 1524896). Work at King’s College London (Grant C313/A14329) and the University of Dundee (Grant C4639/A10822) is supported by Cancer Research UK. Work at King’s College London is further supported by the Wellcome Trust (Grants 101126/Z/13/Z and 101126/B/13/Z) and Natural Environmental Research Council (NE/L006782/1). Work at Charles University is supported by the Grant Agency of Czech Republic (Grant 17-12816S).",
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    Cytochrome b5 impacts on cytochrome P450-mediated metabolism of benzo[a]pyrene and its DNA adduct formation : studies in hepatic cytochrome b5 /P450 reductase null (HBRN) mice. / Reed, Lindsay; Mrizova, Iveta; Barta, Frantisek; Indra, Radek; Moserova, Michaela; Kopka, Klaus; Schmeiser, Heinz H.; Wolf, C. Roland; Henderson, Colin J.; Stiborova, Marie; Phillips, David H.; Arlt, Volker M. (Lead / Corresponding author).

    In: Archives of Toxicology, Vol. 92, No. 4, 04.2018, p. 1625-1638.

    Research output: Contribution to journalArticle

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    AU - Reed, Lindsay

    AU - Mrizova, Iveta

    AU - Barta, Frantisek

    AU - Indra, Radek

    AU - Moserova, Michaela

    AU - Kopka, Klaus

    AU - Schmeiser, Heinz H.

    AU - Wolf, C. Roland

    AU - Henderson, Colin J.

    AU - Stiborova, Marie

    AU - Phillips, David H.

    AU - Arlt, Volker M.

    N1 - Lindsay Reed is supported by a King’s College London Health Faculty PhD Studentship funded by the Medical Research Council (Grant 1524896). Work at King’s College London (Grant C313/A14329) and the University of Dundee (Grant C4639/A10822) is supported by Cancer Research UK. Work at King’s College London is further supported by the Wellcome Trust (Grants 101126/Z/13/Z and 101126/B/13/Z) and Natural Environmental Research Council (NE/L006782/1). Work at Charles University is supported by the Grant Agency of Czech Republic (Grant 17-12816S).

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    N2 - Benzo[a]pyrene (BaP) is an environmental pollutant that, based on evidence largely from in vitro studies, exerts its genotoxic effects after metabolic activation by cytochrome P450s. In the present study, Hepatic Reductase Null (HRN) and Hepatic Cytochrome b 5 /P450 Reductase Null (HBRN) mice have been used to study the role of P450s in the metabolic activation of BaP in vivo. In HRN mice, cytochrome P450 oxidoreductase (POR), the electron donor to P450, is deleted specifically in hepatocytes. In HBRN mice the microsomal haemoprotein cytochrome b 5 , which can also act as an electron donor from cytochrome b 5 reductase to P450s, is also deleted in the liver. Wild-type (WT), HRN and HBRN mice were treated by i.p. injection with 125 mg/kg body weight BaP for 24 h. Hepatic microsomal fractions were isolated from BaP-treated and untreated mice. In vitro incubations carried out with BaP-pretreated microsomal fractions, BaP and DNA resulted in significantly higher BaP-DNA adduct formation with WT microsomal fractions compared to those from HRN or HBRN mice. Adduct formation (i.e. 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP [dG-N2-BPDE]) correlated with observed CYP1A activity and metabolite formation (i.e. BaP-7,8-dihydrodiol) when NADPH or NADH was used as enzymatic cofactors. BaP-DNA adduct levels (i.e. dG-N2-BPDE) in vivo were significantly higher (~ sevenfold) in liver of HRN mice than WT mice while no significant difference in adduct formation was observed in liver between HBRN and WT mice. Our results demonstrate that POR and cytochrome b 5 both modulate P450-mediated activation of BaP in vitro. However, hepatic P450 enzymes in vivo appear to be more important for BaP detoxification than its activation.

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