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,Volker M Arlt,C Roland Wolf,Radek Indra,Klaus Kopka,Michaela Moserova,David H Phillips,Marie Stiborova,Heinz H Schmeiser,Colin J Henderson

doi:10.1007/s00204-018-2162-7

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 b5/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 b5, which can also act as an electron donor from cytochrome b5 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 b5 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.

Highlights

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment as a result of their formation by the incomplete combustion of organic matter (Baird et al 2005; International Agency for Research on Cancer (IARC) 2010)
When NADH was used as cofactor the overall formation of BaP metabolites in hepatic microsomal fractions from BaP-pretreated Hepatic Reductase Null (HRN) mice was significantly higher than with fractions from BaP-pretreated WT mice with all metabolites being formed in higher quantities
The in vitro experiments in this study using pooled hepatic microsomal fractions clearly demonstrated that cytochrome b5 (Cyb5) contributes to the bioactivation of BaP: formation of BaP–DNA adducts and BaP metabolites correlated with Cyp1a enzyme activity, and was lessened by the loss of electron donors when NADPH was used as cofactor to examine the P450 oxidoreductase (POR)-dependent pathway

Summary

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment as a result of their formation by the incomplete combustion of organic matter (Baird et al 2005; IARC 2010). Benzo[a]pyrene (BaP) is the most commonly studied and measured of the PAHs (Arlt et al 2015b; Krais et al 2016; Labib et al 2012, 2016; Siddens et al 2012; Wohak et al 2016; Long et al 2017) and it is classified by the International Agency for Research on Cancer (IARC) as a human carcinogen (Group 1). Archives of Toxicology (2018) 92:1625–1638 effects (Luch and Baird 2005; Phillips 2005). It is activated via cytochrome P450 (P450)-dependent monooxygenases with CYP1A1 and CYP1B1 playing the major role. BPDE reacts with DNA to form principally the pre-mutagenic adduct 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP (dG-N2-BPDE) (Arlt et al 2008; Kucab et al 2015; Long et al 2016)

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Results

Conclusion