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

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.Electronic supplementary materialThe online version of this article (10.1007/s00204-018-2162-7) contains supplementary material, which is available to authorized users.

The impact of p53 function on the metabolic activation of the carcinogenic air pollutant 3-nitrobenzanthrone and its metabolites 3-aminobenzanthrone and N-hydroxy-3-aminobenzanthrone in human cells

“…Tissue distribution of this SULT is restricted mainly to the liver and bladder ( 60 ), although low amounts have been detected in another human colon carcinoma cell line (Caco-2) ( 61 ) and in some human colon samples ( 36 ). Furthermore, the microsomal enzyme NADPH:P450 oxidoreductase (POR), which is the electron donor of CYP enzymes ( 62 ), is also capable of activating 3-NBA through simple nitroreduction in cell-free experimental systems ( 56 ). However, using the Hepatic Reductase Null mouse model, in which POR is specifically deleted in hepatocytes, it was shown that bioactivation of 3-NBA is dependent predominantly on cytosolic NQO1 rather than microsomal POR ( 10 ).…”

Application of hepatic cytochrome b/P450 reductase null (HBRN) mice to study the role of cytochrome b in the cytochrome P450-mediated bioactivation of the anticancer drug ellipticine

“…Hepatic microsomes from ellipticine-treated mice were isolated as described previously (Reed et al, 2018). Microsomes were isolated from 4 pooled livers of each mouse model.…”

“…POR enzyme activity was determined as described previously (Arlt et al, 2003). Western blotting analysis using 4–12% Bis-Tris gradient gels and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) were carried out as described previously (Reed et al, 2018). After migration the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes and the following primary antibodies were used: anti-Cyp1a1 1:1000 (sc-20,772 (H-70), Santa Cruz Biotech); anti-Cyp3a 1:20000 (ab3572, Abcam); anti-Por 1:1000 (ab39995, Abcam), anti-Cyb5 1:750 (ab69801, Abcam); and anti-Cyb5R 1:1000 (ABIN453978, antibodies-online.com).…”

Bulky DNA adducts, microRNA profiles, and lipid biomarkers in Norwegian tunnel finishing workers occupationally exposed to diesel exhaust

“…The nuclease P 1 enrichment version of the thin-layer chromatography 32 P-postlabelling assay was used to determine bulky DNA adduct formation. The procedure was performed essentially as described,10 with minor modifications. All samples were analysed blind and in duplicate.…”

“…PAHs induce cytochrome P450 monooxygenases, which are key metabolising enzymes in PAH-bioactivation to diol-epoxides, radical cations and o-quinones 10. These reactive metabolites can directly affect DNA by forming adducts and initiating carcinogenesis.…”