Carcinogenesis Advance Access originally published online on January 19, 2008
Carcinogenesis 2008 29(3):656-665; doi:10.1093/carcin/bgn002
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Metabolic activation of benzo[a]pyrene in vitro by hepatic cytochrome P450 contrasts with detoxification in vivo: experiments with hepatic cytochrome P450 reductase null mice
Section of Molecular Carcinogenesis, Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK
1 Department of Biochemistry, Faculty of Science, Charles University, 128 40 Prague 2, Czech Republic
2 Cancer Research UK Molecular Pharmacology Unit, Biomedical Research Centre, Dundee DD1 9SY, UK
3 Department of Analytical Chemistry, University of Wuppertal, D-42119 Wuppertal, Germany
4 Division of Molecular Toxicology, German Cancer Research Center, D-69126 Heidelberg, Germany
5 Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester, Leicester LE1 7RH, UK
6 Present address: Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA
* To whom correspondence should be addressed. Tel: +44 208 722 4405; Fax: +44 208 722 4052; Email: volker.arlt{at}icr.ac.uk
Many studies using mammalian cellular and subcellular systems have demonstrated that polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BaP), are metabolically activated by cytochrome P450s (CYPs). In order to evaluate the role of hepatic versus extra-hepatic metabolism of BaP and its pharmacokinetics, we used the hepatic cytochrome P450 reductase null (HRN) mouse model, in which cytochrome P450 oxidoreductase, the unique electron donor to CYPs, is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic CYP function. HRN and wild-type (WT) mice were treated intraperitoneally (i.p.) with 125 mg/kg body wt BaP daily for up to 5 days. Clearance of BaP from blood was analysed by high-performance liquid chromatography with fluorescence detection. DNA adduct levels were measured by 32P-post-labelling analysis with structural confirmation of the formation of 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene by liquid chromatography–tandem mass spectrometry analysis. Hepatic microsomes isolated from BaP-treated and untreated mice were also incubated with BaP and DNA in vitro. BaP–DNA adduct formation was up to 7-fold lower with the microsomes from HRN mice than with that from WT mice. Most of the hepatic microsomal activation of BaP in vitro was attributable to CYP1A. Pharmacokinetic analysis of BaP in blood revealed no significant differences between HRN and WT mice. BaP–DNA adduct levels were higher in the livers (up to 13-fold) and elevated in several extra-hepatic tissues of HRN mice (by 1.7- to 2.6-fold) relative to WT mice. These data reveal an apparent paradox, whereby hepatic CYP enzymes appear to be more important for detoxification of BaP in vivo, despite being involved in its metabolic activation in vitro.
Abbreviations: AhR, aryl hydrocarbon receptor; BaP, benzo[a]pyrene; BPDE, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide; CYP, cytochrome P450; dG-N2-BPDE, 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene; EROD, 7-ethoxyresorufin O-deethylation; GST, glutathione S-transferase; HPLC, high-performance liquid chromatography; HRN, hepatic cytochrome P450 reductase null; i.p., intraperitoneally; LC–MS/MS, liquid chromatography–tandem mass spectrometry; NADH, reduced form of nicotinamide adenine dinucleotide; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NF, naphthoflavone; POR, cytochrome P450 oxidoreductase; PTGS, prostaglandin H synthase; SRM, selected reaction monitoring; TLC, thin-layer chromatography; WT, wild-type
Received August 22, 2007; revised December 11, 2007; accepted December 27, 2007.