The tumour suppressor p53 is one of the most important cancer genes. Previous findings have shown that p53 expression can influence DNA adduct formation of the environmental carcinogen benzo[a]pyrene (BaP) in human cells, indicating a role for p53 in the cytochrome P450 (CYP) 1A1-mediated biotransformation of BaP in vitro. We investigated the potential role of p53 in xenobiotic metabolism in vivo by treating Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice with BaP. BaP-DNA adduct levels, as measured by (32)P-postlabelling analysis, were significantly higher in liver and kidney of Trp53(-/-) mice than of Trp53(+/+) mice. Complementarily, significantly higher amounts of BaP metabolites were also formed ex vivo in hepatic microsomes from BaP-pretreated Trp53(-/-) mice. Bypass of the need for metabolic activation by treating mice with BaP-7,8-dihydrodiol-9,10-epoxide resulted in similar adduct levels in liver and kidney in all mouse lines, confirming that the influence of p53 is on the biotransformation of the parent compound. Higher BaP-DNA adduct levels in the livers of Trp53(-/-) mice correlated with higher CYP1A protein levels and increased CYP1A enzyme activity in these animals. Our study demonstrates a role for p53 in the metabolism of BaP in vivo, confirming previous in vitro results on a novel role for p53 in CYP1A1-mediated BaP metabolism. However, our results also suggest that the mechanisms involved in the altered expression and activity of the CYP1A1 enzyme by p53 in vitro and in vivo are different.

Analytical and Environmental Sciences Division MRC PHE Centre for Environment and Health King's College London Franklin Wilkins Building 150 Stamford Street London SE1 9NH UK

Biochemical Institute for Environmental Carcinogens Prof Dr Gernot Grimmer Foundation 22927 Grosshansdorf Germany

Center for Health Protection National Institute for Public Health and the Environment 3721 MA Bilthoven The Netherlands

Department of Biochemistry Faculty of Science Charles University 12840 Prague 2 Czech Republic

Department of Human Genetics Leiden University Medical Center 2300 RC Leiden The Netherlands

Department of Toxicology School for Nutrition Toxicology and Metabolism Maastricht University Medical Centre 6200 MD Maastricht The Netherlands

Division of Radiopharmaceutical Chemistry German Cancer Research Center Im Neuenheimer Feld 280 69120 Heidelberg Germany

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Ariyoshi N, Imaoka S, Nakayama K, Takahashi Y, Fujita K, Funae Y, Kamataki T. Comparison of the levels of enzymes involved in drug metabolism between transgenic or gene-knockout and the parental mice. Toxicol Pathol. 2001;29(Suppl):161–172. doi: 10.1080/019262301753178573. PubMed DOI

Arlt VM. 3-Nitrobenzanthrone, a potential human cancer hazard in diesel exhaust and urban air pollution: a review of the evidence. Mutagenesis. 2005;20:399–410. doi: 10.1093/mutage/gei057. PubMed DOI

Arlt VM, Bieler CA, Mier W, Wiessler M, Schmeiser HH. DNA adduct formation by the ubiquitous environmental contaminant 3-nitrobenzanthrone in rats determined by (32)P-postlabeling. Int J Cancer. 2001;93:450–454. doi: 10.1002/ijc.1346. PubMed DOI

Arlt VM, Glatt H, Muckel E, Pabel U, Sorg BL, Schmeiser HH, Phillips DH. Metabolic activation of the environmental contaminant 3-nitrobenzanthrone by human acetyltransferases and sulfotransferase. Carcinogenesis. 2002;23:1937–1945. doi: 10.1093/carcin/23.11.1937. PubMed DOI

Arlt VM, Stiborova M, Hewer A, Schmeiser HH, Phillips DH. Human enzymes involved in the metabolic activation of the environmental contaminant 3-nitrobenzanthrone: evidence for reductive activation by human NADPH:cytochrome p450 reductase. Cancer Res. 2003;63:2752–2761. PubMed

Arlt VM, Stiborova M, Henderson CJ, Osborne MR, Bieler CA, Frei E, Martinek V, Sopko B, Wolf CR, Schmeiser HH, Phillips DH. Environmental pollutant and potent mutagen 3-nitrobenzanthrone forms DNA adducts after reduction by NAD(P)H:quinone oxidoreductase and conjugation by acetyltransferases and sulfotransferases in human hepatic cytosols. Cancer Res. 2005;65:2644–2652. doi: 10.1158/0008-5472.CAN-04-3544. PubMed DOI

Arlt VM, Schmeiser HH, Osborne MR, Kawanishi M, Kanno T, Yagi T, Phillips DH, Takamura-Enya T. Identification of three major DNA adducts formed by the carcinogenic air pollutant 3-nitrobenzanthrone in rat lung at the C8 and N2 position of guanine and at the N6 position of adenine. Int J Cancer. 2006;118:2139–2146. doi: 10.1002/ijc.21622. PubMed DOI

Arlt VM, Stiborova M, Henderson CJ, Thiemann M, Frei E, Aimova D, Singh R, Gamboa da Costa G, Schmitz OJ, Farmer PB, Wolf CR, Phillips DH. 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. Carcinogenesis. 2008;29:656–665. doi: 10.1093/carcin/bgn002. PubMed DOI

Arlt VM, Poirier MC, Sykes SE, John K, Moserova M, Stiborova M, Wolf CR, Henderson CJ, Phillips DH. Exposure to benzo[a]pyrene of Hepatic Cytochrome P450 Reductase Null (HRN) and P450 Reductase Conditional Null (RCN) mice: detection of benzo[a]pyrene diol epoxide-DNA adducts by immunohistochemistry and 32P-postlabelling. Toxicol Lett. 2012;213:160–166. doi: 10.1016/j.toxlet.2012.06.016. PubMed DOI PMC

Bauer E, Guo Z, Ueng YF, Bell LC, Zeldin D, Guengerich FP. Oxidation of benzo[a]pyrene by recombinant human cytochrome P450 enzymes. Chem Res Toxicol. 1995;8:136–142. doi: 10.1021/tx00043a018. PubMed DOI

Berger AH, Pandolfi PP. Haplo-insufficiency: a driving force in cancer. J Pathol. 2011;223(2):137–146. doi: 10.1002/path.2800. PubMed DOI

Berger AH, Knudson AG, Pandolfi PP. A continuum model for tumour suppression. Nature. 2011;476:163–169. doi: 10.1038/nature10275. PubMed DOI PMC

Carmichael PL, Mills JJ, Campbell M, Basu M, Caldwell J. Mechanisms of hormonal carcinogenesis in the p53 +/- hemizygous knockout mouse: studies with diethylstilbestrol. Toxicol Pathol. 2001;29(Suppl):155–160. doi: 10.1080/019262301753178564. PubMed DOI

Donehower LA. Insights into wild-type and mutant p53 functions provided by genetically engineered mice. Human Mutat. 2014;35:715–727. doi: 10.1002/humu.22507. PubMed DOI

Ford JM. Regulation of DNA damage recognition and nucleotide excision repair: another role for p53. Mutat Res. 2005;577:195–202. doi: 10.1016/j.mrfmmm.2005.04.005. PubMed DOI

Goldstein I, Rivlin N, Shoshana OY, Ezra O, Madar S, Goldfinger N, Rotter V. Chemotherapeutic agents induce the expression and activity of their clearing enzyme CYP3A4 by activating p53. Carcinogenesis. 2013;34:190–198. doi: 10.1093/carcin/bgs318. PubMed DOI

Güngör N, Haegens A, Knaapen AM, Godschalk RW, Chiu RK, Wouters EF, van Schooten FJ. Lung inflammation is associated with reduced pulmonary nucleotide excision repair in vivo. Mutagenesis. 2010;25:77–82. doi: 10.1093/mutage/gep049. PubMed DOI

Hakura A, Tsutsui Y, Sonoda J, Kai J, Imade T, Shimada M, Sugihara Y, Mikami T. Comparison between in vivo mutagenicity and carcinogenicity in multiple organs by benzo[a]pyrene in the lacZ transgenic mouse (Muta Mouse) Mutat Res. 1998;398:123–130. doi: 10.1016/S0027-5107(97)00248-0. PubMed DOI

Hockley SL, Arlt VM, Brewer D, Giddings I, Phillips DH. Time- and concentration-dependent changes in gene expression induced by benzo(a)pyrene in two human cell lines, MCF-7 and HepG2. BMC Genom. 2006;7:260. doi: 10.1186/1471-2164-7-260. PubMed DOI PMC

Hockley SL, Arlt VM, Jahnke G, Hartwig A, Giddings I, Phillips DH. Identification through microarray gene expression analysis of cellular responses to benzo(a)pyrene and its diol-epoxide that are dependent or independent of p53. Carcinogenesis. 2008;29:202–210. doi: 10.1093/carcin/bgm227. PubMed DOI

Jacks T, Remington L, Williams BO, Schmitt EM, Halachmi S, Bronson RT, Weinberg RA. Tumor spectrum analysis in p53-mutant mice. Curr Biol. 1994;4:1–7. doi: 10.1016/S0960-9822(00)00002-6. PubMed DOI

Kenzelmann Broz D, Attardi LD. In vivo analysis of p53 tumor suppressor function using genetically engineered mouse models. Carcinogenesis. 2010;31:1311–1318. doi: 10.1093/carcin/bgp331. PubMed DOI PMC

Kim JH, Stansbury KH, Walker NJ, Trush MA, Strickland PT, Sutter TR. Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-diol by human cytochrome P450 1B1. Carcinogenesis. 1998;19:1847–1853. doi: 10.1093/carcin/19.10.1847. PubMed DOI

Kondraganti SR, Fernandez-Salguero P, Gonzalez FJ, Ramos KS, Jiang W, Moorthy B. Polycyclic aromatic hydrocarbon-inducible DNA adducts: evidence by 32P-postlabeling and use of knockout mice for Ah receptor-independent mechanisms of metabolic activation in vivo. Int J Cancer. 2003;103:5–11. doi: 10.1002/ijc.10784. PubMed DOI

Kucab JE, Phillips DH, Arlt VM. Linking environmental carcinogen exposure to TP53 mutations in human tumours using the human TP53 knock-in (Hupki) mouse model. FEBS J. 2010;277:2567–2583. doi: 10.1111/j.1742-4658.2010.07676.x. PubMed DOI

Kucab JE, van Steeg H, Luiten M, Schmeiser HH, White PA, Phillips DH, Arlt VM. TP53 mutations induced by BPDE in Xpa-WT and Xpa-Null human TP53 knock-in (Hupki) mouse embryo fibroblasts. Mutat Res, Fundam Mol Mech Mutagen. 2015;773:48–62. doi: 10.1016/j.mrfmmm.2015.01.013. PubMed DOI PMC

Langie SA, Knaapen AM, Brauers KJ, van Berlo D, van Schooten FJ, Godschalk RW. Development and validation of a modified comet assay to phenotypically assess nucleotide excision repair. Mutagenesis. 2006;21:153–158. doi: 10.1093/mutage/gel013. PubMed DOI

Lozano G. Mouse models of p53 functions. Cold Spring Harb Perspect Biol. 2010;2:a001115. doi: 10.1101/cshperspect.a001115. PubMed DOI PMC

Luch A, Baird WM. Metabolic activation and detoxification of polycyclic aromatic hydrocarbons. London: Imperial College Press; 2005.

Maddocks OD, Vousden KH. Metabolic regulation by p53. J Mol Med. 2011;89:237–245. doi: 10.1007/s00109-011-0735-5. PubMed DOI PMC

Mizerovska J, Dracinska H, Frei E, Schmeiser HH, Arlt VM, Stiborova M. Induction of biotransformation enzymes by the carcinogenic air-pollutant 3-nitrobenzanthrone in liver, kidney and lung, after intra-tracheal instillation in rats. Mutat Res. 2011;720:34–41. doi: 10.1016/j.mrgentox.2010.12.003. PubMed DOI

Mori Y, Koide A, Fuwa K, Wanibuchi H, Fukushima S. Lack of change in the levels of liver and kidney cytochrome P-450 isozymes in p53 +/- knockout mice treated with N-butyl-N-(4-hydroxybutyl)nitrosamine. Mutagenesis. 2001;16:377–383. doi: 10.1093/mutage/16.5.377. PubMed DOI

Nebert DW. Comparison of gene expression in cell culture to that in the intact animal: relevance to drugs and environmental toxicants. Focus on “development of a transactivator in hepatoma cells that allows expression of phase I, phase II, and chemical defense genes”. Am J Physiol Cell Physiol. 2006;290:C37–C41. doi: 10.1152/ajpcell.00444.2005. PubMed DOI

Nebert DW, Dalton TP. The role of cytochrome P450 enzymes in endogenous signalling pathways and environmental carcinogenesis. Nat Rev. 2006;6:947–960. doi: 10.1038/nrc2015. PubMed DOI

Nebert DW, Shi Z, Galvez-Peralta M, Uno S, Dragin N. Oral benzo[a]pyrene: understanding pharmacokinetics, detoxication, and consequences–Cyp1 knockout mouse lines as a paradigm. Mol Pharmacol. 2013;84:304–313. doi: 10.1124/mol.113.086637. PubMed DOI PMC

Olivier M, Hollstein M, Hainaut P. TP53 mutations in human cancers: origins, consequences, and clinical use. Cold Spring Harb Perspect Biol. 2010;2:a001008. doi: 10.1101/cshperspect.a001008. PubMed DOI PMC

Phillips DH, Arlt VM. The 32P-postlabeling assay for DNA adducts. Nat Protocol. 2007;2:2772–2781. doi: 10.1038/nprot.2007.394. PubMed DOI

Phillips DH, Arlt VM. (32)P-postlabeling analysis of DNA adducts. Meth Mol Biol. 2014;1105:127–138. doi: 10.1007/978-1-62703-739-6_10. PubMed DOI

Ress NB, Donnelly KC, George SE. The effect of pentachlorophenol on DNA adduct formation in p53 wild-type and knockout mice exposed to benzo[a]pyrene. Cancer Lett. 2002;178:11–17. doi: 10.1016/S0304-3835(01)00810-2. PubMed DOI

Sagredo C, Ovrebo S, Haugen A, Fujii-Kuriyama Y, Baera R, Botnen IV, Mollerup S. Quantitative analysis of benzo[a]pyrene biotransformation and adduct formation in Ahr knockout mice. Toxicol Lett. 2006;167:173–182. doi: 10.1016/j.toxlet.2006.09.005. PubMed DOI

Sagredo C, Mollerup S, Cole KJ, Phillips DH, Uppstad H, Ovrebo S. Biotransformation of benzo[a]pyrene in Ahr knockout mice is dependent on time and route of exposure. Chem Res Toxicol. 2009;22:584–591. doi: 10.1021/tx8003664. PubMed DOI

Sanders JM, Burka LT, Chanas B, Matthews HB. Comparative xenobiotic metabolism between Tg.AC and p53 +/- genetically altered mice and their respective wild types. Toxicol Sci. 2001;61:54–61. doi: 10.1093/toxsci/61.1.54. PubMed DOI

Sengupta S, Harris CC. p53: traffic cop at the crossroads of DNA repair and recombination. Nat Rev Mol Cell Biol. 2005;6:44–55. doi: 10.1038/nrm1546. PubMed DOI

Shimizu Y, Nakatsuru Y, Ichinose M, Takahashi Y, Kume H, Mimura J, Fujii-Kuriyama Y, Ishikawa T. Benzo[a]pyrene carcinogenicity is lost in mice lacking the aryl hydrocarbon receptor. Proc Natl Acad Sci USA. 2000;97:779–782. doi: 10.1073/pnas.97.2.779. PubMed DOI PMC

Simoes ML, Hockley SL, Schwerdtle T, da Costa GG, Schmeiser HH, Phillips DH, Arlt VM. Gene expression profiles modulated by the human carcinogen aristolochic acid I in human cancer cells and their dependence on TP53. Toxicol Appl Pharmacol. 2008;232:86–98. doi: 10.1016/j.taap.2008.06.006. PubMed DOI

Stiborova M, Dracinska H, Hajkova J, Kaderabkova P, Frei E, Schmeiser HH, Soucek P, Phillips DH, Arlt VM. The environmental pollutant and carcinogen 3-nitrobenzanthrone and its human metabolite 3-aminobenzanthrone are potent inducers of rat hepatic cytochromes P450 1A1 and -1A2 and NAD(P)H:quinone oxidoreductase. Drug Metab Dispos. 2006;34:1398–1405. doi: 10.1124/dmd.106.009373. PubMed DOI

Stiborova M, Dracinska H, Mizerovska J, Frei E, Schmeiser HH, Hudecek J, Hodek P, Phillips DH, Arlt VM. The environmental pollutant and carcinogen 3-nitrobenzanthrone induces cytochrome P450 1A1 and NAD(P)H:quinone oxidoreductase in rat lung and kidney, thereby enhancing its own genotoxicity. Toxicology. 2008;247:11–22. doi: 10.1016/j.tox.2008.01.018. PubMed DOI

Stiborova M, Martinek V, Svobodova M, Sístkova J, Dvorak Z, Ulrichova J, Simanek V, Frei E, Schmeiser HH, Phillips DH, Arlt VM. Mechanisms of the different DNA adduct forming potentials of the urban air pollutants 2-nitrobenzanthrone and carcinogenic 3-nitrobenzanthrone. Chem Res Toxicol. 2010;23:1192–1201. doi: 10.1021/tx100052d. PubMed DOI

Stiborova M, Moserova M, Cerna V, Indra R, Dracinsky M, Sulc M, Henderson CJ, Wolf CR, Schmeiser HH, Phillips DH, Frei E, Arlt VM. 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. 2014;318:1–12. doi: 10.1016/j.tox.2014.02.002. PubMed DOI

Taneja P, Zhu S, Maglic D, Fry EA, Kendig RD, Inoue K. Transgenic and knockout mice models to reveal the functions of tumor suppressor genes. Clin Med Insights Oncol. 2011;5:235–257. PubMed PMC

Van Kesteren PCE, Zwart PE, Schaap MM, Pronk TE, van Herwijnen MHM, Kleinjans JCS, Bokkers BGH, Godschalk RWL, Zeilmaker MJ, van Steeg H, Luijten M. Benzo[a]pyrene-induced transcriptomic responses in primary hepatocytes and in vivo liver: toxicokinetics is essential for in vivo-in vitro comparisons. Arch Toxicol. 2013;87:505–515. doi: 10.1007/s00204-012-0949-5. PubMed DOI

Wang T, Gavin HM, Arlt VM, Lawrence BP, Fenton SE, Medina D, Vorderstrasse BA. Aryl hydrocarbon receptor activation during pregnancy, and in adult nulliparous mice, delays the subsequent development of DMBA-induced mammary tumors. Int J Cancer. 2011;128:1509–1523. doi: 10.1002/ijc.25493. PubMed DOI PMC

Whibley C, Pharoah PD, Hollstein M. p53 polymorphisms: cancer implications. Nature Rev. 2009;9:95–107. PubMed

Wiechelman KJ, Braun RD, Fitzpatrick JD. Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation. Anal Biochem. 1988;175:231–237. doi: 10.1016/0003-2697(88)90383-1. PubMed DOI

Wohak LE, Krais AM, Kucab JE, Stertmann J, Ovrebo S, Seidel A, Phillips DH, Arlt VM (2014) Carcinogenic polycyclic aromatic hydrocarbons induce CYP1A1 in human cells via a p53 dependent mechanism. Arch Toxicol, Nov 15 (Epub ahead of print) PubMed PMC

The impact of p53 on aristolochic acid I-induced nephrotoxicity and DNA damage in vivo and in vitro

Exposure to endocrine disruptors 17alpha-ethinylestradiol and estradiol influences cytochrome P450 1A1-mediated genotoxicity of benzo[a]pyrene and expression of this enzyme in rats

The impact of chemotherapeutic drugs on the CYP1A1-catalysed metabolism of the environmental carcinogen benzo[a]pyrene: Effects in human colorectal HCT116 TP53(+/+), TP53(+/-) and TP53(-/-) cells

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

Comparison of human cytochrome P450 1A1-catalysed oxidation of benzo[a]pyrene in prokaryotic and eukaryotic expression systems

Impact of genetic modulation of SULT1A enzymes on DNA adduct formation by aristolochic acids and 3-nitrobenzanthrone

NADH:Cytochrome b5 Reductase and Cytochrome b5 Can Act as Sole Electron Donors to Human Cytochrome P450 1A1-Mediated Oxidation and DNA Adduct Formation by Benzo[a]pyrene

NADPH- and NADH-dependent metabolism of and DNA adduct formation by benzo[a]pyrene catalyzed with rat hepatic microsomes and cytochrome P450 1A1

Induced expression of microsomal cytochrome b5 determined at mRNA and protein levels in rats exposed to ellipticine, benzo[a]pyrene, and 1-phenylazo-2-naphthol (Sudan I)