Extra-hepatic metabolism of xenobiotics by epithelial tissues has evolved as a self-defence mechanism but has potential to contribute to the local activation of carcinogens. Bladder epithelium (urothelium) is bathed in excreted urinary toxicants and pro-carcinogens. This study reveals how differentiation affects cytochrome P450 (CYP) activity and the role of NADPH:P450 oxidoreductase (POR). CYP1A1 and CYP1B1 transcripts were inducible in normal human urothelial (NHU) cells maintained in both undifferentiated and functional barrier-forming differentiated states in vitro. However, ethoxyresorufin O-deethylation (EROD) activity, the generation of reactive BaP metabolites and BaP-DNA adducts, were predominantly detected in differentiated NHU cell cultures. This gain-of-function was attributable to the expression of POR, an essential electron donor for all CYPs, which was significantly upregulated as part of urothelial differentiation. Immunohistology of muscle-invasive bladder cancer (MIBC) revealed significant overall suppression of POR expression. Stratification of MIBC biopsies into "luminal" and "basal" groups, based on GATA3 and cytokeratin 5/6 labeling, showed POR over-expression by a subgroup of the differentiated luminal tumors. In bladder cancer cell lines, CYP1-activity was undetectable/low in basal PORlo T24 and SCaBER cells and higher in the luminal POR over-expressing RT4 and RT112 cells than in differentiated NHU cells, indicating that CYP-function is related to differentiation status in bladder cancers. This study establishes POR as a predictive biomarker of metabolic potential. This has implications in bladder carcinogenesis for the hepatic versus local activation of carcinogens and as a functional predictor of the potential for MIBC to respond to prodrug therapies.

Department of Analytical Environmental and Forensic Sciences MRC PHE Centre for Environment and Health King's College London Franklin Wilkins Building London UK

Department of Urology Frankfurt University Medical Center Johann Wolfgang Goethe University Frankfurt am Main Germany

Department of Urology Regensburg University Medical Centre Regensburg Germany

Faculty of Science Department of Biochemistry Charles University Albertov Prague Czech Republic

Jack Birch Unit of Molecular Carcinogenesis Department of Biology University of York Heslington York UK

NIHR Health Protection Research Unit in Health Impact of Environmental Hazards at King's College London in Partnership with Public Health England Franklin Wilkins Building London UK

St James's Hospital Leeds UK

Zobrazit více v PubMed

Gundert‐Remy U, Bernauer U, Blomeke B, et al. Extrahepatic metabolism at the body's internal‐external interfaces. Drug Metab Rev. 2014; 46:291–324. PubMed

Sutter CH, Yin H, Li Y, et al. EGF receptor signaling blocks aryl hydrocarbon receptor‐mediated transcription and cell differentiation in human epidermal keratinocytes. PNAS. 2009; 106:4266–4271. PubMed PMC

Kiriluk KJ, Prasad SM, Patel AR, Steinberg GD, Smith ND. Bladder cancer risk from occupational and environmental exposures. Urol Oncol. 2012; 30:199–211. PubMed

Rendic S, Guengerich FP. Contributions of human enzymes in carcinogen metabolism. Chem Res Toxicol. 2012; 25:1316–1383. PubMed PMC

Figueroa JD, Malats N, Garcia‐Closas M, et al. Bladder cancer risk and genetic variation in AKR1C3 and other metabolizing genes. Carcinogenesis. 2008; 29:1955–1962. PubMed PMC

Grando JP, Kuasne H, Losi‐Guembarovski R, et al. Association between polymorphisms in the biometabolism genes CYP1A1, GSTM1, GSTT1 and GSTP1 in bladder cancer. Clin Exp Med. 2009; 9:21–28. PubMed

Alexandrov LB, Nik‐Zainal S, Wedge DC, et al. Signatures of mutational processes in human cancer. Nature. 2013; 500:415–421. PubMed PMC

Freedman ND, Silverman DT, Hollenbeck AR, Schatzkin A, Abnet CC. Association between smoking and risk of bladder cancer among men and women. Jama. 2011; 306:737–745. PubMed PMC

Haugen A, Becher G, Benestad C, et al. Determination of polycyclic aromatic hydrocarbons in the urine, benzo(a)pyrene diol epoxide‐DNA adducts in lymphocyte DNA, and antibodies to the adducts in sera from coke oven workers exposed to measured amounts of polycyclic aromatic hydrocarbons in the work atmosphere. Cancer Res. 1986; 46:4178–4183. PubMed

Benhamou S, Laplanche A, Guillonneau B, et al. DNA adducts in normal bladder tissue and bladder cancer risk. Mutagenesis. 2003; 18:445–448. PubMed

Sulc M, Indra R, Moserova M, et al. The impact of individual cytochrome P450 enzymes on oxidative metabolism of benzo[a]pyrene in human livers. Environ Mol Mutagen. 2016; 57:229–235. PubMed PMC

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. PubMed PMC

El‐Serafi I, Afsharian P, Moshfegh A, Hassan M, Terelius Y. Cytochrome P450 oxidoreductase influences CYP2B6 activity in cyclophosphamide bioactivation. PLoS ONE. 2015; 10:e0141979. PubMed PMC

Arlt VM, Stiborova M, Henderson CJ, et al. 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. PubMed

Iyanagi T, Xia C, Kim JJ. NADPH‐cytochrome P450 oxidoreductase: prototypic member of the diflavin reductase family. Arch Biochem Biophys. 2012; 528:72–89. PubMed PMC

Xiao X, Ma G, Li S, et al. Functional POR A503V is associated with the risk of bladder cancer in a Chinese population. Sci Rep. 2015; 5:11751. PubMed PMC

Vanderslice RR, Boyd JA, Eling TE, Philpot RM. The cytochrome P‐450 monooxygenase system of rabbit bladder mucosa: enzyme components and isozyme 5‐dependent metabolism of 2‐aminofluorene. Cancer Res. 1985; 45:5851–5858. PubMed

Guhe C, Degen GH, Schuhmacher US, Kiefer F, Follmann W. Drug metabolizing enzyme activities in porcine urinary bladder epithelial cell cultures (PUBEC). Arch Toxicol. 1996; 70:599–606. PubMed

Rheinwald JG, O'Connell TM. Intermediate filament proteins as distinguishing markers of cell type and differentiated state in cultured human urinary tract epithelia. Ann NY Acad Sci. 1985; 455:259–267. PubMed

Baker SC, Shabir S, Southgate J. Biomimetic urothelial tissue models for the in vitro evaluation of barrier physiology and bladder drug efficacy. Mol Pharm. 2014; 11:1964–1970. PubMed

Southgate J, Hutton KA, Thomas DF, Trejdosiewicz LK. Normal human urothelial cells in vitro: proliferation and induction of stratification. Lab Invest. 1994; 71:583–594. PubMed

Cross WR, Eardley I, Leese HJ, Southgate J. A biomimetic tissue from cultured normal human urothelial cells: analysis of physiological function. Am J Physiol Renal Physiol. 2005; 289:F459–F468. PubMed

Booth C, Harnden P, Trejdosiewicz LK, Scriven S, Selby PJ, Southgate J. Stromal and vascular invasion in an human in vitro bladder cancer model. Lab Invest. 1997; 76:843–857. PubMed

van Oers JM, Wild PJ, Burger M, et al. FGFR3 mutations and a normal CK20 staining pattern define low‐grade noninvasive urothelial bladder tumours. Eur Urol. 2007; 52:760–768. PubMed

Wittekind C. [2010 TNM system: on the 7th edition of TNM classification of malignant tumors]. Pathologe. 2010; 31:331–332. PubMed

Otto W, Denzinger S, Fritsche HM, et al. The WHO classification of 1973 is more suitable than the WHO classification of 2004 for predicting survival in pT1 urothelial bladder cancer. BJU Int. 2011; 107:404–408. PubMed

Southgate J, Varley CL, Garthwaite MA, et al. Differentiation potential of urothelium from patients with benign bladder dysfunction. BJU Int. 2007; 99:1506–1516. PubMed PMC

Smith GC, Tew DG, Wolf CR. Dissection of NADPH‐cytochrome P450 oxidoreductase into distinct functional domains. PNAS. 1994; 91:8710–8714. PubMed PMC

Wohak LE, Krais AM, Kucab JE, et al. Carcinogenic polycyclic aromatic hydrocarbons induce CYP1A1 in human cells via a p53‐dependent mechanism. Arch Toxicol. 2016; 90:291–304. PubMed PMC

Divi RL, Luch A, Verma M, Mahadevan B. CYP1B1 detection. Curr Protoc Toxicol. 2012; Chapter 4:Unit 4:38. PubMed

Phillips DH, Arlt VM. (32)P‐postlabeling analysis of DNA adducts. Methods Mol Biol. 2014; 1105:127–138. PubMed

Kucab JE, van Steeg H, Luijten M, et al. TP53 mutations induced by BPDE in Xpa‐WT and Xpa‐Null human TP53 knock‐in (Hupki) mouse embryo fibroblasts. Mutat Res. 2015; 773:48–62. PubMed PMC

Dadhania V, Zhang M, Zhang L, et al. Meta‐analysis of the luminal and basal subtypes of bladder cancer and the identification of signature immunohistochemical markers for clinical use. EBioMedicine. 2016; 12:105–117. PubMed PMC

Shimada T, Gillam EM, Sutter TR, Strickland PT, Guengerich FP, Yamazaki H. Oxidation of xenobiotics by recombinant human cytochrome P450 1B1. Drug Metab Dispos. 1997; 25:617–622. PubMed

Sutherland M, Gill JH, Loadman PM, et al. Antitumor activity of a duocarmycin analogue rationalized to be metabolically activated by cytochrome P450 1A1 in human transitional cell carcinoma of the bladder. Mol Cancer Ther. 2013; 12:27–37. PubMed

Fichtner I, Monks A, Hose C, Stevens MF, Bradshaw TD. The experimental antitumor agents Phortress and doxorubicin are equiactive against human‐derived breast carcinoma xenograft models. Breast Cancer Res Treat. 2004; 87:97–107. PubMed

Akama T, Shida Y, Sugaya T, Ishida H, Gomi K, Kasai M. Novel 5‐aminoflavone derivatives as specific antitumor agents in breast cancer. J Med Chem. 1996; 39:3461–3469. PubMed

Hunter FW, Young RJ, Shalev Z, et al. Identification of P450 oxidoreductase as a major determinant of sensitivity to hypoxia‐activated prodrugs. Cancer Res. 2015; 75:4211–4223. PubMed

Moore BP, Hicks RM, Knowles MA, Redgrave S. Metabolism and binding of benzo(a)pyrene and 2‐acetylaminofluorene by short‐term organ cultures of human and rat bladder. Cancer Res. 1982; 42:642–648. PubMed

Stoner GD, Daniel FB, Schenck KM, Schut HA, Goldblatt PJ, Sandwisch DW. Metabolism and DNA binding of benzo[a]pyrene in cultured human bladder and bronchus. Carcinogenesis. 1982; 3:195–201. PubMed

Chang TK, Chen J, Yang G, Yeung EY. Inhibition of procarcinogen‐bioactivating human CYP1A1, CYP1A2 and CYP1B1 enzymes by melatonin. J Pineal Res. 2010; 48:55–64. PubMed

Alexandrov LB, Ju YS, Haase K, et al. Mutational signatures associated with tobacco smoking in human cancer. Science. 2016; 354:618–622. PubMed PMC

Adachi J, Mori Y, Matsui S, et al. Indirubin and indigo are potent aryl hydrocarbon receptor ligands present in human urine. J Biol Chem. 2001; 276:31475–31478. PubMed

Varley CL, Stahlschmidt J, Lee WC, et al. Role of PPARgamma and EGFR signalling in the urothelial terminal differentiation programme. J Cell Sci. 2004; 117:2029–2036. PubMed

Cho YC, Zheng W, Yamamoto M, Liu X, Hanlon PR, Jefcoate CR. Differentiation of pluripotent C3H10T1/2 cells rapidly elevates CYP1B1 through a novel process that overcomes a loss of Ah Receptor. Arch Biochem Biophys. 2005; 439:139–153. PubMed

Walsh AA, Szklarz GD, Scott EE. Human cytochrome P450 1A1 structure and utility in understanding drug and xenobiotic metabolism. J Biol Chem. 2013; 288:12932–12943. PubMed PMC

Chopra B, Hinley J, Oleksiewicz MB, Southgate J. Trans‐species comparison of PPAR and RXR expression by rat and human urothelial tissues. Toxicol Pathol. 2008; 36:485–495. PubMed

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