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

Department of Urology Regensburg University Medical Centre Regensburg Germany

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

St James's Hospital Leeds UK

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$a Differentiation-associated urothelial cytochrome P450 oxidoreductase predicates the xenobiotic-metabolizing activity of "luminal" muscle-invasive bladder cancers / $c SC. Baker, VM. Arlt, R. Indra, M. Joel, M. Stiborová, I. Eardley, N. Ahmad, W. Otto, M. Burger, P. Rubenwolf, DH. Phillips, J. Southgate,

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$a 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.

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$a Arlt, Volker M $u Department of Analytical, Environmental and Forensic Sciences, MRC-PHE Centre for Environment and Health, King's College London, Franklin-Wilkins Building, London, 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.

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$a Indra, Radek $u Faculty of Science, Department of Biochemistry, Charles University, Albertov, Prague, Czech Republic.

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$a Joel, Madeleine $u Department of Analytical, Environmental and Forensic Sciences, MRC-PHE Centre for Environment and Health, King's College London, Franklin-Wilkins Building, London, UK.

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$a Stiborová, Marie $u Faculty of Science, Department of Biochemistry, Charles University, Albertov, Prague, Czech Republic.

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$a Otto, Wolfgang $u Department of Urology, Regensburg University Medical Centre, Regensburg, Germany.

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$a Burger, Maximilian $u Department of Urology, Regensburg University Medical Centre, Regensburg, Germany. Department of Urology, Frankfurt University Medical Center, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany.

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$a Phillips, David H $u Department of Analytical, Environmental and Forensic Sciences, MRC-PHE Centre for Environment and Health, King's College London, Franklin-Wilkins Building, London, 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.

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