Distinct subtypes of urinary bladder epithelial cells with inducible and non-inducible cytochrome P450 1A1.
ABSTRACT Cultured primary porcine urinary bladder epithelial cells (PUBEC) represent an adequate and easy to handle in vitro system for studies of urothelial toxicity. PUBEC maintain in vivo-like metabolic activities and physiological functions. They express inducible cytochrome P4501A isoenzymes, which are of particular relevance, since they contribute to activation of bladder carcinogens. A possible drawback of PUBEC is their isolation from common domestic pigs that do not represent an inbred strain. In order to further establish PUBEC as a standard in vitro toxicity test system we analysed possible interindividual differences in CYP1A1 inducibility. Interestingly, we observed by flow cytometry that PUBEC obtained from individual pigs consist of two distinct subpopulations with inducible and non-inducible cells. A strong, concentration-dependent CYP1A1 induction was observed in the responsive subpopulation when incubated with benzo[a]pyrene (B[a]P) in a concentration range between 1 and 10 muM. In contrast, no CYP1A1 induction was obtained in the non-responsive subpopulation up to the highest tested concentrations of 100 muM. The fraction of responsive cells showed large interindividual differences ranging from 10 to 65% of the total cell number. For practical purposes it might be reasonable to analyse pools of PUBEC from five pigs which substantially reduce batch to batch variability. In conclusion, we have identified two functionally distinct subpopulations of urinary bladder epithelial cells. It will be interesting to study whether the CYP1A inducible subtype is more susceptible to bladder carcinogens.
- Archive für Toxikologie 08/2014; · 5.08 Impact Factor
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ABSTRACT: Polycyclic aromatic hydrocarbons (PAHs) are associated with occupational exposure and urban atmospheric pollution. Determination of the genotoxic properties of these compounds is thus of outmost importance. For this purpose a variety of cellular models have been widely used. Reliable results can however only be obtained with models reflecting the specific sensitivity of different organs towards PAHs. In this work, we compared the response to benzo[a]pyrene in cell lines from human lungs (A549) and bladder (T24); two important target organs for PAHs-induced cancer. Human hepatocytes (HepG2) were used as a reference, although liver is not a concern for PAHs carcinogenesis. Adducts arising from the ultimate diol-epoxide metabolite of B[a]P, BPDE, were found to be produced in a dose-dependent manner in HepG2. BPDE DNA adducts were not detected in T24 and in A549 their formation was found to be most efficient at the lowest concentration studied (0.2 µM). These results are probably explained by differences in induction and activity of phase I metabolization enzymes, as well as by proteins eliminating the B[a]P epoxide in A549. In addition to BPDE adducts, oxidative DNA damage, namely strand breaks and oxidized purines were measured and found to be produced only in minute amounts in all three cell lines. In summary, our results emphasize the large differences in the response of cells originating from different organs. Our data also point out the importance of carefully selecting the doses used in in vitro toxicological experiments. The example of A549 shows that working at high doses may lead to an underestimation of the risk. Finally, the choice of method for evaluating genotoxicity appears to be of crucial importance. The comet assay does not seem to be the best method for a compound like B[a]P which induces stable adducts causing limited oxidative damage.PLoS ONE 11/2013; 8(11):e78356. · 3.53 Impact Factor
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ABSTRACT: N-Acetyltransferases (NAT) are important enzymes in the metabolism of certain carcinogenic arylamines, as N-acetylation decreases or prevents their bioactivation via N-hydroxylation. To study such processes in the bladder, cell culture models may be used, but metabolic competence needs to be characterized. This study focused on the N-acetylation capacity of two urothelial cell systems, using p-aminobenzoic acid (PABA) and the hair dye precursor p-phenylenediamine (PPD), two well-known substrates of the enzyme NAT1. The constitutive NAT1 activity was investigated using primary cultures of porcine urinary bladder epithelial cells (PUBEC) and in the human urothelial cell line 5637 to assess their suitability for further in vitro studies on PABA and PPD-induced toxicity. N-Acetylation of PABA and PPD was determined by high-performance liquid chromatography (HPLC) analysis in cytosols of the two cell systems upon incubation with various substrate levels for up to 60 min. The primary PUBEC revealed higher N-acetylation rates (2.5-fold for PABA, 5-fold for PPD) compared to the 5637 cell line, based on both PABA conversion to its acetylated metabolite and formation of mono- and diacetylated PPD. The urothelial cell systems may thus be useful as a tool for further studies on the N-acetylation of aromatic amines via NAT1.Journal of Toxicology and Environmental Health Part A 10/2012; 75(19-20):1206-15. · 1.83 Impact Factor