Porcine Urinary Bladder Epithelial Cells Research Articles

Benzo[a]pyrene mediated time- and dose-dependent alteration in cellular metabolism of primary pig bladder cells with emphasis on proline cycling.

Exposure to xenobiotic such as benzo[a]pyrene (B[a]P) induces metabolic changes, which have a considerable impact on the cellular response. Nevertheless, we are just in the beginning to reach an understanding of these processes. In this study, a gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach was applied to distinguish the metabolic changes that bladder epithelia cells undergo upon B[a]P exposure. To closely reflect the epithelia cell conditions in vivo, freshly isolated primary porcine urinary bladder epithelial cells (PUBEC) were utilized for the current study. An untargeted metabolomics approach was used to characterize the time- (6h, 24h, 48h) and dose-dependent (0.5µM, 5µM, 10µM B[a]P) changes in the metabolome of PUBEC upon B[a]P exposure, which led to the profiling of more than 200 metabolites that differed significantly between control and exposed samples. Multivariate analysis of the data highlighted that in the experimental setup/model used other than the exposure concentration, it is the exposure time which seems to be most important for distinguishing between different groups and hence may have a bigger role in B[a]P-mediated toxicity but may be specific for cell model used and hence requires further investigations. Further, enrichment and pathway analysis using MetaboAnalyst highlighted that exposure to B[a]P mainly alters the cellular amino acid metabolism. Particularly, 1-pyrroline-5-carboxylic acid (P5C), an intermediate of the cycling of the amino acid proline, was identified as a differentially altered metabolite at all concentrations and exposure times used in the experiment. An increase in the activity of proline dehydrogenase/proline oxidase (PRODH/POX), which oxidizes proline to P5C, was also observed, further supporting our metabolomic data. Our findings contribute to an improved knowledge about the reprogramming of metabolism which is a fundamental element of the cellular response to B[a]P and draw attention to the role of proline in this context.

N-Acetylation of p-Aminobenzoic Acid and p-Phenylenediamine in Primary Porcine Urinary Bladder Epithelial Cells and in the Human Urothelial Cell Line 5637

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.

Effects of Cigarette Smoke Condensate on Primary Urothelial Cells in Vitro

Cigarette smoking is a risk factor for bladder cancer. Since urothelial cells express phase I and II enzymes these cells are able to metabolize precarcinogens into DNA reactive intermediates. Cigarette smoke is a complex mixture containing at least 80 known carcinogens. In this context especially aromatic amines and polycyclic aromatic hydrocarbons are discussed as being responsible for bladder-carcinogenicity. Cell cultures of primary porcine urinary bladder epithelial cells (PUBEC) have been useful models for studies on bladder-specific effects. These cells are metabolically competent and found to be a valuable tool for examining effects of cigarette smoke constituents. In the present study PUBEC were utilized to investigate the effects of the complex mixture cigarette smoke condensate total particulate matter (CSC TPM) with emphasis on induction of cytochrome P-450 1A1 (CYP1A1) and genotoxic effects. CYP1A1 induction was investigated by Western blot and flow cytometry. The most pronounced effects were found after 24 h of incubation with 1–10 μg/ml CSC TPM. Maximal induction was observed at 5 μg/ml by flow cytometry and at 10 μg/ml by Western blot analysis. Genotoxic effects were investigated by means of alkaline single-cell gel electrophoresis (“comet assay”) with and without the use of the DNA repair enzyme formamidopyrimidine-DNA glycosylase (Fpg) and the micronucleus (MN) test. A numerical concentration-dependent increase in Fpg-sensitive sites indicating oxidative DNA damage and a quantitative rise in MN formation were noted. The CSC utilized in this study contained low amounts of benzo[a]pyrene, 4-aminobiphenyl, and 2-naphthylamine. With regard to the observed CYP1A1 induction, these substances cannot explain the CYP1A1 inducing effect of CSC TPM. It is possible that other compounds within CSC TPM contribute to CYP1A1 induction in our cellular model.

Synergism of aromatic amines and benzo[a]pyrene in induction of Ah receptor-dependent genes

Aromatic amines have been shown to cause bladder cancer. However, epithelial cells of the urinary bladder, cells of origin of bladder cancer, may be exposed to numerous substances besides aromatic amines. In the present study, we analysed possible interactions between the aromatic amines 4-aminobiphenyl (4-ABP) as well as 2-naphthylamine (2-NA) and the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P). For this purpose we incubated primary porcine urinary bladder epithelial cells (PUBEC) with concentrations of 1 to 50 microM 4-ABP with and without co-exposure to B[a]P. As expected B[a]P increased mRNA expression of cytochrome P450 1A1 (CYP1A1), whereas 4-ABP had no effect. However, when co-exposed 4-ABP enhanced the induction of CYP1A1 by B[a]P. This result was confirmed by Western blot analysis of CYP1A1 protein expression. A similar effect as for CYP1A1 was also observed for cyclooxygenase-2 (COX-2) and UDP-glucuronosyltransferase 1 (UGT1). Next, we studied co-exposures of 2-NA and B[a]P. Similar as for 4-ABP also 2-NA enhanced B[a]P-mediated induction of CYP1A1. Our results demonstrate that some aromatic amines may enhance the influence of B[a]P on Ah receptor-dependent genes.

Analysis of CYP1A1 induction in single cells of urothelial cell populations by flow cytometry

As carcinogenesis is a process starting at the single-cell level it is desirable to study carcinogen-mediated effects in individual cells. A primary step in chemically induced carcinogenesis is the formation of reactive DNA-binding metabolites by cytochromes P450 (CYP). We applied indirect immunofluorescence to stain CYP1A1 in urothelial cells for quantification by flow cytometry. Our studies were carried out with metabolically competent primary porcine urinary bladder epithelial cells (PUBECs) and the human urothelial cell line 5637 for which we have previously demonstrated CYP1A1 mRNA induction by the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (B[a]P) applying real-time RT-PCR. Flow cytometric analysis revealed that for PUBEC and 5637 cells the fraction of CYP1A1-induced cells increased with B[a]P concentration. Furthermore, in 5637 cells this effect was time-dependent, being more pronounced after 48 h than after 24 h. However, CYP1A1 induction could not be detected in all analyzed PUBEC and 5637 cells after treatment with up to 50 muM B[a]P. The reason for this remains unknown at the moment. Overall, B[a]P-treated cells could be divided into fractions of clearly CYP1A1-induced and clearly uninduced cells. Another fraction of "unclear" CYP1A1-induced cells and one of unclassifiable cells remained, as quantification of CYP1A1 induction by flow cytometry was hampered by B[a]P-related fluorescence. This is ascribed to phenolic B[a]P metabolites formed by CYP1A1 and which are known to fluoresce at wavelengths above 500 nm, whereas B[a]P does not. Overall, the method permits the detection of CYP1A1 protein level in large numbers of individual cells, thereby providing an adequate basis for statistical analyses. Flow cytometric detection of CYP1A1 induction in individual cells allows further insight into the metabolic competence of single cells and therefore could be a valuable tool for toxicological studies.

Distinct subtypes of urinary bladder epithelial cells with inducible and non-inducible cytochrome P450 1A1

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.

Evaluation of Time Dependence and Interindividual Differences in Benzo[a]pyrene-Mediated CYP1A1 Induction and Genotoxicity in Porcine Urinary Bladder Cell Cultures*

Exposure to tobacco smoke is an established cause of cancer in humans and cigarette smoking is a risk factor for urinary bladder cancer development. Aromatic amines are believed responsible for the bladder-specific carcinogenic effect, but polycyclic aromatic hydrocarbons (PAHs) are also of potential relevance. Urothelial cells contain a number of xenobiotic-metabolizing enzymes, which enable them to convert pro-carcinogens into reactive intermediates. In a preceding study, it was demonstrated using cultured porcine urinary bladder epithelial cells (PUBEC) that CYP1A1 mRNA is induced in a potent manner by treatment with benzo[a]pyrene (BaP). In the present study, the time dependence of these effects was evaluated and whether PUBEC cultures derived from individual donors respond differently to BaP treatment was determined. CYP1A1 induction was analyzed by quantitative reverse-transcription polymerase chain reaction (RT-PCR), and genotoxic effects were studied using the Comet assay. Incubation of PUBEC with BaP increased CYP1A1 expression and induction of DNA strand breaks in a time-dependent manner. Interindividual differences were found between PUBEC cultures derived from several donor animals with respect to the response to BaP, such that the extent of CYP1A1 induction and magnitude of DNA damage was interrelated. Hence, individual differences in metabolic capacities and responsiveness to xenobiotics of urothelial cells from individual donors may be factors in susceptibility to genotoxic effects induced by PAHs.

Miniaturization of Primary Porcine Urinary Bladder Epithelial Cell Cultures and Application for Gene Expression Studies after Exposure to Benzo[a]Pyrene*

Benzo[a]pyrene (BaP) is an environmental pollutant used as a key marker substance for polycyclic aromatic hydrocarbons (PAHs). PAHs are believed to play a prominent role in the development of bladder cancer. A test system based on primary porcine urinary bladder epithelial cells (PUBEC) has been utilized as an in vitro model for urinary bladder epithelium. Recently in PUBEC cultures derived from pools of several bladders potent induction of CYP1A1 was detected after BaP treatment. Results from a modified approach using miniaturized PUBEC cultures for the analysis of individual bladder specimens with regard to cell growth and to BaP-mediated induction of CYP1A1 mRNA expression are presented herein. Two types of responses, low and high CYP1A1 induction among individual bladder specimens from eight donor animals, were detected. All of these tissue samples expressed the wild-type genotype of CYP1A1.

Primary Cultures of Human Urothelial Cells for Genotoxicity Testing

A cell culture system with human-derived urothelial cells was established based upon previous experience with cultures of porcine urinary bladder epithelial cells. Human tissue specimens used were derived from urinary bladders (n = 17) or ureters (n = 50) of patients undergoing urological operations. The epithelial origin and differentiation status was evaluated by an immunohistochemical staining for cytokeratins 7, 8, 18, 19, and 20 for isolated and cultured cells. Specimens from human ureters were better suited for primary cell cultures of the urothelium than specimens from human urinary bladders. Successful attachment and proliferation were reached by 98% of the ureter specimens (urinary bladder: 71%) and confluency was reached by 78% of the ureter cultures (urinary bladder: 18%). In the first 14 d of culture the cytokeratin patterns of cultured cells were comparable to those of native mucosa cells. During prolonged cell culture the cytokeratin patterns of the human urothelial cells (HUC) changed into a beginning dedifferentiation: Cytokeratin (CK) 18 was only detectable in cell cultures cultured for more than 29 d, whereas CK 19 was not detectable at d 29. Cell cultures of primary human urothelial cells may be used for in vitro testing of cytotoxic or genotoxic effects. The authors thank Dr. M. Albrecht for providing samples from pediatric surgery. Present address for Dr. Angelika Flieger is Federal Office of Civil Protection and Disaster Assistance, Bonn Federal Office of Civil Protection and Disaster Assistance, Provinzialstr. 93, D-53127 Bonn, Germany. Prof. Harald Schulze is deceased.

Repair of ochratoxin A-induced DNA damage and modulation of OTA-related genotoxicity by co-incubation with bile acids and methotrexatein vitro

The mycotoxin ochratoxin A (OTA) induced DNA strand breaks in porcine urinary bladder epithelial cells (PUBEC) and in Madin Darby canine kidney (MDCK) cells. A co-incubation with bile acids or methotrexate reduced or even prevented this adverse effect of OTAin vitro. The protective effect is possibly attributable to a decreased OTA uptake in cells, since bile acids and methotrexate are known to share common transport systems such as organic anion transporters (OAT) and/or organic anion transporting polypeptides (OATP) with the mycotoxin. OTA uptake in cells and its modulation can be one factor which determines the extent of adverse effects in different cell types. Another aspect of interest in this regard relates to repair of DNA damage: PUBEC cells are sensitive to OTA-induced damage which is more pronounced when DNA repair is blocked (by cytosine β-D-arabino-furanosid/hydroxyurea). On the other hand, when cells are kept in fresh (toxin-free) medium for 3 h, OTA-induced DNA damage decreased to control levels.

The effect of benzo(a)pyrene on porcine urinary bladder epithelial cells analyzed for the expression of selected genes and cellular toxicological endpoints

Consumption of tobacco products is the most relevant risk factor for the development of bladder cancer beside occupational contributions. In order to investigate mechanisms of tobacco smoke components in bladder carcinogenesis we have introduced a primary epithelial cell culture system derived from porcine urinary bladder as a suitable representative for the corresponding human tissue under physiological conditions. Two independent readouts were selected as markers for genotoxic events. Changes in the expression level of several toxicologically relevant genes should serve as indicators for early response, while classical genotoxic endpoints monitored manifested damages. Here, we present the first results of our study with benzo(a)pyrene (BaP) as a member of polycyclic aromatic hydrocarbons (PAHs) found in tobacco smoke. Cells treated with BaP show a dramatic increase in the expression of CYP1A1 that appears to be both indicator of and contributor for BaP toxicity. Genes coding for other proteins relevant in xenobiotic metabolism, signal transduction or tumor suppression show moderate effects or no enhancement of their expression levels. Comet assay and micronucleus test did show a significant, dose-dependent increase in DNA damages or aberrations after cell division. While these effects are conforming to the response at the mRNA expression level, they are less pronounced and require rather higher dosages of the chemical.

Comparative metabolic activation of benzidine and N-acetylbenzidine by prostaglandin H synthase

Benzidine and N-acetylbenzidine are activated to genotoxic metabolite(s) within the urothelial target tissue, with phase-I and phase-II enzymes being relevant. In principle, both benzidine and N-acetylbenzidine are activated by prostaglandin H synthase (PHS) to reactive intermediates. However, the relative impacts of benzidine and N-acetylbenzidine in this process remain unclear. Two experimental in vitro systems were used in the present comparative investigation: ram seminal vesicle microsomes rich in PHS and porcine urinary bladder epithelial cells (PUBEC) as a model system mimicking the general metabolic situation within the human urothelium. Benzidine, N-acetylbenzidine and N, N′-diacetylbenzidine were incubated with ram seminal vesicle microsomes and arachidonic acid and control incubations were performed with heat-inactivated microsomes. The metabolic disappearance of benzidine, N-acetylbenzidine or N, N′-diacetylbenzidine indicated a rapid turnover by PHS of benzidine and a slower turnover of N-acetylbenzidine. There was almost no PHS-associated metabolism of N, N′-diacetylbenzidine, suggesting that diacetylation of benzidine could represent a pathway of biological inactivation. Under similar conditions, incubations were performed with ram seminal vesicles and benzidine or N-acetylbenzidine upon addition of calf thymus DNA. After re-isolation of the DNA and 32 P -postlabeling, with benzidine 2 distinct adducts were found of unknown nature, and with N-acetylbenzidine a single adduct appeared with co-migrated with the N′-(3′-monophosphodeoxyguanosin-8-yl)- N-acetylbenzidine. PUBEC cells were also incubated with benzidine or N-acetylbenzidine. No DNA adduct was found with benzidine, but a total of five adducts was produced from N-acetylbenzidine. The major adduct again co-migrated with N′-(3′-monophosphodeoxyguanosin-8-yl)- N-acetylbenzidine. When benzidine was incubated with PUBEC cells N-acetylbenzidine and, with some delay, N, N′-diacetylbenzidine were formed. Application of Lineweaver–Burk plots for the formation of N-acetylbenzidine from benzidine revealed a K m of 56.4 μM and a V max of 7.05 nmol/h per 10 6 PUBEC cells. The investigations generally support a key role of N-acetylbenzidine at the target site of the urothelium.

Uptake and genotoxic effects of ochratoxin A in cultured porcine urinary bladder epithelial cells

Uptake of radiolabelled ochratoxin A (OTA) into porcine urinary bladder epithelial cells (PUBEC) was measured at neutral (pH 7.5) or acidic (pH 5.0) conditions. Genotoxicity of OTA was evaluated with the Comet assay and cytotoxicity with the neutral red uptake assay.At acidic pH-conditions, the bladder cells were able to take up more OTA than at neutral conditions. Cytotoxic effects were not increased at pH 5.0 compared to pH 7.5, but higher OTA uptake correlated with stronger genotoxic effects in the Comet assay at pH 5.0 compared to pH 7.5.These results demonstrate that uptake of OTA has to be regarded as an important factor for the toxicity of OTA as adverse effects depend on the amount of OTA taken up by the cells.

Toxicogenomics: an emerging discipline.

Toxicogenomics: an emerging discipline.

Effects of ochratoxin A on DNA evaluatedin vitro with the single cell gel electrophoresis (Comet assay).

In cell cultures of Madin Darby canine kidney (MDCK) cells, the mycotoxin ochratoxin A (OTA) induced single strand breaks (ssb) in a concentration dependent manner detected with the single cell gel electrophoresis (Comet assay). When an external metabolizing enzyme system (S9-mix from rat liver) was added, this genotoxic effect was significantly stronger. By addition of methotrexate (MT), a substrate of the hepatic organic anion transporter, the effect of OTA can be completely blocked at concentrations >100 μM methotrexate.When DNA repair was inhibited by addition of cytosine arabinose (araC) and hydroxyurea (HU), the tail length in the Comet assay increased dramatically and all treated cells showed ssb. A further culture of the damaged cells in the absence of any supplement resulted in a complete repair of the damaged DNA within three hours.Compared with MDCK cells, primary cultured porcine urinary bladder epithelial cells (PUBECs) showed weaker effects in the Comet assay if treated with OTA. The presence of S9-mix did not significantly enhance the response. Methotrexate only partially reduced the OTA-induced effects, because in PUBECs methotrexate induced ssb at high concentrations. If DNA repair was inhibited, also in PUBECs clearly more ssb were induced by OTA, an effect which was reversible.These results demonstrate that OTA induces single strand breaks in vitro. The damaged DNA can be repaired more effectively in primary cultured epithelial cells (PUBECs) compared to cells of a cell line (MDCK cells). By competitive inhibition of OTA uptake, DNA damage can be prevented with suitable substrates.

Cultured porcine urinary bladder epithelial cells as a screening model for genotoxic effects of aromatic amines: characterisation and application of the cell culture model.

Isolated epithelial cells from porcine urinary bladders were maintained in dividing long-term monolayer cultures, and were used as a model system for the urinary bladder in toxicological studies in vitro. To examine the state of differentiation during the culture period, the culture system was characterised morphologically by light and transmission electron microscopy and by immune fluorescence labelling with antibodies against cytokeratins 7,13 and pan. The cultured cells were identified as urothelial epithelium by their polarised structure, and by their expression of several uroepithelial specific morphological features, such as fusiform vesicles, tight junctions and an asymmetric apical cell membrane. Additionally, the cells were labelled with anti-cytokeratin 7,13 and pan antibodies, and negatively with anti-vimentin antibodies. The maintenance of suitable culture conditions was shown by the stable enzyme activities of (gamma-glutamyltranspeptidase, alkaline phosphatase and acid phosphatase over a culture period of 4 weeks. A good viability of the cultured cells under the chosen culture conditions was shown by the presence of low amounts of lactate dehydrogenase (< of = 5%) in the culture medium. The activities of the chosen marker enzymes for cell differentiation (gamma-glutamyltranspeptidase), lysosomes (acid phosphatase) and luminal membranes (alkaline phosphatase) were relatively stable over the observed culture period. Enzyme activities involved in metabolism of xenobiotics were determined, to define the ability for metabolism in cultured cells compared with bladder tissue in situ. Several constitutive phase I and II enzyme activities were found to be stable during the culture period, indicating that the cultured cells should be able to metabolise xenobiotics in a comparable manner to the urothelium in vivo. The cytotoxic effects of xenobiotics were investigated and IC50 values were determined by means of lactate dehydrogenase leakage and inhibition of neutral red uptake. The induction of sister chromatid exchanges was used as a parameter for the genotoxic effects of several xenobiotics. This cell culture system was found to be a very good screening system for the testing of substances that affect the bladder, especially aromatic amines.

Cytotoxicity of ochratoxin A and citrinin in different cell typesin vitro

Cytotoxic effects resulting from the two mycotoxins ochratoxin A (OTA) and citrinin were evaluatedin vitro using cell cultures of different origin. Cytotoxicity was estimated by the neutral red (NR) uptake assay which allows to determine the viability of cells by detecting dye uptake into the cells and its storage in the lysosomes. The assay was performed with primary cell cultures derived from isolated porcine urinary bladder epithelial cells (PUBEC), which are competent in metabolism of xenobiotics, and with V79 hamster fibroblasts, a well established and frequently used cell line. In both systems, OTA was more cytotoxic compared to citrinin. When both mycotoxins were applied simultaneously no additive or synergistic effects were detected. Obviously, the primary cell culture system which represents a target tissue of the mycotoxins was more susceptible to the toxins, expressed in lower IC50-values. These results indicate that origin of a cell system and its competence in metabolism of xenobiotics have to be considered inin vitro investigations particularly when different systems were used.

Regulation of sorbitol content in cultured porcine urinary bladder epithelial cells.

Sorbitol content was determined in porcine urinary bladder epithelial cells immediately after death of the animals and after primary culture of the cells at different osmolalities. In both instances, sorbitol content increased with urine and medium osmolality, respectively. For example, at 300 mosmol/kg the cultured cells contained 0.84 +/- 0.02 nmol/mg protein, at 600 mosmol/kg contained 21.7 +/- 0.95 nmol/mg protein, and at 900 mosmol/kg contained 59.5 +/- 2.8 nmol/mg protein. Similarly, aldose reductase activity rose from 0.27 +/- 0.04 mumol.h-1.mg protein-1 at 300 mosmol/kg to 1.81 +/- 0.16 at 600 mosmol/kg and to 3.02 +/- 0.33 at 900 mosmol/kg. These changes were, however, only observed when NaCl but not when urea was used to augment the medium osmolality, since urea equilibrated across the cell membrane. In contrast, sorbitol release from cells cultured at 900 mosmol/kg was slowest into a 900 mosmol/kg medium and fastest into a 300 mosmol/kg medium (63 +/- 16 nmol/10 min compared with 389 +/- 52 nmol/10 min). These studies demonstrate that the sorbitol content of porcine urinary bladder epithelium is regulated by changes both in sorbitol synthesis and sorbitol release. Thus the regulatory mechanisms in the urinary bladder seem to be similar to those present in the embryological related collecting duct.

An In Vitro Model of the Urinary Bladder: Cultured Porcine Urinary Bladder Epithelial Cells

Epithelial cells from urinary bladders of pigs were isolated and cultured under serum-free conditions. For these cells it was previously shown that they developed morphologic polarity resembling the epithelium in vivo. Lactate dehydrogenase release was low, chromosome set and activities of marker enzymes (alkaline phosphatase, acid phosphatase, g-glutamyltranspeptidase) were stable over a period of 4 wk. In this study, metabolic competence was evaluated by measuring activities of phase I and phase II enzymes. Activity of prostaglandin H-synthase was expressed in freshly isolated cells as well as in cultured cells, as were activities of the conjugating enzymes glutathione transferase, UDP-glucuronyltransferase and N-acetyltransferase. Cytochrome P4501A1 activity in freshly isolated cells amounted to 10–15% of the respective activity in the porcine liver, this activity was not detectable in cultured cells. No activity was seen in cultured cells after induction with methylcholanthrene and benz[ a]anthracene. This cell culture system was used to detect genotoxic effects of substances suspected to induce bladder cancer by measuring the induction of sister chromatid exchanges (SCE). The aromatic amines 4-aminobiphenyl and 2-aminofluorene induced a concentration dependent increase of SCEs at non-cytotoxic concentrations. These results imply that urinary bladder epithelial cells are capable to perform metabolic activation which is required to generate genotoxic effects of aromatic amines. Therefore, this new cell culture system, representing the urinary bladder epithelium, is an effective tool in in vitro toxicology to investigate adverse effects of compounds, regarded or suspected to induce toxic effects in the bladder.

Effects of ochratoxin A on DNA repair in cultures of rat hepatocytes and porcine urinary bladder epithelial cells

In cultured rat hepatocytes the mycotoxin ochratoxin A (OTA) induced unscheduled DNA synthesis (UDS) only in a narrow concentration range. Using a culture medium supplemented with 1% fetal calf serum, at 750 nM OTA a weak induction and at 1 microM OTA a marked induction of DNA repair was observed (15 +/- 11 and 38 +/- 24% cells in repair, respectively). Concentrations > 1 microM OTA were cytotoxic, and <750 nM no induction occurred. In cultures of cells from the urinary bladder (porcine urinary bladder epithelial cells; PUBEC), a target organ of the mycotoxin, OTA induced UDS in a concentration-dependent manner. To inhibit the proliferation of the cultured epithelial cells, which would counteract the detection of DNA repair, epidermal growth factor was omitted and an arginine-deficient medium (ADM) was used. Under these serum-free culture conditions the amount of cells undergoing DNA repair in PUBEC control cultures was approximately 7 +/- 4%, a value also comparable to those of control cultures of rat hepatocytes. At concentrations between 250 nM and 1 microM OTA a concentration-dependent increase of cells in repair was observed. Above 1 microM OTA was cytotoxic. At this concentration a maximum of approximately 61 +/- 9% of the cells undergo DNA repair. This amount is comparable to control cultures incubated with 5 or 10 mM ethylmethane-sulphonate (EMS) (49 +/- 9 and 69 +/- 10% cells in repair, respectively), used as a positive control. These results show that in cultured rat hepatocytes induction of UDS is relatively weak whereas in urothelial cells this effect was significant. Whether this effect is due to OTA metabolites formed locally in the urothelium cannot be excluded since PUBEC have been shown to be able to metabolize xenobiotics independently from the liver.