Primary rat hepatocytes as in vitro system for gene expression studies: comparison of sandwich, Matrigel and 2D cultures.

Recent studies have presented evidence that in vivo obtained gene expression data can be used for carcinogen classification, for instance to differentiate between genotoxic and non-genotoxic carcinogens. However, although primary rat hepatocytes represent a well-established in vitro system for drug metabolism and enzyme induction, they have not yet been systematically optimized for toxicogenomic studies. The latter may be confounded by the fact that cultured hepatocytes show strong spontaneous alterations in gene expression patterns. Therefore, we addressed the following questions: (1) which culture system is optimal, comparing sandwich, Matrigel and 2D cultures, (2) how critical is the impact of culture period on substance-induced alterations in gene expression and (3) do these substance-induced alterations in cultured hepatocytes occur already at in vivo relevant concentrations? For this purpose we analyzed the expression of four genes, namely Abat, Gsk3beta, Myd116 and Sult1a1 that recently have been reported to be influenced by the antihistamine and non-genotoxic carcinogen methapyrilene (MPy). The most reproducible effects of MPy were observed in sandwich cultures. Induction factors of Gsk3beta and Myd116 at 100 microM MPy were 2 and 4 (medians), respectively, whereas expression of Abat and Sult1a1 were inhibited by factors of 7 and 5, respectively. Similar results were observed in hepatocytes maintained for 24 h or 3 weeks in sandwich culture with respect to the influence of MPy on the expression of Abat, Gsk3beta, Myd116 and Sult1a1. To determine whether MPy influences gene expression at in vivo relevant concentrations, 3.5 mg/kg MPy were administered to male Wistar rats intraperitoneally, resulting in plasma concentrations ranging between 1.72 and 0.32 microM 5 and 80 min after injection. Inhibition of Abat and Sult1a1 expression in vitro already occurred at in vivo relevant concentrations of 0.39 microM MPy. Induction of Myd116 was observed at 6.25 microM which is higher but in the same order of magnitude as in vivo relevant concentrations. In conclusion, the presented data strongly suggest that sandwich cultures are most adequate for detection of MPy-induced gene expression alterations and the effect of MPy was detected at in vivo relevant concentrations.

Induction of micronuclei by ochratoxin A is a sensitive parameter of its genotoxicity in cultured cells.

In order to better characterize the ochratoxin A (OTA)-induced DNA damage and to further investigate factors which may modulate dose-effect relationships in cells, the induction of micronuclei was studied in V79 Chinese hamster fibroblast cells and in primary cultures of porcine urothelial bladder epithelial cells (PUBEC). OTA was able to induce micronuclei in PUBEC and V79 cells at concentrations below those which were overtly cytotoxic. OTA concentrations between 0.03 and 1 µM caused a dose-dependent increase of micronuclei in V79 cells (up to 3-fold compared to controls); but the lowest tested concentration of 0.01 µM OTA did not induce a higher frequency of micronuclei than in the solvent control, indicative of an apparent threshold. Clear evidence for genotoxic effects was also found in PUBEC cultures treated with OTA concentrations of 1 µM and more, although the dose-effect relationship in PUBEC was more variable for several freshly isolated cell batches, pointing to differences in susceptibility to OTA between bladder cells from different donor animals. The chromosomal genotoxicity of OTA demonstrated in this study is in general accord with previous findings on the induction of clastogenic effects and oxidative DNA damage by OTA. In both cases, the shape of the dose-response curve at very low OTA concentrations supports the existence of a threshold for its genotoxicity.

Expression of cytochrome P450 enzymes CYP1A1, CYP1B1, CYP2E1 and CYP4B1 in cultured transitional cells from specimens of the human urinary tract and from urinary sediments.

Expression of cytochromes P450 CYP1A1, CYP1B1, CYP2E1 and CYP4B1 was analysed on the transcript level in human urothelial cells obtained by various methods. As a source of urothelial cells, exfoliated cells in urine samples were used. Their expression profiles were determined either immediately after centrifugal enrichment (n=4) or after their cultivation and propagation (n=8). Another source of urothelial cells were ureter specimens from surgical subjects (n=4). Generally, expression was most prominent for CYP1B1 and CYP4B1 among the CYP transcripts analysed. CYP1B1 mRNA was detected in all samples investigated except for one ureter specimen. CYP4B1 mRNA was present in cell cultures from three out of eight healthy subjects, in three out of four directly investigated urinary sediments and in the cells of all five ureter specimens of four donors investigated after resection and subsequent cell culture. In most cases, CYP2E1 transcript levels were lower than those of CYP1B1 and CYP4B1. CYP2E1 mRNA was detected in cell cultures of six out of eight healthy subjects, in one out of four urinary sediments and in three out of five ureter specimens. CYP1A1 mRNA was clearly observed only in cells from resected ureters. In cell cultures the relative mRNA expression levels varied with subjects interindividually, intraindividually and also during the time of cell culture. The study demonstrates constitutive mRNA expressions of xenobiotic metabolising CYP enzymes in human urothelial cells obtained by different methods. In particular, transcripts of CYP1B1 and CYP4B1 are present, coding for enzymes which are active in the metabolism of polycyclic aromatic hydrocarbons and arylamines, respectively.

Investigation of cytotoxic, genotoxic, mutagenic, and estrogenic effects of the flame retardants tris-(2-chloroethyl)-phosphate (TCEP) and tris-(2-chloropropyl)-phosphate (TCPP) in vitro.

The organophosphorus esters tris-(2-chloroethyl)-phosphate (TCEP) and tris-(2-chloropropyl)-phosphate (TCPP) have been widely used as flame retardants and fire preventing agents, e.g. in polyurethane foams. We investigated the cytotoxic, genotoxic, mutagenic, and estrogenic potentials of TCEP and TCPP, using different in vitro models. Cytotoxic effects were evaluated by neutral red uptake and genotoxicity with the alkaline single cell gel electrophoresis (Comet assay), both in V79 (hamster fibroblasts) cells. Mutagenicity was tested in the Ames assay with Salmonella typhimurium using the strains TA 97 a, 98, 100, 102, 104, 1535, 1537, and 1538, with and without metabolic activation by S9-rat liver homogenate. Estrogenic or anti-estrogenic effects were examined with the recombinant yeast reporter gene assay, and in human endometrial cancer Ishikawa cells by induction of alkaline phosphatase. In V79 cells TCEP was weakly cytotoxic at concentrations above 10 microM in the presence of S9-rat liver homogenate whereas TCPP showed cytotoxicity above 1mM in the presence of S9. Both substances did not induce DNA strand breaks in the alkaline version of the Comet assay neither without an external enzymatic metabolizing system, nor in the presence of S9-mix. Additionally, no mutagenic potential could be detected for TCEP and TCPP in eight Salmonella strains using concentrations up to 1mM in the presence and absence of S9. Hormonal activity shown as induction of estrogenic or anti-estrogenic effects could not be detected in the two in vitro test systems.

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 ß-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.

Modulation of ochratoxin A induced DNA-damage in urothelial cell cultures.

Despite good evidence for a genotoxic potential of ochratoxin A (OTA), the mechanism of OTA-induced genotoxicity (direct or indirect?) is still unclear. This calls for a further characterization of OTA-related DNA damage, and investigations of factors that may modulate dose-effect relationships in cells.Since bladder epithelium is a target tissue for the toxicity of OTA, its effects were studied in cultures of human bladder carcinoma (H5637) cells. Cytotoxicity of OTA, assessed by Neutral red (NR) uptake or Alamar-Blue assay, is concentration- and time-dependent: Upon 24 h treatment of 5637 cells, NR uptake is reduced by 50% with OTA concentrations of ≥0.2 microM, but not with 3 h treatment of the cells. Since cytotoxicity of OTA was not affected by addition of xenobiotic metabolizing enzymes (S-9 mix), it appears to be unrelated to biotransformation of the mycotoxin. Also, addition of S-9 mix did not significantly affect the genotoxicity of OTA as studied by alkaline single cell gel electrophoresis (Comet assay). DNA damage was detectable after 3 h treatment of cells at OTA concentrations between 0.1 and 1 microM, and increased further at higher concentrations. The magnitude of OTA-induced DNA damage did not increase with longer treatment times (18, 24 h), probably due to repair processes in the cells. Repair of OTA-induced lesions is quite efficient in kidney (Arch Toxicol 2002, 75, 734-741) and in porcine bladder cells (Föllmann and Lebrun, 2005, Mycotoxin Research, this volume). Interestingly, the genotoxicity of OTA is modulated by the pH of the culture medium, with higher damage at pH 5 compared to pH 7.5. In line with this, uptake studies with tritiated OTA show a higher cellular accumulation of the mycotoxin at pH 5 than in buffer of pH 7.5. Thus, bladder cells exposed to OTA in slightly acidic urine (which facilitates reabsorption) may be at higher risk.

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.

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.

Primary cell cultures of bovine colon epithelium: isolation and cell culture of colonocytes.

Epithelial cells from bovine colon were isolated by mechanical preparation combined with an enzymatic digestion from colon specimens derived from freshly slaughtered animals. After digestion with collagenase I, the isolated tissue was centrifuged on a 2% D-sorbitol gradient to separate epithelial crypts which were seeded in collagen I-coated culture flasks. By using colon crypts and omitting the seeding of single cells a contamination by fibroblasts was prevented. The cells proliferated under the chosen culture conditions and formed monolayer cultures which were maintained for several weeks, including subcultivation steps. A population doubling time of about 21 hr was estimated in the log phase of the corresponding growth curve. During the culture period the cells were characterized morphologically and enzymatically. By using antibodies against cytokeratine 7 and 13 the isolated cells were identified as cells of epithelial origin. Antibodies against vimentin served as negative control. Morphological features such as microvilli, desmosomes and tight junctions, which demonstrated the ability of the cultured cells to restore an epithelial like monolayer, were shown by ultrastructural investigations. The preservation of the secretory function of the cultured cells was demonstrated by mucine cytochemistry with alcian blue staining. A stable expression of enzyme activities over a period of 6 days in culture occurred for gamma-glutamyltranspeptidase, acid phosphatase and NADH-dehydrogenase activity under the chosen culture conditions. Activity of alkaline phosphatase decreased to about 50% of basal value after 6 days in culture. Preliminary estimations of the metabolic competence of these cells revealed cytochrome P450 1A1-associated EROD activity in freshly isolated cells which was stable over 5 days in cultured cells. Then activity decreased completely. This culture system with primary epithelial cells from the colon will be used further as a model for the colon epithelium in toxicological studies in vitro.

Induction of unscheduled DNA synthesis in primary human urothelial cells by the mycotoxin ochratoxin A.

Ochratoxin A (OTA) is a widespread contaminant in human staple food. Exposure of humans to this mycotoxin is a matter of concern because OTA is a known rodent carcinogen. As the urothelium is one target tissue of this mycotoxin, primary cultured human urothelial cells (HUC) from adults and children were used to analyze the induction of unscheduled DNA synthesis (UDS) by OTA. HUC were isolated from the ureters or renal pelves of two nephrectomized adults and of two children with ureteropelvic junction stenosis and cultured under serum-free conditions. After a confluency of 70-80% was reached, cell proliferation was suppressed by arginine-deficient medium (ADM), and UDS was assessed autoradiographically by 3H-thymidine incorporation upon exposure to OTA (10-2000 nM), ethyl methanesulfonate (EMS, 5 mM, positive control), or dimethyl sulfoxide (DMSO, 0.2%, solvent control). In control cultures the level of UDS was low. Exposure to EMS resulted in an induction of UDS (2-to 5-fold compared to control), thus allowing the sensitive detection of repair resulting from induction of DNA lesions in all four specimens, and demonstrating that repair of EMS-induced DNA lesions can take place under the chosen culture conditions. In two HUC cultures derived from adults, a significant induction of UDS was observed in the concentration range of 50-500 nM OTA. The highest fraction of cells in repair (CIR) was found at 50 nM OTA for the HUC from the older male (50% CIR). The maximum response in the other specimens from the adult female and the 7-year-old boy were seen at OTA concentrations of 500 and 250 nM, respectively. In contrast to all other specimens, no significant induction of UDS by OTA was found in the HUC cultures derived from an infant's urothelium. Signs of cytotoxicity were observed above 500 nM OTA in all cultures. The varying susceptibility toward OTA observed in vitro may hint at varying predispositions of individuals in vivo.

Cultures of exfoliated epithelial cells from different locations of the human urinary tract and the renal tubular system.

Exfoliated human urinary tract epithelial cells and renal tubular cells from urinary sediments of healthy adults, of urological patients and of internal patients were isolated and cultured. Cells started proliferating within 1 week after seeding a sediment. Proliferating cells formed colonies of different morphologies, designated as type-1 or type-2 cell colonies. Type-1 cell colonies showed irregular contours and spindle-like cells within the colonies. Subcultivation of type-1 cells for up to six passages was possible. Type-2 cell colonies showed smooth-edged contours and subcultivation was not possible. The epithelial character of type-1 cells was demonstrated by positive immunohistochemical staining for cytokeratin-7. In contrast to carbonic anhydrase-positive stained Madin Darby canine kidney cells (MDCK), which were used as positive controls for renal tubular cells, type-1 cells were carbonic anhydrase-negative on staining with the cobalt phosphate method. This indicates that type-1 cells were not of renal tubular origin. Type-2 cells were positively stained for carbonic anhydrase, indicating that type-2 cells were renal tubular cells. Type-2 cell colonies could be assigned to two subgroups with different cell forms. Colonies of cobblestone-like cells more often occurred than type-2 cell colonies with spindle-like cells, which are described in this study for the first time. Colonies with cobblestone-like cells formed domes (hemicysts), whereas spindle-like type-2 cell colonies did not. Cultures of urinary sediments from healthy adults, elderly multimorbid patients treated with furosemide, and urological patients with urolithiasis treated with sulfamethoxazole/trimethoprim and/or with a percutaneous nephrostomy catheter were compared. In 52% of all cultured sediments from healthy adults, in 30% of those from multimorbid patients, and in 75-80% of those from urological patients cells proliferated to colonies. The ratios of type-1 to type-2 cell colonies were 3.3:1 (healthy adults), 1.4:1 (urological patients with urolithiasis), and 1.8:1 (urological patients with urolithiasis, urine was directly collected from the renal pelvis with a percutaneous nephrostomy catheter). Successful cultures of the urinary sediments from these three groups revealed means of 3 or 4 colonies, 14 colonies, and 21 colonies, respectively. Differences in the number of colonies in relation to sex were observed only for the group of urological patients. It was shown that type-1 cells were urothelial cells, which did not show morphological differences due to their locations of origin within the urinary tract, whereas type-2 cells were probably renal tubular cells. These findings offer new aspects in the culturing of human urothelial or kidney epithelial cells with a method based on noninvasive collecting of specimens and requiring only minimal culture effort. The cultures obtained by this method can be used for in vitro studies in toxicological and clinical research.

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.

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.

Determination of degradation rates of transfer and ribosomal ribonucleic acids in cultured rat hepatocytes by measuring N6-threoninocarbonyladenosine, dihydrouridine, and pseudouridine in medium using high-performance liquid chromatography.

Modified ribonucleic acid catabolites excreted into the medium by primary cultures of rat hepatocytes (2.3 +/- 0.42 x 10(6) cells/dish) during a 24-h cultivation period were quantified by reversed-phase high-performance liquid chromatography (fmol/10(3) cells): 613 +/- 81 dihydrouridine, 46 +/- 6 N6-threoninocarbonyladenosine, 1879 +/- 220 pseudouridine. On the basis of these excretion rates and the average frequency of occurrence of these modified ribonucleosides per cytoplasmic transfer ribonucleic acid (residues: 2.6 dihydrouridine, 0.22 N6-threoninocarbonyladenosine, 3 pseudouridine) as well as per cytoplasmic ribosomal ribonucleic acid (residues: 95 pseudouridine), the degradation rates of transfer and ribosomal ribonucleic acids were calculated. The degradation rate of transfer ribonucleic acid (fmol/10(3) cells/24 h) was 236 +/- 31 (via dihydrouridine) and 211 +/- 28 (via N6-threoninocarbonyladenosine) and that of ribosomal ribonucleic acid (fmol/10(3) cells/24 h) was 13.1 +/- 1.7 (via pseudouridine and N6-threoninocarbonyladenosine).

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.

Drug metabolizing enzyme activities in porcine urinary bladder epithelial cell cultures (PUBEC).

Drug metabolizing enzyme activities have been determined in cultured porcine urinary bladder epithelial cells (PUBEC) in order to evaluate this system as an in vitro model for studies of urinary bladder carcinogens. Activities of several phase I and II enzymes were measured in cells cultured for various periods and compared with the activities determined in freshly isolated PUBEC. Prostaglandin H synthase mediated production of prostaglandin E2 was found both in freshly isolated and in cultured PUBEC, whereas cytochrome P450 1A1-associated EROD activity was only detectable in freshly isolated bladder cells. The latter activity was not inducible by benz(a)anthracene or 3-methylcholanthrene in PUBEC cultures. N-acetyltransferase (NAT) activity measured with p-aminobenzoic acid, a diagnostic substrate for human NAT-1, was stable and even higher during the culture period compared to freshly isolated cells. In contrast, isoniazid (a substrate for NAT-2) was not acetylated either in fresh or cultured PUBEC. Glutathione S-transferases activity determined with 1-chloro-2,4-dinitrobenzene decreased gradually to 50% after 1 week and to 20% after 4 weeks in culture compared to fresh cells. A similar decline was also observed for UDP-glucuronyltransferase activities measured with 1-naphthol. In accordance with the reported lack of sulfotransferases in pigs, no sulfation of 1-naphthol or 2-naphthylamine was detected in PUBEC. Our results show that cultured porcine urinary bladder epithelial cells maintain several enzyme activities required for the biotransformation of xenobiotics. In future investigations on the mechanism of action of bladder carcinogens PUBEC cultures may thus provide a useful in vitro model for this target tissue.

What we have learned about bumetanide and the concept of multispecific bile acid/drug transporters from the liver.

Bumetanide is a weak organic acid which is transported into hepatocytes by a transport system that is related neither to the cloned sodium-dependent taurocholate cotransporting polypeptide Ntcp nor to the cloned organic anion transporting polypeptide oatp. Bumetanide is known to be transported in the kidney by a multispecific organic anion transporter which is the pAH-transporter from the proximal tubule cell. In the liver, bumetanide uptake competes with bile acid uptake, indicating a functionally related multispecific transporter for bile acids and drugs in hepatocytes. This multispecific bile acid transporter MBAT has not been cloned yet. When basolateral membranes were photoaffinity labeled with [3H]bumetanide, several bumetanide binding proteins were separated and identified after protein sequencing from two-dimensional electrophoresis gels.

Sister chromatid exchange frequency in cultured isolated porcine urinary bladder epithelial cells (PUBEC) treated with ochratoxin A and alpha.

The mycotoxin ochratoxin A (OTA) and its metabolite ochratoxin alpha (OT-alpha) were investigated, to examine their potency to induce sister chromatid exchanges (SCE) in cultured porcine urinary bladder epithelial cells (PUBEC) (primary culture). Serum-free cultured PUBEC were incubated for 5 h with either OTA or OT-alpha, respectively, and subsequently cultured in the presence of 5-bromo-2-deoxyuridine (BrdU). After two cell cycles, mitosis was inhibited by the colchicine derivative Colcemid, cells were fixed and chromosomes were prepared for SCE analysis. For OTA, a dose-dependent increase in SCE frequency was measured in concentrations between 100 pM and 100 nM OTA. At 100 nM OTA, SCE frequency increased by about 41%, compared to the base SCE level (7.27 SCEs per chromosome set, solvent control). Higher concentrations of OTA were cytotoxic. The metabolite OT-alpha also increased SCE frequency, but at higher concentrations. At a concentration of 10 microM OT-alpha, an increase of about 55% was detected. OT-alpha showed no cytotoxic effect. These results indicate that OTA is genotoxic in this in vitro system, which represents the urinary bladder epithelium, a target organ of OTA in vivo. It could also be shown that OT-alpha, which is said to be non-toxic, is genotoxic in this assay at higher concentrations.