Rat Urinary Bladder Epithelium Research Articles

Examination of in vivo mutagenicity of sodium arsenite and dimethylarsinic acid in gpt delta rats

Arsenic is a well-known human bladder and liver carcinogen, but its exact mechanism of carcinogenicity is not fully understood. Dimethylarsinic acid (DMAV) is a major urinary metabolite of sodium arsenite (iAsIII) and induces urinary bladder cancers in rats. DMAV and iAsIII are negative in in vitro mutagenicity tests. However, their in vivo mutagenicities have not been determined. The purpose of present study is to evaluate the in vivo mutagenicities of DMAV and iAsIII in rat urinary bladder epithelium and liver using gpt delta F344 rats. Ten-week old male gpt delta F344 rats were randomized into 3 groups and administered 0, 92mg/L DMAV, or 87mg/L iAsIII (each 50mg/L As) for 13weeks in the drinking water. In the mutation assay, point mutations are detected in the gpt gene by 6-thioguanine selection (gpt assay) and deletion mutations are identified in the red/gam genes by Spi− selection (Spi− assay). Results of the gpt and Spi− assays showed that DMAV and iAsIII had no effects on the mutant frequencies or mutation spectrum in urinary bladder epithelium or liver. These findings indicate that DMAV and iAsIII are not mutagenic in urinary bladder epithelium or liver in rats.

Mineralocorticoid receptor stimulation induces urinary storage dysfunction via upregulation of epithelial sodium channel expression in the rat urinary bladder epithelium

We aimed to evaluate mineralocorticoid receptor (MR) expression in rat bladder and the physiological role of the MR-epithelial sodium channel (ENaC) pathway in controlling bladder function in 10–12-week-old, male Sprague–Dawley rats. First, we examined the mRNA expression of MR and localization of MR and ENaC-α proteins in the urinary bladder. MR mRNA expression was observed in untreated-rat urinary bladders, and MR and ENaC-α proteins were localized in the epithelium. Next, rats were treated with vehicle (controls) or fludrocortisone (an MR agonist) for 3 days, and ENaC-α protein expression levels and bladder function were evaluated on day 4. ENaC-α protein expression was significantly higher in fludrocortisone-treated rats than in controls. In addition, cystometry was performed during intravesical infusion of saline and amiloride (an ENaC inhibitor). While intercontraction intervals (ICIs) during saline infusion were significantly shorter in the fludrocortisone group than in the controls, infusion of amiloride normalized the ICIs in the fludrocortisone group. However, no intra- or inter-group differences in maximum intravesical pressure were observed. Taken together, MR protein is localized in the rat urinary bladder epithelium, and may regulate ENaC expression and bladder afferent input. The MR-ENaC pathway may be a therapeutic target for ameliorating storage symptoms.

Dietary administration of sodium arsenite to rats: Relations between dose and urinary concentrations of methylated and thio-metabolites and effects on the rat urinary bladder epithelium

Based on epidemiological data, chronic exposure to high levels of inorganic arsenic in drinking water is carcinogenic to humans, inducing skin, urinary bladder and lung tumors. In vivo, inorganic arsenic is metabolized to organic methylated arsenicals including the highly toxic dimethylarsinous acid (DMA III) and monomethylarsonous acid (MMA III). Short-term treatment of rats with 100 μg/g trivalent arsenic (As III) as sodium arsenite in the diet or in drinking water induced cytotoxicity and necrosis of the urothelial superficial layer, with increased cell proliferation and hyperplasia. The objectives of this study were to determine if these arsenic-induced urothelial effects are dose responsive, the dose of arsenic at which urothelial effects are not detected, and the urinary concentrations of the arsenical metabolites. We treated female F344 rats for 5 weeks with sodium arsenite at dietary doses of 0, 1, 10, 25, 50, and 100 ppm. Cytotoxicity, cell proliferation and hyperplasia of urothelial superficial cells were increased in a dose-responsive manner, with maximum effects found at 50 ppm As III. There were no effects at 1 ppm As III. The main urinary arsenical in As III-treated rats was the organic arsenical dimethylarsinic acid (DMA V). The thio-metabolites dimethylmonothioarsinic acid (DMMTA V) and monomethylmonothioarsinic acid (MMMTA V) were also found in the urine of As III-treated rats. The LC 50 concentrations of DMMTA V for rat and human urothelial cells in vitro were similar to trivalent oxygen-containing arsenicals. These data suggest that dietary As III-induced urothelial cytotoxicity and proliferation are dose responsive, and the urothelial effects have a threshold corresponding to the urinary excretion of measurable reactive metabolites.

Effects of Co-administration of Antioxidants and Arsenicals on the Rat Urinary Bladder Epithelium

Oxidative stress has been increasingly recognized as a possible mechanism in the toxicity and carcinogenicity of various chemicals, including arsenic. Therefore, treatment with antioxidants may afford a protective effect against arsenic-induced cytotoxicity and carcinogenesis. Dimethylarsinic acid (DMAV) has been shown to be a bladder carcinogen in rats when administered at high doses (100 ppm) in the diet or in the drinking water. The main purpose of the present study was to evaluate the effects of co-administration of antioxidants with arsenicals on the rat urinary bladder epithelium in vitro and in vivo. In a previous experiment, treatment with 1000 ppm melatonin for two weeks did not inhibit cell proliferation induced in the rat urothelium by 100 ppm DMAV. In the current study, we examined the effects of five antioxidants that act via different mechanisms, on the in vitro cytotoxicity of various arsenicals, for the purpose of determining which antioxidants might have protective effects against arsenic-induced cytotoxicity. The antioxidants that inhibited cytotoxicity in vitro were then studied also in vivo. Melatonin showed slight inhibition of the cytotoxicity of arsenite, but had no effect on the other arsenicals. N-acetylcysteine (NAC) inhibited the cytotoxicity of monomethylarsonous acid (MMAIII), DMAV, dimethylarsinous acid (DMAIII), and trimethylarsine oxide (TMAO). Vitamin C inhibited cytotoxicity induced by arsenate, arsenite, MMAIII) and DMAIII. Tiron and Trolox had no effect on the cytotoxicity of any arsenical. The in vitro inhibitory effects of NAC and vitamin C on DMAV and on DMAIII, suggested that these antioxidants might afford preventive effects on DMAV-induced bladder cytotoxicity and carcinogenesis in rats. To test this hypothesis, a 10-week rat bioassay was conducted. Melatonin was also included to clarify the results of the previous two-week experiment. The sodium salt of vitamin C (Na-Asc), but not melatonin or NAC, inhibited the proliferative effects of DMAV on the bladder epithelium in rats. These results suggest that oxidative stress is at least in part involved in DMAV-induced rat bladder toxicity and proliferation, and therefore, vitamin C may afford inhibitory effects in DMAV-induced bladder carcinogenesis in rats. Microarray analysis of DMAV-responsive genes revealed that DMAV did not have a consistent modifying effect on gene expression in the rat bladder epithelium, suggesting that proteins and/or lipids may be the targets of damage by DMAV-induced oxidative stress.

Indomethacin-induced morphologic changes in the rat urinary bladder epithelium

Objectives To evaluate the morphologic changes in rat urothelium induced by indomethacin. Nonsteroidal anti-inflammatory drug-induced cystitis is a poorly recognized and under-reported condition. In addition to tiaprofenic acid, indomethacin has been reported to be associated with this condition. Methods Three groups were established: a control group (n = 10), a high-dose group (n = 10), treated with one intraperitoneal injection of indomethacin 20 mg/kg, and a therapeutic dose group (n = 10) in which oral indomethacin was administered 3.25 mg/kg body weight daily for 3 weeks. The animals were then killed and the bladders removed for light and electron microscopic studies. Results The light microscopic findings showed some focal epithelial degeneration that was more prominent in the high-dose group. When compared with the control group, both indomethacin groups revealed statistically increased numbers of mast cells in the mucosa ( P <0.0001) and penetration of lanthanum nitrate through intercellular areas of the epithelium. Furthermore, the difference in mast cell counts between the high and therapeutic dose groups was also statistically significant ( P <0.0001). Conclusions Indomethacin resulted in histopathologic findings typical of interstitial cystitis, such as leaky bladder epithelium and mucosal mastocytosis. The true incidence of nonsteroidal anti-inflammatory drug-induced cystitis in humans must be clarified by prospective clinical trials.

Aberrant expression of p27(Kip1) is associated with malignant transformation of the rat urinary bladder epithelium.

Alteration in cell cycle regulators is considered to play an important role in carcinogenesis. In order to cast light on changes in reversible hyperplastic and irreversible tumorigenic lesions in the rat urinary bladder, expression of p27(Kip1), cyclin D1 and cyclin E proteins was sequentially compared. In the first study, 3% uracil was fed for 4 weeks to cause urinary calculi and consequent hyperplasia and papillomatosis, both regressing after withdrawal of the insult. Compared with normal bladder epithelium, in papillomatosis at week 4, the BrdU index and immunohistochemical positivities for cyclin D1 and cyclin E were significantly elevated, whereas values for p27(Kip1) tended to be reduced. One week after withdrawal of uracil, the BrdU index and positivities for cyclin D1 and cyclin E were decreased to below the control levels, while positivity for p27(Kip1) was dramatically increased, with a strong staining intensity. In a second study, rats were initiated with a bladder carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine for 4 weeks, then fed 3% uracil for 8 weeks. During this latter period, expression of cyclin D1, cyclin E and p27(Kip1) in hyperplastic urothelium were comparable with those in the first study. One week after withdrawal of uracil, most urothelial lesions regressed, showing high p27(Kip1) and low cyclin D1 and cyclin E staining. Two weeks after uracil withdrawal, transitional cell carcinomas, with a low p27(Kip1) and high cyclin D1 and cyclin E staining pattern, could be easily distinguished from surrounding regressing epithelium. These data indicate that during regression of papillomatosis after cessation of a proliferative stimulus, expression of p27(Kip1)is elevated, accompanied by a lowering of cyclin D1 and cyclin E. In irreversible tumorous bladder lesions, on the other hand, persistent low expression of p27(Kip1) and elevated cyclin D1 and cyclin E are characteristic.

A Simple Method for the Preparation of Cells from Rat Urinary Bladder Epithelium for Flow Cytometry.

A simple method of preparing rat bladder epithelial cells for flow cytometry was developed, which involves the procedure of (1) eversion of urinary bladder, (2) dissociation of the urothelial cells from the underlying connective tissue using an enzyme cocktail (collagenase, dispase, and trypsin) and (3) subsequent treatment with EDTA. The use of trypsin was essential to remove erythrocytes from cell suspension. This method allowed us to obtain at least 5×105 cells per bladder which were sufficient to measure DNA content distribution more than 10 times per animal. Despite use of trypsin, this method yielded DNA histograms with a sharp G0/G1-peak whose coefficient of variation was approximately 5%. The percentages of cells in G0/G1, S, and G2/M phases (mean±standard deviation) were 96.0±1.3%, 0.5±0.1%, and 3.2±1.1%, respectively. Thus, the results indicate that this method in a good isolation procedure to obtain a number of single urothelial cells for flow cytometry.

Paraneoplastic Pemphigus: Report of a Case

A 56-year-old male with chronic lymphocytic leukemia developed extensive erosive mucocutaneous lesions with histologic acantholysis. Immunopathologic studies showed IgG deposition at the intercellular space, C3 deposition at both the intercellular space and the dermo-epidermal junction, and reactivity of the serum to rat urinary bladder epithelium. Autoantibodies in the serum to human epidermal proteins of 210 kD and 190 kD were shown by Western blotting and to proteins of 250 kD, 210 kD, and 190 kD by immunoprecipitation. All these data suggest the diagnosis of paraneoplastic pemphigus. Repeated plasmapheresis resulted in re-epithelialization of the mucocutaneous lesions and reduction in antibody titer from 1:1280 to 1:20. Although this mucocutaneous disease was established as a new autoimmune bullous disease by Anhalt et al. (1990), cases have rarely been reported from Japan. The present patient demonstrates the major characteristics of paraneoplastic pemphigus.

Lysosomal cysteine and aspartic proteinases and ubiquitin in rat and human urinary bladder epithelium.

To examine localization of cysteine and aspartic proteinases, and ubiquitin in rat and human urinary bladders, immunocytochemistry was applied to the tissues. In semi-thin sections, immunoreactivity for cathepsins B and D was densely localized throughout epithelial layers of rats and humans, while that for cathepsins H and L was mainly localized in rat superficial and human intermediate cells. Immunoreactivity for cathepsin C was relatively high in rat and human epithelia, especially in humans. Immunoreactivity for ubiquitin was detected in rat and human epithelial cells. By electron microscopy, vesicular or heterogeneously dense lysosomes labeled with immunogold particles indicating cathepsin B were seen in rat and human epithelial cells; particularly, they often appeared near fusiform vesicles in rat superficial cells and in human intermediate and superficial cells. By double immunostaining, lysosomes with or without vesicular structures were co-labeled with immunogold particles showing both cathepsin B and ubiquitin. The results suggest that cathepsins B, C, H, and L, and cathepsin D are involved in the lysosomal system of rat and human bladder epithelia. Moreover, considering that ubiquitin is a cofactor in the soluble ATP-dependent proteolysis, the results may also indicate that epithelial cells actively form autophagolysosomes.

The role of alkalizing and neutral potassium salts in urinary bladder carcinogenesis in rats.

Using an initiation-promotion rat model, we have previously shown that the alkalizing salt KHCO3 is a strong and the neutral salt KCl a weak promoter of urinary bladder carcinogenesis. We have now studied the effects of these salts on rat urinary bladder epithelium without prior exposure to a bladder tumour initiator. In four studies ranging in duration from 4 to 130 weeks, (equimolar) amounts of K+ were administered in the diet to male and female rats (85 rats/sex/group) as KHCO3 or KCl. Comparable increases in urinary volume and potassium levels were found with both KHCO3 and KCl, but only KHCO3 induced an elevated urinary pH. The feeding of KHCO3 resulted in simple epithelial hyperplasia and, after prolonged administration, in papillary/nodular hyperplasia, papillomas and transitional cell carcinomas of the urinary bladder. With KCl, only a slight increase in proliferative urothelial lesions was found; one male showed papillary hyperplasia and one female exhibited nodular hyperplasia and a papilloma. Our results allow the conclusion that KHCO3, a strong promoter of bladder carcinogenesis, is capable of inducing urinary bladder cancer in rats without prior application of an initiator, whereas KCl, a weak tumour promoter, induced only a few (pre)neoplastic lesions.

Effect of coffee drinking on cell proliferation in rat urinary bladder epithelium

A possible effect of freshly brewed drip coffee on urinary bladder carcinogenesis was investigated in male Wistar rats using cell proliferation in urinary bladder epithelium as the indicator of tumour promotion. Male rats were given either undiluted coffee brew (100% coffee), coffee diluted 10 times (10% coffee) or tap water (controls), as their only source of drinking fluid for 2 or 6 wk. Uracil, known to induce cell proliferation in urinary bladder epithelium, was included in the study as a positive control. In rats receiving 100% coffee, body weights, liquid intake and urinary volume were decreased. Neither histopathological examination of urinary bladder tissue nor the bromodeoxyuridine labelling index revealed biologically significant differences between rats receiving coffee and the tap water controls. Uracil increased the labelling index and induced hyperplasia of the urinary bladder epithelium, as expected. It was concluded that these results produced no evidence that drinking coffee predisposes to tumour development in the urinary bladder.

12-O-tetradecanoylphorbol-13-acetate induces selective desquamation of superficial cells in rat urinary bladder epithelium.

12-O-tetradecanoylphorbol-13-acetate (TPA) is known to affect the proliferation and/or differentiation of several types of cells. We injected TPA directly into the lumen of rat bladder to determine, using scanning and transmission electron microscopy, its effects on the bladder epithelium in vivo. At 1 h after TPA injection (1 microgram/ml), the superficial cells of the epithelium had changed their morphology, and large spherical vacuoles occupied their cytoplasm. In some areas, the underlying intermediate cells were exposed by the desquamation of the superficial cells. During the next few hours, TPA was excreted from the bladder lumen by voluntary micturition, but the desquamation of the superficial cells proceeded further. All the superficial cells were lost from the luminal surface by 24 h after TPA injection. The changes noted were specific for the superficial cells and were not observed in the intermediate or basal cells. After 24 h, part of the epithelium had a three-layer structure, indicating that regeneration was taking place. These results demonstrate that TPA selectively affects and desquamates superficial cells in a short period of time. This experimental system may be useful for studying in vivo cell proliferation and/or differentiation.

Change of membrane potential in rat urinary bladder epithelium treated with sodium l-ascorbate

We previously showed that 5% sodium l-ascorbate (Na-AsA) in the diet promotes rat urinary bladder carcinogenesis, whereas 5% ascorbic acid (AsA) and 1% sodium chloride (NaCl) in the diet do not. In order to cast light on basic properties of these compounds regarding the rat urinary bladder, we examined membrane potential levels in the bladder epithelium of F344 rats treated with 5% Na-AsA, 5% AsA or 1% NaCl in the diet for 2, 4 or 8 weeks. Microelectrode measurement showed Na-AsA to induce hyperpolarization of the membrane potential, which was, however, lacking with AsA and NaCl from week 2 until the end of experiment. Thus a good correlation between the effects on membrane potential and tumor promoting activity could be established.

The modifying effects of indomethacin or ascorbic acid on cell proliferation induced by different types of bladder tumor promoters in rat urinary bladder and forestomach mucosal epithelium.

The effects of indomethacin (IM) or L‐ascorbic acid (AsA) on cell proliferation induced by bladder tumor promoters such as butylated hydroxyanisole (BHA), sodium L‐ascorbate (Na‐AsA), sodium citrate (Na‐Cit), and diphenyl (DP) in rat bladder and forestomach epithelium were investigated. Treatment with IM in combination with BHA or Na‐AsA diminished DNA synthesis levels of bladder epithelium as compared to the BHA or Na‐AsA alone values. On the other hand, AsA further amplified the increase of bladder epithelial DNA synthesis caused by Na‐Cit treatment. Histopathologically, administration of Na‐AsA in combination with IM reduced the incidence of simple hyperplasia. In contrast, simultaneous treatment with Na‐Cit and AsA caused an increase of the hyperplasia development. No apparent combination effects were observed in the DP‐treated groups. In forestomach epithelium, AsA enhanced the BHA‐induced increase in DNA synthesis and epithelial hyperplasia, characterized by marked basal cell proliferation. The present results thus suggested that IM may exert inhibitory effects on promotion of bladder carcinogenesis by certain tumor promoter types, and AsA may enhance BHA forestomach carcinogenesis.

Susceptibility of rodent cell lines to rat coronaviruses and differential enhancement by trypsin or DEAE-dextran

SummaryCell lines of rodent origin were tested for susceptibility to infection with rat coronavirus (RCV), including sialodacryoadenitis virus (SDAV) and Parker's rat coronavirus (PRCV). LBC rat mammary adenocarcinoma cells were susceptible only if the cells were treated with diethylaminoethyl-dextran (DEAE-D). A recent report that RCVs grow well in L2 mouse fibroblast cells was confirmed and expanded. RCV infection of L2 cells was substantially enhanced by treatment of cells with trypsin but not by treatment with DEAE-D. Primary isolation of SDAV from experimentally infected rats was accomplished using trypsin-treated L2 cells. One of 13 additional cell lines tested (rat urinary bladder epithelium, RBL-02) supported growth of RCVs, and growth was slightly enhanced by DEAE-D, but not by trypsin. These refinements of in vitro growth conditions for RCVs should facilitate further studies of their basic biology and improve options for primary isolation.

Immunofluorescence microscopic study on the cytoskeletal filaments in the separated superficial cells of rat urinary bladder epithelium.

We observed by immunofluorescence microscopy the distribution of cytoskeletal filaments in the superficial cells (SP cells) of rat urinary bladder epithelium in a distending state. To precisely detect SP cell fluorescence, the cells were separated from the distended bladder epithelium by scraping with a glass slide after paraformaldehyde fixation. Each cell appeared characteristically polygonal and flattened. In these cells, F-actins were predominantly distributed at the lateral cell border, marking the cell boundary. They also formed fine networks in the inner cytoplasm. Cytokeratin filaments were distributed in two characteristic patterns: they marked cell boundaries as did F-actin, and they formed thick circular bundles between the nucleus and cell margin. These two types of keratin filaments were also connected by thin filaments. These cytokeratins contained at least cytokeratin 18 subunit. Numerous microtubules were distributed evenly in the cytoplasm forming fine networks, except near the cell periphery.These observations show that SP cells are supported mainly by cytokeratin networks.

Bacterial infection and acid mucopolysaccharides in epithelium of rat urinary bladder.

The distribution of acid mucopolysaccharides in bladder epithelium was studied by colloidal iron staining in experimental cystitis of rats. In normal bladders the staining was mainly confined to the superficial (luminal) layer, but observed uniformly in all layers of epithelium in cystitis. Enzyme digestion (hyaluronidase, heparitinase, chondroitinase AC, chondroitinase ABC) disclosed the existence of hyaluronic acid, heparan sulphate and dermatan sulphate in the bladder epithelium. The possible roles of these mucopolysaccharides glycosaminogycans) were discussed.

Analysis of membrane structure in the transitional epithelium of rat urinary bladder. 3. Localization of cholesterol using filipin and digitonin

Filipin (a polyene antibiotic) and digitonin (a saponin) interact with cholesterol in membranes, producing characteristic lesions detectable by electron microscopy. Using these agents in conjunction with the techniques of freeze-fracture, negative staining, and thin-sectioning, we have investigated the distribution of cholesterol in the membrane systems of superficial urothelial cells, with particular reference to the luminal plasma membrane. This membrane is structurally differentiated into plaques (composed of arrays of subunits) and interplaque regions, which possess markedly different physical properties. Plaques were sensitive to digitonin but not to filipin whereas interplaque regions responded to both agents. These results are interpreted with reference to the factors that influence lesion formation, and to data on the structure and composition of the luminal membrane. It is concluded that both interplaque regions and plaques are cholesterol rich, and the significance of this in relation to the physical properties of the luminal membrane is discussed. Intra- and intermembrane variations in response of the other membrane systems of the cell, and the nature of the lesions induced by the agents are examined. Technical aspects of the application of filipin and digitonin to the localization of membrane cholesterol are also considered. The results obtained with bladder luminal membrane plaques emphasise that caution should be exercised in the interpretation of a negative response to filipin, particularly in membrane domains containing closely packed protein subunits.

Effects of sodium saccharin and caffeine on the urinary bladder of rats treated with n-butyl-n-(4-hydroxybutyl)nitrosamine.

The effects of sodium saccharin and caffeine on urinary bladder carcinogenesis in Wistar strain rats treated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) were studied. Animals were given 0.01% BBN as an initiator for 4 weeks and then sodium saccharin and/or caffeine as promoters for 32 weeks (Experiment I), or were treated simultaneously with 0.001% BBN, sodium saccharin and/or caffeine for 40 weeks (Experiment II). The urinary bladders were then removed and examined by light and electron microscopy. Sequential administration of sodium saccharin after BBN significantly enhanced the induction of hyperplasias compared with administration of BBN alone (Experiment I), and simultaneous administration of sodium saccharin with BBN significantly enhanced the induction of hyperplasia and papillomas compared with BBN alone (Experiment II). Two types of hyperplasias developed in the urinary bladder of rats treated with sodium saccharin alone in both experiments. Caffeine alone had no effect on the rat urinary bladder epithelium, and either sequential or simultaneous administration of caffeine with BBN caused no marked enhancement of carcinogenesis in these experiments.

Analysis of membrane structure in the transitional epithelium of rat urinary bladder. 2. The discoidal vesicles and golgi apparatus: their role in luminal membrane biogenesis

The luminal plasma membrane of the rat urothelium is composed of plaques seen in freeze-fracture as hexagonal arrays of 12 nm-diameter subunits on the E-face. In the present freeze-fracture study, the structure of the discoidal vesicle and Golgi membranes of superficial urothelial cells was investigated and the relationship between discoidal vesicle and luminal membrane structure examined by comparative analysis of subunit number and density. The vesicle membranes exhibit a striking variation in subunit disposition and number, forming a complete spectrum from those appearing similar to luminal membrane to those with smooth E- and P-faces devoid of subunits. Golgi membranes also display smooth zones on both fracture faces, situated between regions bearing irregularly distributed intramembrane particles of the usual size and polarity. Subunits are not observed in Golgi membranes. The results are interpreted as sequential stages in assembly of luminal membrane and are discussed in relation to current concepts of membrane biogenesis.