* Drug Safety Research Laboratory and
Research Technology Center, Daiichi Pharmaceutical Co., Ltd., 1-16-13 Kita-Kasai, Edogawa-ku, Tokyo 134-8630, Japan
Received October 2, 2002; accepted December 3, 2002
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ABSTRACT |
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Key Words: nefiracetam; metabolites; canine urinary bladder; primary culture; uroepithelial cells; transepithelial electric resistance; morphology.
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INTRODUCTION |
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More recently, a method to evaluate the barrier function using primary uroepithelial cells of the rabbit urinary bladder has been reported (Truschel et al., 1999). This system had physiological characteristics of intact urinary bladder epithelia including the presence of an apical umbrella cell layer, impermeability to water and urea, and development of high transepithelial electrical resistance (TER; >8000 ohm-cm2). To the best of our knowledge, there have been no reports dealing with the barrier function in the primary urinary bladder cells of dogs. Therefore, we developed primary cultured uroepithelial cells of the canine urinary bladder and used them to assess the effects of nefiracetam and its five main metabolites (M-3, M-10, M-11, M-18, and M-20) on TER measurement and immunofluorescence for ZO-1 as a marker of the tight junction.
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MATERIALS AND METHODS |
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Animals and housing conditions.
Male beagle dogs who were 31 months of age and purchased from Toyota Tsusho Corporation (Tokyo, Japan) were used for the investigation. They were individually housed at an environmental temperature of 23 ± 2°C and a relative humidity of 60 ± 20% with a 12 h light/dark cycle. The animals were allowed access to a commercial laboratory diet (DS, Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water ad libitum. All dogs were treated humanely, and the study protocol was in accordance with the Institutional Guidelines of Daiichi Pharmaceutical Co., Ltd. for use of laboratory animals.
Isolation of uroepithelial cells of the urinary bladder.
Dogs from a control group used in a toxicological study were killed by exsanguination under sodium pentobarbital anesthesia (25 mg/kg, iv; Dainippon Pharmaceutical Co., Ltd., Osaka, Japan), and the bladder was aseptically excised. An incision was then made lengthwise along the bladder, and the opened bladder was washed three times with the Krebs solution (110 mM NaCl, 5.8 mM KCl, 25 mM NaHCO3, 1.2 mM KH2PO4, 2.0 mM CaCl2, 1.2 mM MgSO4, and 11.1 mM glucose, pH 7.4) at 4°C. Afterward, the bladder was trimmed to remove excess fatty tissues and transferred, mucosal side down, to a metal rack with 10 sharp metal pins along each edge (Lewis and Hanrahan, 1990), which was placed in the same solution at 4°C, and the smooth muscle layer was carefully removed. The tissue was stretched, mucosal side up, across the metal pins on a 10 x 10-cm square plate, and then incubated for 24 h at 4°C in the minimum essential medium (MEM; Life Technologies Inc., Grand Island, NY) containing 1% (v/v) penicillin/streptomycin/fungizon (PSF; Life Technologies Inc.), 2.5 mg/ml dispase (Life Technologies Inc.), and 20 mM HEPES (MEM/PSF/dispase solution, pH 7.4).
After incubation, the MEM/PSF/dispase solution was aspirated, the stripped mucosa was transferred to a sterile 150-mm culture dish, and uroepithelial cells were scraped from the connective tissues with cell scrapers. The scraped cells were suspended in 20 ml of trypsin-EDTA (0.25% trypsin and 1 mM EDTA4Na, Life Technologies Inc.), and incubated for 30 min at 37°C. Later, the single cell suspension was brought up to 50 ml with MEM containing 1% PSF, 5% fetal bovine serum (FBS; Life Technologies Inc.) and 20 mM HEPES (MEM/PSF/FBS solution, pH 7.4) in a sterile tube and spun down with a centrifuge (KUBOTA 8800, KUBOTA Corporation, Tokyo, Japan) at 1000 rpm for 5 min at 4°C. The resulting supernatant was aspirated carefully and the cells were suspended in 50 ml of the same MEM/PSF/FBS solution. This washing process was repeated two more times. The cells were then rewashed in 50 ml of the keratinocyte medium (defined keratinocyte-SFM, Life Technologies Inc.) and resuspended in the appropriate volume of the keratinocyte medium to make a final concentration of 6.07.0 x 105 cells/ml, as determined by counting the cells in a hemocytometer chamber.
Cell culture.
The collagen solution was prepared by mixing 5 mg type IV collagen (Sigma), 100 µl glacial acetic acid, and 50 ml distilled water, and kept overnight at 4°C without stirring. The collagen solution was sterilized with a 0.22-µm bottle top filter (Asahi Techno Grass Corporation, Tokyo, Japan) and stored at 4°C. The keratinocyte medium was added to both chambers with a 12-mm transwell filter (Corning Coaster Corporation, Cambridge, MA) and incubated for 2 h at 37°C. Prior to use, the collagen solution was diluted 1:9 with 10 mM Na2CO3-HCl (pH 9.0), and 500 µl of the resultant solution was added to each apical chamber after aspirating the keratinocyte medium and incubated for 1 h at 37°C.
Before plating, the collagen solution was aspirated, 0.5 ml of the cell suspension was added to the apical chamber, 1.5 ml of keratinocyte medium was added to the basal chamber, and cells were incubated at 37°C. Three days later, the apical and basal media were aspirated and replaced with 0.5 and 1.5 ml of the keratinocyte medium containing 1 mM CaCl2 (KM/Ca solution), respectively, in case the TER reached levels of approximately 1000 ohm-cm2 or higher.
TER measurement.
TER was measured using an epithelial voltohmmeter (EVOM, World Precision Instruments, Sarasota, FL). The electrodes were sterilized by immersing them in 70% ethanol, and they were washed with sterile PBS prior to use. Calculations for ohm-cm2 were made by subtracting values of blank inserts from all samples and multiplying by the area seeded with cells.
Light and electron microscopy.
Cells cultured on a 12-mm transwell filter were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), rinsed with 8.2% sucrose, postfixed in 1% OsO4 in the buffer, dehydrated through an ascending alcohol series, and embedded in epoxy resin. Semi-thin sections were made and stained with 1% toluidine blue for light microscopic examination. Ultrathin sections were stained with uranyl acetate and lead citrate, and were observed with a transmission electron microscope (H-500, Hitachi Ltd., Tokyo, Japan).
Immunofluorescence staining and confocal laser scanning microscopy.
For immunofluorescence staining, cells cultured on a 12-mm transwell filter were fixed with a HISTOCHOICE (Amresco, Solon, OH) for 1 min at room temperature, followed by acidic methanol (95% methanol and 5% glacial acetic acid) for 15 min at 20°C. The fixed cells were incubated for 1 h with either anti-ZO-1 rabbit polyclonal antibody (Zymed Laboratories, Inc., San Francisco, CA) or anti-E-cadherin mouse monoclonal antibody (Transduction Laboratories, San Diego, CA) as the first antibody being diluted 1:50 or 1:100 with PBS, respectively. Then, the cells were incubated for 30 min with the second antibody, either FITC-conjugated donkey antirabbit IgG (Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, England) diluted 1:40 or FITC-conjugated goat antimouse IgG (DAKO JAPAN Co., Ltd., Kyoto, Japan) diluted 1:10 with PBS. The cells were washed three times for 5 min with PBS, and the cell-grown transwell filters were cut, transferred to a slide glass and mounted with the mounting medium (Vectashield, Vector Laboratories, Inc., Burlingame, CA). Thereafter, the cells were observed with a confocal laser scanning microscope (TCS-SP, Leica Microsystems AG, Wetzlar, Germany).
Confirmation of the property of the cultured uroepithleial cells.
After plating on a 12-mm transwell filter, the changes in TER were serially measured for 20 days. The medium was replaced every three days. When TER reached 10,000 ohm-cm2 or more, the cultured cells were observed by light and electron microscopy, and their ZO-1 and E-cadherin were checked with a confocal microscope. Furthermore, effects of cytochalasin-B, which depolymerized actin microfilaments and consequently opened the tight junction, on TER and ZO-1 were examined to validate this culture system. Cytochalasin-B was first dissolved in DMSO, diluted with the KM/Ca solution to make concentrations of 1.6, 4, and 10 µM, and sterilized with a 0.22-µm membrane filter (Millex-GV, Millipore Co., Bedford, MA). The final DMSO concentration was 0.08% in the 1.6 µM solution, 0.2% in the 4 µM solution, or 0.5% in the 10 µM solution. DMSO diluted 1:200 with KM/Ca solution served as a negative control solution. TER was measured 0, 1, 2, 4, 8, 24, and 48 h after exposure, and immunofluorescence for ZO-1 was observed 48 h after exposure.
Effect of nefiracetam and its five main metabolites on cultured uroepithelial cells.
Cells whose TER reached 10,000 ohm-cm2 or more were used for this investigation. In a preliminary study, the metabolite M-18 at 2 mM induced a significant decrease in TER 48 h after exposure. Based on these data, nefiracetam and its five metabolites were dissolved in DMSO, diluted with KM/Ca solution to make concentrations of 0.8 and 2 mM, and sterilized with a 0.22-µm membrane filter (Millex-GV). The final DMSO concentration ranged between 0.08 and 0.2%, and DMSO diluted acted as a negative control. TER was measured 0, 1, 2, 4, 8, 24, 48, 72, and 120 h after exposure, and immunofluorescence for ZO-1 was observed 48 and 120 h after exposure.
Statistical analysis.
The data are expressed as the mean ± SD, and statistical analysis was performed using the software package, EXSAS ver. 5.10 (Arm Corp., Osaka, Japan). The data of TER were evaluated by Dunnett's multiple comparison test, and a p value of < 0.05 was considered significant.
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RESULTS |
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DISCUSSION |
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First of all, we evaluated time-course changes in TER and morphology of cultured uroepithelial cells to substantiate the barrier function in the naive state. The epithelium of the urinary bladder is shown to have one of the highest TERs in the whole body with values ranging from 10,000 to 75,000 ohm-cm2 (Lewis and Diamond, 1976). In the present study, as a device to prevent keratinocyte stratification and differentiation, the medium was formulated to have a low calcium concentration (< 0.1 mM). TER was monitored during the 20-day observation periods. On day 3, TER achieved 10003000 ohm-cm2, in which values were high as compared with those of rabbit cultured uroepithelial cells (about 200 ohm-cm2, Truschel et al., 1999
). When TER was over 10003000 ohm-cm2, the culture medium was replaced with the keratinocyte medium containing 1 mM CaCl2. In a case that uroepithelial cells were grown in the medium without 1 mM CaCl2, or the medium replacement was delayed by only one day, uroepithelial cells failed to achieve high TER. From days 5 to 10, TER reached 16,000 ohm-cm2 or more, suggesting that the canine uroepithelial cells have the tight junction, since high TER emerged as the feature of culture cells forming a tight junction-like function to ion flux (Schneeberger and Lynch, 1992
).
Morphologically, the uroepithelium of the urinary bladder comprised three cell layers with a superficial cell layer consisting of cells forming junctional complex (containing tight and adherence junctions). Furthermore, the immunofluorescence for both ZO-1 and E-cadherin was observed around the respective cultured cells. ZO-1 is an essential protein associated with the tight junction, and E-cadherin is necessary to form the adherence junction (Denker and Nigam, 1998). Our results demonstrated that the culture system possessed functional and morphological characteristics of uroepithelial cells of the urinary bladder such as high TER and three cell layers with junctional complex.
Cytochalasin-B induced decreases in TER, disruption of actin microfilaments, and marked increases in paracellular permeability (Ma et al., 1995). The decrease in TER with the disruption of ZO-1 band is thought to be due to an alteration of the actin structure, because actin microfilaments closely correlate with the tight junction and the localization of ZO-1 proteins. In addition, since actin microfilaments and microtubules play a critical role in cytoskeletal formation, it is proposed that depolymerization of these elements may be related to cell deformation. Hence, effects of cytochalasin-B on TER and immunofluorescence for ZO-1 were examined to validate the culture system developed. Cytochalasin-B decreased TER from 1.6 µM in a concentration-dependent manner. In the immunofluorescence, this compound displayed induced a slight reduction of the ZO-1 band at 4 µM and a disruption of ZO-1 band and cell disappearance at 10 µM. These findings indicate that the culture system can be successfully performed and evaluate disturbed tight junction arising from the new chemical entity.
Among nefiracetam and its five main metabolites employed, only M-18 elicited a significant decrease in TER from 0.8 mM. In immunofluorescence appearances, M-18 exhibited a slight reduction of ZO-1 band and deformation of the uroepithelial cells. Meanwhile, M-10, a monohydroxylated derivative of nefiracetam with a sulfate-conjugating metabolite of M-18, also induced deformation of the uroepithelial cells in the late phase (120 h later). This implied that the effect of M-18 on uroepithelial cells was stronger than that of M-10. M-18 is reported to show a high concentration in canine serum as compared with other species (unpublished data). Because the measurement of M-18 in urine has been extremely difficult, its quantitative data have not been procured. The possibility is raised that a high concentration of M-18 is present in urine as well as in serum. According to the previous report from Kashida et al.(1996), it was suggested that the direct action of nefiracetam or its metabolites in urine induced degeneration of epithelial cells of the urinary bladder. On the other hand, it is reported that cyclophosphamide, a typical toxicant of the urinary bladder, caused hemorrhagic cystitis, preceded by contact with acrolein, a metabolite of cyclophosphamide (Fraiser and Kehrer, 1992
; Pohl et al., 1989
). Likewise, cystitis has been confirmed to be brought about by the direct toxicity of a carbonic anhydrase inhibitor (Durand-Cavagna et al., 1992
) or trimethyl imidazopyrazolopyrimidine (Macallum and Albassam, 1994
) to the urinary bladder wall. Their mechanisms remain unclear, but it is speculated that their metabolites excreted into urine play an important role. In urinalyses from a previous dog study, although increases in protein excretion and positive occult blood were observed from two-week oral treatment of nefiracetam, neither the fluctuation of urinary pH and osmotic pressure nor the presence of medicine-like crystals was noted (unpublished data). In rats and monkeys, no effects of nefiracetam on urinalyses were seen (unpublished data). In the present study, pH and osmotic pressure in the culture medium containing nefiracetam, M-10, and M-18 at 2 mM were similar to those in the control medium. Further, M-18 showed no cytotoxic effect on Mardin-Darby canine kidney cells or canine renal papilla slices at a concentration of up to 5 mM in a previous in vitro study (unpublished data). Taken together, M-18 in urine may cause alterations of actin microfilaments and microtubules leading to cell deformation in the uroepithelium of the canine urinary bladder, preceded by reductions in TER and ZO-1 band.
In conclusion, the canine uroepithelial culture system possessed both functional and morphological features of the uroepithelium reflected in vivo. In the urinary bladder lesion in dogs due to nefiracetam, the metabolite M-18 may strongly contribute to the process of its occurrence.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Chang, A., Hammond, T. G., Sun, T. T., and Zeidel, M. L. (1994). Permeability properties of the mammalian bladder apical membrane. Am. J. Physiol. 267, C1483C1492.
Chlapowski, F. J., and Haynes, L. (1979). The growth and differentiation of transitional epithelium in vitro. J. Cell. Biol. 83, 605614.[Abstract]
Denker, B. M., and Nigam, S. K. (1998). Molecular structure and assembly of the tight junction. Am. J. Physiol. 274, F1F9.[ISI][Medline]
Durand-Cavagna, G., Owen, R. A., Gordon, L. R., Peter, C. P., and Boussiquet-Leroux, C. (1992). Urothelial hyperplasia induced by carbonic anhydrase inhibitors (CAIs) in animals and its relationship to urinary Na and pH. Fundum. Appl. Toxicol. 18, 137143.[CrossRef][ISI][Medline]
Fraiser, L., and Kehrer, J. P. (1992). Murine strain differences in metabolism and bladder toxicity of cyclophosphamide. Toxicology 75, 257272.[CrossRef][ISI][Medline]
Guhe, C., and Follmann, W. (1994). Growth and characterization of porcine urinary bladder epithelial cells in vitro. Am. J. Physiol. 266, F298F308.
Gumbiner, B. (1987). Structure, biochemistry, and assembly of epithelial tight junctions. Am. J. Physiol. 253, C749C758.
Hicks, R. M. (1975). The mammalian urinary bladder: An accommodating organ. Biol. Rev. Camb. Philos. Soc. 50, 215246.[ISI][Medline]
Howlett, A. R., Hodges, G. M., and Rowlatt, C. (1986). Epithelial-stromal interactions in adult bladder: Urothelial growth, differentiation, and maturation on culture facsimiles of bladder stroma. Dev. Biol. 118, 403415.[ISI][Medline]
Jindo, T., Shimizu, Y., Kato, M., and Takayama, S. (1994). Thirteen-week oral toxicity study of the new cognition-enhancing agent nefiracetam in rats. Arzneim.-Forsch./Drug Res. 44, 214216.
Hiramatsu, M., Koide, T., Ishihara, S., Shiotani, T., Kameyama, T., and Nabeshima, T. (1992). Involvement of the cholinergic system in the effects of nefiracetam (DM-9384) on carbon monoxide (CO)-induced acute and delayed amnesia. Eur. J. Pharmacol. 216, 279285.[CrossRef][ISI][Medline]
Kashida, Y., Yoshida, M., Ishii, Y., Nomura, M., and Kato, M. (1996). Examination of lesions in the urinary bladder and kidney of dogs induced by nefiracetam, a new nootropic agent. Toxicol. Pathol. 24, 549557.[ISI][Medline]
Lewis, S. A., and Diamond, J. M. (1976). Na+ transport by rabbit urinary bladder, a tight epithelium. J. Membr. Biol. 28, 140.[ISI][Medline]
Lewis, S. A., and Hanrahan, J. W. (1990). Physiological approaches for studying mammalian urinary bladder epithelium. In Methods in Enzymology (S. Fleischer and B. Fleischer, Eds.), Vol. 192, pp. 632650. Academic Press.
Ma, T. Y., Hollander, D., Tran, L. T., Nguyen, D., Hoa, N., and Bhalla, D. (1995). Cytoskeletal regulation of Caco-2 intestinal monolayer paracellular permeability. J. Cell. Physiol. 164, 533545.[ISI][Medline]
Macallum, G. E., and Albassam, M. A. (1994). Renal toxicity of nondopaminergic antipsychotic agent, trimethyl imidazopyrazolopyrimidine, in rats. Toxicol. Pathol. 22, 3947.[ISI][Medline]
Murasaki, M., Inami, M., Ishigooka, J., Wakatabe, H., Utsumi, M., Matsumoto, T., Fukuyama, Y., Miura, S., Tachizawa, H., Sudo, K., and Fujimaki, Y. (1994). Phase I study on DM-9384 (nefiracetam). Jpn. Pharmacol. Ther. 22, 35393587.
Nabeshima, T., Nakayama, S., Ichihara, K., Yamada, K., Shiotani, T., and Hasegawa, T. (1994). Effects of nefiracetam on drug-induced impairment of latent learning in mice in a water finding task. Eur. J. Pharmacol. 255, 5765.[CrossRef][ISI][Medline]
Negrete, H. O., Lavelle, J. P., Berg, J., Lewis, S. A., and Zeidel, M. L. (1996). Permeability properties of the intact mammalian bladder epithelium. Am. J. Physiol. 271, F886F894.
Nybom, P., and Magnusson, K. (1996). Modulation of the junctional integrity by low or high concentration of cytochalasin B and dihydrocytochalasin B is associated with distinct changes in F-actin and ZO-1. Biosci. Rep. 16, 313326.[ISI][Medline]
Otomo, E., Kogure, K., Hirai, S., Goto, F., Hasegawa, K., Tazaki, Y., Ito, E., Nishimura, T., Fujishima, M., Inanaga, K., and Ogawa, N. (1994). Clinical evaluation of DM-9384 in the treatment of cerebrovascular disorders: Early Phase II study. Jpn. Pharmacol. Ther. 22, 35893624.
Parsons, C. L., Lilly, J. D., and Stein, P. (1991). Epithelial dysfunction in nonbacterial cystitis (interstitial cystitis). J. Urol. 145, 732735.[ISI][Medline]
Perrone, R. D., Johns, C., Grubman, S. A., Moy, E., Lee, D. W., Alroy, J., Sant, G. R., and Jefferson, D. M. (1996). Immortalized human bladder cell line exhibits amiloride-sensitive sodium absorption. Am. J. Physiol. 270, F148F153.
Pohl, J., Stekar, J., and Hilgard, P. (1989). Chloroacetaldehyde and its contribution to urotoxicity during traeatment with cyclophosphamide or ifosfamide. An experimental study/short communication. Arzneim.-Forsch./Drug Res. 39, 704705.
Sakurai, T., Ojima, H., Yamasaki, T., Kojima, H., and Akashi, A. (1989). Effects of N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl) acetamide (DM-9384) on learning and memory in rats. Jpn. J. Pharmacol. 50, 4753.[ISI][Medline]
Schneeberger, E. E., and Lynch, R. D. (1992). Structure, function, and regulation of cellular tight junctions. Am. J. Physiol. 262, L647L661.
Smith, P. R., Mackler, S. A., Weiser, P. C., Brooker, D. R., Ahn, Y. J., Harte, B. J., McNulty, K. A., and Kleyman, T. R. (1998). Expression and localization of epithelial sodium channel in mammalian urinary bladder. Am. J. Physiol. 274, F91F96.
Stevenson, B. R., Anderson, J. M., and Bullivant, S. (1988). The epithelial tight junction: Structure, function and preliminary biochemical characterization. Mol. Cell. Biochem. 83, 129145.[ISI][Medline]
Sudo, K., Hashimoto, K., Fujimaki, Y., and Tachizawa, H. (1988). Abstracts of the Collegium International Neuro-Phychopharmacologium Congress No. 32.02.25.
Surya, B., Yu, J., Manabe, M., and Sun, T. T. (1990). Assessing the differentiation state of cultured bovine urothelial cells: Elevated synthesis of stratification-related K5 and K6 keratins and persistent expression of uroplakin I. J. Cell Sci. 97, 419432.[Abstract]
Truschel, S. T., Ruiz, W. G., Shulman, T., Pilewski, J., Sun, T. T., Zeidel, M. L., and Apodaca, G. (1999). Primary uroepithelial cultures. J. Biol. Chem. 274, 1502015029.
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