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ToxSci Advance Access originally published online on February 18, 2003
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Toxicological Sciences 72, 164-170 (2003)
Copyright © 2003 by the Society of Toxicology

SAFETY EVALUATION

Effect of Nefiracetam, a Neurotransmission Enhancer, on Primary Uroepithelial Cells of the Canine Urinary Bladder

Koichi Goto*,1, Yoshikazu Ishii{dagger}, Toshimasa Jindo* and Kazuhisa Furuhama*

* Drug Safety Research Laboratory and {dagger} Research Technology Center, Daiichi Pharmaceutical Co., Ltd., 1-16-13 Kita-Kasai, Edogawa-ku, Tokyo 134-8630, Japan

Repeated oral treatment of dogs with a high dose of nefiracetam is reported to induce hemorrhagic lesions in the urinary bladder. To delineate its pathogenesis, we established the primary culture of uroepithelial cells of the canine urinary bladder, and then explored the effect of nefiracetam on the cultured cells. Uroepithelial cells scraped from the connective tissues of the urinary bladder of naive dogs were suspended in the minimum essential medium containing dispase, and then resuspended in the keratinocyte medium to be 6.0–7.0 x 105 cells/ml. Afterward, they were added to an apical chamber with a 12-mm transwell filter, cultured for three days, and recultured in the keratinocyte medium containing 1 mM CaCl2 for 20 days. Microscopically, these cultured cells consisted of three cell layers with high transepithelial electric resistance (TER; > 10,000 ohm-cm2). Immunofluorescence observations revealed ZO-1 and E-cadherin bands, and electron microscopic examinations displayed the superficial cells with the assembly of tight junctions. When the effect of nefiracetam and its five main metabolites (M-3, M-10, M-11, M-18, and M-20) on TER and the ZO-1 band was assessed using cultured cells, only M-18 significantly reduced TER in the coculture for 48 h or more. Both M-10 and M-18 exhibited a deformation of uroepithelial cells and a slight reduction of the ZO-1 band from 120 h later. In conclusion, this culture system possesses both functional and morphological features of the uroepithelium reflected in vivo, and M-18 may play a pivotal role in the impairment of uroepithelial cells, leading to the onset of the urinary bladder lesion in dogs due to nefiracetam.

Key Words: nefiracetam; metabolites; canine urinary bladder; primary culture; uroepithelial cells; transepithelial electric resistance; morphology.


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