|Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2001: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2000: ¥2,100,000 (Direct Cost: ¥2,100,000)
(A) As tri-n-butyltin (TBT), one of environmental pollutants, is accumulated in wild animals, concern regarding the toxicity of TBT in both wildlife and humans is increasing. TBT has been reported to increase intracellular Ca2+ concentration in several types of cells. In order to examine how Ca2+ is involved in TBT-induced cell death, the effect of TBT on rat thymocytes has been compared with that of A23187, a calcium ionophore, under various concentrations of external Ca2+ using a flow cytometer and fluorescent probes. Although both TBT and A23187 were toxic to cells under normal Ca2+ condition, under external Ca2+-free condition the cytotoxic action of TBT was potentiated without changing the threshold concentration while that of A23187 was completely abolished. A23187 attenuated the TBT-induced descent in cell viability under normal Ca2+ concentration despite intracellular Ca2+ concentration was increased. As external Ca2+ concentration increased, the TBT-induced increase in number
of dead cells gradually decreased whereas the number of cells in an early stage of apoptosis increased. Results suggest that Ca2+ has contradictory actions on the process of TBT-induced cell death in rat thymocytes.
(B) The effects of tri-n-butyltin (TBT), an environmental pollutant, on membrane currents elicited by N-methyl-D-asparlate (NMDA) were examined in the neurons acutely dissociated from the dorsal motor nucleus of vagus of rats, using a nystatin-perforated patch recording mode under the voltage-clamp conditions, in order to predict a neurotoxic action of TBT on humans and wild animals. TBT at an environmentally-relevant concentration (l00 nM) greatly increased the amplitude of NMDA-induced outward current, but not that of NMDA-induced inward current. The reversal potential of the NMDA-induced outward current was close to the theoretical K+ equilibrium potential of -82 mV. The NMDA-induced outward current augmented by TBT was markedly inhibited by 5 mM tetraethylammonium chloride and 300 nM charybdotoxin. The outward current was abolished by removal of extracellular Ca2+, suggesting that the outward current was due to the activation of Ca2+-activated K+ channels by Ca2+ passing through NMDA channels. The NMDA-induced outward current was completely blocked by Cs+-internal solution. Under this condition, TBT had no effect on the NMDA-induced inward current. The present results suggest that TBT potentiates Ca2+-activated K+ current induced by NMDA without augmenting the NMDA-induced inward current. Less