Increase in intracellular Cd(2+) concentration of rat cerebellar granule neurons incubated with cadmium chloride: cadmium cytotoxicity under external Ca(2+)-free condition.

In order to examine the cadmium cytotoxicity unrelated to external Ca(2+), the effects of micromolar CdCl(2) on intracellular Cd(2+) concentration, cellular content of glutathione, and cell viability of rat cerebellar granule neurons were examined under normal Ca(2+) and external Ca(2+)-free conditions, using a laser confocal microscope with fluorescent probes, fluo-3-AM, 5-chloromethylfluorescein (CMF) diacetate, and propidium iodide. CdCl(2) (10-300 microM) dose-dependently increased the intensity of fluo-3 fluorescence. Exposure to CdCl(2) equally enhanced the fluo-3 fluorescence under both Ca(2+) conditions and MnCl(2) did not quench the CdCl(2)-enhanced fluorescence. The results indicate that the enhancement of fluo-3 fluorescence is due to the increase in intracellular Cd(2+) concentration. CdCl(2) at 100-300 microM decreased the intensity of CMF fluorescence, indicating the decrease in cellular content of glutathione. The population of cells stained with propidium (dead cells) was increased by 100-300 microM CdCl(2). Similar results described above were also observed under external Ca(2+)-free condition. It is suggested that some of cytotoxic actions of CdCl(2) on neurons are unrelated to external Ca(2+), one of main sources for increasing intracellular Ca(2+) concentration.

Cytometric analysis of lidocaine-induced cytotoxicity: a model experiment using rat thymocytes.

Lidocaine is a local anesthetic possessing both lipophilic and hydrophilic properties. It also acts as a surfactant. Thus, the disruption of membranes, resulting in necrosis, is one of possible mechanisms for lidocaine-induced cytotoxicity. However, lidocaine is reported to induce apoptosis. Therefore, in order to compare two mechanisms for cell death induced by lidocaine, the effects of millimolar lidocaine were examined on rat thymocytes by a flow cytometer with appropriate fluorescent probes. Lidocaine decreased the population of living cells with phosphatidylserine-exposed membranes, one of markers for early stage of apoptosis, and increased the population of dead cells without increasing that of cells with hypodiploidal DNA. Lidocaine at millimolar concentrations may deteriorate the membranes of such apoptotic living cells rather than those of intact living cells, resulting in necrosis. It is suggested that the process of apoptosis is not completed in the presence of millimolar lidocaine.

Tri-n-butyltin-induced blockade of store-operated calcium influx in rat thymocytes.

Tri-n-butyltin (TBT), one of environmental pollutants, disturbs intracellular Ca(2+) homeostasis by increasing intracellular Ca(2+) concentration ([Ca(2+)]i). Effect of TBT on oscillatory change in [Ca(2+)]i (Ca(2+) oscillation) of rat thymocytes was examined using a laser microscope with fluo-3-AM in order to further elucidate the TBT toxicity related to intracellular Ca(2+). The Ca(2+) oscillation was completely attenuated by 300nM TBT. Since store-operated Ca(2+) channels are involved in the generation of Ca(2+) oscillation, the action of TBT on an increase in [Ca(2+)]i by Ca(2+) influx through store-operated Ca(2+) channels was examined. The increase in [Ca(2+)]i by the store-operated Ca(2+) influx was not affected by 3nM TBT. However, TBT at 10nM or more significantly reduced the increase in [Ca(2+)]i. It is likely that TBT attenuates the Ca(2+) oscillation by reducing the Ca(2+) influx through store-operated Ca(2+) channels.

Modification of vulnerability to dodecylbenzenesulfonate, an anionic surfactant, by calcium in rat thymocytes.

We have previously reported that cremophor EL, a nonionic surfactant, at clinical concentrations significantly decreases the cell viability of rat thymocytes with phosphatidylserine-exposed (PS-exposed) membranes under in vitro condition. It is reminiscent of a possibility that sodium dodecylbenzenesulfonate (DCBS), an anionic surfactant world-widely used for detergents, also affects the cells in the similar manner. To test the possibility, the effect of DCBS on rat thymocytes has been examined using a flow cytometer with fluorescent probes. Exposure of PS on outer surface of cell membranes was induced by A23187, a calcium ionophore to increase intracellular Ca(2+) concentration ([Ca(2+)](i)). DCBS at 1µg/mL (2.87µM) significantly decreased the viability of cells with PS-exposed membranes, but not with intact membranes. DCBS also significantly decreased the viability of cells exposed to H(2)O(2), an oxidative stress increasing the [Ca(2+)](i). On the other hand, the decrease in extracellular Ca(2+) concentration ([Ca(2+)](e)) increased the cell vulnerability to DCBS and vice versa. Intact membrane lipid bilayer and extracellular Ca(2+) are required to maintain membrane integrity. Therefore, the change of membrane property by manipulation of [Ca(2+)](i) and [Ca(2+)](e) is one of causes for the augmentation of DCBS cytotoxicity.

Cremophor EL, a non-ionic surfactant, promotes Ca(2+)-dependent process of cell death in rat thymocytes.

Cremophor EL, a surfactant for pharmaceutical products, augments the cytotoxicity of hydrogen peroxide in rat thymocytes [Iwase, K., Oyama, Y., Tatsuishi, T., Yamaguchi1, J., Nishimura1, Y., Kanada, A., Kobayashi, M., Maemura, Y., Ishida, S., Okano, Y., 2004. Cremophor EL augments the cytotoxicity of hydrogen peroxide in lymphocytes dissociated from rat thymus glands. Toxicol. Lett. 154, 143-148]. The effect of cremophor EL on Ca(2+)-dependent process of cell death has been examined using a flow cytometer since hydrogen peroxide increases intracellular Ca2+ concentration. Cremophor EL at clinically-relevant concentrations greatly increased the population of dead cells in rat thymocytes simultaneously treated with A23187, a calcium ionophore increasing intracellular Ca2+ concentration. Removal of Ca2+ from external solution diminished the cremophor EL-induced increase in the dead cell population. Result suggests that Ca(2+)-dependent process is involved in the cremophor EL-induced decrease in the cell viability in the simultaneous presence of A23187. The population of cells with hypodiploidal DNA was not increased by the application of cremophor EL and A23187 although the cell viability was greatly decreased, indicating that the type of cell death is necrosis. It is suggested that cremophor EL at clinically-relevant concentrations augments the Ca(2+)-dependent process of necrosis.

Extract of Ginkgo biloba leaves attenuates kainate-induced increase in intracellular Ca2+ concentration of rat cerebellar granule neurons.

In order to reveal one of possible mechanisms for neuronal protective action of extract of Ginkgo biloba leaves (EGBL), the effect of EGBL on kainate- and KCl-induced increases in intracellular Ca(2+) concentration ([Ca(2+)]i) of rat cerebellar neurons was examined using a confocal laser microscope with appropriate fluorescent probes. EGBL at 3 microg/ml started to attenuate kainate-induced increase of [Ca(2+)]i and further increase in EGBL concentration (up to 30 microg/ml) concentration-dependently and significantly inhibited the kainate response. The complete inhibition by EGBL was observed in some neurons when the concentration was 10-30 microg/ml. The kainate-induced increase in [Ca(2+)]i was mainly due to Ca(2+) influx through voltage-dependent Ca(2+) channel opened by membrane depolarization via activation of kainate receptor-channel. However, the increase in [Ca(2+)]i by KCl was not significantly affected by EGBL at concentrations where the kainate response was greatly inhibited. EGBL consisting of flavone glycosides and terpene lactones is known to be an antioxidant. Furthermore, in this study, it is shown that EGBL exerts an inhibitory action on kainate receptor (a subtype of glutamate receptor). Since some of neurodegenerative diseases are due to cell death induced by glutamate excitotoxicity and oxidative stress, EGBL may be very suitable for preventing and/or treating such diseases.

Polysorbate 80 increases the susceptibility to oxidative stress in rat thymocytes.

Effect of simultaneous application of polysorbate 80, a nonionic surfactant widely used in pharmaceutical products, and hydrogen peroxide on rat thymocytes was examined to see if polysorbate 80 increases the susceptibility to oxidative stress because this surfactant decreases the cellular content of glutathione. Polysorbate 80 at clinically-relevant concentrations increases the cytotoxicity of hydrogen peroxide under the in vitro condition. Result suggests that polysorbate 80 may increase the susceptibility of cells to oxidative stress.

Cytometric analysis of adverse action of diphenyl ditelluride on rat thymocytes: cell shrinkage as a cytotoxic parameter.

Despite the growing use of organotellurium compounds in the chemical and biomedical fields, there has been no great concern about their toxicity until now. To test the possibility that diphenyl ditelluride (DPDT) and tellurium chloride (TeCl2), organic and inorganic tellurium compounds, may exert adverse action on mammals, their effects on rat thymocytes were examined under in vitro conditions using a flow cytometer with fluorescent probes. Incubation of thymocytes with DPDT at 300 nM or more for 24 h significantly increased the populations of shrunken cells and of cells with hypodiploidal DNA. Z-VAD-FMK, a paninhibitor of caspases, greatly suppressed the DPDT-induced increase in the hypodiploidal cell population, suggesting the involvement of caspase activation in DPDT toxicity. Hence, it is possible that DPDT would increase the population of thymocytes undergoing apoptosis if the blood concentration in mammals reached at least 300 nM or more. TeCl2 was much less potent than DPDT in increasing the population of hypodiploidal cells.

Cremophor EL augments the cytotoxicity of hydrogen peroxide in lymphocytes dissociated from rat thymus glands.

The pharmaceutical uses of cremophor EL, a non-ionic surfactant, are similar to those of polysorbate 80. In our previous study, polysorbate 80 exerted some adverse actions on rat thymocytes under in vitro condition. Therefore, the effects of cremophor EL on thymic lymphocytes were examined using a flow cytometer with appropriate fluorescent dyes. Cremophor EL at 10 microg/ml or more (up to 300 microg/ml) concentration-dependently decreased cellular content of glutathione. The cell viability of thymocytes under control condition was 95.4 +/- 1.2% (n = 7, mean +/- S.D.). The incubation of thymocytes with 300 microg/ml cremophor EL or 3 mM hydrogen peroxide for 2 h, respectively, decreased the cell viability to 90.8 +/- 2.8% or 91.2 +/- 2.6%. However, the simultaneous incubation with cremophor EL and hydrogen peroxide decreased the cell viability to 28.7 +/- 8.2%. Cremophor EL at 100 microg/ml accelerated the process of cell death induced by hydrogen peroxide. Results suggest that cremophor EL increases the susceptibility to oxidative stress. Cremophor EL at clinically relevant concentrations may increase the therapeutic potential of some anticancer agents to produce oxidative stress.