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Polysorbate 80, a non-ionic surfactant, is used in the formula of water-insoluble anticancer agents for intravenous application. In our recent studies, this surfactant decreased cellular thiol content and the chemicals decreasing cellular thiol content increased intracellular Zn(2+) concentration. In this study using rat thymocytes, the effect of polysorbate 80 on FluoZin-3 fluorescence, an indicator for intracellular Zn(2+), and the influence of ZnCl(2) on cytotoxicity of polysorbate 80 were examined in order to test the possibility that Zn(2+) is involved in cytotoxic action of polysorbate 80. The surfactant at concentrations of 10 microg/ml or more significantly augmented FluoZin-3 fluorescent in a concentration-dependent manner, indicating an increase in intracellular Zn(2+) concentration. The increase by polysorbate 80 was also observed after removing extracellular Zn(2+), suggesting an intracellular Zn(2+) release. The simultaneous application of polysorbate 80 (30 microg/ml) and ZnCl(2) (10-30 microM) significantly increased cell lethality. The simultaneous application of ZnCl(2) accelerated the process of cell death induced by polysorbate 80 and the combination increased oxidative stress. Results may indicate that the cytotoxicity of polysorbate 80 at clinical concentrations is modified by micromolar zinc. Although there is no clinical report that polysorbate 80 and zinc salt are simultaneously applied to human as far as our knowledge, it may be speculated that zinc induces some diverse actions in cancer treatment with water-insoluble anticancer agent including nanoparticle drug of which the solvent is polysorbate 80., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Thimerosal (TMR), an ethylmercury-containing preservative in pharmaceutical products, was recently reported to increase intracellular Zn(2+) concentration. Therefore, some health concerns about the toxicity of TMR remain because of physiological and pathological roles of Zn(2+). To reveal the property of TMR-induced increase in intracellular Zn(2+) concentration, the effect of TMR on FluoZin-3 fluorescence, an indicator of intracellular Zn(2+), of rat thymocytes was examined. TMR at concentrations ranging from 0.3 microM to 10 microM increased the intensity of FluoZin-3 fluorescence in a concentration-dependent manner under external Ca(2+)- and Zn(2+)-free condition. The threshold concentration was 0.3-1 microM. The increase in the intensity was significant when TMR concentration was 1 microM or more. N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a chelator for intracellular Zn(2+), completely attenuated the TMR-induced augmentation of FluoZin-3 fluorescence. Hydrogen peroxide (H(2)O(2)) and N-ethylmaleimide, reducing cellular thiol content, significantly increased FluoZin-3 fluorescence intensity and decreased 5-chloromethylfluorescein (5-CMF) fluorescence intensity, an indicator for cellular thiol. The correlation coefficient between TMR-induced augmentation of FluoZin-3 fluorescence and attenuation of 5-CMF fluorescence was -0.882. TMR also attenuated the 5-CMF fluorescence in the presence of TPEN. Simultaneous application of H(2)O(2) and TMR synergistically augmented the FluoZin-3 fluorescence. It is suggested that TMR increases intracellular Zn(2+) concentration via decreasing cellular thiol content.

Econazole, one of imidazole antifungals, has been reported to exhibit an inhibitory action on Mycobacterium tuberculosis and its multidrug-resistant strains under in vitro and ex vivo conditions. There is a chemotherapeutic potential of econazole against tuberculosis. We have revealed that Zn(2+) at micromolar concentrations potentiates the cytotoxicity of imidazole antifungals by increasing membrane Zn(2+) permeability. It is reminiscent of a possibility that econazole exhibits harmful action on human in the presence of Zn(2+) at a physiological range when the agent is systemically administered. Because it is necessary to characterize the cytotoxic action of econazole in the presence of Zn(2+), we have cytometrically examined the effects of econazole, ZnCl(2), and their combination on rat thymocytes. ZnCl(2) at concentrations ranging from 1 microM to 30 microM significantly increased the lethality induced by 10 microM econazole in a concentration-dependent manner. Econazole at a sublethal concentration of 1 microM significantly augmented the intensity of side scatter in the presence of micromolar ZnCl(2), suggesting the change in an intracellular circumstance by the combination of econazole and ZnCl(2). Econazole at 0.3 microM or more in the presence of ZnCl(2) increased the intensity of Fluo-3 fluorescence, an indicator for intracellular Ca(2+). Furthermore, the intensity of FluoZin-3 fluorescence, an indicator for intracellular Zn(2+), was also augmented by econazole at 0.1 microM or more in the presence of ZnCl(2). Results suggest that the combination of submicromolar econazole with micromolar ZnCl(2) may increase the intracellular concentration of Ca(2+) and Zn(2+), leading to disturbance of intracellular Ca(2+) and Zn(2+) homeostasis that triggers cytotoxic action.

A23187, a calcium ionophore, is used to induce Ca(2+)-dependent cell death by increasing intracellular Ca(2+) concentration ([Ca(2+)](i)) under in vitro condition. Since this ionophore also increases membrane permeability of metal divalent cations such as Zn(2+) and Fe(2+) rather than Ca(2+), trace metal cations in cell suspension may affect Ca(2+)-dependent cell death induced by A23187. Therefore, the effects of chelators for divalent metal cations, EDTA and TPEN, on the A23187-induced cytotoxicity were cytometrically examined in rat thymocytes. The cytotoxicity of A23187 was attenuated by 1mM EDTA while it was augmented by 50 microM EDTA and 10 microM TPEN. These changes were statistically significant. The A23187-induced increase in Fluo-3 fluorescence intensity, a parameter for [Ca(2+)](i), was significantly reduced by 1mM EDTA while it was not the case for 50 microM EDTA and 10 microM TPEN. The intensity of FluoZin-3 fluorescence, a parameter for [Zn(2+)](i), increased by A23187 was respectively reduced by 50 microM EDTA and 10 microM TPEN. It is suggested that the attenuation of A23187-induced cytotoxicity by 1mM EDTA is due to the chelation of extracellular Ca(2+) and Zn(2+) while the augmentation by 50 microM ETDA or 10 microM TPEN is due to the chelation of extracellular Zn(2+). The Tyrode's solution without thymocytes contained 32.4 nM of zinc while it was 216.9 nM in the cell suspension. In conclusion, trace Zn(2+), derived from cell preparation, partly attenuates the Ca(2+)-dependent cell death induced by A23187.

Quercetin, a flavonoid found in fruits and vegetables, exerts beneficial effects that contribute to human health. Therefore, quercetin preparation is expected as complementary or alternative medicine used by general population. The plausible criterion for such medicines is to exert no toxic action on normal cells. In this study, the effects of quercetin on normal cells were examined using rat thymocytes in RPMI-1640 medium. Significant cytotoxic actions of quercetin were observed at 30 microM. Quercetin increased the populations of propidium-stained cells, shrunken cells, annexin V-positive cells, and the cells with hypodiploidal DNA. Thus, the type of cell death induced by quercetin was apoptosis. Z-VAD-FMK, a pan-inhibitor for caspases, partly attenuated the process of quercetin-induced apoptosis. It can be suggested that plasma concentration of quercetin should be below 30 microM after the digestion when quercetin preparation as complementary or alternative medicine is used.

In previous study, clotrimazole, an antifungal drug, exerted potent cytotoxic action on rat thymocytes in presence of metal divalent cations such as Cd(2+) and Pb(2+). To reveal one of toxicological characteristics of clotrimazole, we examined the effect of clotrimazole on intracellular concentration of metal divalent cations by flow cytometer with fluo-3, a fluorescent. Simultaneous application of clotrimazole and CdCl(2) significantly decreased the cell viability although their concentrations were not cytotoxic, respectively. Clotrimazole alone increased the intensity of fluo-3 fluorescence, suggesting an increased concentration of intracellular Ca(2+). The intensity of fluo-3 fluorescence augmented by the combination of clotrimazole and CdCl(2) was much higher than that by respective agents. Removal of external Ca(2+) further increased the intensity of fluorescence augmented by the combination. Furthermore, the application of MnCl(2) did not attenuate the intensity in the presence of CdCl(2). Therefore, it is suggested that the augmentation of fluo-3 fluorescence in the simultaneous presence of clotrimazole and CdCl(2) is Cd(2+)-dependent. Clotrimazole may increase membrane permeation of Cd(2+).