Apoptosis-inducing action of two products from oxidation of sesamol, an antioxidative constituent of sesame oil: a possible cytotoxicity of oxidized antioxidant.

Many effects of sesamol, an antioxidative constituent of sesame oil, have been reported for human health benefits due to its antioxidative action. However, we recently isolated two cytotoxic products, trimer and tetramer of sesamol, from oxidation of sesamol by an assay-guided purification. In this study, we have revealed some cytotoxic characteristics of these products in rat thymocytes and human leukemia K562 cells. Incubation of cells with trimer or tetramer at 10-30 microM for 24h significantly increased cell lethality and population of rat thymocytes containing hypodiploid DNA, suggesting cell death with DNA fragmentation, while it was not the case for 30 microM sesamol. The cytotoxic action of tetramer was more potent than that of trimer in rat thymocytes when their concentrations were 10-30 microM. The incubation of cells with 10 microM tetramer for 24h increased the population of cells with exposed phosphatidylserine, the activity of caspases, and the nick of DNA. These results indicate tetramer-induced apoptosis. In K562 cells, the incubation with tetramer at 10 microM for 72 h significantly inhibited the growth without affecting the lethality. However, tetramer at 30 microM significantly increased cell lethality. It is likely that tetramer exerts more cytotoxic action on normal non-proliferative cells (rat thymocytes) rather than proliferative cancer cells (human leukemia K562 cells). It may be necessary to consider the condition for preservation of sesamol and the safety of products from in vivo oxidation of sesamol for human health.

Zinc at clinically-relevant concentrations potentiates the cytotoxicity of polysorbate 80, a non-ionic surfactant.

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.

Tri-n-butyltin increases intracellular Zn(2+) concentration by decreasing cellular thiol content in rat thymocytes.

Effect of tri-n-butyltin (TBT), an environmental pollutant, on intracellular Zn(2+) concentration was tested in rat thymocytes to reveal one of cytotoxic profiles of TBT at nanomolar concentrations using a flow cytometer and appropriate fluorescent probes. TBT at concentrations of 30 nM or more (up to 300 nM) significantly increased the intensity of FluoZin-3 fluorescence, an indicator for intracellular Zn(2+) concentration, under external Ca(2+)- and Zn(2+)-free condition. Chelating intracellular Zn(2+) completely attenuated the TBT-induced augmentation of FluoZin-3 fluorescence. Result suggests that nanomolar TBT releases Zn(2+) from intracellular store site. Oxidative stress induced by hydrogen peroxide also increased the FluoZin-3 fluorescence intensity. The effects of TBT and hydrogen peroxide on the fluorescence were additive. TBT-induced changes in the fluorescence of FluoZin-3 and 5-chloromethylfluorescein, an indicator for cellular thiol content, were correlated with a coefficient of -0.962. Result suggests that the intracellular Zn(2+) release by TBT is associated with TBT-induced reduction of cellular thiol content. However, chelating intracellular Zn(2+) potentiated the cytotoxicity of TBT. Therefore, the TBT-induced increase in intracellular Zn(2+) concentration may be a type of stress responses to protect the cells.

Increase in intracellular Zn2+ concentration by thimerosal in rat thymocytes: intracellular Zn2+ release induced by oxidative stress.

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.

Zn(2+), derived from cell preparation, partly attenuates Ca(2+)-dependent cell death induced by A23187, calcium ionophore, in rat thymocytes.

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.

Possible use of quercetin, an antioxidant, for protection of cells suffering from overload of intracellular Ca2+: a model experiment.

Quercetin is known to protect the cells suffering from oxidative stress. The oxidative stress elevates intracellular Ca(2+) concentration, one of the phenomena responsible for cell death. Therefore, we hypothesized that quercetin would protect the cells suffering from overload of intracellular Ca(2+). To test the hypothesis, the effects of quercetin on the cells suffering from oxidative stress and intracellular Ca(2+) overload were examined by using a flow cytometer with appropriate fluorescence probes (propidium iodide, fluo-3-AM, and annexin V-FITC) and rat thymocytes. The concentrations (1-30 microM) of quercetin to protect the cells suffering from intracellular Ca(2+) overload by A23187, a calcium ionophore, were similar to those for the cells suffering from oxidative stress by H(2)O(2). The cell death respectively induced by H(2)O(2) and A23187 was significantly suppressed by removal of external Ca(2+). Furthermore, quercetin greatly delayed the process of Ca(2+)-dependent cell death although it did not significantly affect the elevation of intracellular Ca(2+) concentration by H(2)O(2) and A23187, respectively. It is concluded that quercetin can protect the cells from oxidative injury in spite of increased concentration of intracellular Ca(2+). Results suggest that quercetin is also used for protection of cells suffering from overload of intracellular Ca(2+).

Imidazole antifungals, but not triazole antifungals, increase membrane Zn2+ permeability in rat thymocytes Possible contribution to their cytotoxicity.

The use of zinc as a nutritional supplement has become common in many countries. Since zinc has diverse actions, it may be difficult to predict its synergistic and/or antagonistic action in simultaneous presence of drug(s). The combination of imidazole antifungals, but not triazole antifungals, with 3-30 microM ZnCl2 significantly increased the lethality of rat thymocytes. Since intracellular Zn2+ exerts various actions on the process of cell death, there is a possibility that imidazole antifungals, but not triazole antifungals, increases concentration of intracellular Zn2+ ([Zn2+]i). To test the possibility, we examined the effects of imidazole and triazole antifungals on [Zn2+]i of rat thymocytes in absence and presence of extracellular Zn2+ by the use of FluoZin-3, a fluorescent Zn2+ indicator. Imidazole antifungals (clotrimazole, econazole, and oxiconazole) increased the [Zn2+]i in the presence of extracellular Zn2+ while it was not the case for triazole antifungals (itraconazole and fluoconazole). Thus, it is suggested that imidazole antifungals increase the membrane permeability of Zn2+. The potency order in the augmentation of FluoZin-3 fluorescence by imidazole antifungals in the presence of extracellular Zn2+ was the same as that in their cytotoxic action. Therefore, the cytotoxic action of imidazole antifungals may be related to their action on membrane Zn2+ permeability.

Some characteristics of quercetin-induced cytotoxicity on rat thymocytes under in vitro condition.

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.

Clotrimazole, an antifungal drug possessing diverse actions, increases membrane permeation of cadmium in rat thymocytes.

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+).

Clotrimazole, an antifungal drug possessing diverse actions, increases the vulnerability to cadmium in lymphocytes dissociated from rat thymus.

Since clotrimazole, known as an antifungal drug, exerts diverse actions on cellular functions, it is expected that clotrimazole can be used for other purposes. This antifungal drug protects the cells overloaded with Ca(2+) by A23187, a calcium ionophore. Therefore, the agent may prevent the cells from death induced by heavy metals such as CdCl(2), PbCl(2), or HgCl(2) that are respectively proposed to increase intracellular Ca(2+) concentration. To test this possibility, we have examined the effect of clotrimazole on the cells simultaneously treated with CdCl(2), PbCl(2), or HgCl(2) using rat thymocytes and a flow cytometer with fluorescent probes. The simultaneous application of clotrimazole and CdCl(2) significantly decreased cell viability, even though the concentrations of both were ineffective at affecting the viability. The significant decrease in cell viability was not due to the inhibition of Ca(2+)-ATPase and Ca(2+)-dependent K(+) channels that were induced by clotrimazole. The simultaneous application increased the population of cells with phosphatidylserine exposed on membrane surface, indicating the change in asymmetrical distribution of membrane phospholipids. Furthermore, the cytotoxicity induced by the combination of clotrimazole and CdCl(2) under nominally Ca(2+)-free condition was more profound than that under normal Ca(2+) condition. Therefore, the membrane may be a target for the cytotoxic action of clotrimazole and CdCl(2) that were simultaneously applied. It is also the case for PbCl(2), but not the case for HgCl(2). It is concluded that clotrimazole can modulate the cytotoxicity of some heavy metals.

Reciprocal effects of glucose on the process of cell death induced by calcium ionophore or H2O2 in rat lymphocytes.

We have examined the effects of glucose at high concentrations on the process of cell death induced by excessive increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) or oxidative stress in rat lymphocytes. The cell death elicited by the excessive increase in [Ca(2+)](i) seemed to be induced by an activation of Ca(2+)-dependent K(+) channels because the inhibitors for Ca(2+)-dependent K(+) channels attenuated the decrease in cell viability. Glucose at 30-50mM augmented the decrease in cell viability by the excessive increase in [Ca(2+)](i). It was not specific for glucose because it was the case for sucrose or NaCl, suggesting an involvement of increased osmolarity in adverse action of glucose. On the contrary, glucose protected the cells suffering from oxidative stress induced by H(2)O(2), one of reactive oxygen species. It was also the case for fructose or sucrose, but not for NaCl. The process of cell death induced by H(2)O(2) started, being independent from the presence of glucose. Glucose delayed the process of cell death induced by H(2)O(2). Sucrose and fructose also protected the cells against oxidative stress. The reactivity of sucrose to reactive oxygen species is lower than those of glucose and fructose. The order in the reactivity cannot explain the protective action of glucose. Glucose at high concentrations exerts reciprocal actions on the process of cell death induced by the oxidative stress and excessive increase in [Ca(2+)](i).