Tomohiro M. Oyama

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+).

Low micromolar zinc exerts cytotoxic action under H2O2-induced oxidative stress: Excessive increase in intracellular Zn2+ concentration

The ability of zinc to retard oxidative processes has been recognized for many years. However, zinc is cytotoxic under certain oxidative stress. In this study, we investigated the effect of H2O2 on intracellular Zn2+ concentration of rat thymocytes and its relation to the cytotoxicity. Experiments were cytometrically performed by the use of fluorescent probes, propidium iodide, FluoZin-3-AM, and 5-chloromethylfluorescein diacetate. ZnCl2 potentiated cytotoxicity of H2O2 while TPEN, a chelator for intracellular Zn2+, attenuated it. Results suggested an involvement of intracellular Zn2+ in the cytotoxicity of H2O2. H2O2 at concentrations of 30microM or more (up to 1000microM) significantly increased intracellular Zn2+ concentration. There were two mechanisms. (1) H2O2 decreased cellular content of nonprotein thiols, possibly resulting in release of Zn2+ from thiols as cellular Zn2+ binding sites. (2) H2O2 increased membrane Zn2+ permeability because external ZnCl2 application further elevated intracellular Zn2+ concentration. Micromolar H2O2 may induce excessive elevation of intracellular Zn2+ concentration that is harmful to cellular functions. However, the incubation with micromolar ZnCl2 alone increased cellular content of nonprotein thiols, one of the factors protecting cells against oxidative stress. Though zinc is generally considered to be protective with its antioxidant property, this study reveals the toxic effect of zinc even in micromolar range under oxidative stress induced by H2O2.

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.

Zn2+, derived from cell preparation, partly attenuates Ca2+-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 Tyrodes 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.

Putative role of intracellular Zn2+ release during oxidative stress: a trigger to restore cellular thiol content that is decreased by oxidative stress

Although the ability of zinc to retard the oxidative process has been recognized for many years, zinc itself has been reported to induce oxidative stress. In order to give some insights into elucidating the role of intracellular Zn2+ in cells suffering from oxidative stress, the effects of N-ethylmaleimide (NEM) and ZnCl2 on cellular thiol content and intracellular Zn2+ concentration were studied by use of 5-chloromethylfluorescein diacetate (5-CMF-DA) and FluoZin-3 pentaacetoxymethyl ester (FluoZin-3-AM) in rat thymocytes. The treatment of cells with NEM attenuated 5-CMF fluorescence and augmented FluoZin-3 fluorescence in a dose-dependent manner. These NEM-induced phenomena were observed under external Zn2+-free conditions. Results suggest that NEM decreases cellular thiol content and induces intracellular Zn2+ release. Micromolar ZnCl2 dose-dependently augmented both FluoZin-3 and 5-CMF fluorescences, suggesting that the elevation of intracellular Zn2+ concentration increases cellular thiol content. Taken together, it is hypothesized that intracellular Zn2+ release during oxidative stress is a trigger to restore cellular thiol content that is decreased by oxidative stress.

Tri-n-butyltin increases intracellular Zn2+ 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.

Nanomolar concentrations of zinc pyrithione increase cell susceptibility to oxidative stress induced by hydrogen peroxide in rat thymocytes.

Zinc pyrithione is used as an antifouling agent. However, the environmental impacts of zinc pyrithione have recently been of concern. Zinc induces diverse actions during oxidative stress; therefore, we examined the effect of zinc pyrithione on rat thymocytes suffering from oxidative stress using appropriate fluorescent probes. The cytotoxicity of zinc pyrithione was not observed when the cells were incubated with 3 μM zinc pyrithione for 3 h. However, zinc pyrithione at nanomolar concentrations (10 nM or more) significantly increased the lethality of cells suffering from oxidative stress induced by 3 mM H(2)O(2). The application of zinc pyrithione alone at nanomolar concentrations increased intracellular Zn(2+) level and the cellular content of superoxide anions, and decreased the cellular content of nonprotein thiols. The simultaneous application of nanomolar zinc pyrithione and micromolar H(2)O(2) synergistically increased the intracellular Zn(2+) level. Therefore, zinc pyrithione at nanomolar concentrations may exert severe cytotoxic action on cells simultaneously exposed to chemicals that induce oxidative stress. If so, zinc pyrithione leaked from antifouling materials into surrounding environments would be a risk factor for aquatic ecosystems. Alternatively, zinc pyrithione under conditions of oxidative stress may become more potent antifouling ingredient.

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.

Dithiocarbamate fungicides increase intracellular Zn2+ levels by increasing influx of Zn2+ in rat thymic lymphocytes

Dithiocarbamate fungicides are used as alternative antifouling agents to highly toxic organotin antifouling agents, such as tri-n-butyltin and triphenyltin. There are some concerns regarding their environmental and health risks. It has been shown that tri-n-butyltin increases intracellular Zn(2+) levels of mammalian lymphocytes. Therefore, we examined the effects of dithiocarbamate fungicides (Ziram, Thiram, and Zineb) on rat thymic lymphocytes using a flow-cytometric technique to elucidate how these fungicides affect intracellular Zn(2+) levels. We further determined whether the agents increase intracellular Zn(2+) and/or Ca(2+), because both Zn(2+) and Ca(2+) are intracellular signals in lymphocytes, and excessive increases in their intracellular concentrations can have adverse effects. Dithiocarbamate fungicides increased intracellular Zn(2+) levels, without affecting intracellular Ca(2+) levels. Ziram was the most potent compound, increasing intracellular Zn(2+) levels via Zn(2+) influx. Ziram (1μM) greatly decreased the cellular nonprotein thiol content, and Zn(2+) chelators attenuated the Ziram-induced decrease. Ziram increased the population of annexin V-positive cells in a Zn(2+)-dependent manner. Therefore, we propose that dithiocarbamate fungicides induce Zn(2+) influx, resulting in an excessive elevation of intracellular Zn(2+) levels, leading to the induction of apoptosis. This study gives a basic insight into the mechanisms of dithiocarbamate fungicide-induced adverse events.

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.