Keisuke 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+).

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.

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.

Clioquinol, a lipophilic Zn2+ chelator, augments and attenuates the cytotoxicity of H2O2: a bell-shaped response curve of the effects of the drug

Clioquinol, a lipophilic Zn2+ chelator, has emerged as a potential novel therapeutic agent for several diseases such as cancer and Alzheimers disease. Clioquinol has different effects on the intracellular Zn2+ concentrations in rat thymocytes, depending on its concentration and extracellular Zn2+ levels. In this study, we examined the effect of clioquinol on cells under oxidative stress induced by hydrogen peroxide (H2O2) by using a conventional flow cytometric technique with appropriate fluorescent probes. We observed a bell-shaped relationship between the clioquinol concentration and changes in H2O2 cytotoxicity; H2O2-induced cytotoxicity was the highest at a clioquinol concentration of 100 nM. Zn2+ chelators significantly decreased the clioquinol-induced increase in H2O2 cytotoxicity. A bell-shaped curve was observed between the increase in H2O2-induced cytotoxicity by clioquinol and the intracellular Zn2+ concentrations, although the maximal increases in Zn2+ levels were induced by 300 nM clioquinol. In addition, clioquinol at concentrations ≥100 nM exerted antioxidant activity by decreasing the cellular oxidant levels. Thus, clioquinol can exert pro-oxidant or antioxidant effects, depending on its concentration and the extracellular concentration of Zn2+. Because of the unique Zn2+-dependent effects and toxicological profile of clioquinol, clioquinol should be considered for clinical use.

Zinc-dependent and independent actions of hydroxyhydroquinone on rat thymic lymphocytes

Abstract Coffee contains hydroxyhydroquinone (HHQ). HHQ is one of the by-products released during bean roasting. Therefore, it is important to elucidate the bioactivity of HHQ to predict its beneficial or adverse effects on humans. We studied zinc-dependent and independent actions of commercially procured synthetic HHQ in rat thymocytes using flow cytometric techniques with propidium iodide, FluoZin-3-AM, 5-chloromethylfluorescein diacetate, and annexin V-FITC. HHQ at 1050 µM elevated intracellular Zn2+ levels by releasing intracellular Zn2+. HHQ at 10 µM increased cellular thiol content in a zinc-dependent manner. However, HHQ at 30–50 µM reduced cellular thiol content. Although the latter actions of HHQ (30–50 µM) were suggested to increase cell vulnerability to oxidative stress, HHQ at 0.3–100 µM significantly protected cells against oxidative stress induced by H2O2. The process of cell death induced by H2O2 was delayed by HHQ, although both H2O2 and HHQ increased the population of annexin V-positive living cells. However, HHQ at 10–30 µM promoted cell death induced by A23187, a calcium ionophore. HHQ at 10–30 µM exerted contrasting effects on cell death caused by oxidative stress and Ca2+ overload. Because HHQ is considered to possess diverse cellular actions, coffee with reduced amount of HHQ may be preferable to avoid potential adverse effects.

N-(3-oxododecanoyl)- l -homoserine-lactone, a quorum sensing molecule, affects cellular content of nonprotein thiol content in rat lymphocytes: Its relation with intracellular Zn 2+

Cellular actions of N-(3-oxododecanoyl)-l-homoserine-lactone (ODHL), a quorum sensing molecule of bacteria, were studied on rat thymocytes using a flow cytometer with appropriate fluorescent dyes to elucidate the effects of ODHL on host cells. A bell-shaped concentration-response relation was observed in the ODHL-induced changes in cellular glutathione content ([GSH]i). ODHL concentration-dependently increased intracellular Zn2+ levels ([Zn2+]i) and cellular O2- content ([O2-]i). The bell-shaped relation induced by ODHL can be explained as follows: a low concentration of ODHL is expected to induce moderate oxidative stress that intracellularly releases Zn2+ by converting thiols to disulfides. A slight elevation of [Zn2+]i may increase the [GSH]i. On the other hand, it is likely that a high concentration of ODHL causes severe oxidative stress that further causes both the decrease in [GSH]i and the increase in [Zn2+]i. Excessive increase in [Zn2+]i may augment oxidative stress that further decreases the [GSH]i. Other notable actions induced by ODHL included the elevation of [Zn2+]i by Zn2+ influx and the increase in [GSH]i under Zn2+-free conditions. Therefore, it is suggested that ODHL elicits diverse actions on host cells.

Captan-induced increase in the concentrations of intracellular Ca2+ and Zn2+ and its correlation with oxidative stress in rat thymic lymphocytes

Captan, a phthalimide fungicide, is considered to be relatively nontoxic to mammals. There is a possibility that captan affects membrane and cellular parameters of mammalian cells, resulting in adverse effects, because of high residue levels. To test the possibility, we examined the effects of captan on rat thymic lymphocytes using flow-cytometry with appropriate fluorescent probes. Treatment with 10 and 30 μM captan induced apoptotic and necrotic cell death. Before cell death occurred, captan elevated the intracellular concentrations of Ca2+ and Zn2+ and decreased the concentration of cellular thiol compounds. These captan-induced phenomena are shown to cause cell death and are similar to those caused by oxidative stress. Captan also elevated the cytotoxicity of hydrogen peroxide. Results indicate that 10 and 30 μM captan cause cytotoxic effects on mammalian cells. Despite no report on the significant environmental toxicity hazard of captan in humans, it may exhibit adverse effects, described above, on wild organisms.

Ziram, a dithiocarbamate fungicide, exhibits pseudo-cytoprotective actions against oxidative stress in rat thymocytes: Possible environmental risks

Abstract Ziram, a dithiocarbamate fungicide, protects various vegetables and fruits against infections by fungus. Recently, there have been increasing anxieties about the risks in the use of dithiocarbamate fungicides. Our previous studies showed that Zn2+ was a determinant of Ziram cytotoxicity. In addition, Zn2+ is linked to H2O2 cytotoxicity. Therefore, in this study, we aimed to test the hypothesis that Ziram could augment the cytotoxicity of H2O2 by examining the changes induced by Ziram in some cellular parameters in rat thymic lymphocytes subjected to H2O2‐induced oxidative stress using flow‐cytometric methods with fluorescent dyes. Ziram significantly attenuated H2O2‐induced cell death at sublethal concentrations. However, in the cells under oxidative stress elicited by H2O2, Ziram promoted the changing over from intact cells to living cells with exposed phosphatidylserine (PS) on plasma membranes, whereas it inhibited the transition from PS‐exposed living cells to dead cells. Ziram significantly augmented H2O2 actions, including reduction of cellular glutathione levels and elevation of intracellular Zn2+ concentrations. Conversely, it attenuated H2O2‐induced depolarization of mitochondrial membrane potential. Ziram at sublethal concentrations seems to exhibit promotive and suppressive actions on the process of cell death caused by H2O2. Ziram increased the number of living cells with exposed PS, a phenomenon characteristic of early stages of apoptosis. Thus, it is concluded that Ziram exhibits pseudo‐cytoprotective actions against H2O2‐induced oxidative stress. Ziram at sublethal concentrations exerts promotive and suppressive actions on the process of cell death caused by oxidative stress. Graphical abstract Figure. No Caption available. HighlightsZiram exerted reciprocal actions on H2O2‐induced cell death.Ziram augmented H2O2‐induced decrease in cellular glutathione levels.Ziram enhanced H2O2‐induced increase in intracellular Zn2+ levels.Ziram attenuated H2O2‐induced depolarization of mitochondrial membrane potential.Ziram exhibited pseudo‐cytoprotective actions against oxidative stress induced by H2O2.

Hyperpolarization by N-(3-oxododecanoyl)- l -homoserine-lactone, a quorum sensing molecule, in rat thymic lymphocytes

To study the adverse effects of N-(3-oxododecanoyl)-l-homoserine-lactone (ODHL), a quorum sensing molecule, on mammalian host cells, its effect on membrane potential was examined in rat thymic lymphocytes using flow cytometric techniques with a voltage-sensitive fluorescent probe. As 3-300 μM ODHL elicited hyperpolarization, it is likely that it increases membrane K+ permeability because hyperpolarization is directly linked to changing K+ gradient across membranes, but not Na+ and Cl- gradients. ODHL did not increase intracellular Ca2+ concentration. ODHL also produced a response in the presence of an intracellular Zn2+ chelator. Thus, it is unlikely that intracellular Ca2+ and Zn2+ are attributed to the response. Quinine, a non-specific K+ channel blocker, greatly reduced hyperpolarization. However, because charybdotoxin, tetraethylammonium chloride, 4-aminopyridine, and glibenclamide did not affect it, it is pharmacologically hypothesized that Ca2+-activated K+ channels, voltage-gated K+ channels, and ATP-sensitive K+ channels are not involved in ODHL-induced hyperpolarization. Although the K+ channels responsible for ODHL-induced hyperpolarization have not been identified, it is suggested that ODHL can elicit hyperpolarization in mammalian host cells, disturbing cellular functions.