Triclocarban-induced change in intracellular Ca²âº level in rat thymocytes: cytometric analysis with Fluo-3 under Zn²âº-free conditions.

Triclocarban (TCC) is an antimicrobial used in personal hygiene products. Recent health concerns arose after TCC was detected in the blood of human subjects who showered with soap containing TCC. In this study, the effect of TCC on intracellular Ca(2+) concentration in rat thymocytes was examined using Fluo-3, an indicator of intracellular Ca(2+). TCC at concentrations ranging from 0.1 µM to 3 µM increased intracellular Ca(2+) concentration biphasically: first by releasing Ca(2+) from intracellular Ca(2+) stores and then inducing Ca(2+) influx through store-operated Ca(2+) channels. The threshold TCC concentration to increase intracellular Ca(2+) concentration in this study was lower than the maximum TCC concentrations reported in human blood samples. Therefore, we anticipate that TCC at concentrations reported in human blood samples might disturb intracellular Ca(2+) signaling in human lymphocytes.

Synergistic increase in cell lethality by dieldrin and H2O2 in rat thymocytes: effect of dieldrin on the cells exposed to oxidative stress.

Dieldrin, one of persistent pesticides, is highly resistant to biotic and abiotic degradation. It is accumulated in organisms. Recent studies suggest that dieldrin exerts a potent cytotoxic action on cells exposed to oxidative stress. In this study, the effect of dieldrin on rat thymocytes exposed to hydrogen peroxide (H2O2)-induced oxidative stress was examined. Dieldrin at 5µM and H2O2 at 300µM slightly increased cell lethality from a control value of 5.4±0.5% (mean±standard deviation of four experiments) to 7.8±1.3% and 9.0±0.3%, respectively. Simultaneous application of dieldrin and H2O2 significantly increased cell lethality to 46.2±1.8%. The synergistic increase in cell lethality was dependent on dieldrin concentration (0.3-5µM) but not on H2O2 concentration (30-300µM). Dieldrin accelerated H2O2-induced cell death, which was estimated with the help of annexin V-FITC and propidium iodide. Presence of either dieldrin or H2O2 decreased the cellular content of nonprotein thiol and increased intracellular Zn(2+) concentration. The combination of dieldrin and H2O2 further pronounced these effects. TPEN, a chelator of intracellular Zn(2+), significantly attenuated the synergistic increase in cell lethality induced by dieldrin and H2O2. It is, therefore, suggested that dieldrin augments the cytotoxicity of H2O2 in a Zn(2+)-dependent manner.

Nanomolar concentration of triclocarban increases the vulnerability of rat thymocytes to oxidative stress.

It was recently reported that triclocarban was absorbed significantly from soap used during showering in human subjects and that its C(max) in their whole blood ranged from 23 nM to 530 nM. We revealed that a nanomolar concentration (300 nM) of triclocarban potentiated the cytotoxicity of 300 µM H(2)O(2) in rat thymocytes by using cytometric techniques with appropriate fluorescent probes. Although 300 nM triclocarban did not itself increase the population of dead cells (cell lethality), it facilitated the process of cell death induced by H(2)O(2), resulting in a further increase in the population of dead cells. Nanomolar concentrations (300 nM or higher) of triclocarban significantly decreased the cellular content of nonprotein thiol (glutathione), which has a protective role against oxidative stress. Triclocarban at 300 nM or higher increased the cell vulnerability to oxidative stress. The results may suggest that nanomolar concentration (300 nM or higher) of triclocarban affects some cellular functions although there is no evidence for adverse effects of triclocarban in humans at present.

Triclosan, an antibacterial agent, increases intracellular Zn(2+) concentration in rat thymocytes: its relation to oxidative stress.

Triclosan is used as an antibacterial agent in household items and personal care products. Since this compound is found in maternal milk of humans and bodies of wild animals, there is growing concern among some consumer groups and scientific community that triclosan is adverse for humans and wild animals. In order to estimate adverse actions of triclosan, the effects of triclosan on intracellular Zn(2+) concentration and cellular thiol content were studied in rat thymocytes by the use of flow cytometer with appropriate fluorescent probes. Triclosan at 1-3 µM (sublethal concentrations) increased the intensity of FluoZin-3 fluorescence (intracellular Zn(2+) concentration) and decreased the intensity of 5-chloromethylfluorescein (5-CMF) fluorescence (cellular thiol content). Negative correlation (r=-0.985) between triclosan-induced changes in FluoZin-3 and 5-CMF fluorescences was found. Removal of external Zn(2+) did not significantly affect the triclosan-induced augmentation of FluoZin-3 fluorescence, suggesting an intracellular Zn(2+) release by triclosan. These actions of triclosan were similar to those of H(2)O(2) and triclosan significantly potentiated the cytotoxicity of H(2)O(2). Therefore, the results may suggest that triclosan at sublethal concentrations induces oxidative stress that decreases cellular thiol content, resulting in an increase in intracellular Zn(2+) concentration by Zn(2+) release from intracellular store(s). Since recent studies show many physiological roles of intracellular Zn(2+) in cellular functions, the triclosan-induced disturbance of cellular Zn(2+) homeostasis may induce adverse actions on the cells.

Putative role of intracellular Zn(2+) 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 Zn(2+) in cells suffering from oxidative stress, the effects of N-ethylmaleimide (NEM) and ZnCl(2) on cellular thiol content and intracellular Zn(2+) 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 Zn(2+)-free conditions. Results suggest that NEM decreases cellular thiol content and induces intracellular Zn(2+) release. Micromolar ZnCl(2) dose-dependently augmented both FluoZin-3 and 5-CMF fluorescences, suggesting that the elevation of intracellular Zn(2+) concentration increases cellular thiol content. Taken together, it is hypothesized that intracellular Zn(2+) release during oxidative stress is a trigger to restore cellular thiol content that is decreased by oxidative stress.

Some characteristics of membrane Cd²+ transport in rat thymocytes: an analysis using Fluo-3.

Although cadmium-induced apoptosis of lymphocytes is one of common features in the immunotoxicity of cadmium, the membrane pathway for intracellular cadmium accumulation is not fully elucidated. To characterize membrane Cd(2+) transport of rat thymocytes, the change in intracellular Cd(2+) concentration under various conditions was examined by the use of Fluo-3, a fluorescent probe for monitoring the change in intracellular concentration of divalent metal cations. The membrane Cd(2+) transport was estimated by the augmentation of Fluo-3 fluorescence induced by bath application of CdCl(2). Lowering temperature strongly suppressed the augmentation of Fluo-3 fluorescence by CdCl(2), suggesting that the metabolic process can be involved in membrane Cd(2+) transport. External acidification (decreasing pH) and membrane depolarization by adding KCl attenuated the augmentation, indicating the requirement of electrochemical driving force for membrane Cd(2+) transport into the cells. Bath application of CaCl(2) and ZnCl(2) equally decreased the augmentation, suggesting their competition with Cd(2+) at the membrane transport. The augmentation by CdCl(2) was lesser in the cells treated with N-ethylmaleimide inducing chemical depletion of cellular thiols. The result suggests the contribution of sulfhydryl groups to membrane Cd(2+) transport. Taken together, it is suggested that the cells possess a temperature-sensitive membrane Cd(2+) pathway, driven by electrochemical gradient of Cd(2+) and transmembrane potential, with competitive binding site. Based on the characteristics described above, it is unlikely that the membrane Cd(2+) transport in rat thymocytes is attributed to a single transport system although it has characteristics that are similar to those of divalent cation transporter 1.

Methylmercury elicits intracellular Zn2+ release in rat thymocytes: its relation to methylmercury-induced decrease in cellular thiol content.

We have previously revealed that thimerosal, an organomercurial preservative, increases intracellular Zn(2+) concentration in rat thymocytes. Because thimerosal contains ethylmercury that confers the toxicity, it is a possibility that methylmercury (MetHg), an environmental pollutant, also increases intracellular Zn(2+) concentration. This possibility was tested by measuring intracellular Zn(2+) level with FluoZin-3, a fluorescent probe for intracellular Zn(2+). MetHg at concentrations ranging from 100 nM to 1 microM significantly increased the intensity of FluoZin-3 fluorescence, an indicator for intracellular Zn(2+) concentration, under external Ca(2+)- and Zn(2+)-free condition in a concentration-dependent manner. TPEN, a chelator for intracellular Zn(2+), completely diminished the MetHg-induced augmentation of FluoZin-3 fluorescence. MetHg at 100 nM or more significantly decreased the intensity of 5-chlormethylfluorescein fluorescence, an indicator for cellular thiol content. Such MetHg-induced changes in the fluorescence were correlated with a coefficient of -0.917. Taken together, it is suggested that submicromolar MetHg releases Zn(2+) from intracellular thiol, resulting in the increase in intracellular Zn(2+) concentration. However, it is unlikely that MetHg at critical maternal blood concentration (27 nM) affects intracellular Zn(2+) homeostasis.

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 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.

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.

Increase in number of annexin V-positive living cells of rat thymocytes by intracellular Pb(2+).

Lead is ubiquitous in our environment and lead poisoning is a major public health problem worldwide. In this study, to see if intracellular Pb(2+) induces the exposure of phosphatidylserine in rat thymocyte membranes, we have examined the effect of PbCl(2) on rat thymocytes treated with A23187 using a flow cytometer with appropriate fluorescent indicators under nominally-Ca(2+)-free condition. PbCl(2) at 1-30 µM dose-dependently induced the exposure of phosphatidylserine on outer membranes, associated with increasing the concentration of intracellular Pb(2+). The potency of intracellular Pb(2+) to induce the apoptotic change in thymocyte membranes seems to be greater than those of intracellular Ca(2+) and Cd(2+). Results suggest that intracellular Pb(2+) triggers apoptosis of rat thymocytes. This action of Pb(2+) may be one of mechanisms for the lead-induced changes in immunity.

Cytotoxic effects of triphenylbismuth on rat thymocytes: comparisons with bismuth chloride and triphenyltin chloride.

The biomedical and industrial uses of organobismuth compounds have become widespread, although there is limited information concerning their cytotoxicity. Therefore, the actions of triphenylbismuth on rat thymocytes were examined using a flow cytometer with ethidium bromide, annexin V-FITC, fluo-3-AM, and 5-chloromethylfluorescein (5CMF) diacetate. Triphenylbismuth at 3-30 microM increased the population of cells stained with ethidium, indicating a decrease in cell viability. Organobismuth at 30 microM increased the population of cells positive to annexin V, suggesting an increase in the population of apoptotic cells. Triphenylbismuth at 3 microM or more decreased cellular glutathione content (5CMF fluorescence intensity) and increased intracellular Ca(2+) concentration ([Ca(2+)](i), fluo-3 fluorescence intensity) in a dose-dependent manner. Because an increase in [Ca(2+)](i) is linked to cell death or cell injury and a decrease in cellular glutathione content increases cell vulnerability to oxidative stress, the triphenylbismuth-induced changes in cellular parameters may be responsible for triphenylbismuth-induced cytotoxicity. Bismuth chloride at 10-30 microM did not significantly affect cell viability. These results suggest that triphenylbismuth at micromolar concentrations exerts cytotoxic action on rat thymocytes, possibly related to a health hazard. Although the cytotoxicity of triphenylbismuth was less than that of triphenyltin, one of the environmental pollutants, it is necessary to direct our attention to the use and disposal of organobismuth compounds.