Masaya Satoh

Flow cytometric analysis on tri-n-butyltin-induced increase in annexin V binding to membranes of rat thymocytes

Effects of tri-n-butyltin chloride (TBT) on rat thymocytes were examined by using a flow cytometer and three fluorescent dyes (annexin V-FITC, ethidium bromide and fluo-3-AM) to further characterize its cytotoxic action. TBT at concentrations of 100 nM or greater, time- and dose-dependently increased the population of annexin V-positive live cells in the cell suspension. Most of cells became to be annexin V-positive within 60 min after the start of application of 300 nM TBT. Some of annexin V-positive live cells were further stained with ethidium, indicating that some of the cells were killed, in continued presence of TBT at 300 nM or greater. When the cells were exposed to 300 nM TBT only for 15 min, the population of annexin V-positive live cells increased after removal of TBT from incubation medium. TBT-induced increase in the population of annexin V-positive live cells was partly attenuated under Ca(2+)-free condition, although that was not the case for the dead cells. TBT at 30 nM or greater increased [Ca(2+)]i in a dose-dependent manner. Triethyltin and trimethyltin even at 1 μM did not increase the [Ca(2+)]i and the population of annexin V-positive live cells. The population of annexin V-positive live cells increased as the [Ca(2+)]i was increased by ionomycin, a calcium ionophore. Results suggest an involvement of Ca(2+) in some of TBT-induced cytotoxicity.

Organization of heterogeneous mitochondrial DNA molecules in mitochondrial nuclei of cultured tobacco cells

SummaryThe molecular size of mitochondrial DNA (mtDNA) molecules and the number of copies of mtDNA per mitochondrion were evaluated from cultured cells of the tobacco BY-2 line derived fromNicotiana tabacum L. cv. Bright Yellow-2. To determine the DNA content per mitochondrion, protoplasts of cultured cells were stained with 4′,6-diamidino-2-phenylindole (DAPI), and the intensity of the fluorescence emitted from the mitochondrial nuclei (mt-nuclei) was measured with a video-intensified photon counting microscope system (VIM system). Each mitochondrion except for those undergoing a division contained one mt-nucleus. The most frequently measured size of the DNA in the mitochondria was between 120 and 200 kilobase pairs (kbp) throughout the course of culture of the tobacco cells. Mitochondria containing more than 200 kbp of DNA increased significantly in number 24 h after transfer of the cells into fresh medium but their number fell as the culture continued. Because division of mitochondria began soon after transfer of the cells into fresh medium and continued for 3 days, the change of the DNA content per mitochondrion during the culture must correspond to DNA synthesis of mitochondria in the course of mitochondrial division. By contrast, the analyses of products of digestion by restriction endonucleases indicated that the genome size of the mtDNA was at least 270 kbp. Electron microscopy revealed that mtDNAs were circular molecules and their length ranged from 1 to 35 μm, and 60% of them ranged from 7 to 11 μrn. These results indicate that the mitochondrial genome in tobacco cells consists of multiple species of mtDNA molecules, and mitochondria do not contain all the mtDNA species. Therefore, mitochondria are heterogeneous in mtDNA composition.

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.

Triclosan, an antibacterial agent, increases intracellular Zn2+ 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.

Tri-n-butyltin-induced change in cellular level of glutathione in rat thymocytes: a flow cytometric study.

Since some of organotins, accumulated in edible mollusks of aquatic environments, exert a variety of toxic actions on experimental animals, it causes concern for the health of humans. We examined the effects of tri-n-butyltin chloride (TBT) and other organotins (triethyltin chloride, trimethyltin chloride, triphenyltin chloride and tetrabutyltin) on cellular content of glutathione (GSH) in rat thymocytes using a flow cytometer to further characterize the toxicity of TBT. When the cells were incubated with TBT at concentrations of 3 nM or more for 15 min, the cellular content of GSH dose-dependently decreased. However, it completely or partly recovered until 180 min even in the continued presence of TBT. This recovery was temperature-sensitive, suggesting an involvement of metabolic process. The efficacy of TBT to decrease the cellular content of GSH was greater than those of other organotins. Results suggest that TBT and some organotins at environmentally relevant (nanomolar) concentrations significantly reduce the cellular content of GSH, suggesting that they increase the vulnerability to some biological and chemical insults.

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 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.

DETERMINATION OF CELLULAR LEVELS OF NONPROTEIN THIOLS IN PHYTOPLANKTON AND THEIR CORRELATIONS WITH SUSCEPTIBILITY TO MERCURY1

We determined the intracellular contents and concentrations of cysteine and glutathione in five species of marine phytoplankton, Tetraselmis tetrathele (West) Butcher (Prasinophyceae), Porphyridium purpureum (Bory) Drew et Ross (Rhodophyceae), Pavlova sp. (Haptophyceae), Isochrysis sp. (Haptophyceae), and Pleurochrysis carterae (Braarud et Fagerl) Christensen (Haptophyceae), and examined relationships to mercury susceptibility. Intracellular contents (concentrations) of nonprotein thiols in the five species ranged from 119 to 1210 amol (0.66–12.0 mM) for cysteine, 78 to 719 amol (0.65–2.52 mM) for cystine, 31 to 677 amol (0.13–1.25 mM) for reduced glutathione (GSH), and 12 to 123 amol (0.15–0.26 mM) for oxidized glutathione (GSSG). The intracellular contents of the nonprotein thiols were not proportional to the intracellular concentrations because the cell sizes differed. Oxidation ratios of cysteine:cystine and GSH:GSSG were also wide ranging in the five species, and the higher the concentration of the reduced form of nonprotein thiols, the less they tended to be oxidized. Flow cytometric analyses with fluorescein diacetate were used to monitor the effect of HgCl2 on esterase, and the 50% effect concentrations (EC50) were compared in the five species. The EC50 after 3 h exposure to HgCl2 correlated well with the GSH concentrations but not with those of cysteine. These results indicate that the intracellular concentrations of the nonprotein thiols reflect antioxidant activity and susceptibility to heavy metals.

Toxicity of methylmercury conjugated with L-cysteine on rat thymocytes and human leukemia K562 cells in comparison with that of methylmercury chloride.

In order to reveal the implication of use of methylmercury chloride (MeHgCl) in in vitro study, the effects of 10 µM MeHgCl on rat thymocytes and human leukemia K562 cells were compared with those of methylmercury conjugated with L-cysteine (10 µM MeHg-Cys) using a flow cytometer and fluorescent probes to monitor cellular physiological and pathological parameters. MeHgCl hyperpolarized membranes of thymocytes, followed by depolarization within a few minutes after the application, while MeHg-Cys persistently hyperpolarized them. MeHgCl increased intracellular concentration of Ca(2+), decreased cellular content of glutathione and increased generation of superoxide anion in the cells. The effects of MeHg-Cys were much less than those of MeHgCl. MeHgCl greatly increased both numbers of the cells undergoing apoptosis and dead cells in cell suspension containing thymocytes, while this was not the case for MeHg-Cys. MeHgCl reduced the cell viability of human leukemia K562 cells and completely inhibited the cell growth. The effects of MeHg-Cys on K562 cells were less than those of MeHgCl. It can be concluded that the effects of MeHgCl on rat thymocytes and K562 cells are different from those of MeHg-Cys. The results obtained from the in vitro studies using MeHgCl may be less implicit to elucidate the mechanism of MeHg intoxication in humans and experimental animals because MeHg are present in forms of MeHg-Cys and/or MeHg-S conjugate under the in vivo conditions.

Increase in number of annexin V-positive living cells of rat thymocytes by intracellular Pb2+

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.