Eri Fukunaga

Elevation of the intracellular Zn2+ level by 2-n-octyl-4-isothiazolin-3-one in rat thymocytes: an involvement of a temperature-sensitive Zn2+ pathway

High amounts of 2-n-octyl-4-isothiazolin-3-one (OIT), an antimicrobial, are found in wet polyvinyl alcohol towels with cooling properties. Although the diverse actions of OIT are of concern, information on its cellular actions is limited. In this study, we examined the effects of OIT on Zn2+ levels in rat thymocytes by flow cytometric analysis with FluoZin-3, and assessed the cytotoxicity of this agent. OIT (1–3 μM) significantly increased the intensity of the FluoZin-3 fluorescence. Removal of extracellular Zn2+ almost completely inhibited the OIT-induced increase in FluoZin-3 fluorescence. Furthermore, the increase in OIT-induced FluoZin-3 fluorescence was attenuated at cold temperatures. The effect of OIT on the FluoZin-3 fluorescence was dependent on the transmembrane Zn2+ gradient. Based on our data, we concluded that OIT activates a temperature-sensitive, bi-directional Zn2+ pathway, resulting in alterations to the intracellular Zn2+ levels. Importantly, these changes are dependent on a transmembrane Zn2+ gradient. These results may provide insight into the cytotoxicity of OIT, because Zn2+ has physiological and pathological roles in cellular functions.

Zn2+-dependence of the synergistic increase in rat thymocyte cell lethality caused by simultaneous application of 4,5-dichloro-2-octyl-4-isothiazolin-3-one (DCOIT) and H2O2

4,5-Dichloro-2-octyl-4-isothiazolin-3-one (DCOIT) is an antifouling agent that is an alternative to organotins such as tributyltin (TBT). Because DCOIT decreases catalase activity, it may increase the susceptibility of cells to oxidative stress. We examined the effects of DCOIT on rat thymocytes suffering from oxidative stress induced by H2O2. The simultaneous application of DCOIT and H2O2 induced a synergistic increase in cell lethality that was completely suppressed by chelating intracellular Zn(2+). Intracellular Zn(2+) concentration was increased by DCOIT at concentrations ranging from 0.1 μM to 3 μM. Although the increase in cell lethality produced by DCOIT alone was less than that produced by TBT alone, a synergistic increase was not induced by the combination of TBT and H2O2. Therefore, these results suggest that DCOIT increases vulnerability to oxidative stress and is more cytotoxic than TBT when oxidative stress is induced by H2O2.

Changes in cellular thiol content and intracellular Zn2+ level by 1,4-naphthoquinone in rat thymocytes

1,4-Naphthoquinone is an active metabolite of naphthalene and it is also found in diesel exhaust particles. It is known to cause oxidative stress. In this study, we characterized 1,4-naphthoquinone-mediated cytotoxicity and its effects on the levels of non-protein thiols and intracellular Zn(2+) in rat thymocytes (thymic lymphocytes) by using 5-chloromethylfluorescein (5-CMF) fluorescence and FluoZin-3 fluorescence, respectively. Low concentrations of 1,4-naphthoquinone (0.3μM) increased the intensity of 5-CMF fluorescence, which is used to measure non-protein thiols. In contrast, 5-CMF intensity decreased at higher concentrations (1-3μM) of 1,4-naphthoquinone. Removal of intracellular Zn(2+) attenuated the 1,4-naphthoquinone-induced augmentation of 5-CMF fluorescence. Additionally, 1,4-naphthoquinone (0.3-3μM) increased FluoZin-3 fluorescence, which is used to assess intracellular Zn(2+), in a concentration-dependent manner. The augmentation of FluoZin-3 fluorescence by 1,4-naphthoquinone was due to the release of intracellular Zn(2+), because the removal of extracellular Zn(2+) did not affect the augmentation of FluoZin-3 fluorescence. These results suggest that sublethal concentrations of 1,4-naphthoquinone (0.3-1μM) affect the cellular levels of non-protein thiols and intracellular Zn(2+). The difference in the observed decrease in cellular thiol content due to 1,4-naphthoquinone treatment and increase due to Zn(2+) release following 1,4-naphthoquinone treatment likely confers the change in cellular thiol content. Further, the increase in intracellular Zn(2+) concentration after 1,4-naphthoquinone exposure may change the activity of thymocytes because thymulin, a thymus-specific hormone, requires Zn(2+) for its biological activity.

Increase in intracellular Ca2+ level by phenylsulfamide fungicides, tolylfluanid and dichlofluanid, in rat thymic lymphocytes

Tolylfluanid, a phenylsulfamide fungicide, is one of the many pesticides that are frequently detected in crops. Therefore, its health risk is a concern. Micromolar concentrations of tolylfluanid induce chromosomal aberrations and micronuclei in mammalian lymphocytes. The findings prompted us to study the cellular actions of tolylfluanid and another frequently detected pesticide, dichlofluanid, at submicromolar and micromolar concentrations. Of the cellular actions of chemicals, the action on cellular Ca(2+) homeostasis is important since Ca(2+) is involved in cell signaling and death. Consequently, in this study, the effects of phenylsulfamide fungicides were examined on rat thymocytes by using fluorescent probes in order to further characterize the cellular actions of phenylsulfamide fungicides. Both phenylsulfamide fungicides exhibited biphasic, early and late, increase in intracellular Ca(2+) levels. The early phase was dependent on intracellular Ca(2+) release and increased membrane Ca(2+) permeability. The late phase was owing to Ca(2+) influx via activation of store-operated Ca(2+) channels and the further increase of membrane ionic permeability. Voltage-dependent Ca(2+) channels were not involved. The increases in intracellular Ca(2+) levels by phenylsulfamide fungicides were observed at drug concentrations of 0.1 μM or more (up to 10 μM). Thus, it is plausible that micromolar concentrations of phenylsulfamide fungicides deregulate intracellular Ca(2+) homeostasis in rat thymocytes. Both phenylsulfamide fungicides at 10 μM promoted the transition from intact living cells to living cells with phosphatidylserine-exposed membranes. This was not the case for phenylsulfamide fungicides at 3 μM. The potency of tolylfluanid was similar to that of dichlofluanid. Although the information on residual concentrations of tolylfluanid and dichlofluanid is very limited, their residual concentrations do not reach micromolar levels. It is unlikely that humans will develop adverse effects on exposure to phenylsulfamide fungicides under present environmental conditions.

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.

Bisabololoxide A, One of the Constituents in German Chamomile Extract, Attenuates Cell Death Induced by Calcium Overload

Bisabololoxide A (BSBO), main constituents in German chamomile extract, is responsible for antipruritic effect. In previous study, the incubation with 30–100 μM BSBO for 24 h exerted cytotoxic and proapoptotic effects on rat thymocytes. To further characterize BSBO cytotoxicity, the effect on the cells suffering from calcium overload by calcium ionophore A23187 was examined. A23187 induced Ca2+‐dependent cell death. Contrary to our expectation, 1–10 μM BSBO inhibited A23187‐induced increase in cell lethality of rat thymocytes. BSBO attenuated A23187‐induced increases in populations of shrunken living cells, phosphatidylserine‐exposed living cells, and dead cells, without affecting the increase in intracellular Ca2+ concentration and the Ca2+‐dependent hyperpolarization. The effect of BSBO on A23187‐treated cells may be unique because the activation of Ca2+‐dependent K+ channels is required for cell shrinkage, externalization of phosphatidylserine, and cell death in some cells. The cell death induced by A23187 was not inhibited by Z‐VAD‐FMK, a pan‐inhibitor of caspases. Thus, the cell death may be a necrosis with some features observed during an early stage of apoptosis. These results suggest that BSBO at low micromolar concentrations is cytoprotective against calcium overload. Copyright