Modification of cell vulnerability to oxidative stress by N-(3-oxododecanoyl)-L-homoserine-lactone, a quorum sensing molecule, in rat thymocytes

N-(3-oxododecanoyl)-L-homoserine-lactone (ODHL), a quorum sensing molecule, affects intracellular Zn2+ concentration ([Zn2+]i) and cellular levels of nonprotein thiols ([NPT]i) of rat thymic lymphocytes, both of which are assumed to affect cell vulnerability to oxidative stress. Therefore, it is interesting to examine the effects of ODHL on the cells under oxidative stress. ODHL augmented the cytotoxicity of H2O2, but not calcium ionophore A23187. ODHL potentiated the H2O2-induced elevation of [Zn2+]i, wherein, it greatly attenuated the H2O2-induced increase in intracellular Ca2+ concentration. ODHL did not affect [NPT]i in the presence of H2O2. Therefore, we conclude that the elevation of [Zn2+]i is involved in the ODHL-induced potentiation of H2O2 cytotoxicity. Our findings suggest that ODHL modifies cell vulnerability to oxidative stress in host cells

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

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

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

DCOIT and Neurotransmission

4,5-Dichloro-2-octyl-4-isothiazolin-3-one (DCOIT) is an alternative to organotin antifoulants, such as tributyltin and triphenyltin. Since DCOIT is found in harbors, bays, and coastal areas worldwide, this chemical compound may have some impacts on ecosystems. To determine whether DCOIT possesses neurotoxic activity by modifying synaptic transmission, we examined the effects of DCOIT on synaptic transmission in a ‘synaptic bouton’ preparation of rat brain. DCOIT at concentrations of 0.03–1 μM increased the amplitudes of evoked synaptic currents mediated by GABA and glutamate, while it reduced the amplitudes of these currents at 3–10 μM. However, the currents elicited by exogenous applications of GABA and glutamate were not affected by DCOIT. DCOIT at 1–10 μM increased the frequency of spontaneous synaptic currents mediated by GABA. It also increased the frequency of glutamate-mediated spontaneous currents at 0.3–10 μM. The frequencies of miniature synaptic currents mediated by GABA and glutamate, observed in the presence of tetrodotoxin under external Ca2+-free conditions, were increased by 10 μM DCOIT. With the repetitive applications of DCOIT, the frequency of miniature synaptic currents mediated by glutamate was not increased by the second and third applications of DCOIT. Voltage-dependent Ca2+ channels were not affected by DCOIT, but DCOIT slowed the inactivation of voltage-dependent Na+ channels. These results suggest that DCOIT increases Ca2+ release from intracellular Ca2+ stores, resulting in the facilitation of both action potential-dependent and spontaneous neurotransmission, possibly leading to neurotoxicity

Further analysis on lidocaine-induced increase in intracellular Zn2+ concentration : Cytometric model study using FluoZin-3, 5-chloromethylfluorescein, and rat thymocytes

Lidocaine augmented FluoZin-3 fluorescence of rat thymocytes, suggesting the increase in intracellular Zn2+ concentration. However, the mechanism for lidocaine-induced increase in the intensity of FluoZin-3 fluorescence was not elucidated. In this study, in order to reveal the mechanism, the effects of lidocaine on FluoZin-3 and 5-chloromethylfluorescein (5-CMF) fluorescence were examined under various conditions. 5-CMF fluorescence was used as a parameter for cellular content of nonprotein thiols. The lidocaine-induced augmentation of FluoZin-3 fluorescence was observed under external Ca2+- and/or Zn2+-free conditions, suggesting that lidocaine induced intracellular Zn2+ release. Lidocaine attenuated 5-CMF fluorescence, suggesting the decrease in cellular content of nonprotein thiols. Since some peptides consisting of SH-group have been proposed to store Zn2+, the decrease in nonprotein thiol content b

Effects of tetracaine and lidocaine on intracellular Zn2+ levels in rat neurons : Preliminary analysis with FluoZin-3 fluorescence

There are some differences in cytotoxic actions between local anesthetics. In a study performed using rat thymocytes, lidocaine increased intracellular Zn2+ concentration whereas tetracaine decreased it. However, thymocytes are not subject to local anesthetics. This study was conducted to confirm the actions of tetracaine and lidocaine on neurons. Tetracaine and lidocaine changed intracellular Zn2+ levels in rat cerebellar granule neurons, as had been observed in rat thymocytes. The changes in intracellular Zn2+ levels by these local anesthetics were statistically significant, but small. The mechanism of lidocaine-induced increase in intracellular Zn2+ levels in neurons was similar to that in rat thymocytes. The mechanism of tetracaine-induced decrease in intracellular Zn2+ levels in neurons did not seem to be consistent with that in rat thymocytes. Further studies will be needed to see if the changes in intracellular Zn2+ levels by local anesthetics correspond to differences in cytotoxic activities since their actions on intracellular Zn2+ levels of neurons were weak

Cell death process induced by hydrogen peroxide is accelerated by clioquinol in rat thymocytes

We examined the effect of clioquinol on the process of cell death induced by hydrogen peroxide (H2O2) using a flow cytometric technique with propidium iodide and annexin V-FITC in order to see if clioquinol augments the toxicity caused by oxidative stress. Clioquinol (100 nM) alone did not change the process of spontaneous cell death. However, the agent accelerated the process of cell death induced by 300 μM H2O2. Result indicates that clioquinol augments the cytotoxicity induced by H2O2. Therefore, the use of clioquinol may be inadequate for the treatment of some diseases related to oxidative stress

Membrane hyperpolarization and depolarization of rat thymocytes by azoxystrobin, a post harvest fungicide

Azoxystrobin, a broad-spectrum fungicide, has been increasingly used in the agricultural industry. In Japan in 2018, azoxystrobin at five times the normal limit was detected in a shipment of Australian barley that had been used in food products. Therefore, the effects of azoxystrobin need to be carefully examined to predict potential adverse reactions in humans. In this study, the effects of azoxystrobin on the membrane potential and intracellular Ca2+ levels of thymocytes have been photochemically examined using flow cytometry. Azoxystrobin hyperpolarized plasma membrane potential. This hyperpolarization appeared to be due to the activation of Ca2+-dependent K+ channels, as both the removal of extracellular Ca2+ and addition of charybdotoxin attenuated the observed hyperpolarization. In the presence of quinine, an anti-malarial drug that blocks Ca2+-dependent K+ channels, azoxystrobin depolarized the membranes instead. Azoxystrobin increased intracellular Ca2+ levels in a concentration-dependent manner through the influx of extracellular Ca2+ and intracellular release of Ca2+, as confirmed by reduction in azoxystrobin-induced response in the absence of extracellular Ca2+. It appears likely that azoxystrobin at micromolar concentrations modifies membrane ion permeability in thymocytes. Since changes in membrane potential and intracellular Ca2+ levels occur during typical physiological lymphocyte responses, azoxystrobin may disturb lymphocyte function

Cytotoxic actions of lidocaine at sublethal concentrations : A model in vitro experiment using rat thymocytes

In order to reveal some cytotoxic features of lidocaine, the effects of lidocaine at sublethal concentrations (10 mM or less) on rat thymocytes were examined by the use of flow-cytometric technique with fluorescent probes. Treatment with lidocaine at 3-10 mM for 1-3 hr significantly increased intracellular Ca2+ and Zn2+ concentrations under normal condition. After the removal of external Ca2+ and Zn2+, lidocaine still increased intracellular Ca2+ and Zn2+ concentrations, suggesting the lidocaine-induced release of intracellular Ca2+ and Zn2+. Treatment with lidocaine at 0.3-1 mM for 24 hr significantly increased the populations of dead cells and shrunken cells. Lidocaine at sublethal concentrations exerts some cellular actions that are supposed to be linked to the cytotoxicity. The mechanism of lidocaine-induced cell death varies in time- and concentration-dependent manners