Dithiocarbamate fungicides increase intracellular Zn2+ levels by increasing influx of Zn2+ in rat thymic lymphocytes

Dithiocarbamate fungicides increase intracellular Zn2+ levels by increasing influx of Zn2+ in rat thymic lymphocytes

Chemico-Biological Interactions 237 (2015) 80–86 Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.els...

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Chemico-Biological Interactions 237 (2015) 80–86

Contents lists available at ScienceDirect

Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint

Dithiocarbamate fungicides increase intracellular Zn2+ levels by increasing influx of Zn2+ in rat thymic lymphocytes Yumiko Kanemoto-Kataoka 1, Tomohiro M. Oyama 2, Hitoshi Ishibashi 3, Yasuo Oyama ⇑ Laboratory of Cellular Signaling, Graduate School of Integrated Arts and Sciences, The University of Tokushima, Tokushima 770-8502, Japan

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Article history: Received 18 November 2014 Received in revised form 16 May 2015 Accepted 20 May 2015 Available online 27 May 2015 Keywords: Ziram Thiram Zineb Intracellular Zn2+ Lymphocytes

a b s t r a c t Dithiocarbamate fungicides are used as alternative antifouling agents to highly toxic organotin antifouling agents, such as tri-n-butyltin and triphenyltin. There are some concerns regarding their environmental and health risks. It has been shown that tri-n-butyltin increases intracellular Zn2+ levels of mammalian lymphocytes. Therefore, we examined the effects of dithiocarbamate fungicides (Ziram, Thiram, and Zineb) on rat thymic lymphocytes using a flow-cytometric technique to elucidate how these fungicides affect intracellular Zn2+ levels. We further determined whether the agents increase intracellular Zn2+ and/or Ca2+, because both Zn2+ and Ca2+ are intracellular signals in lymphocytes, and excessive increases in their intracellular concentrations can have adverse effects. Dithiocarbamate fungicides increased intracellular Zn2+ levels, without affecting intracellular Ca2+ levels. Ziram was the most potent compound, increasing intracellular Zn2+ levels via Zn2+ influx. Ziram (1 lM) greatly decreased the cellular nonprotein thiol content, and Zn2+ chelators attenuated the Ziram-induced decrease. Ziram increased the population of annexin V-positive cells in a Zn2+-dependent manner. Therefore, we propose that dithiocarbamate fungicides induce Zn2+ influx, resulting in an excessive elevation of intracellular Zn2+ levels, leading to the induction of apoptosis. This study gives a basic insight into the mechanisms of dithiocarbamate fungicide-induced adverse events. Ó 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction In agriculture, dithiocarbamate fungicides, such as Ziram and Thiram, are used to protect various fruits and vegetables from fungal infection [1,31,44]. Furthermore, dithiocarbamate fungicides are employed as antifouling agents, because the use of highly toxic organotins, such as tri-n-butyltin and triphenyltin, was banned [12,47]. There are increasing concerns regarding the environmental and health risks of dithiocarbamate biocide use [2–4,32,44]. Several papers have evaluated the cellular toxicity of dithiocarbamate fungicides. For example, these fungicides can induce oxidative stress [5,13,19,34]. The cellular thiol content was also reduced in preparations treated with these fungicides [6,18]. The modification of thiols to disulfides releases Zn2+ from protein and nonprotein sources [33]. Thus, fungicides may cause a secondary increase in intracellular Zn2+ levels. Furthermore, these fungicides ⇑ Corresponding author. Tel.: +81 88 656 7256. E-mail address: [email protected] (Y. Oyama). Present address: Bayer Yakuhin, Ltd., Osaka 530-0001, Japan. 2 Present address: Medical Co. LTA Kyushu Clinical Pharmacology Research Clinic, Fukuoka 810-0064, Japan. 3 Present address: Kitasato University, Kanagawa 252-0373, Japan. 1

http://dx.doi.org/10.1016/j.cbi.2015.05.014 0009-2797/Ó 2015 Elsevier Ireland Ltd. All rights reserved.

could also directly induce Zn2+ influx into cells, resulting in an increase in intracellular Zn2+ levels. This excessive increase in intracellular Zn2+ could also cause oxidative stress, leading to the reduction of cellular thiol content [38,41,46]. Zn2+ is an intracellular messengers in lymphocytes [11,22,20]. Therefore, it is important to elucidate how dithiocarbamate fungicides affect intracellular Zn2+ levels. In addition, it is necessary to determine whether the fungicides enhance Zn2+, Ca2+, or both, because Zn2+ augments the fluorescence of some Ca2+ indicators, resulting in misleading observations regarding intracellular Ca2+ levels. In this study, we examined the effects of Ziram, Thiram, and Zineb on rat thymic lymphocytes using a flow-cytometric technique with appropriate fluorescent probes. The study may provide cellular data regarding the mechanism of dithiocarbamate fungicide-induced adverse effects.

2. Materials and methods 2.1. Chemicals Ziram and Thiram were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Zineb was obtained from Wako

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Pure Chemicals (Osaka, Japan). The purity of Ziram, Thiram, and Zineb was 99.9%, 100%, and 96.7%, respectively. Propidium iodide, FluoZin-3-tetra(acetoxymethyl)ester (FluoZin-3-AM), and 5-chloromethylfluorescein diacetate (5-CMF-DA) were obtained from Molecular Probes Inc., Invitrogen (Eugene, OR, USA). Fluo-3-AM and the Zn2+ chelators, diethylenetriamine-N,N,N0 ,N00 , N00 -pentaacetic acid (DTPA) and N,N,N0 ,N0 -tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), were obtained from Dojin Chemical Laboratory (Kumamoto, Japan). A23187, an ionophore for divalent metal cations, was purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). Other chemicals were obtained from Wako Pure Chemicals unless mentioned. 2.2. Animals and cell preparation This study was approved by the Committee for Animal Experiments at the University of Tokushima (No. 05279). The cell suspension was prepared as previously reported [8,35]. In brief, thymus glands dissected from ether-anesthetized rats were sliced under cold conditions (2–4 °C). The slices were triturated in chilled Tyrode’s solution to dissociate the thymocytes. The cell-containing solution was then passed through a 56-lM diameter mesh to prepare the cell suspension. The cell suspension was incubated at 36–37 °C for 1 h before the experiment. Importantly, the zinc concentration in Tyrode’s solution was 32.4 ± 4.0 nM in the case of Tyrode’s solution [40]. This significant increase in zinc concentration was probably due to the reagents, probably containing very trace zinc, that were used to prepare the solution. Furthermore, the zinc concentration in the solution that was obtained after removing the cells from cell suspension by a filtration (a pore diameter: 0.22 lM) was 216.9 ± 14.4 nM [40]. Thus, it is likely that the cell suspension contains trace zinc derived from the cell preparation. Various concentrations of dithiocarbamate fungicides (0.03– 3 mM fungicide in 2 lL DMSO) were added to cell suspensions (2 mL per test tube) and incubated at 36–37 °C for 1–3 h. A sample from each cell suspension (100 lL) was analyzed by flow cytometry to assess the fungicide-induced changes in cellular parameters. Data acquisition from 2  103 cells or 2.5  103 cells required 10– 15 s. 2.3. Fluorescence measurements of cellular parameters Cell and membrane parameters were measured using a flow cytometer equipped with an argon laser (CytoACE-150; JASCO, Tokyo, Japan) and fluorescent probes. The excitation wavelength for the fluorescent probes used in this study was 488 nM. The emissions were detected at 530 ± 20 nm for FluoZin-3, Fluo-3, and 5-CMF, and at 600 ± 20 nm for propidium iodide. Fluorescence was analyzed by JASCO software (Version 3.06; JASCO, Tokyo, Japan). FluoZin-3, Fluo-3, and 5-CMF fluorescence were monitored in cells that did not exhibit propidium fluorescence because the cells exhibiting propidium fluorescence were supposed to be dead cells. No fluorescence was produced by the reagents used in the study under the present experimental conditions, with the exception of the fluorescent probes. To assess cell viability using propidium iodide, the dye was added to the cell suspension at a final concentration of 5 lM. Since propidium stains dead cells and/or cells with compromised membranes, the measurement of propidium fluorescence in cells can be used to assess viability. To estimate the change in intracellular Zn2+ levels, FluoZin-3-AM was used [16,35]. The cells were incubated with 500 nM FluoZin-3-AM for 60 min at least before the fluorescence measurement and drug application. To estimate changes in intracellular Ca2+ levels, Fluo-3-AM was used [7,27]. The cells were incubated with 500 nM Fluo-3-AM for 60 min at

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least before the fluorescence measurement and drug application. 5-CMF-DA was used to monitor changes in the cellular non-protein thiol content, presumably glutathione [8]. The cells were incubated with 1 lM 5-CMF-DA for 30 min before the fluorescence measurements. It is noted that 5-CMF-DA was applied to the cells 30 min after the start of Ziram application. Therefore, the intensity of 5-CMF fluorescence reflects the drug-induced change of cellular glutathione content. The relationship between the intensity of 5-CMF fluorescence monitored from rat thymocytes and the cellular content of glutathione and the correlation coefficient between them was 0.965 in rat thymocytes [8]. Therefore, it is possible to estimate the change in cellular content of glutathione by the use of 5-CMF fluorescence. Exposure of phosphatidylserine on outer surface of cell membranes, a phenomenon during early stage of apoptosis, was detected using annexin V-FITC [29]. The cells were incubated with annexin V-FITC (10 lL/mL) for 30 min before the measurement. Thus, the cells were initially incubated with Ziram for 30 min and then annexin V-FITC was added to the cell suspension in the continued presence of Ziram. 2.4. Statistical analysis and figure presentation Statistical analyses were performed by ANOVA, with post doc Tukey’s multivariate analysis. A P-value less than 0.05 was considered significant. In the results, values (columns and bars in figures) were expressed as the mean and the standard deviation of four samples. Each experiment was repeated three times unless noted otherwise. 3. Results 3.1. Changes in cell lethality and FluoZin-3 fluorescence by dithiocarbamate fungicides Incubation of cells with 0.3–1 lM Ziram, 0.3–1 lM Thiram, or 1–3 lM Zineb for 3 h did not induce cell death in rat thymocytes. The concentrations of dithiocarbamate fungicides used in this study did not increase the population of dead cells. As shown in Fig. 1A, the incubation of cells with 0.3–1 lM Ziram, 0.3–1 lM Thiram, and 3 lM Zineb shifted the histogram of FluoZin-3 fluorescence to a direction of higher intensity, indicating the drug-induced increased intracellular Zn2+ levels. A rapid increase in the intensity of FluoZin-3 fluorescence was observed after the application of Ziram and Thiram. The fluorescence attained a peak and steady state within 30 min of application. Therefore, the effects of dithiocarbamate fungicides on the fluorescence were examined 30 min after application. The potency of dithiocarbamate fungicides varied from agent to agent. Ziram (0.03 lM) initially increased the intensity of FluoZin-3 fluorescence, and further increases in Ziram concentration (0.1–1 lM) exhibited further augmentation of FluoZin-3 fluorescence in a concentration-dependent manner. Thiram was less potent than Ziram at concentrations of 0.3–1 lM. Further, Zineb (1–3 lM) exhibited a very weak effect on FluoZin-3 fluorescence. These results are summarized in Fig. 1B. 3.2. Augmentation of Fluo-3 fluorescence by dithiocarbamate fungicides Dithiocarbamate fungicides, such as Thiram and Ziram, were reported to increase intracellular Ca2+ levels in neuronal cells [21]. Therefore, their effects on Fluo-3 fluorescence, an indicator of intracellular Ca2+, were tested. Incubation of cells with Ziram and Thiram (0.3–1 lM) slightly, but significantly, increased the

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Fig. 2. Changes in the mean intensity of Fluo-3 fluorescence by dithiocarbamate fungicides (Upper panel) and A23187 (Lower panel) in the presence or absence of 10 lM TPEN. Their effects were examined 30 min after application. Symbol (⁄⁄) indicates a significant difference (P < 0.01) between the control group and drugtreated group.

TPEN completely suppressed the augmentation of Fluo-3 fluorescence by Ziram and Thiram, suggesting that Zn2+ mediated the enhancement of Fluo-3 fluorescence. 3.3. Ziram-induced augmentation of FluoZin-3 fluorescence in the presence of ZnCl2, DTPA, or TPEN

Fig. 1. Changes in FluoZin-3 fluorescence by dithiocarbamate fungicides. (A) Changes in the histograms of FluoZin-3 fluorescence induced by Ziram, Thiram, and Zineb. Each histogram was constructed with 2000 cells. The effects of the fungicides were examined 30 min after application. (B) Changes in the mean intensity of FluoZin-3 fluorescence using 0.03–1 lM Ziram, 0.1–1 lM Thiram, and 0.3–3 lM Zineb. Their effects were examined 30 min after application. Symbol (⁄⁄) indicates a significant difference (P < 0.01) between control group and drug-treated group.

intensity of Fluo-3 fluorescence (Fig. 2). However, Zineb (0.3– 1 lM) did not enhance intracellular Ca2+. It is possible that Ziram and Thiram augmented the Fluo-3 fluorescence via increasing intracellular Zn2+ concentrations. To test this possibility, the effects of dithiocarbamate fungicides were examined in the presence of 10 lM TPEN, a chelator of intracellular Zn2+. As shown in Fig. 2,

To identify the Zn2+ source mediating the Ziram-induced augmentation of FluoZin-3 fluorescence, the effects of 0.3 lM Ziram were examined under various conditions. As mentioned earlier, the cell suspension contained 200–230 nM Zn2+ derived from cell preparation under control conditions [40]. As shown in Fig. 3, Ziram increased the intensity of FluoZin-3 fluorescence under control conditions. Addition of 3 lM ZnCl2 into the cell suspension elevated the control level of FluoZin-3 fluorescence, and greatly boosted the Ziram-induced augmentation of FluoZin-3 fluorescence. In contrast, the chelation of extracellular Zn2+ by 10 lM DTPA almost completely suppressed the augmentation of FluoZin-3 fluorescence by Ziram. TPEN (10 lM), a chelator of intracellular Zn2+, completely blocked this augmentation. Thus, it is likely that the Ziram-induced augmentation of FluoZin-3 fluorescence is dependent on extracellular Zn2+. 3.4. Ziram-induced augmentation of FluoZin-3 fluorescence under cold conditions Zn2+ influx into cells under control conditions was significantly reduced at low temperatures [14]. To determine if the Ziram-induced influx of Zn2+ is sensitive to temperature, the effect of 0.3–1 lM Ziram on FluoZin-3 fluorescence was examined under

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Fig. 3. Changes in the mean intensity of FluoZin-3 fluorescence by Ziram in the presence of ZnCl2 and Zn2+ chelators. The cells were treated with 3 lM ZnCl2, 10 lM DTPA, or 10 lM TPEN for 10 min before the application of 0.3 lM Ziram. Effects were examined at 30 min after Ziram treatment. Symbol (⁄⁄) indicates a significant difference (P < 0.01) between the control group and drug-treated group.

cold conditions (3–4 °C). The increased intensity of FluoZin-3 fluorescence by Ziram was greatly decreased under cold conditions (Fig. 4A). However, the Ziram-induced increase was still observed under cold conditions. Rewarming (36–37 °C) induced the recovery of FluoZin-3 fluorescence augmented by Ziram (Fig. 4A). The Zn2+ influx induced by Ziram was partly sensitive to a temperature. The Zn2+ influx induced by the addition of 3 lM ZnCl2 was completely suppressed under cold conditions (Fig. 4B). Therefore, it is likely that there are two pathways regulating Zn2+ influx induced by Ziram with distinct temperature sensitivity. 3.5. Ziram-induced changes in 5-CMF fluorescence Excessive increases in intracellular Zn2+ levels are reported to induce oxidative stress [41,46]. To determine if Ziram induces oxidative stress, the effect of 0.3–1 lM Ziram on 5-CMF fluorescence, a parameter of cellular glutathione content, was examined. Strong oxidative stress that induces cell death greatly reduces cellular content of glutathione. However, weak oxidative stress increases cellular content of glutathione. Oxidative stress induces an increase in intracellular Zn2+ concentration and an elevation of intracellular Zn2+ levels increases cellular glutathione content [28]. Therefore, the cellular content of glutathione (5-CMF fluorescence) is used to estimate cytotoxic level of oxidative stress in our study. As shown in Fig. 5A, the intensity of 5-CMF fluorescence after the incubation of cells with 1 lM Ziram for 60 min was much lower than the control. However, 0.3 lM Ziram had no effect on 5-CMF fluorescence. In the presence of 10 lM DTPA, a chelator of extracellular Zn2+, the Ziram-induced decrease in 5-CMF fluorescence intensity was not observed (Fig. 5A). TPEN, a chelator of intracellular Zn2+, exhibited a similar effect (Fig. 5B). 3.6. Zn2+-dependent increase in the population of apoptotic living cells by Ziram The Ziram-induced changes in FluoZin-3 and 5-CMF fluorescence suggested the possibility that Ziram initiated the process of

Fig. 4. Effects of Ziram and ZnCl2 under cold conditions. (A) Change in the mean intensity of FluoZin-3 fluorescence augmented by 0.3–1 lM Ziram under cold conditions. The effects were examined 30 min after Ziram application. (B) Changes in the mean intensity of FluoZin-3 fluorescence augmented by 3 lM ZnCl2 under cold conditions. The effects were examined 30 min after ZnCl2 application. In the case of the cold conditions, the cell suspension was incubated in iced water baths for 10 min before the application of Ziram or ZnCl2. Symbol (⁄⁄) indicates a significant difference (P < 0.01) between the control group and drug-treated group.

cell death. Therefore, the effect of Ziram, ZnCl2, and their combination on cell population (Fig. 6) classified with annexin V-FITC and propidium iodide were examined. As shown in Fig. 6A and B, under control conditions, a large population of intact living cells in area N was observed. The incubation of cells with 1 lM Ziram for 1 h decreased the population of normal cells in area N and increased the population of annexin V-positive living cells in area A (Fig. 6A). The Ziram-induced changes were not observed in the presence of 10 lM DTPA (Fig. 6A). Thus, it was likely that Ziram promoted the transition from intact living cells to annexin V-positive living cells, the cells during early stage of apoptosis, in a Zn2+-dependent manner. As shown in Fig. 6B, 0.3 lM Ziram or 10 lM ZnCl2 (ZINC in Fig. 6B) did not change the population. However, the combination of 0.3 lM Ziram and 10 lM ZnCl2 significantly promoted the transition from intact living cells to annexin V-positive living cells. Results are summarized in Fig. 7.

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Fig. 5. Effects of Zn2+ chelators. (A) Changes in the mean intensity of 5-CMF fluorescence by 0.3–1 lM Ziram, in the presence or absence of 10 lM DTPA. (B) Changes in the mean intensity of 5-CMF fluorescence by 0.3–1 lM Ziram in the presence or absence of 10 lM TPEN. DTPA or TPEN was applied to the cell suspension 10 min before the application of Ziram. Effects were examined 60 min after Ziram application. Symbol (⁄⁄) indicates a significant difference (P < 0.01) between the control group and drug-treated group.

4. Discussion 4.1. Mechanism for the increase in intracellular Zn2+ level by dithiocarbamate fungicides Dithiocarbamate fungicides increased the intensity of FluoZin-3 fluorescence (Fig. 1), suggesting that these fungicides increase intracellular Zn2+ levels. Chelation of external Zn2+ by DTPA almost completely attenuated the Ziram-induced augmentation of FluoZin-3 fluorescence by Ziram, while external application of Zn2+ significantly potentiated it (Fig. 3). Furthermore, TPEN, a chelator of intracellular Zn2+, completely suppressed the Ziram response (Fig. 3). Thus, it is likely that dithiocarbamate fungicides increase intracellular Zn2+ levels via Zn2+ influx. How Zn2+ influx is enhanced by dithiocarbamate fungicides remains unclear. There are many zinc transporters (for a review; [25]). Of these transporters, some zinc transporters, such as Zip2, Zip12, and Zip14, are sensitive to temperature [9,15,39]. Approximately two-thirds of the Ziram-induced FluoZin-3 fluorescence augmentation was suppressed by cold temperatures (3–4 °C), whereas the augmentation of FluoZin-3 fluorescence by external application of ZnCl2 was completely attenuated (Fig. 4). Therefore, there may be two Zn2+ pathways activated by Ziram. The possibility that Ziram produces a non-specific increase in membrane permeability was ruled out,

Fig. 6. Changes in cell populations, classified by annexin V-FITC and propidium iodide, by Ziram, ZnCl2, DTPA, and their combination. (A) Changes in fluorescence cytogram (propidium fluorescence versus FITC fluorescence) by 1 lM OIT, 10 lM DTPA, and their combination (ZIRAM + DTPA). Effects were examined 1 h after application. Areas of N, A, P, and AP show the population of intact living cells, annexin V-positive living cells, dead cells, and annexin V-positive dead cells, respectively. Each cytogram consisted of 2000 cells. (B) Changes in cell population by 0.3 lM Ziram, 10 lM ZnCl2 (ZINC) and their combination (ZIRAM + ZINC).

because an increase in intracellular Ca2+ levels was not observed, as discussed below. Ziram and Thiram were reported to increase intracellular Ca2+ levels in neuronal cells [21]. In the present study, Ziram and Thiram augmented the Fluo-3 fluorescence, an indicator of intracellular Ca2+ (Fig. 2). The increase in Fluo-3 fluorescence intensity by dithiocarbamate fungicides suggests that the fungicides increase intracellular Ca2+ levels. However, this is unlikely in rat thymic lymphocytes, because the augmentation of Fluo-3 fluorescence was not observed in the presence of TPEN, a chelator

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Fig. 7. Changes in percentage cell populations by Ziram, ZnCl2, DTPA, and their combination. Values (columns and bar) were expressed as the mean and standard deviation of four experiments, respectively. The dead cell population consisted of areas of P and AP as shown in Fig. 6. Asterisks (⁄⁄) indicate a significant difference (P < 0.01) between the control and test group.

of intracellular Zn2+ (Fig. 2). It can be argued that TPEN may chelate intracellular Ca2+, resulting in the attenuation of Fluo-3 fluorescence. This possibility is also unlikely, because the augmentation of Fluo-3 fluorescence by A23187, an ionophore of divalent metal cations, was not reduced by TPEN (Fig. 2). Therefore, it is unlikely that dithiocarbamate fungicides increase intracellular Ca2+ levels in rat thymocytes. However, Ziram was suggested to increase intracellular Ca2+ level in PC12 cells and baby hamster kidney (BHK) cells transfected with rat brain NCX3 (the third isoform of the sodium–calcium exchanger family) [21,26]. Therefore, Zn2+-related action of Ziram may be specific for thymocytes. 4.2. Toxicological implications

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neurodegenerative diseases, cardiovascular diseases, and metabolic diseases [17,30,37]. In addition, it is clearly shown that Ziram initiates the process of apoptosis (Figs. 6 and 7). It is necessary to consider the contributions of Zn2+-related events to the toxicity induced by dithiocarbamate fungicides. However, some cautions may be necessary to interpret Zn2+-mediated actions. We showed that small increase in intracellular Zn2+ concentrations increased cellular content of nonprotein thiols [14,28,42]. The Zn2+-induced increase in cellular content of nonprotein thiols (glutathione) by chemical compounds may be cytoprotective against oxidative stress. In this study using rat thymocytes, the combination of 0.3 lM Ziram and 10 lM ZnCl2 increased the population of annexin V-positive living cells (Fig. 6). This concentration of Ziram to increase the cells at early stage of apoptosis in the external presence of Zn2+ is lower than those to affect cellular functions of immune cells [43,45]. Therefore, this in vitro study has a toxicological implication. The in vivo implication of our in vitro findings is not clear although we show that Ziram exerts Zn2+-dependent cytotoxicity in rat thymocytes. There are many in vivo studies on the toxicity of Ziram (for a review, [24]) and other dithiocarbamate fungicides (for a review, [23]). However, Zn2+-related toxicity was not reported in previous animal experiments. In addition, there is also no information on Ziram-induced immunotoxicity in humans [10] although immunotoxic actions were suggested only by in vitro studies [43,45]. It may be necessary to specifically study Zn2+-dependent immune functions in Ziram-treated mammals. Conflict of interest All authors affirm that there are no conflicts of interest to declare. Transparency Document

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Ziram, Thiram, and Zineb increase intracellular Zn levels in rat thymic lymphocytes (Fig. 1). Among them, Ziram increases the intracellular Zn2+ level most potently. Furthermore, Ziram decreases the population of intact living cells and increases that of annexin V-positive living cells, the cells during early stage of apoptosis (Fig. 6). Therefore, there are some environmental and toxicological implications of the Ziram-induced elevation of intracellular Zn2+ level in lymphocytes. Dithiocarbamate fungicides protect plants from fungal infection [1,31,44]. Furthermore, the use of dithiocarbamate fungicides has expanded because of their utility as an antifouling agent [12,47]. Therefore, the environmental and health risks of dithiocarbamate biocides are of concern [32,44]. Zn2+ is an intracellular signal in lymphocytes [22,36,20]. Excessive influx of Zn2+ may disturb cellular Zn2+ homeostasis in lymphocytes, possibly resulting in cellular malfunction or cell death. There is some evidence of pesticide-induced immunotoxicity in humans (for a review; [10]). Ziram (0.125–2.5 lM) decreased the activity of natural killer lymphocytes and cytotoxic T lymphocytes in vitro [45]. This concentration range corresponds to the increase in intracellular Zn2+ levels in this study. Furthermore, excessive elevation of intracellular Zn2+ levels induces oxidative stress and/or increases vulnerability to oxidative stress [38,41,46]. The results of our study are consistent with this, because Ziram (1 lM) decreased the intensity of 5-CMF fluorescence, an indicator of cellular thiol content, and the Ziram-induced attenuation of 5-CMF fluorescence was completely suppressed in the presence of DTPA or TPEN, chelators of Zn2+ (Fig. 5). Zn2+-dependent oxidative stress is partly caused via the impairment of mitochondrial function and signaling [41,46]. Oxidative stress is implicated in the progression of

The Transparency document associated with this article can be found in the online version.

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