Tri-n-butyltin increases intracellular Zn2+ concentration by decreasing cellular thiol content in rat thymocytes

Tri-n-butyltin increases intracellular Zn2+ concentration by decreasing cellular thiol content in rat thymocytes

Toxicology 262 (2009) 245–249 Contents lists available at ScienceDirect Toxicology journal homepage: www.elsevier.com/locate/toxicol Tri-n-butyltin...

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Toxicology 262 (2009) 245–249

Contents lists available at ScienceDirect

Toxicology journal homepage: www.elsevier.com/locate/toxicol

Tri-n-butyltin increases intracellular Zn2+ concentration by decreasing cellular thiol content in rat thymocytes Toshihisa B. Oyama 1 , Keisuke Oyama 2 , Takuya Kawanai, Tomohiro M. Oyama 3 , Erika Hashimoto, Masaya Satoh, Yasuo Oyama ∗ Laboratory of Cellular Signaling, Faculty of Integrated Arts and Sciences, The University of Tokushima, Tokushima 770-8502, Japan

a r t i c l e

i n f o

Article history: Received 10 May 2009 Received in revised form 11 June 2009 Accepted 17 June 2009 Available online 25 June 2009 Keywords: Tri-n-butyltin Intracellular zinc Intracellular thiol

a b s t r a c t Effect of tri-n-butyltin (TBT), an environmental pollutant, on intracellular Zn2+ 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 Zn2+ concentration, under external Ca2+ - and Zn2+ -free condition. Chelating intracellular Zn2+ completely attenuated the TBT-induced augmentation of FluoZin-3 fluorescence. Result suggests that nanomolar TBT releases Zn2+ 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 Zn2+ release by TBT is associated with TBT-induced reduction of cellular thiol content. However, chelating intracellular Zn2+ potentiated the cytotoxicity of TBT. Therefore, the TBT-induced increase in intracellular Zn2+ concentration may be a type of stress responses to protect the cells. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Tri-n-butyltin (TBT), an environmental pollutant in edible mollusks (Yamamoto, 1994; Kannan et al., 1996; Inoue et al., 2006; Choi et al., 2009), exerts a variety of cytotoxic actions at environmentally relevant nanomolar concentrations (Kannan et al., 1998; Nakata et al., 1999; Whalen et al., 1999; Okada et al., 2000). TBT is immunotoxic and induces apoptosis of thymocytes (Aw et al., 1990; Raffray et al., 1993), leading to thymus atrophy in rats (Raffray and Cohen, 1993). The toxic action of TBT has been partly explained by TBT-induced decrease or depletion of cellular thiol content (Cima and Ballarin, 2004; Powell et al., 2008; Tada-Oikawa et al., 2008). Cellular thiols such as glutathione and metallothionein are complexed with Zn2+ (Diaz-Cruz et al., 1998; Jacob et al., 1998; Maret and Vallee, 1998; Gelinsky et al., 2003). Thus, oxidative stress releases Zn2+ from thiols via interchange between thiol and disulfide (Maret, 1994;

∗ Corresponding author. Tel.: +81 88 656 7256; fax: +81 88 656 7256. E-mail address: [email protected] (Y. Oyama). 1 Present address: Faculty of Engineering, Okayama University, Okayama 7008530, Japan. 2 Present address: Faculty of Medicine, Saga University, Saga 849-8501, Japan. 3 Present address: Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8512, Japan. 0300-483X/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2009.06.016

Quesada et al., 1996). Therefore, it is reminiscent of a possibility that TBT increases intracellular Zn2+ concentration via decreasing cellular thiol content. To test the possibility, the effect of TBT on FluoZin-3 fluorescence, a fluorescent indicator for intracellular Zn2+ , has been examined in rat thymocytes because of following reasons. Zn2+ is the second most prevalent trace element and it is involved in the structure and function of over 300 enzymes (Prasad, 1995). Zn2+ stimulates the activity of approximately 100 enzymes (Sandstead, 1994). Therefore, an abnormal increase in intracellular Zn2+ concentration by TBT may cause cytotoxic phenomena. 2. Materials and methods 2.1. Chemicals Tri-n-butyltin (TBT) chloride was purchased from Tokyo Kasei Co. (Tokyo, Japan). Chelators for Zn2+ , diethylenetriamine-N,N,N ,N ,N -pentaacetic acid (DTPA), N,N,N ,N -tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), and ethylenediamineN,N,N ,N -tetraacetic acid (EDTA), were obtained from Dojin Chemical Laboratory (Kumamoto, Japan). Fluorescent probes, propidium iodide, FluoZin-3 acetoxymethyl ester (FluoZin-3-AM), and 5-chloromethylfluorescein diacetate (5-CMF-DA), were products of Molecular Probes Inc. (Eugene, Oregon, USA). NaCl, MgCl2 , KCl, glucose, HEPES, NaOH, and ZnCl2 were also obtained from Woko Pure Chemicals. Hydrogen peroxide (H2 O2 ) was purchased from Sumitomo Chemical Industry (Osaka, Japan). Dimethyl sulfoxide (DMSO) was purchased from Wako Pure Chemicals (Osaka, Japan). Final concentration of DMSO as a solvent for TPEN, FluoZin-3AM, 5-CMF-DA, and TBT in cell suspension was 0.3% or less. The incubation with

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DMSO at 0.3% or less did not affect the viability of rat thymocytes during experiments. 2.2. Animals and cell preparation This study was approved by the Committee for Animal Experiments in the University of Tokushima (No. 05279 for Y. Oyama). The procedure to prepare cell suspension was similar to that previously reported (Chikahisa and Oyama, 1992; Chikahisa et al., 1996). In brief, thymus glands dissected from ether-anaesthetized rats (Wistar strain) were sliced at a thickness of 400–500 ␮m with razor under an ice-cold condition (1–4 ◦ C). The slices were triturated by gently shaking in chilled normal Tyrode’s solution (in mM: NaCl 150, KCl 5, CaCl2 2, MgCl2 1, glucose 5, HEPES 5, with an appropriate amount of NaOH to adjust pH to 7.3–7.4) or chilled Ca2+ -free Tyrode’s solution (in mM: NaCl 150, KCl 5, MgCl2 3, glucose 5, HEPES 5, with an appropriate amount of NaOH to adjust pH to 7.3–7.4) to dissociate thymocytes. The purities of chemicals for preparing Ca2+ -free Tyrode’s solution were greater than 99.999%. Thereafter, both Tyrode’s solutions containing the cells were respectively passed through a mesh (a diameter of 10 ␮m) to prepare cell suspension (about 5 × 105 cells/ml). The beaker containing the cell suspension was water-bathed at 36 ◦ C for 1 h before the start of experiment.

Fig. 1. TBT-induced change in histogram of FluoZin-3 fluorescence. Effect of TBT was tested at 1 h after application. Each histogram was constructed with 2000 cells. The shift of histogram to a direction of higher intensity of FluoZin-3 fluorescence indicates an increase in intracellular Zn2+ concentration.

2.3. Fluorescence measurements of cellular and membrane parameters The methods for measurements of cellular and membrane parameters using a flow cytometer equipped with an argon laser (CytoACE-150, JASCO, Tokyo, Japan) and fluorescent probes were similar to those previously described (Chikahisa and Oyama, 1992; Chikahisa et al., 1996; Matsui et al., 2008). The fluorescence was analyzed by JASCO software (Ver.3XX, JASCO). As to chemicals used in this study, there was no fluorescence detected under our experimental condition. To estimate cell size for identifying shrunken cells, the intensity of forward scatter obtained from each cells in cytogram (forward scatter versus side scatter) was measured. Reduction of forward scatter intensity indicates the decrease in cell size. To assess cell lethality, propidium iodide was added to cell suspension to achieve a final concentration of 5 ␮M. Since propidium stains dead cells, the measurement of propidium fluorescence from cells provides a clue to estimate the lethality. The fluorescence was measured at 2 min after the application of propidium iodide by a flow cytometer. Excitation wavelength for propidium was 488 nm and emission was detected at 600 ± 20 nm. FluoZin-3-AM (Gee et al., 2002) is used as an indicator for intracellular Zn2+ . The cells were incubated with 500 nM FluoZin-3-AM for 60 min before any fluorescence measurements. FluoZin-3 fluorescence was measured from the cells that were not stained with 5 ␮M propidium iodide to estimate the change in intracellular Zn2+ concentration of rat thymocytes with intact membranes (Matsui et al., 2008). Excitation wavelength for FluoZin-3 was 488 nm and emission was detected at 530 ± 15 nm. 5-CMF-DA was used to monitor the change in cellular content of nonprotein thiols (Chikahisa et al., 1996). The cells were incubated with 1 ␮M 5-CMF-DA for 30 min before any fluorescence measurements. 5-CMF fluorescence was measured from the cells that were not stained with 5 ␮M propidium iodide. Excitation wavelength for 5-CMF was 488 nm and emission was detected at 530 ± 15 nm. 2.4. Statistics Values were expressed as the mean ± standard deviation of four experiments. Statistical analysis was performed by using Tukey’s multivariate analysis. A P value of <0.05 was considered significant.

3. Results 3.1. Increase in the intensity of FluoZin-3 fluorescence by TBT As shown in Fig. 1, the histogram of FluoZin-3 fluorescence monitored from rat thymocytes incubated with normal Tyrode’s solution was shifted to a direction of higher intensity by 10–30 nM TBT. The effect of TBT on FluoZin-3 fluorescence seemed to reach a steady state level within 1 h because the further change in mean intensity was less than 5% during next 0.5–1 h incubation. Therefore, the effect of TBT on FluoZin-3 fluorescence was tested at 1 h after the application of TBT in the experiments described below. The TBT-induced augmentation of FluoZin-3 fluorescence was not observed in the presence of TPEN, a chelator for extracellular and intracellular Zn2+ . The results indicate the increase in intracellular Zn2+ concentration by TBT. Nanomolar TBT increases membrane permeability of Ca2+ (Chikahisa and Oyama, 1992; Chow et al., 1992). To rule out the possibility that external Ca2+ and Zn2+ contribute to TBT-induced increase in FluoZin-3 fluorescence intensity, the effect of TBT was examined under external Ca2+ - and Zn2+ -free condition. The cells were incubated with Ca2+ -free Tyrode’s solution containing 10 ␮M DTPA. The threshold concentration of TBT to increase mean intensity of FluoZin-3 fluorescence was 1–10 nM. As shown in Fig. 2, the increases were statistically significant when the concentration of TBT was 30 nM or more (up to 300 nM). Thus, it is suggested that nanomolar TBT induces the release of intracellular Zn2+ , resulting in

Fig. 2. Concentration-dependent change in mean intensity of FluoZin-3 fluorescence by TBT. Column and bar respectively indicate average and standard deviation of four experiments. Asterisks (* and **) show significant increase (P < 0.05 and P < 0.01, respectively) in comparison with control.

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Fig. 3. Effect of simultaneous application of TBT and H2 O2 on FluoZin-3 fluorescence. Effect was examined at 60 min after respective application. Column and bar respectively indicate average and standard deviation of four experiments. Asterisks (* and **) show significant increase (P < 0.05 and P < 0.01, respectively) in comparison with CONTROL. Symbol (# ) indicates significant increase in comparison with the group of cells incubated with H2 O2 alone.

the increase in intracellular Zn2+ concentration. The concentrationdependent change in FluoZin-3 fluorescence by TBT under external Ca2+ - and Zn2+ -free condition was similar to that under normal condition (not shown). 3.2. Decrease in the intensity of 5-CMF fluorescence by TBT Since it has been reported that intracellular Zn2+ concentration is modulated via the interchange between thiol and disulfide (Maret, 1994; Quesada et al., 1996), the effect of simultaneous application of H2 O2 and TBT was tested on FluoZin-3 fluorescence. H2 O2 was used to decrease cellular thiol content, resulting in an increase of cellular disulfide content. H2 O2 at 30 ␮M attenuated the 5-CMF fluorescence by less than 40% without affecting the cell viability of rat thymocytes (Chikahisa et al., 1996). The concentration of TBT was 10–30 nM to observe further augmentation of the FluoZin-3 fluorescence by 30 ␮M H2 O2 . As shown in Fig. 3, H2 O2 significantly increased the intensity of FluoZin-3 fluorescence. However, the effects of H2 O2 and TBT seemed to be additive. TBT decreases cellular content of thiols under normal condition (Okada et al., 2000). The effect of TBT on intensity of 5-CMF fluorescence was tested under external Ca2+ - and Zn2+ -free condition to reveal a correlation between TBT-induced increase in intracellular Zn2+ concentration (Fig. 2) and TBT-induced change in cellular thiol content. As shown in Fig. 4, TBT at concentrations ranging from

Fig. 4. Concentration-dependent change in mean intensity of 5-CMF fluorescence by TBT. Column and bar respectively indicate average and standard deviation of four experiments. Asterisks (* and **) show significant increase (P < 0.05 and P < 0.01, respectively).

30 nM to 300 nM significantly decreased mean intensity of 5-CMF fluorescence. The result of Fig. 4 was compared with those of Fig. 2 to see if there is a correlation between them. The correlation coefficient was −0.962 (Fig. 5). In the presence of 10 ␮M TPEN (under external and internal Zn2+ -free condition), TBT at 300 nM also significantly decreased the intensity of 5-CMF fluorescence. Thus, TBT is supposed to decrease cellular thiol content, being independent from Zn2+ . 3.3. Effect of TPEN on the cells treated with TBT under normal condition The result of Fig. 5 suggests that TBT increases intracellular Zn2+ concentration by decreasing cellular thiol content. To see if the TBTinduced increase in intracellular Zn2+ concentration contributes to the TBT cytotoxicity, the effect of 300 nM TBT on rat thymocytes incubated with normal Tyrode’s solution was examined in absence and presence of 10 ␮M TPEN. The cells were incubated with and

Fig. 5. Correlation between the TBT-induced changes in FluoZin-3 and 5-CMF fluorescence. Results are obtained from Figs. 2 and 4.

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Fig. 6. Effect of TPEN on TBT-induced cytotoxicity. Cell lethality was examined at 1 h after respective application. Asterisks (* and **) show significant increase (P < 0.05 and P < 0.01, respectively) in comparison with CONTROL. Symbol (# ) indicates significant increase in comparison with both groups of cells incubated with TBT alone and TPEN alone.

without TBT and/or TPEN for 1 h. Although the incubation with 300 nM TBT or 10 ␮M TPEN for 1 h did not significantly increase cell lethality, the simultaneous application of TBT with TPEN induced significant increase in cell lethality (Fig. 6). Thus, the removal of intracellular Zn2+ is supposed to augment the cytotoxicity of TBT. Cell shrinkage is one of characteristics during an early stage of apoptosis (Klassen et al., 1993; Beauvais et al., 1995; Bortner and Cidlowski, 1998). As to the population of shrunken cells, 300 nM TBT significantly increased it under normal condition while it was not the case under external Ca2+ -free condition (Fig. 6). The combination of TBT and TPEN further increased the population although TPEN alone did not so (Fig. 6). This increase is statistically significant in comparison with the control group and the group of cells treated with 300 nM TBT. 4. Discussion It is likely that TBT releases Zn2+ from intracellular stores, resulting in the increase in intracellular Zn2+ concentration, via decreasing cellular thiol content because of following reasons. First, the TBT-induced increase in FluoZin-3 fluorescence intensity was observed under external Ca2+ - and Zn2+ -free condition. The augmentation of FluoZin-3 fluorescence by TBT was not observed in the presence of TPEN. These results suggest that TBT increases intracellular Zn2+ concentration by releasing intracellular Zn2+ from intracellular stores. Second, the TBT-induced changes in fluorescence of FluoZin-3, an indicator for intracellular Zn2+ concentration, and 5-CMF, an indicator for cellular thiol content, are correlated

with a coefficient of −0.962. Zn2+ makes a complex with thiol group of protein and nonprotein such as metallothionein and glutathione (Diaz-Cruz et al., 1998; Jacob et al., 1998; Maret and Vallee, 1998; Gelinsky et al., 2003). The modification from thiol to disulfide has been reported to release Zn2+ (Maret, 1994; Quesada et al., 1996). In fact, N-ethylmaleimide, inducing a chemical depletion of intracellular thiol, decreased intensity of 5-CMF fluorescence and increased that of FluoZin-3 fluorescence (Hashimoto et al., in press). These results suggest that TBT increase in intracellular Zn2+ concentration from intracellular stores by decreasing cellular thiol content. TBT exerts an immunotoxic action on mammals (Snoeij et al., 1987). Nanomolar TBT induces some toxic actions on immune system cells under in vitro condition. TBT inhibits tumorkilling capacity of human and murine natural killer lymphocytes (Ghoneum et al., 1990; Whalen et al., 1999), elevates intracellular Ca2+ concentration (Chikahisa and Oyama, 1992; Chow et al., 1992; Oyama et al., 1994) and increases the population of apoptotic cells in murine thymocytes (Nakata et al., 1999). In addition, TBT reduces the cellular content of GSH in rat thymocytes (Okada et al., 2000). However, the increase in intracellular Zn2+ concentration by nanomolar TBT may be not toxic since the removal of intracellular Zn2+ by TPEN augmented the cytotoxicity of TBT. Intracellular Zn2+ partly attenuates Ca2+ -dependent cell death induced by A23187, a calcium ionophore, in rat thymocytes (Sakanashi et al., 2009). Thus, the removal of intracellular Zn2+ by TPEN may potentiate Ca2+ -dependent cell death induced by TBT since TBT increases intracellular Ca2+ concentration (Chikahisa and Oyama, 1992; Chow et al., 1992; Oyama et al., 1994). The increase in population of

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shrunken cells by TBT was reduced under external Ca2+ -free condition, suggesting the involvement of Ca2+ in the cell shrinkage. The combination of TBT and TPEN further increased the population of shrunken cells. Therefore, the TBT-induced increase in intracellular Zn2+ concentration in this study may be a type of stress responses to protect the cells. In fact, TBT delays the process of cell death induced by H2 O2 in rat thymocytes (Sakai et al., 2001). Both TBT and H2 O2 increased intracellular Zn2+ concentration in this study. The cell death induced by H2 O2 is dependent on Ca2+ (Sakanashi et al., 2008). Cellular Zn2+ partly attenuates Ca2+ -dependent cell death (Sakanashi et al., 2009). Thus, the TBT-induced increase in intracellular Zn2+ concentration may delay the cell death induced by H2 O2 . Zinc pyrithione at nanomolar concentrations induces apoptosis and the addition of zinc chelator protects the cells against apoptosis induced by zinc pyrithione in murine thymic and splenic lymphocytes and human Ramos B and Jurkat T cells (Mann and Fraker, 2005). However, the chelation of intracellular zinc triggers apoptosis in murine and human mature thymocytes (McCabe et al., 1993). Thus, the role of intracellular Zn2+ itself in cell death may vary from cell to cell or may be complex. In addition, intracellular Zn2+ affects the process of Ca2+ -dependent cell death in rat thymocytes (Sakanashi et al., 2009). Therefore, the TBT-induced increase in intracellular Zn2+ concentration may induce various effects on the process of cell death, being dependent on the type of cells. Conflict of interest statement All authors declare that there are no conflicts of interest in this study. References Aw, T.Y., Nicotera, P., Manzo, L., Orrenius, S., 1990. Tributyltin stimulates apoptosis in rat thymocytes. Archives of Biochemistry and Biophysics 283, 46–50. Beauvais, F., Michel, L., Dubertret, L., 1995. Human eosinophils in culture undergo a striking and rapid shrinkage during apoptosis. Role of K+ channels. Journal of Leukocyte Biology 57, 851–855. Bortner, C.D., Cidlowski, J.A., 1998. A necessary role for cell shrinkage in apoptosis. Biochemical Pharmacology 56, 1549–1559. Chikahisa, L., Oyama, Y., 1992. Tri-n-butyltin increases intracellular Ca2+ in mouse thymocytes: a flow-cytometric study using fluorescent dyes for membrane potential and intracellular Ca2+ . Pharmacology and Toxicology 71, 190– 195. Chikahisa, L., Oyama, Y., Okazaki, E., Noda, K., 1996. Fluorescent estimation of H2 O2 induced changes in cell viability and cellular nonprotein thiol level of dissociated rat thymocytes. Japanese Journal of Pharmacology 71, 299–305. Choi, M., Choi, H.G., Moon, H.B., Kim, G.Y., 2009. Spatial and temporal distribution of tributyltin (TBT) in seawater, sediments and bivalves from coastal areas of Korea during 2001–2005. Environmental Monitoring and Assessment 151, 301–310. Chow, S.C., Kass, G.E., McCabe, M.J., Orrenius, S., 1992. Tributyltin increases cytosolic free Ca2+ concentration in thymocytes by mobilizing intracellular Ca2+ , activating a Ca2+ entry pathway, and inhibiting Ca2+ efflux. Archives of Biochemistry and Biophysics 298, 143–149. Cima, F., Ballarin, L., 2004. Tributyltin-sulfhydryl interaction as a cause of immunotoxicity in phagocytes of tunicates. Ecotoxicology and Environmental Safety 58, 386–395. Diaz-Cruz, M.S., Mendiete, J., Monjonell, A., Tauler, R., Esteban, M., 1998. Study of the zinc-binding properties of glutathione by differential pulse polarography and multivariate curve resolution. Journal of Inorganic Biochemistry 70, 91–98. Gee, K.R., Zhou, Z.L., Qian, W.J., Kennedy, R., 2002. Detection and imaging of zinc secretion from pancreatic beta-cells using a new fluorescent zinc indicator. Journal of the American Chemical Society 124, 776–778. Gelinsky, M., Vogler, R., Vahrenkamp, H., 2003. Zinc complexation of glutathione and glutathione-derived peptides. Inorganica Chimica Acta 344, 230–238. Ghoneum, M., Hussein, A.E., Gill, G., Alfred, L.J., 1990. Suppression of murine natural killer cell activity by tributyltin: in vivo and in vitro assessment. Environmental Research 52, 178–186. Hashimoto, E., Oyama, T.B., Oyama, K., Nishimura, Y., Oyama, T.M., Ueha-Ishibashi, T., Okano, Y., Oyama, Y., in press. Increase in intracellular Zn2+ concentration by

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