Toxicology in Vitro 65 (2020) 104782
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Triphenyltin disrupts intracellular Zn2+ homeostasis in rat thymic lymphocytes Toshiya Ueno1,2, Keisuke Oyama1,3, Youn Jae Hyung, Shinya Ueno4, Yasuo Oyama
Laboratory of Cell Signaling, Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima 770-8513, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Triphenyltin Zinc Cytotoxicity Flow cytometry Fluorescent dyes
Triphenyltin (TPT), previously used as an agricultural fungicide and industrial antifoulant, is now considered an environmental pollutant. The eﬀect of TPT on human health is concerning due to its presence as a contaminant in seafood. In this study, the changes in intracellular Zn2+ concentration ([Zn2+]i) and cellular content of nonprotein thiols ([NPT]i) induced by triphenyltin chloride (TPTCH), were measured in rat thymic lymphocytes. This was studied by ﬂow-cytometry using the ﬂuorescent probes FluoZin-3-AM and 5-chloromethylﬂuorescein diacetate (5-CMF-DA). Incubation with TPTCH, at 0.1 μM or more (up to 3 μM), increased [Zn2+]i in a concentration-dependent manner. The TPTCH-induced elevation in [Zn2+]i was due to the increase in membrane Zn2+ permeability and intracellular Zn2+ release. Incubation with TPTCH at 0.3 μM signiﬁcantly increased [NPT]i levels, whereas the addition of an intracellular Zn2+ chelator had no eﬀect on the same. TPT at higher concentrations (1 or 3 μM) reduced [NPT]i. TPT may disturb intracellular Zn2+ signaling in lymphocytes that disturbs cellular functions.
1. Introduction Triphenyltin (TPT) is a biocide previously used as an agricultural fungicide and industrial antifoulant (Yi et al., 2012). Although, the use of TPT is banned in many countries, pollution of coastal areas by TPT is still reported in Asia (Ho and Leung, 2014). The risk assessment of organotin intake from seafood has been carried out in Taiwan (Lee et al., 2016) and Mainland China (Chen et al., 2018). The study of Lee et al. (2016) indicates that phenyltin levels in seafood (ﬁsh, shellﬁsh, sea vegetables) are higher than butyltin levels. Chen et al. (2018) have showed an increased occurrence of TPT in commercial and wild oysters. Thus, the health impact of TPT is still a matter of concern in Asia. In previous studies (Oyama and Akaike, 1990; Oyama, 1992), it has been shown that TPT increases membrane Na+ permeability by inhibiting the inactivation of voltage-gated Na+ channels. It also decreases membrane K+ permeability by reducing the activation of delayed K+ channels. Furthermore, TPT elevates intracellular Ca2+ levels ([Ca2+]i) in lymphocytes and neurons by increasing membrane permeability of Ca2+ and releasing Ca2+ from intracellular organelles (Oyama et al., 1991, 1992b, 1992c). Thus, TPT is considered to
modulate membrane ionic permeability and intracellular ionic concentrations. Recently, intracellular Zn2+ signaling has been shown to drive many physiological functions (Hojyo and Fukada, 2016; Nishida and Uchida, 2017; Levaot and Hershﬁnkel, 2018). Therefore, it is important to see if TPT modiﬁes intracellular Zn2+ levels in order to gain insights into the cellular mechanism of TPT-induced adverse actions. In this study, we examined the eﬀects of triphenyltin chloride (TPTCH) on the intracellular Zn2+ level ([Zn2+]i) and cellular content of nonprotein thiols ([NPT]i) in lymphocytes isolated from the thymus of young rats using ﬂow-cytometry with appropriate ﬂuorescent probes. As the thymus is most active during neonatal and pre-adolescent periods (Zdrojewicz et al., 2016), this study may give some insight on the health impact of TPT in neonates and pre-adolescents. 2. Materials and methods 2.1. Chemical reagents TPTCH was obtained from Tokyo Chemical Industry (Tokyo, Japan). Triphenyltin hydride (TPTHY) was a product of Sigma Aldrich Co. Ltd.
Corresponding author. E-mail address: [email protected]
(Y. Oyama). These authors equally contribute to this study. 2 Faculty of Agriculture, Kagawa University, Kagawa 761-0701, Japan. 3 Sakai City Medical Center, Sakai 593-8304, Japan. 4 Hirosaki University School of Medicine, Hirosaki 036-8562, Japan. 1
https://doi.org/10.1016/j.tiv.2020.104782 Received 25 November 2018; Received in revised form 18 January 2020; Accepted 21 January 2020 Available online 23 January 2020 0887-2333/ © 2020 Elsevier Ltd. All rights reserved.
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(St. Louis, Missouri, USA). Fluorescent probes (Invitrogen, Eugene, Oregon, USA) were propidium iodide, 5-chloromethylﬂuorescein diacetate (5-CMF-DA), and FluoZin-3-AM. Zn2+ chelators, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid (DTPA), were purchased from Dojin Chemical (Kumamoto, Japan). Other chemical reagents were obtained from Wako Pure Chemicals (Osaka, Japan). The composition of Ca2+-free Tyrode's solution was as follows: 150 mM NaCl, 5 mM KCl, 3 mM MgCl2, and 5 mM glucose. The pH of Ca2+-free Tyrode's solution was adjusted to 7.4 using 5 mM HEPES and an appropriate amount of NaOH (approximately 2 mM). The Tyrode's solution contained small amounts (approximately 230 nM) of zinc obtained from salts and cellular preparations (Sakanashi et al., 2009). 2.2. Cell preparation
Fig. 1. Change in ﬂuorescence intensity of FluoZin-3 by 0.3 μM and 1 μM TPTCH. The change was examined at 10 min after TPTCH application. Each histogram was obtained by counting 2500 cells. Control histogram (left histogram) is superimposed with open circles. Middle histogram with open squares and ﬁlled histogram (right one) were obtained from the cells treated with 0.3 μM and 1 μM TPTCH, respectively.
The committee of Tokushima University approved this study using experimental animals (T29–52). Thymus was isolated from Wistar male rats (8–12 weeks), intraperitoneally anesthetized with thiopental (50 mg/kg). Isolated thymi were immersed in Ca2+-free Tyrode's solution and sliced with a razor under ice-cold conditions. Sliced thymi were dispersed in Tyrode's solution to dissociate thymic lymphocytes (thymocytes) and obtain a cell suspension under ice-cold conditions. The cell suspension was passed through a mesh (with a diameter of 50 μm) and incubated at a temperature of 36–37 °C for 60 min before using for experiments. The cell density was 5–7 × 105 cells/mL. Since TPTCH increases membrane Ca2+ permeability, resulting in increased [Ca2+]i (Oyama et al., 1992b, 1992c), a Ca2+-free Tyrode's solution was used in this study. 2.3. Experimental protocol To photochemically estimate the change in [Zn2+]i, cells were incubated with 1 μM FluoZin-3-AM (Gee et al., 2002) for at least 60 min before the measurement. FluoZin-3 ﬂuorescence was monitored only from living cells with intact membranes by a ﬂow cytometer (CytoACE150, JASCO, Tokyo, Japan). Dead cells and cells with deteriorated membranes were stained with propidium iodide and cells exhibiting propidium iodide ﬂuorescence were not considered for the measurements. Cells were incubated with 500 nM 5-CMF-DA for 20 min before the measurement of 5-CMF ﬂuorescence. The 5-CMF ﬂuorescence measurement allows to estimate the changes in [NPT]i (Poot et al., 1991; Chikahisa et al., 1996). Although, 5-CMF ﬂuorescence attains a steady intensity in 20–30 min post-incubation with 5-CMF-DA (Chikahisa et al., 1996), cells were further incubated, with the test agent (TPT in this study), for 20–30 min in the experiments using 5-CMF-DA. The excitation wave length for all ﬂuorescent probes was 488 nm and the emission was detected at 600 ± 20 nm for propidium ﬂuorescence and at 530 ± 20 nm for 5-CMF and FluoZin-3 ﬂuorescence.
Fig. 2. Time-dependent changes in the intensity of FluoZin-3 ﬂuorescence after incubation with 1 μM TPTCH. Column and bar indicate mean and standard deviation of four samples. Asterisks (**) show signiﬁcant change (P < .01) between control group (Control) and test groups (10 min, 20 min, and 30 min).
treatment with 0.3 μM TPTCH attained a peak at 10 min after incubation (Fig. 2). While the threshold concentration of TPTCH to signiﬁcantly augment the FluoZin-3 ﬂuorescence was found to be 0.1 μM, a drastic augmentation of FluoZin-3 ﬂuorescence was observed at a concentration of 1 μM (Fig. 3). Further increase in the intensity of FluoZin-3 ﬂuorescence by 3 μM TPTCH was also conﬁrmed (not shown). It is likely that TPTCH at a concentration of ≥0.1 μM increases the [Zn2+]i.
2.4. Statistical analyses Statistical analyses was performed using Tukey's post-hoc test and/or a paired t-test. P-values ˂ 0.05 were considered statistically signiﬁcant. Data shows the mean ± standard deviation of four samples. Each series of experiments were conducted in triplicate, unless otherwise speciﬁed. 3. Results 3.1. Changes in [Zn2+]i by triphenyltin chloride (TPTCH)
Fig. 3. Concentration-dependent changes in the intensity of FluoZin-3 ﬂuorescence at 30 min post-incubation with 1 μM TPTCH. Column and bar indicate mean and standard deviation of four samples. Asterisks (**) show signiﬁcant change (P < .01) between control group (Control) and test groups (0.1 μM, 0.3 μM, and 1 μM).
The incubation of cells with TPTCH (0.3–1 μM) for 30 min led to an increase in FluoZin-3 ﬂuorescence intensity, as suggested by a shift in the histogram (Fig. 1). The increase in the FluoZin-3 intensity upon 2
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Table 1 Reagents and ﬂuorescent probes used in this study. A. Chemical Reagents Chemical Name Triphenyltin Chloride (TPTCH) Triphenyltin Hydride (TPTHY) N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) Diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid (DTPA)
Manufacturer Tokyo Chemical Industry (Tokyo, Japan) Sigma Aldrich (St. Louis, Missouri, USA) Dojin Chemical (Kumamoto, Japan) Dojin Chemical
B. Fluorescent Probes Chemical Name
Propidium Iodide 5-Chloromethylﬂuorescein Diacetate (5-CMF-DA) FluoZin-3-AM
Invitrogen (Eugene, Oregon, USA) Invitrogen Invitrogen
448 nm 448 nm 448 nm
600 ± 20 nm 530 ± 20 nm 530 ± 20 nm
3.2. Eﬀects of Zn2+ chelators on the TPTCH-induced augmentation of FluoZin-3 ﬂuorescence Eﬀects of Zn2+ chelators (DTPA and TPEN as shown in Table 1) were examined to determine the contribution of Zn2+ to the TPTCHinduced response. In the presence of 10 μM DTPA, a membrane-impermeable Zn2+ chelator (to remove extracellular Zn2+), the basal level of FluoZin-3 ﬂuorescence was reduced. Although, the application of 1 μM TPTCH still increased the intensity of FluoZin-3 ﬂuorescence (Fig. 4), the increase in ﬂuorescence in the presence of DTPA was much lesser than that under control conditions. The application of 10 μM TPEN, a membrane-permeable Zn2+ chelator (to remove intracellular Zn2+), greatly attenuated the FluoZin-3 ﬂuorescence and completely diminished the TPTCH-induced response (Fig. 4). Therefore, the increase in [Zn2+]i by TPTCH seems to be dependent on both intra- and extracellular Zn2+.
Fig. 5. Comparison of FluoZin-3 ﬂuorescence intensity in TPTCH (Chloride) and TPTHY (Hydride) treated cells. Column and bar indicate mean and standard deviation of four samples. Asterisks (**) show signiﬁcant change (P < .01) between control group (Control) and test groups. Pounds (##) show signiﬁcant diﬀerence (P < .01) between the groups of cells treated with TPTCH (Chloride) and TPTHY (Hydride).
3.3. Comparison of FluoZin-3 ﬂuorescence in the presence of TPTCH and triphenyltin hydride (TPTHY)
signiﬁcantly increased the intensity of FluoZin-3 ﬂuorescence (Fig. 5). The potency of TPTHY to augment FluoZin-3 ﬂuorescence was lesser than that of TPTCH when the concentration was 1 μM. It is likely that both TPTCH and TPTHY increase [Zn2+]i.
TPTCH is an electrophilic compound while TPTHY is a nucleophilic one. Therefore, there is a diﬀerence in the chemical reactivity of TPTCH and TPTHY. The incubation with TPTHY at 1 μM for 10 min also
3.4. Changes in 5-CMF ﬂuorescence by TPTCH Intracellular Zn2+ forms a complex with nonprotein thiols, leading to the oxidation of thiols to disulﬁdes, thereby releasing Zn2+ (Maret, 1994). Furthermore, Zn2+ increases the synthesis of glutathione, the major cellular nonprotein thiol (Ha et al., 2006; Cortese et al., 2008). Therefore, it is important to examine the eﬀect of TPTCH on [NPT]i. The intensity of 5-CMF ﬂuorescence on cells treated with 0.3 μM TPTCH for 10–30 min was higher than that of control cells (Fig. 6). Further increase in the concentration of TPTCH to 1 μM decreased the intensity of 5-CMF ﬂuorescence in three series of experiments and increased that in one series of experiment. In the latter case, incubation with 3 μM TPTCH also reduced the 5-CMF ﬂuorescence intensity (Fig. 6). Thus, TPTCH seems to reciprocally change the [NPT]i, in a concentration dependent manner. It is proposed that an increase in [Zn2+]i, by oxidative stress, acts as a trigger to restore cellular thiol content that is decreased by oxidative stress (Kinazaki et al., 2011). As shown in Fig. 7, the incubation with 3 μM ZnCl2 for 10–30 min increased the intensity of 5-CMF ﬂuorescence. To test the sequence of events (decrease in [NPT]i and an increase in [Zn2+]i) post TPTCH treatment, the change in 5-CMF ﬂuorescence intensity by 0.3 μM TPTCH was examined in the presence of 10 μM TPEN. Incubation with 0.3 μM TPTCH attenuated the 5-CMF ﬂuorescence in cells treated with TPEN. It is likely that the increase in [NPT]i by TPTCH is caused by elevated [Zn2+]i (also due to TPTCH), and that TPT reduces [NPT]i in the absence of intracellular Zn2+.
Fig. 4. Eﬀects of Zn2+ chelators, DTPA and TPEN, on the TPTCH-induced change in the intensity of FluoZin-3 ﬂuorescence. Eﬀect was examined at 30 min post-incubation with 1 μM TPTCH in the presence of respective Zn2+ chelators. Column and bar indicate mean and standard deviation of four samples. Asterisks (**) show signiﬁcant diﬀerence (P < .01) between control group (Control as also indicated by the dotted line) without Zn2+ chelator and other groups. Pounds (##) show signiﬁcant diﬀerence (P < .01) between control group (Control) and the group of cells treated with TPTCH in the presence and absence of DTPA. 3
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4. Discussion 4.1. Changes in [Zn2+]i by TPTCH The treatment of cells with TPTCH increases the [Zn2+]i by increasing Zn2+ inﬂux and releasing intracellular Zn2+, as suggested by the following observations. DTPA, a chelator of extracellular Zn2+, reduces the TPTCH-induced increase in [Zn2+]i (Fig. 4). Since TPTCH increases Ca2+ inﬂux (Oyama et al., 1992b, 1992c), it is suggested that it may increase membrane permeability of divalent metal cations such as Zn2+. Micromolar concentrations of TPTCH reduces [NPT]i (Fig. 6) and TPT induces oxidative stress (Yi et al., 2016; Clasen et al., 2017). Therefore, oxidative conversion from thiols to disulﬁdes releases Zn2+, resulting in the elevation of [Zn2+]i. 4.2. Changes in [NPT]i by TPTCH The estimation of [NPT]i by 5-CMF-DA has technical limitations. It requires 20–30 min to attain a steady peak intensity of 5-CMF ﬂuorescence (Chikahisa et al., 1996). Therefore, the cells were further treated with TPTCH for 20 min for the measurement of 5-CMF ﬂuorescence. The TPTCH-induced change in [NPT]i is complicated by the fact that an increase in [Zn2+]i increases [NPT]i (Kinazaki et al., 2011). Thus, if TPT induces the conversion from thiols to disulﬁde (the reduction of [NPT]i), releasing Zn2+ that elevates [Zn2+]i, TPT would cause reciprocal changes (decrease, increase, or their combination) in [NPT]i. Furthermore, the [NPT]i initially reduced by tributyltin chloride recovers during a prolonged incubation of 180 min (Okada et al., 2000). If this is the case for TPTCH, the TPTCH-induced change in [NPT]i would be modiﬁed in a complex manner, determined by the concentration of TPTCH, the time of incubation with TPTCH, and the amount of released Zn2+ by TPTCH.
Fig. 6. Changes in the intensity of 5-CMF ﬂuorescence by TPTCH. The 5-CMF ﬂuorescence was measured at 20 min post-incubation with 5-CMF-DA. 5-CMFDA was applied to the cells at 10 min after TPTCH application. Column and bar indicate mean and standard deviation of four samples. Asterisks (**) show signiﬁcant change (P < .01) between control group (Control) and the groups of cells treated with TPTCH. Dotted line indicates the control level.
4.3. Toxicological implications TPT is found in seafood in Taiwan and Mainland China (Lee et al., 2016; Chen et al., 2018) even though the agricultural and industrial use of TPT is already banned in many countries. The concentrations of TPT found in seafood are not low. Mean levels of TPT in seafood are 0.145 mg/kg fresh weight in fresh water ﬁsh, 0.089 mg/kg in saltwater ﬁsh, 0.673 mg/kg in crustacean, 0.166 mg/kg in bivalves/univalves, and 0.089 mg/kg in ammonites (Lee et al., 2016). The ngSn/g range of TPT in dried commercial oysters from seafood market in Shanghai, China (Chen et al., 2018) is similar to those in dried bivalves/univalves of Taiwan (Lee et al., 2016). If the inhabitants consume high amounts of such seafood, they might be at a risk of health issues, because TPTCH, even at nanomolar concentrations, aﬀects [Zn2+]i. It is likely that the mechanism of action of TPTCH on [Zn2+]i and [NPT]i is similar to those of organometallic compounds such as methylmercury chloride and TBTCH (Kawanai et al., 2009; Oyama et al., 2009). Therefore, the amount of contamination by organometallic compounds should be of concern, due to its impact on the health of humans and wildlife. Declaration of Competing Interest
Fig. 7. Changes in the intensity of 5-CMF ﬂuorescence by TPTCH in the presence of TPEN. Upper panel shows the comparison of 5-CMF ﬂuorescence in cells incubated with ZnCl2 and TPTCH treated cells. Lower panel shows the comparison of 5-CMF ﬂuorescence of TPTCH treated cells in absence and presence of TPEN. Column and bar indicate mean and standard deviation of four samples. Dotted line indicates the control level. In both panels, asterisks (**) show signiﬁcant change (P < .01) between control group (Control) and test groups. Pounds (##) show signiﬁcant diﬀerence (P < .01) between control group and test group in the presence of TPEN.
All authors declare no conﬂicts of interest. Acknowledgements This study was supported by Grant-in-Aids for Scientiﬁc Research (C26340039) awarded to Y. Oyama from the Japan Society for the Promotion of Science (Tokyo, Japan). References Chen, C., Chen, L., Huang, Q., Chen, Z., Zhang, W., 2018. Organotin contamination in
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