Toxicology Letters 215 (2012) 208–213
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Elevation of intracellular Zn2+ level by nanomolar concentrations of triclocarban in rat thymocytes Junpei Morita 1 , Aoi Teramachi 1 , Yosuke Sanagawa 1 , Saramaiti Toyson 1 , Hiroshi Yamamoto, Yasuo Oyama ∗ Division of Environmental Symbiosis Studies, Graduate School of Integrated Arts and Sciences, The University of Tokushima, Tokushima 770-8502, Japan
h i g h l i g h t s I I I I
Triclocarban, an antimicrobial agent, is contained in personal care products. Triclocarban was reported to be detected in human blood after showering. Triclocarban at relevant concentrations increased cellular Zn2+ level in rat thymocytes. Triclocarban may affect immunity because zinc has physiological roles in lymphocytes.
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Article history: Received 23 August 2012 Received in revised form 14 October 2012 Accepted 15 October 2012 Available online 22 October 2012 Keywords: Intracellular Zn2+ Oxidative stress Personal care product Thiol Triclocarban
a b s t r a c t It was recently reported that nanomolar concentrations of triclocarban, an antimicrobial agent, were detected in human blood after the use of soap containing triclocarban. Due to the widespread use of triclocarban in adult and infant personal care products, the report prompted us to study its cytotoxicity. The cytotoxicity of triclocarban was examined in rat thymocytes by using a cytometric technique with propidium iodide for examining cell lethality, FluoZin-3-AM for monitoring the intracellular Zn2+ level, and 5-chloromethylﬂuorescencein diacetate for estimating the cellular content of non-protein thiol. The incubation with triclocarban at nanomolar concentrations (50–500 nM) for 1 h did not affect cell lethality but signiﬁcantly elevated the intracellular Zn2+ level. The elevation of the intracellular Zn2+ level by triclocarban was not signiﬁcantly dependent on external Zn2+ level. There was a negative correlation (r = −0.9225) between the effect on the intracellular Zn2+ level and that on the cellular content of nonprotein thiol. These results suggest that nanomolar concentrations of triclocarban decrease the cellular content of non-protein thiol, leading to intracellular Zn2+ release. Since zinc plays physiological roles in mammalian cells, the percutaneous absorption of triclocarban from soap may, therefore, affect some cellular functions. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Triclocarban (3,4,4 -trichlorocarbanilide, CAS 101-20-2) is commonly used as an antimicrobial agent in personal care products. It is present in surface waters and bioaccumulates in aquatic animals (Coogan and La Point, 2008; Zhao et al., 2010; Snyder et al., 2011; Schebb et al., 2011a). Triclocarban has attracted some public attention because of reports that it acts as an endocrine disruptor in a cell-based androgen receptor-mediated bioassay (Chen et al.,
∗ Corresponding author. Tel.: +81 88 656 7256; fax: +81 88 656 7256. E-mail address: [email protected]
(Y. Oyama). 1 These authors (graduate students) contributed equally to this work because experiments and manuscript preparation were carried out during the graduate class of Environmental Symbiosis Studies. 0378-4274/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2012.10.012
2008) and in freshwater mudsnails (Giudice and Young, 2010). Its endocrine disrupting actions have, therefore, been well evaluated in other preparations under in vitro and in vivo conditions (Christen et al., 2011; Chung et al., 2011; Duleba et al., 2011; Hinther et al., 2011). In contrast, there is very limited information concerning the cytotoxic actions of triclocarban in mammalian cells, although these actions have been examined, to some extent, in aquatic and terrestrial organisms (Lawrence et al., 2009; Snyder et al., 2011). In this study, we examined the cellular actions of triclocarban in rat thymocytes by using a cytometric technique with appropriate ﬂuorescent probes for various reasons. Firstly, triclocarban is highly lipophilic and its log Kow (4.2) indicates that its potential to bioaccumulate is high. Triclocarban may, therefore, directly affect cell membranes and permeate into cells, resulting in changes in intracellular functioning. Secondly, in recent studies, nanomolar concentrations of triclocarban were detected in
J. Morita et al. / Toxicology Letters 215 (2012) 208–213
human blood following the use of soap containing triclocarban (Schebb et al., 2012) and in rat blood after the topical application of triclocarban-containing cream (Schebb et al., 2011b). If the triclocarban in personal care products reaches the blood in infants as well as in adults, evaluations of its cytotoxicity should be a priority given that adverse effects of drug-containing personal care products have been reported previously in neonates and infants (Schmalz, 1961; Larrègue et al., 1986; Turpeinen et al., 1986). Thirdly, thymocytes are known to be susceptible to various chemicals, and thymus atrophy has been experimentally induced in vivo by chemical exposure (Pieters et al., 1989; Enan et al., 1996; Cuff et al., 1996; Wijnen et al., 1997; Nohara et al., 2008). The processes of cell death (apoptosis and necrosis) have been extensively studied in murine thymocytes (McConkey et al., 1994; Quaglino and Ronchetti, 2001). Furthermore, the thymus is most active during the neonatal and pre-adolescent periods. Taken together, rat thymocytes are highly appropriate for examining the cytotoxic actions of triclocarban. 2. Material and methods 2.1. Chemicals Triclocarban was purchased from Wako Pure Chemicals (Osaka, Japan). FluoZin-3 pentaacetoxymethyl ester (FluoZin-3-AM), 5-chloromethylﬂuorescein diacetate (5-CMFDA), and propidium iodide were purchased from Molecular Probes Inc. (Eugene, OR, USA). The chelators for the divalent metal cations, ethylenediaminetetraacetic acid (EDTA), N,N,N ,N -tetrakis[2-pyridylmethyl]ethylenediamine (TPEN), and diethylenetriamine-N,N,N ,N ,N -pentaacetic acid (DTPA) were obtained from Dojin Chemical Laboratory (Kumamoto, Japan). The buffer was 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Nacalai Tesque, Kyoto, Japan). The other chemicals (NaCl, CaCl2 , MgCl2 , KCl, glucose, and NaOH) were purchased from Wako Pure Chemicals. Triclocarban, FluoZin-3-AM, 5-CMF-DA, and TPEN were initially dissolved in dimethyl sulfoxide (Sigma, St. Louis, MO, USA). Dimethyl sulfoxide as a solvent at 0.1–0.3% did not affect the cell viability. 2.2. Animals and cell preparation The study was approved by the Committee for Animal Experiments of the University of Tokushima (Registration No. 05279). The cell suspension was prepared in a similar manner to that previously reported (Chikahisa et al., 1996). In brief, thymus glands dissected from ether-anesthetized rats were sliced at a thickness of 400–500 m with a razor blade under cold conditions (3–4 ◦ C). The slices were triturated by gentle shaking in chilled Tyrode’s solution (NaCl 150 mM, KCl 5 mM, CaCl2 2 mM, MgCl2 1 mM, glucose 5 mM, and HEPES 5 mM, with an appropriate amount of NaOH to adjust the pH to 7.3–7.4) to dissociate the thymocytes. Thereafter, the Tyrode’s solution containing the cells was passed through a mesh (size: 10 m) to prepare the cell suspension. The beaker containing the cell suspension was incubated in a water bath at 36–37 ◦ C for 1 h before the experiment. Although Tyrode’s solution did not contain ZnCl2 , the cell suspension generally contained 200–230 nM zinc derived from the cell preparation (Sakanashi et al., 2009). 2.3. Fluorescence measurements of cellular and membrane parameters The methods for the measurements of the cellular and membrane parameters by using a ﬂow cytometer equipped with an argon laser (CytoACE-150, JASCO, Tokyo, Japan) and ﬂuorescent
probes were similar to those previously described (Chikahisa et al., 1996; Matsui et al., 2008). The ﬂuorescence was analyzed using JASCO software (version 3XX, JASCO). There was no ﬂuorescence from the reagents used in the study, except for the ﬂuorescent probes, under our experimental conditions. To assess cell lethality, propidium iodide was added to the cell suspension to achieve a ﬁnal concentration of 5 M. Since propidium stains dead cells, the measurement of propidium ﬂuorescence from cells provided an estimation of cell lethality. The ﬂuorescence was measured with a ﬂow cytometer 2 min after the application of propidium iodide. The excitation wavelength used for propidium was 488 nm and emission was detected at 600 ± 20 nm. FluoZin-3-AM (Gee et al., 2002) was used as an indicator of intracellular Zn2+ . The cells were incubated with 500 nM FluoZin-3AM for 60 min before any ﬂuorescence measurements were taken. FluoZin-3 ﬂuorescence was measured in cells that were not stained with 5 M propidium iodide (Matsui et al., 2008). The excitation wavelength used for FluoZin-3 was 488 nm and emission was detected at 530 ± 20 nm. 5-CMF-DA was used to monitor changes in the cellular content of non-protein thiol (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 in the cells that were not stained with 5 M propidium iodide. The excitation wavelength used for 5-CMF was 488 nm and emission was detected at 530 ± 15 nm. 2.4. Protocols Triclocarban in 2 L dimethyl sulfoxide was added to the cell suspension (2 mL in test tube). The cells were incubated with triclocarban at 36–37 ◦ C for 1 h. The data acquisition of ﬂuorescence from 2 × 103 cells using a ﬂow cytometer after the 1 h incubation required at least 10 s. Each experiment was performed with 4 control and test samples at least. Result was conﬁrmed by the experiments that were repeated twice or three times. 2.5. Statistics Values were expressed as the mean ± standard deviation of 4 samples. Statistical analysis on the difference between control group and test groups (triclocarban-treated group) was performed with Tukey’s multivariate analysis. A P value of <0.05 was considered signiﬁcant. Relationships between parameters were estimated with a correlational analysis. 3. Results 3.1. The effect of triclocarban on cell lethality The incubation of cells with triclocarban at concentrations ranging from 30 nM to 500 nM for 1 h did not affect cell lethality. The level of lethality in the control group was 5.7 ± 0.5% compared with 5.4 ± 1.0% after incubation with 500 nM triclocarban for 1 h. A signiﬁcant increase in cell lethality was observed when the concentration of triclocarban was increased to 10 M. 3.2. The effect of triclocarban on FluoZin-3 ﬂuorescence As shown in Fig. 1, the incubation of cells with 500 nM triclocarban shifted the histogram of the FluoZin-3 ﬂuorescence to a higher intensity, suggesting an increase in the intracellular Zn2+ level as a result of triclocarban exposure. Triclocarban at 500 nM increased FluoZin-3 ﬂuorescence in a time-dependent manner (Fig. 2A). The ﬂuorescence seemed to reach a maximum (or steady-state) level 30–60 min after the start of the incubation. Control level was
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Fig. 1. Triclocarban-induced increase of FluoZin-3 ﬂuorescence. Each histogram consisted of 2500 cells. The effect of triclocarban was examined 60 min after the start of drug application.
Fig. 3. Alteration of the triclocarban-induced change in FluoZin-3 ﬂuorescence as a result of simultaneous application with DTPA, ZnCl2 , or TPEN. Effects were examined 60 min after the start of drug application. The columns and bars indicate the mean and standard deviation, respectively, of 4 samples. The results of one representative experiment are shown.
not changed during the experiment (60 min). In order to identify concentration-dependent changes in FluoZin-3 ﬂuorescence as a result of exposure to 30–500 nM triclocarban, the effect was examined 60 min after the start of drug incubation. Triclocarban at 30 nM did not change FluoZin-3 ﬂuorescence. The lowest concentration of triclocarban that increased the ﬂuorescence was 50 nM. Further increase in the drug concentration (up to 500 nM) more profoundly increased the FluoZin-3 ﬂuorescence in a concentration-dependent manner (Fig. 2B). 3.3. Role of Zn2+ in the increase of FluoZin-3 ﬂuorescence by triclocarban In order to reveal the source of Zn2+ for triclocarban-induced increase of FluoZin-3 ﬂuorescence, the effect of triclocarban was examined in the presence of an appropriate Zn2+ chelator. In the presence of 10 M DTPA, a chelator of extracellular Zn2+ , triclocarban at 500 nM signiﬁcantly increased the FluoZin-3 ﬂuorescence (Fig. 3). The increase by triclocarban in the presence of DTPA was slightly less than that observed under the control condition. The application of 3 M ZnCl2 signiﬁcantly increased the FluoZin-3 ﬂuorescence under the control condition. Triclocarban at 500 nM also increased the FluoZin-3 ﬂuorescence (Fig. 3). The difference in the FluoZin-3 ﬂuorescence between the control group and the triclocarban-treated group in the presence of 3 M ZnCl2 was slightly more (presumably 200–230 nM zinc) than that under the control condition. In the presence of 10 M TPEN, a membrane-permeable Zn2+ chelator, the control level of FluoZin-3 ﬂuorescence was greatly attenuated. Triclocarban at 500 nM did not induce changes in ﬂuorescence (Fig. 3). The increase of FluoZin-3 ﬂuorescence by triclocarban seemed to be a result of an triclocarban-induced increase in the intracellular Zn2+ concentration. Fig. 2. Time- (A) and concentration- (B) dependent changes in the intensity of FluoZin-3 ﬂuorescence as a result of triclocarban application. Time-dependent effects were examined at 10, 20, 30, and 60 min after the start of drug application. Dose-dependent effects were tested at 60 min after the start of drug application. The columns and bars indicate the mean and standard deviation, respectively, of 4 samples. Open columns are control groups (without triclocarban) and shaded columns are triclocarban-treated groups. The results of one representative experiment are shown.
3.4. Role of Ca2+ in the increase of FluoZin-3 ﬂuorescence by triclocarban Removal of extracellular Ca2+ increased FluoZin-3 ﬂuorescence in rat thymocytes (Nishimura and Oyama, 2012). To examine the contribution of Ca2+ to the increase of FluoZin-3 ﬂuorescence by triclocarban, the effect of triclocarban was examined under Ca2+ free conditions.
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Fig. 4. Alteration of the triclocarban-induced change in FluoZin-3 ﬂuorescence as a result of simultaneous application with EDTA or CaCl2 , under nominally Ca2+ free conditions. Effects were examined 60 min after the start of drug application. The columns and bars indicate the mean and standard deviation, respectively, of 4 samples. The results of one representative experiment are shown.
Triclocarban at 500 nM increased FluoZin-3 ﬂuorescence under a nominally Ca2+ -free condition (Fig. 4). EDTA at 500 M chelated both Ca2+ and Zn2+ in nominally Ca2+ -free Tyrode’s solution. Triclocarban further increased FluoZin-3 ﬂuorescence under Ca2+ and Zn2+ -free conditions (Fig. 4). The application of 2 mM CaCl2 attenuated the FluoZin-3 ﬂuorescence. The increase of FluoZin3 by 500 nM triclocarban under normal Ca2+ conditions (2 mM CaCl2 ) was less than that under Ca2+ - and Zn2+ -free conditions. These results suggested that Ca2+ had a negative effect on the triclocarban-induced increase of FluoZin-3 ﬂuorescence. 3.5. The effect of triclocarban on 5-CMF ﬂuorescence To determine whether triclocarban decreased the cellular content of non-protein thiol (glutathione), the effect of triclocarban on 5-CMF ﬂuorescence was examined. Incubation of the cells with 500 nM triclocarban shifted the histogram of 5-CMF ﬂuorescence to a lower intensity (Fig. 5). This suggested a triclocarban-induced decrease in the cellular content of non-protein thiol.
Fig. 6. Alteration of the triclocarban-induced change of 5-CMF ﬂuorescence as a result of simultaneous application with EDTA or CaCl2 , under nominally Ca2+ -free conditions (A), and the correlation between 5-CMF ﬂuorescence and FluoZin-3 ﬂuorescence (B). Effects were examined 60 min after the start of drug application. The columns and bars indicate the mean and standard deviation, respectively, of 4 experiments.
Since FluoZin-3 ﬂuorescence was affected by both triclocarban and Ca2+ (Fig. 4), the effect of triclocarban on 5-CMF ﬂuorescence was examined under both normal Ca2+ and Ca2+ -free conditions. Incubation of the cells with 500 nM triclocarban for 60 min signiﬁcantly attenuated the 5-CMF ﬂuorescence under both conditions (Fig. 6A). The changes in the FluoZin-3 and 5-CMF ﬂuorescence induced by triclocarban (Figs. 4 and 6A) were negatively correlated, with a correlation coefﬁcient of −0.9225 (Fig. 6B). These results suggested that the decrease in the cellular content of non-protein thiol led to the increase in the intracellular Zn2+ level. 4. Discussion
Fig. 5. Triclocarban-induced attenuation of 5-CMF ﬂuorescence. Each histogram consisted of 2500 cells. The effect of triclocarban was examined 60 min after the start of drug application.
FluoZin-3 ﬂuorescence was used to monitor changes in the intracellular Zn2+ concentration (Gee et al., 2002). The increase of FluoZin-3 ﬂuorescence observed as a result of triclocarban treatment signiﬁed an increase in the intracellular Zn2+ level. Thus, the incubation of rat thymocytes with 50–500 nM triclocarban increased intracellular Zn2+ concentration (Figs. 1 and 2). 5-CMF ﬂuorescence was employed to detect changes in the cellular content of non-protein thiol (mainly glutathione) (Chikahisa et al., 1996). The attenuation of 5-CMF ﬂuorescence by triclocarban indicated that triclocarban decreased the cellular content of non-protein thiol (Figs. 5 and 6). The increase in intracellular Zn2+
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concentration by triclocarban was observed under extracellular Zn2+ -free condition (Fig. 3). The release of intracellular Zn2+ has been elicited by N-ethylmaleimide (Kinazaki et al., 2011), tri-nbutyltin (Oyama et al., 2009), methylmercury (Kawanai et al., 2009), and thimerosal (Hashimoto et al., 2009). These chemical compounds greatly decreased the cellular content of non-protein thiol. A negative correlation between the cellular thiol content and the intracellular Zn2+ concentration (Fig. 6B) is understandable because intracellular Zn2+ is known to form a complex with the thiol group of protein and non-protein (Jacob et al., 1998) and modiﬁcation from thiol to disulﬁde releases Zn2+ (Maret, 1994). Triclocarban may also induce oxidative stress, resulting in the modiﬁcation from thiol to disulﬁde and thus eliciting intracellular Zn2+ release. Removal of external Ca2+ modiﬁed the potency of triclocarban to increase intracellular Zn2+ concentration (Fig. 4). Extracellular Ca2+ has a role to maintain membrane integrity and permeability (Gary-Bobo, 1970; Delamere and Paterson, 1978; Crevey et al., 1978; McNeil and Kirchhausen, 2005). The removal of external Ca2+ may increase the membrane permeability of triclocarban, resulting in the modiﬁcation of triclocarban potency. Zinc plays physiological roles in many types of cells (Beyersmann and Haase, 2001; Prasad, 2008; Sensi et al., 2009). Furthermore, zinc dyshomeostasis is recognized as an important mechanism for cellular malfunction, cell injury, and cell death (Truong-Tran et al., 2001; Kröncke, 2007; Sensi et al., 2009; Haase and Rink, 2009). Abnormal increases in intracellular Zn2+ concentrations are associated with a variety of health problems (Frederickson et al., 2004; Maret and Sandstead, 2006; Cummings and Kovacic, 2009). The highest concentrations of triclocarban in blood samples collected from human subjects after exposure were 240 nM and 530 nM (Schebb et al., 2012). Therefore, we cannot rule out the possibility that triclocarban, which is percutaneously absorbed, can elevate the intracellular Zn2+ level of thymic lymphocytes. There are no reports concerning effects of triclocarban on human thymus at present. However, zinc homeostasis is crucial for the normal development and functioning of cells mediating immunity (Shankar and Prasad, 1998; Ibs and Rink, 2003). In the future, therefore, it may be necessary to examine the immune functions of humans or animals exposed to triclocarban. Conﬂict of interest We have no conﬂict of interest. Acknowledgements This study was supported by a Grant-in-Aid for Scientiﬁc Research (C23510078) from the Japan Society for the Promotion of Science. It was also supported by the project expenditure for promoting education and research in environmental symbiosis studies provided from the Ofﬁce of the Dean (Faculty of Integrated Arts and Sciences, The University of Tokushima). References Beyersmann, D., Haase, H., 2001. Functions of zinc in signaling, proliferation and differentiation of mammalian cells. BioMetals 14, 331–341. Chen, J., Ahn, K.C., Gee, N.A., Ahmed, M.I., Duleba, A.J., Zhao, L., Gee, S.J., Hammock, B.D., Lasley, B.L., 2008. Triclocarban enhances testosterone action: a new type of endocrine disruptor? Endocrinology 149, 1173–1179. 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. Christen, V., Crettaz, P., Oberli-Schrämmli, A., Fent, K., 2011. Some ﬂame retardants and the antimicrobials triclosan and triclocarban enhance the androgenic activity in vitro. Chemosphere 81, 1245–1252. Chung, E., Genco, M.C., Megrelis, L., Ruderman, J.V., 2011. Effects of bisphenol A and triclocarban on brain-speciﬁc expression of aromatase in early zebraﬁsh
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