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Triclosan, an environmental pollutant from health care products, evokes charybdotoxin-sensitive hyperpolarization in rat thymocytes
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 2 ( 2 0 1 1 ) 417–422
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Triclosan, an environmental pollutant from health care products, evokes charybdotoxin-sensitive hyperpolarization in rat thymocytes Takuya Kawanai ∗ 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
a b s t r a c t
Article history:
The effects of triclosan, an environmental pollutant from household items and health
Received 9 May 2011
care products, on membrane potential and intracellular Ca2+ concentrations of rat thymo-
Received in revised form
cytes were examined by a flow cytometry with fluorescent probes, di-BA-C4 and fluo-3-AM,
8 August 2011
because triclosan is often found in humans and wild animals. Triclosan at a concentration
Accepted 13 August 2011
of 3 M decreased the intensity of di-BA-C4 fluorescence, indicating the triclosan-induced
Available online 22 August 2011
hyperpolarization. The application of charybdotoxin, a specific inhibitor of Ca2+ -dependent K+ channels, and the removal of external Ca2+ eliminated the triclosan-attenuation of di-
Keywords:
BA-C4 fluorescence. Furthermore, triclosan augmented the fluo-3 fluorescence under normal
Triclosan
Ca2+ condition, indicating that triclosan increased intracellular Ca2+ concentration. These
Thymocytes
results suggest that triclosan induces membrane hyperpolarization by increasing intracel-
Membrane potential
lular Ca2+ concentration that activates Ca2+ -dependent K+ channels. Since the change in
Flow cytometer
membrane potential of lymphocytes influence cellular immune functions, triclosan may exert adverse actions on immune system in human and wild animals. © 2011 Elsevier B.V. All rights reserved.
1.
Introduction
Triclosan is widely used as an antibacterial agent in a number of common household items including pharmaceutical and personal care products (Perencevich et al., 2001; Schweizer, 2001). Significant amount of triclosan is found in terrestrial and aquatic environment (Reiss et al., 2002; Heidler and Halden, 2008). The widespread use of household items containing triclosan has raised concerns regarding the compound’s impacts on the environment and human health (Rodricks et al., 2010). Triclosan inhibits plant growth and soil respiration (Liu et al., 2009). This bactericidal agent increases the rate of metamorphosis and tail fin gene expression in North American bullfrog (Veldhoen et al., 2006). Furthermore, its structural resemblance with non-steroidal estrogens
∗
Corresponding author. E-mail address: [email protected] 1382-6689/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2011.08.009
makes the compound a probable endocrine disruptor (Zorrilla et al., 2009; James et al., 2010). Although the metabolic activity of triclosan is not fully understood in humans, several studies have demonstrated probable biotransformation pathways (Fang et al., 2010). Recently, triclosan is considered to exert adverse actions on human immune functions (Clayton et al., 2011). Therefore, it is necessary to investigate the effect of triclosan on lymphocytes in order to reveal cellular basis of immunotoxicity of triclosan. The membrane potential is controlled by membrane ion permeability and transmembrane ion gradient that are influenced by diverse factors. Therefore, the membrane potential would be changed if the chemicals including environmental pollutants affect one of diverse factors. The changes in membrane potential are associated with physiological functions of lymphocytes because of the following observations.
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e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 2 ( 2 0 1 1 ) 417–422
The exposure of lymphocytes to mitogenic lectins or antibodies induces the change in membrane potential (Gallin and Livengood, 1981; Tsien et al., 1982; DeCoursey et al., 1984; Gallin, 1986; Gelfand et al., 1987). The alteration of membrane potential affects the activation process of lymphocytes and monocytes (Oettgen et al., 1985; Gelfand et al., 1987). Thus, the manipulation of membrane potential by triclosan may be one of events in the immunotoxic action. However, there is no analysis on the effect of triclosan on membrane potential of lymphocytes. In this study, we have examined the effect of triclosan on membrane potential of rat thymocytes using a flow cytometer with appropriate fluorescent indicators.
2.
Materials and methods
2.1.
Chemicals
Triclosan was purchased from Wako Pure Chemicals (Osaka, Japan). Triclosan (0.3–10 mM) was dissolved in distilled water and added to achieve final concentrations of 0.3–10 M in the cell suspension. A23187, a calcium ionophore, was used as a reference agent to increase intracellular Ca2+ . A23187 induces a hyperpolarization in rat thymocytes by an activation of Ca2+ dependent K+ channels (Oyama et al., 1992). Charybdotoxin, a specific inhibitor of Ca2+ -dependent K+ conductance, were products of Peptide Institution (Osaka, Japan). Other chemicals except for fluorescent probes were purchased from Wako Pure Chemicals (Osaka, Japan). Fluorescent probes were purchased from Molecular Probe Inc. (Eugene, Oregon, USA). A23187 at concentrations ranging from 30 nM to 300 nM was reported to induce steady Ca2+ -dependent hyperpolarization in murine thymic lymphocytes (Oyama et al., 1992; Nishizaki et al., 2003). Therefore, 100 nM A23187 was used to elicit the hyperpolarization via activation of Ca2+ -dependent K+ channel. Charybdotoxin is a specific inhibitor for Ca2+ -dependent + K channels (Miller, 1995) and it was reported that the toxin at 30 nM or more strongly suppressed the hyperpolarization induced by ionomycin, a calcium ionophore, in rat thymocytes (Grinstein and Smith, 1989). Nishizaki et al. (2003) also showed the inhibition of A23187-induced attenuation of di-BA-C4 fluorescence (hyperpolarization) by micromolar charybdotoxin. Therefore, 300 nM charybdotoxin was used in this study.
2.2.
Animals and cell preparation
This study was approved by the Committee for Animal Experiments in the University of Tokushima (No. 05279). The procedure to prepare cell suspension was similar to that previously reported (Oyama et al., 1991; Chikahisa and Oyama, 1992). In brief, thymus glands dissected from ether-anesthetized Wistar rats 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 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 Ca2+ -free Tyrode’s solution (in mM: NaCl 150, KCl 5, MgCl2 2, EDTA 1, glucose 5, HEPES 5, with an appropriate amount of NaOH to adjust pH to 7.3–7.4) to dissociate
thymocytes. Thereafter, Tyrode’s solutions containing the cells were passed through a mesh to prepare the cell suspension (about 5 × 105 cells/ml). The cell suspension was incubated at 36 ◦ C for 45–60 min before any fluorescence measurements.
2.3. Fluorescence measurements of cellular and membrane parameters Experimental methods were similar to those previously described (Oyama et al., 1991, 1992, 1995; Chikahisa and Oyama, 1992). In brief, the measurements of membrane potential and intracellular concentration of Ca2+ were made with bis-(1,3-dibutylbarbituric acid)trimethine oxonol (di-BA-C4 ) (Rink et al., 1980; Wilson and Chused, 1985) and pentaacetoxymethyl ester of fluo-3 (fluo-3-AM) (Kao et al., 1989; Minta et al., 1989), respectively. Fluorescent measurements were performed with a flow cytometer equipped with an argon laser (Cyto-ACE 150, JASCO, Tokyo, Japan). To monitor the change in membrane potential of living cells with intact membranes, di-BA-C4 was used in the combination with propidium iodide for staining dead cells and/or the cells with compromised membranes (Chikahisa and Oyama, 1992). Di-BA-C4 and propidium iodide were respectively dissolved in dimethyl sulfoxide (DMSO) and distilled water. These solutions were added into the cell suspension to achieve a final concentration of 300 nM for di-BA-C4 and 5 M for propidium iodide. The cells were incubated with di-BA-C4 for 10 min and propidium iodide for 2 min before any fluorescence measurements. Excitation wavelength for di-BA-C4 and propidium was 488 nm and the emissions were detected at 530 ± 20 nm for di-BA-C4 fluorescence and 600 ± 20 nm for propidium fluorescence. Di-BA-C4 fluorescence was measured from the cells that were not stained with propidium (living cells with intact membranes). To estimate the change in intracellular Ca2+ concentration of rat thymocytes, fluo-3-AM was used. Fluo-3-AM was also dissolved in DMSO. The cells were incubated with 500 nM fluo-3-AM for 60 min before any fluorescence measurements. Fluo-3 fluorescence was also measured from the cells that were not stained with 5 M for propidium iodide. Excitation wavelength for fluo-3 was 488 nm and the emission was detected at 530 ± 20 nm. Shifts toward increased and decreased fluorescent intensities, correspond to depolarization and hyperpolarization of membrane potential for di-BA-C4 fluorescence and increasing and decreasing intracellular Ca2+ concentration for fluo-3 fluorescence, respectively. The fluorescence histogram was obtained from 2000 cells. The sheath flow rate was adjusted to set the measurement of 195–205 cells/s and the interval of 180 s between measurements of forward and side scatters. The measurement started after achieving constant flow of cells. The time of about 10 s was required for data acquisition in the case of 2000 cells. The mean intensity of fluorescence obtained from 2000 cells was similar to that from 10,000 cells.
2.4.
Numerical expression and statistics
Statistical analysis was performed with Tukey multivariate analysis. A P value of <0.05 was considered significant. Since there was no statistical difference in mean intensity of diBA-C4 and fluo-3 fluorescence between control groups (cells
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 2 ( 2 0 1 1 ) 417–422
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Fig. 2 – Concentration-dependent change in mean intensity of di-BA-C4 fluorescence by triclosan. Each column and bar respectively indicate mean value and S.D. of four experiments. Symbol (*) indicates significant difference (P < 0.05) between control group (CONTROL) and test groups (TRICLOSAN). The intensity of di-BA-C4 fluorescence attenuated by 100 nM A23187 is assumed to be near an equilibrium potential for K+ (Wilson and Chused, 1985).
Fig. 1 – Effects of triclosan and A23187 on the histogram of di-BA-C4 fluorescence monitored from rat thymocytes under normal external Ca2+ concentration. Each histogram was constructed with 2000 living cells. Effects of triclosan and A23187 were tested at 3 min after the start of respective applications.
without a solvent, cells treated with 0. 1% DMSO, and cells treated with 0.1% ethanol), the values in figure and text were relative to the control with respective solvent (DMSO or ethanol). Values were expressed as the mean ± standard deviation of 4 experiments.
3.
Results
3.1. Triclosan-induced change in membrane potential of thymocytes As shown in Fig. 1, the histogram of di-BA-C4 fluorescence shifted toward a lower intensity in presence of 3 M triclosan or 100 nM A23187, indicating that both triclosan and A23187 induced hyperpolarization. A steady state of both of triclosan and A23187 action were attained within 1–3 min after the applications. Triclosan at concentrations of 1 M or lower did not significantly affect the di-BA-C4 fluorescence histogram of
thymocytes incubated with normal Tyrode’s solution (Fig. 2), indicating no effect on membrane potential. A significant decrease in intensity of di-BA-C4 fluorescence occurred and reached its maximal level within 1–3 min after adding 3 M triclosan. The largest attenuation of di-BA-C4 fluorescence (largest hyperpolarization) was observed in the case of 3 M triclosan (Fig. 2). However, the degree of hyperpolarization induced by triclosan was much smaller than that induced by 100 nM A23187 (Figs. 1 and 2), where the membrane potential of thymocytes seems to reach an equilibrium potential for K+ (Wilson and Chused, 1985). On the other hand, the application of 10 M triclosan greatly increased the intensity of di-BA-C4 fluorescence, indicating depolarization. In the continued presence of 10 M triclosan, the cell lethality was increased. It is thought that the depolarization induced by 10 M triclosan may be due to non-specific increase in membrane permeability. In the experiments below, therefore, the property of triclosan-induced hyperpolarization was studied. In the presence of 300 nM charybdotoxin, 3 M triclosan and 100 nM A23187 significantly increased the intensity of diBA-C4 fluorescence, indicating the depolarization induced by triclosan and A23187 (Fig. 3). Thus, charybdotoxin completely inhibited the attenuation of di-BA-C4 fluorescence respectively induced by 3 M triclosan and 100 nM A23187. Since charybdotoxin is a specific inhibitor for Ca2+ -dependent K+ channels (Garcia et al., 1995; Miller, 1995) and A23187 is a calcium ionophore (Reed and Lardy, 1972), the experiment was performed to see if external Ca2+ is involved in the triclosan-induced attenuation of di-BA-C4 fluorescence. Under
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e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 2 ( 2 0 1 1 ) 417–422
Fig. 3 – Effects of triclosan and A23187 on di-BA-C4 fluorescence in absence (upper panel) and presence (lower panel) of charybdotoxin under normal Ca2+ condition. Each column and bar respectively indicate mean value and S.D. of four experiments. Symbol (*) indicates significant difference (P < 0.05) between control group (CONTROL) and test group of cells (TRICLOSAN or A23187).
external Ca2+ -free condition, the application of 3 M triclosan significantly increased the intensity of di-BA-C4 fluorescence (Fig. 4), suggesting the involvement of external Ca2+ in the triclosan-induced hyperpolarization. A23187 at 100 nM also increased the intensity of di-BA-C4 fluorescence under external Ca2+ -free condition (Fig. 4). However, the increase was not statistically significant. Taken together, it is suggested that Ca2+ -dependent K+ channels is involved in the triclosaninduced hyperpolarization.
3.2. Triclosan-induced change in intracellular Ca2+ concentration of thymocytes The activation of Ca2+ -dependent K+ channels requires the increase in intracellular Ca2+ concentration (Meech, 1978). The results described above suggest that hyperpolarization is linked to the increase in intracellular Ca2+ concentration of rat thymocytes by triclosan. Therefore, the effect of triclosan on fluo-3 fluorescence was examined to see if triclosan increases intracellular Ca2+ concentration. The application of 3 M triclosan shifted the histogram of fluo-3 fluorescence to the direction of higher intensity (Fig. 5A). It was also the case for 1 M triclosan (Fig. 5B). Removal of external Ca2+ greatly attenuated the triclosan-induced augmentation of fluo-3 fluorescence (not shown). The results suggest that micromolar triclosan increases the intracellular Ca2+ concentration of lymphocytes by promoting Ca2+ influx.
Fig. 4 – Effects of triclosan and A23187 on di-BA-C4 fluorescence under normal Ca2+ (upper panel) and external Ca2+ -free (lower panel) conditions. Each column and bar indicates mean value and S.D. of four experiments. Symbol (*) indicates significant difference (P < 0.05) between control group (CONTROL) and test group (TRICLOSAN or A23187).
4.
Discussion
4.1.
Concentration of triclosan
The threshold concentration of triclosan to affect membrane potential of rat thymocytes seems to be 1–3 M (Fig. 2). The plasma concentrations of triclosan in humans were reported to be 0.4–38 g/l (Bagley and Lin, 2000; Allmyr et al., 2006). Thus, it is unlikely that plasma triclosan directly affects cell membranes in humans. However, this compound is lipophilic (Kow = 4.8), the concentration in fatty tissues may be much higher than the plasma concentration. The concentrations of triclosan were reported to be 60–300 g/kg lipid weight in some samples of maternal milk and 240–900 g/kg fresh weight in bile of wild living fishes, respectively (Adolfsson-Erici et al., 2002). The concentration of triclosan may reach micromolar concentrations in fatty tissues of wild animals resided in aquatic environment.
4.2.
Effect of triclosan on membrane potential
From the results of Figs. 2–4, it is likely that triclosan at 3 M hyperpolarizes membranes of rat thymocytes by activation of Ca2+ -dependent K+ channels. Membranes of thymocytes possess Ca2+ -dependent K+ conductance (Wilson and Chused, 1985; Mahaut-Smith and Schlichter, 1989; Mahaut-Smith and Manson, 1991). The activation of Ca2+ -dependent K+ channels requires an increase in intracellular Ca2+ concentration
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 2 ( 2 0 1 1 ) 417–422
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and 3 M triclosan under external Ca2+ -free concentration. At present, the mechanism of triclosan-induced depolarization is not elucidated. Triclosan at 10 M increased the population of cells stained with propidium iodide. Propidium cannot pass through intact cell membranes, but freely enters cells with compromised cell membranes (Coder, 1997). Thus, the treatment with 10 M triclosan non-specifically increases membrane ionic permeability. The non-specific increase in membrane ionic permeability decreases transmembrane ionic gradient, leading to the shift of membrane potential to depolarizing direction. Although some further studies are necessary to elucidate the mechanism for the depolarization induced by 3 M triclosan under external Ca2+ -free condition, it is interesting because of the following reason. Since the hyperpolarization induced by triclosan is dependent on the activation of Ca2+ -dependent K+ channels, triclosan at low micromolar concentrations may depolarize the membranes of cell lacking Ca2+ -dependent K+ channels that are enough to hyperpolarize membranes. Therefore, it is plausibly suggested that the action of triclosan on membrane potential varies in different cells.
Conflicts of interest The authors declare that there are no conflicts of interest. Fig. 5 – Effect of triclosan on fluo-3 fluorescence under normal Ca2+ condition. (A) Change in the histogram of fluo-3 fluorescence at 3 min after applying 3 M triclosan. (B) Triclosan-induced increase in mean intensity of fluo-3 fluorescence. Each column and bar respectively indicates mean value and S.D. of four experiments. Symbols (* and **) indicate significant difference (P < 0.05 and P < 0.01, respectively) between control group (CONTROL) and test group (TRICLOSAN).
references
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Triclosan, an environmental pollutant from health care products, evokes charybdotoxin-sensitive hyperpolarization in rat thymocytes Takuya Kawanai ∗ 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
a b s t r a c t
Article history:
The effects of triclosan, an environmental pollutant from household items and health
Received 9 May 2011
care products, on membrane potential and intracellular Ca2+ concentrations of rat thymo-
Received in revised form
cytes were examined by a flow cytometry with fluorescent probes, di-BA-C4 and fluo-3-AM,
8 August 2011
because triclosan is often found in humans and wild animals. Triclosan at a concentration
Accepted 13 August 2011
of 3 M decreased the intensity of di-BA-C4 fluorescence, indicating the triclosan-induced
Available online 22 August 2011
hyperpolarization. The application of charybdotoxin, a specific inhibitor of Ca2+ -dependent K+ channels, and the removal of external Ca2+ eliminated the triclosan-attenuation of di-
Keywords:
BA-C4 fluorescence. Furthermore, triclosan augmented the fluo-3 fluorescence under normal
Triclosan
Ca2+ condition, indicating that triclosan increased intracellular Ca2+ concentration. These
Thymocytes
results suggest that triclosan induces membrane hyperpolarization by increasing intracel-
Membrane potential
lular Ca2+ concentration that activates Ca2+ -dependent K+ channels. Since the change in
Flow cytometer
membrane potential of lymphocytes influence cellular immune functions, triclosan may exert adverse actions on immune system in human and wild animals. © 2011 Elsevier B.V. All rights reserved.
1.
Introduction
Triclosan is widely used as an antibacterial agent in a number of common household items including pharmaceutical and personal care products (Perencevich et al., 2001; Schweizer, 2001). Significant amount of triclosan is found in terrestrial and aquatic environment (Reiss et al., 2002; Heidler and Halden, 2008). The widespread use of household items containing triclosan has raised concerns regarding the compound’s impacts on the environment and human health (Rodricks et al., 2010). Triclosan inhibits plant growth and soil respiration (Liu et al., 2009). This bactericidal agent increases the rate of metamorphosis and tail fin gene expression in North American bullfrog (Veldhoen et al., 2006). Furthermore, its structural resemblance with non-steroidal estrogens
∗
Corresponding author. E-mail address: [email protected] 1382-6689/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2011.08.009
makes the compound a probable endocrine disruptor (Zorrilla et al., 2009; James et al., 2010). Although the metabolic activity of triclosan is not fully understood in humans, several studies have demonstrated probable biotransformation pathways (Fang et al., 2010). Recently, triclosan is considered to exert adverse actions on human immune functions (Clayton et al., 2011). Therefore, it is necessary to investigate the effect of triclosan on lymphocytes in order to reveal cellular basis of immunotoxicity of triclosan. The membrane potential is controlled by membrane ion permeability and transmembrane ion gradient that are influenced by diverse factors. Therefore, the membrane potential would be changed if the chemicals including environmental pollutants affect one of diverse factors. The changes in membrane potential are associated with physiological functions of lymphocytes because of the following observations.
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The exposure of lymphocytes to mitogenic lectins or antibodies induces the change in membrane potential (Gallin and Livengood, 1981; Tsien et al., 1982; DeCoursey et al., 1984; Gallin, 1986; Gelfand et al., 1987). The alteration of membrane potential affects the activation process of lymphocytes and monocytes (Oettgen et al., 1985; Gelfand et al., 1987). Thus, the manipulation of membrane potential by triclosan may be one of events in the immunotoxic action. However, there is no analysis on the effect of triclosan on membrane potential of lymphocytes. In this study, we have examined the effect of triclosan on membrane potential of rat thymocytes using a flow cytometer with appropriate fluorescent indicators.
2.
Materials and methods
2.1.
Chemicals
Triclosan was purchased from Wako Pure Chemicals (Osaka, Japan). Triclosan (0.3–10 mM) was dissolved in distilled water and added to achieve final concentrations of 0.3–10 M in the cell suspension. A23187, a calcium ionophore, was used as a reference agent to increase intracellular Ca2+ . A23187 induces a hyperpolarization in rat thymocytes by an activation of Ca2+ dependent K+ channels (Oyama et al., 1992). Charybdotoxin, a specific inhibitor of Ca2+ -dependent K+ conductance, were products of Peptide Institution (Osaka, Japan). Other chemicals except for fluorescent probes were purchased from Wako Pure Chemicals (Osaka, Japan). Fluorescent probes were purchased from Molecular Probe Inc. (Eugene, Oregon, USA). A23187 at concentrations ranging from 30 nM to 300 nM was reported to induce steady Ca2+ -dependent hyperpolarization in murine thymic lymphocytes (Oyama et al., 1992; Nishizaki et al., 2003). Therefore, 100 nM A23187 was used to elicit the hyperpolarization via activation of Ca2+ -dependent K+ channel. Charybdotoxin is a specific inhibitor for Ca2+ -dependent + K channels (Miller, 1995) and it was reported that the toxin at 30 nM or more strongly suppressed the hyperpolarization induced by ionomycin, a calcium ionophore, in rat thymocytes (Grinstein and Smith, 1989). Nishizaki et al. (2003) also showed the inhibition of A23187-induced attenuation of di-BA-C4 fluorescence (hyperpolarization) by micromolar charybdotoxin. Therefore, 300 nM charybdotoxin was used in this study.
2.2.
Animals and cell preparation
This study was approved by the Committee for Animal Experiments in the University of Tokushima (No. 05279). The procedure to prepare cell suspension was similar to that previously reported (Oyama et al., 1991; Chikahisa and Oyama, 1992). In brief, thymus glands dissected from ether-anesthetized Wistar rats 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 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 Ca2+ -free Tyrode’s solution (in mM: NaCl 150, KCl 5, MgCl2 2, EDTA 1, glucose 5, HEPES 5, with an appropriate amount of NaOH to adjust pH to 7.3–7.4) to dissociate
thymocytes. Thereafter, Tyrode’s solutions containing the cells were passed through a mesh to prepare the cell suspension (about 5 × 105 cells/ml). The cell suspension was incubated at 36 ◦ C for 45–60 min before any fluorescence measurements.
2.3. Fluorescence measurements of cellular and membrane parameters Experimental methods were similar to those previously described (Oyama et al., 1991, 1992, 1995; Chikahisa and Oyama, 1992). In brief, the measurements of membrane potential and intracellular concentration of Ca2+ were made with bis-(1,3-dibutylbarbituric acid)trimethine oxonol (di-BA-C4 ) (Rink et al., 1980; Wilson and Chused, 1985) and pentaacetoxymethyl ester of fluo-3 (fluo-3-AM) (Kao et al., 1989; Minta et al., 1989), respectively. Fluorescent measurements were performed with a flow cytometer equipped with an argon laser (Cyto-ACE 150, JASCO, Tokyo, Japan). To monitor the change in membrane potential of living cells with intact membranes, di-BA-C4 was used in the combination with propidium iodide for staining dead cells and/or the cells with compromised membranes (Chikahisa and Oyama, 1992). Di-BA-C4 and propidium iodide were respectively dissolved in dimethyl sulfoxide (DMSO) and distilled water. These solutions were added into the cell suspension to achieve a final concentration of 300 nM for di-BA-C4 and 5 M for propidium iodide. The cells were incubated with di-BA-C4 for 10 min and propidium iodide for 2 min before any fluorescence measurements. Excitation wavelength for di-BA-C4 and propidium was 488 nm and the emissions were detected at 530 ± 20 nm for di-BA-C4 fluorescence and 600 ± 20 nm for propidium fluorescence. Di-BA-C4 fluorescence was measured from the cells that were not stained with propidium (living cells with intact membranes). To estimate the change in intracellular Ca2+ concentration of rat thymocytes, fluo-3-AM was used. Fluo-3-AM was also dissolved in DMSO. The cells were incubated with 500 nM fluo-3-AM for 60 min before any fluorescence measurements. Fluo-3 fluorescence was also measured from the cells that were not stained with 5 M for propidium iodide. Excitation wavelength for fluo-3 was 488 nm and the emission was detected at 530 ± 20 nm. Shifts toward increased and decreased fluorescent intensities, correspond to depolarization and hyperpolarization of membrane potential for di-BA-C4 fluorescence and increasing and decreasing intracellular Ca2+ concentration for fluo-3 fluorescence, respectively. The fluorescence histogram was obtained from 2000 cells. The sheath flow rate was adjusted to set the measurement of 195–205 cells/s and the interval of 180 s between measurements of forward and side scatters. The measurement started after achieving constant flow of cells. The time of about 10 s was required for data acquisition in the case of 2000 cells. The mean intensity of fluorescence obtained from 2000 cells was similar to that from 10,000 cells.
2.4.
Numerical expression and statistics
Statistical analysis was performed with Tukey multivariate analysis. A P value of <0.05 was considered significant. Since there was no statistical difference in mean intensity of diBA-C4 and fluo-3 fluorescence between control groups (cells
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Fig. 2 – Concentration-dependent change in mean intensity of di-BA-C4 fluorescence by triclosan. Each column and bar respectively indicate mean value and S.D. of four experiments. Symbol (*) indicates significant difference (P < 0.05) between control group (CONTROL) and test groups (TRICLOSAN). The intensity of di-BA-C4 fluorescence attenuated by 100 nM A23187 is assumed to be near an equilibrium potential for K+ (Wilson and Chused, 1985).
Fig. 1 – Effects of triclosan and A23187 on the histogram of di-BA-C4 fluorescence monitored from rat thymocytes under normal external Ca2+ concentration. Each histogram was constructed with 2000 living cells. Effects of triclosan and A23187 were tested at 3 min after the start of respective applications.
without a solvent, cells treated with 0. 1% DMSO, and cells treated with 0.1% ethanol), the values in figure and text were relative to the control with respective solvent (DMSO or ethanol). Values were expressed as the mean ± standard deviation of 4 experiments.
3.
Results
3.1. Triclosan-induced change in membrane potential of thymocytes As shown in Fig. 1, the histogram of di-BA-C4 fluorescence shifted toward a lower intensity in presence of 3 M triclosan or 100 nM A23187, indicating that both triclosan and A23187 induced hyperpolarization. A steady state of both of triclosan and A23187 action were attained within 1–3 min after the applications. Triclosan at concentrations of 1 M or lower did not significantly affect the di-BA-C4 fluorescence histogram of
thymocytes incubated with normal Tyrode’s solution (Fig. 2), indicating no effect on membrane potential. A significant decrease in intensity of di-BA-C4 fluorescence occurred and reached its maximal level within 1–3 min after adding 3 M triclosan. The largest attenuation of di-BA-C4 fluorescence (largest hyperpolarization) was observed in the case of 3 M triclosan (Fig. 2). However, the degree of hyperpolarization induced by triclosan was much smaller than that induced by 100 nM A23187 (Figs. 1 and 2), where the membrane potential of thymocytes seems to reach an equilibrium potential for K+ (Wilson and Chused, 1985). On the other hand, the application of 10 M triclosan greatly increased the intensity of di-BA-C4 fluorescence, indicating depolarization. In the continued presence of 10 M triclosan, the cell lethality was increased. It is thought that the depolarization induced by 10 M triclosan may be due to non-specific increase in membrane permeability. In the experiments below, therefore, the property of triclosan-induced hyperpolarization was studied. In the presence of 300 nM charybdotoxin, 3 M triclosan and 100 nM A23187 significantly increased the intensity of diBA-C4 fluorescence, indicating the depolarization induced by triclosan and A23187 (Fig. 3). Thus, charybdotoxin completely inhibited the attenuation of di-BA-C4 fluorescence respectively induced by 3 M triclosan and 100 nM A23187. Since charybdotoxin is a specific inhibitor for Ca2+ -dependent K+ channels (Garcia et al., 1995; Miller, 1995) and A23187 is a calcium ionophore (Reed and Lardy, 1972), the experiment was performed to see if external Ca2+ is involved in the triclosan-induced attenuation of di-BA-C4 fluorescence. Under
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Fig. 3 – Effects of triclosan and A23187 on di-BA-C4 fluorescence in absence (upper panel) and presence (lower panel) of charybdotoxin under normal Ca2+ condition. Each column and bar respectively indicate mean value and S.D. of four experiments. Symbol (*) indicates significant difference (P < 0.05) between control group (CONTROL) and test group of cells (TRICLOSAN or A23187).
external Ca2+ -free condition, the application of 3 M triclosan significantly increased the intensity of di-BA-C4 fluorescence (Fig. 4), suggesting the involvement of external Ca2+ in the triclosan-induced hyperpolarization. A23187 at 100 nM also increased the intensity of di-BA-C4 fluorescence under external Ca2+ -free condition (Fig. 4). However, the increase was not statistically significant. Taken together, it is suggested that Ca2+ -dependent K+ channels is involved in the triclosaninduced hyperpolarization.
3.2. Triclosan-induced change in intracellular Ca2+ concentration of thymocytes The activation of Ca2+ -dependent K+ channels requires the increase in intracellular Ca2+ concentration (Meech, 1978). The results described above suggest that hyperpolarization is linked to the increase in intracellular Ca2+ concentration of rat thymocytes by triclosan. Therefore, the effect of triclosan on fluo-3 fluorescence was examined to see if triclosan increases intracellular Ca2+ concentration. The application of 3 M triclosan shifted the histogram of fluo-3 fluorescence to the direction of higher intensity (Fig. 5A). It was also the case for 1 M triclosan (Fig. 5B). Removal of external Ca2+ greatly attenuated the triclosan-induced augmentation of fluo-3 fluorescence (not shown). The results suggest that micromolar triclosan increases the intracellular Ca2+ concentration of lymphocytes by promoting Ca2+ influx.
Fig. 4 – Effects of triclosan and A23187 on di-BA-C4 fluorescence under normal Ca2+ (upper panel) and external Ca2+ -free (lower panel) conditions. Each column and bar indicates mean value and S.D. of four experiments. Symbol (*) indicates significant difference (P < 0.05) between control group (CONTROL) and test group (TRICLOSAN or A23187).
4.
Discussion
4.1.
Concentration of triclosan
The threshold concentration of triclosan to affect membrane potential of rat thymocytes seems to be 1–3 M (Fig. 2). The plasma concentrations of triclosan in humans were reported to be 0.4–38 g/l (Bagley and Lin, 2000; Allmyr et al., 2006). Thus, it is unlikely that plasma triclosan directly affects cell membranes in humans. However, this compound is lipophilic (Kow = 4.8), the concentration in fatty tissues may be much higher than the plasma concentration. The concentrations of triclosan were reported to be 60–300 g/kg lipid weight in some samples of maternal milk and 240–900 g/kg fresh weight in bile of wild living fishes, respectively (Adolfsson-Erici et al., 2002). The concentration of triclosan may reach micromolar concentrations in fatty tissues of wild animals resided in aquatic environment.
4.2.
Effect of triclosan on membrane potential
From the results of Figs. 2–4, it is likely that triclosan at 3 M hyperpolarizes membranes of rat thymocytes by activation of Ca2+ -dependent K+ channels. Membranes of thymocytes possess Ca2+ -dependent K+ conductance (Wilson and Chused, 1985; Mahaut-Smith and Schlichter, 1989; Mahaut-Smith and Manson, 1991). The activation of Ca2+ -dependent K+ channels requires an increase in intracellular Ca2+ concentration
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and 3 M triclosan under external Ca2+ -free concentration. At present, the mechanism of triclosan-induced depolarization is not elucidated. Triclosan at 10 M increased the population of cells stained with propidium iodide. Propidium cannot pass through intact cell membranes, but freely enters cells with compromised cell membranes (Coder, 1997). Thus, the treatment with 10 M triclosan non-specifically increases membrane ionic permeability. The non-specific increase in membrane ionic permeability decreases transmembrane ionic gradient, leading to the shift of membrane potential to depolarizing direction. Although some further studies are necessary to elucidate the mechanism for the depolarization induced by 3 M triclosan under external Ca2+ -free condition, it is interesting because of the following reason. Since the hyperpolarization induced by triclosan is dependent on the activation of Ca2+ -dependent K+ channels, triclosan at low micromolar concentrations may depolarize the membranes of cell lacking Ca2+ -dependent K+ channels that are enough to hyperpolarize membranes. Therefore, it is plausibly suggested that the action of triclosan on membrane potential varies in different cells.
Conflicts of interest The authors declare that there are no conflicts of interest. Fig. 5 – Effect of triclosan on fluo-3 fluorescence under normal Ca2+ condition. (A) Change in the histogram of fluo-3 fluorescence at 3 min after applying 3 M triclosan. (B) Triclosan-induced increase in mean intensity of fluo-3 fluorescence. Each column and bar respectively indicates mean value and S.D. of four experiments. Symbols (* and **) indicate significant difference (P < 0.05 and P < 0.01, respectively) between control group (CONTROL) and test group (TRICLOSAN).
references
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