Elevation of the intracellular Zn²⁺ level by 2-n-octyl-4-isothiazolin-3-one in rat thymocytes: an involvement of a temperature-sensitive Zn²⁺ pathway

Abstract

High amounts of 2-n-octyl-4-isothiazolin-3-one (OIT), an antimicrobial, are found in wet polyvinyl alcohol towels with cooling properties. Although the diverse actions of OIT are of concern, information on its cellular actions is limited. In this study, we examined the effects of OIT on Zn²⁺ levels in rat thymocytes by flow cytometric analysis with FluoZin-3, and assessed the cytotoxicity of this agent. OIT (1–3 μM) significantly increased the intensity of the FluoZin-3 fluorescence. Removal of extracellular Zn²⁺ almost completely inhibited the OIT-induced increase in FluoZin-3 fluorescence. Furthermore, the increase in OIT-induced FluoZin-3 fluorescence was attenuated at cold temperatures. The effect of OIT on the FluoZin-3 fluorescence was dependent on the transmembrane Zn²⁺ gradient. Based on our data, we concluded that OIT activates a temperature-sensitive, bi-directional Zn²⁺ pathway, resulting in alterations to the intracellular Zn²⁺ levels. Importantly, these changes are dependent on a transmembrane Zn²⁺ gradient. These results may provide insight into the cytotoxicity of OIT, because Zn²⁺ has physiological and pathological roles in cellular functions.

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Introduction

2-n-Octyl-4-isothiazolin-3-one (OIT; CAS 26530-20-1) is an isothiazolinone antimicrobial agent known to cause both occupational and non-occupational contact dermatitis.^1–3 OIT is used as a preservative in polyvinyl alcohol (PVA) products, including the PVA water-absorbing towel, an eco-friendly product with cooling properties.⁴ PVA towels are supplied to schoolchildren to avoid heatstroke in some areas of Japan. A recent report indicates that PVA towels contain 208–531 μg g⁻¹ wet weight and 9.9–281 μg g⁻¹ wet weight of OIT before and after washing, respectively.⁵ OIT is readily absorbed through the skin both in vivo and in vitro.⁶ Since infants can be exposed to OIT by sucking wet PVA towels, the possibility of adverse effects is a concern. However, there is limited information on the cellular effects of OIT exposure, although high micromolar levels of OIT were tested in Chinese hamster ovary cells and in cultured hepatocytes from Sprague–Dawley rats.⁶

In this study, we examined the effects of OIT on rat thymocytes by flow cytometry, with appropriate fluorescent probes to characterize the cytotoxicity of OIT. Thymocytes were used as an experimental model for a number of reasons. First, since thymocytes are dissociated fresh without enzymatic treatment, the cell membranes remain intact. Second, the mechanisms of cell death (apoptosis, necrosis, and autophagy) in thymocytes have been studied in detail.^7–9 Finally, several hormones, biological compounds, and chemicals, including environmental pollutants, can induce apoptosis and necrosis in thymocytes.¹⁰

The effect of OIT on the intracellular Zn²⁺ level was examined because Zn²⁺ is involved in thymocyte cell death.¹¹ Furthermore, our previous studies indicate that Zn²⁺ can potentiate the cytotoxicity of non-ionic surfactants, imidazole antifungals, and hydrogen peroxide.^12–14 We propose that low micromolar levels of OIT activate a temperature-sensitive Zn²⁺ pathway in thymocyte membranes, resulting in an increase in intracellular Zn²⁺ levels under normal conditions. This study may provide insight into the toxicity of OIT because Zn²⁺ plays a number of physiological and pathological roles in the cell^15–17 and can affect several aspects of the human immune system by acting as a molecular signal for immune cells.¹⁷

Materials and methods

Chemicals

OIT was purchased from Tokyo Kasei Kogyo Co., Ltd (Tokyo, Japan). The purity of OIT was 99.4%. Propidium iodide, FluoZin-3-AM, and bis-(1,3-dibutylbarbituric acid)trimethine oxonol (Oxonol) were obtained from Molecular Probes Inc., Invitrogen (Eugene, OR, USA). Fluo-3-AM and the Zn²⁺ chelators, diethylenetriamine-N,N,N′,N′′,N′′-pentaacetic acid (DTPA) and N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), were obtained from Dojin Chemical Laboratory (Kumamoto, Japan). A23187, an ionophore for divalent metal cations, such as Ca²⁺, Cd²⁺, Zn²⁺, and Mn²⁺, was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Other chemicals were obtained from Wako Pure Chemicals (Osaka, Japan).

Animals and cell preparation

This study was approved by the Committee for Animal Experiments at the University of Tokushima (no. 05279).

The cell suspension was prepared as previously reported.^12,18 In brief, thymus glands dissected from ether-anesthetized rats were sliced under cold conditions (2–4 °C). The slices were triturated in chilled Tyrode's solution to dissociate the thymocytes. The cell-containing solution was then passed through a 10 μm diameter mesh to prepare the cell suspension. The cell suspension was incubated at 36–37 °C for 1 h before the experiment. Importantly, cell suspension contained 200–230 nM zinc derived from the cell preparation, although the Tyrode's solution does not contain zinc salts.¹⁹

Various concentrations of OIT (0.03–3 mM of OIT in 2 μL of DMSO) were added to cell suspensions (2 mL per test tube) and incubated at 36–37 °C. The incubation was prolonged with the agent for 1–3 h. Thereafter, 100 μL of each cell suspension was analyzed by flow cytometry to assess the 1,4-naphthoquinone-induced changes in cellular parameters. Fluorescence data acquisition from 2 × 10³ cells or 2.5 × 10³ cells required 10–15 s.

Fluorescence measurements of cellular and membrane parameters

Cellular and membrane parameters were measured using a flow cytometer equipped with an argon laser (CytoACE-150; JASCO, Tokyo, Japan). The fluorescent probes were similar to those previously described.^12,18 Fluorescence was analyzed using the JASCO software package (Version 3.06; JASCO, Tokyo, Japan). No fluorescence was detected from the reagents used in the study under experimental conditions, with the exception of the fluorescent probes.

To assess cell death, propidium iodide (5 μM) was added to the cell suspensions. The propidium fluorescence, a marker of dead cell, was measured at least 2 min after the application of propidium iodide using a flow cytometer. The excitation wavelength used for propidium iodide was 488 nm and the fluorescence emission wavelength was detected at 600 ± 20 nm.

FluoZin-3-AM²⁰ was used as an indicator of intracellular Zn²⁺ levels. The cells were incubated with 500 nM FluoZin-3-AM for 60 min before evaluating changes in intracellular Zn²⁺ in intact thymocytes. Importantly, FluoZin-3 fluorescence was measured in the cells that were not stained with propidium iodide.¹² The excitation wavelength used for FluoZin-3 was 488 nm and the fluorescence emission was detected at 530 ± 20 nm.

Fluo-3-AM was used to monitor changes in intracellular Ca²⁺ levels.²¹ The cells were incubated with 500 nM Fluo-3-AM for 60 min before assaying fluorescence. Fluo-3 fluorescence was measured in cells that were not stained with propidium iodide. The excitation wavelength used for Fluo-3 was 488 nm, and the fluorescence emission was detected at 530 ± 15 nm.

To monitor changes in membrane potential in intact living cells, Oxonol was used in combination with propidium iodide.²² The final concentration of Oxonol was 300 nM. The excitation wavelength for Oxonol was 488 nm and the fluorescence emission was detected at 530 ± 20 nm. Oxonol fluorescence was measured in propidium negative cells.

Statistical analysis

Values were expressed as the mean ± standard deviation of 4 samples. Statistical analysis was performed with Tukey's multivariate analysis. Each experiment was repeated 2–3 times. A p value < 0.05 was considered significant.

Results

Changes in cell lethality and FluoZin-3 fluorescence by OIT

We first assessed the cell lethality in the presence of OIT. The incubation of cells with OIT (0.03–3 μM) for 1–3 h did not change the population of cells stained with propidium iodide. Thus, the cell viability was not changed after incubation with 0.03–3 μM OIT for 1–3 h. In this study, the effects induced by OIT occurred at sublethal concentrations.

As shown in Fig. 1, the application of 3 μM OIT shifted the histogram of FluoZin-3 fluorescence to a higher intensity in a time-dependent manner. The peak fluorescence intensity was attained 45–60 min after OIT application (Fig. 2A). No additional increase in FluoZin-3 fluorescence was observed 90 min after application of OIT (0.3 μM) as compared to 60 min. Further increases in FluoZin-3 fluorescence were not observed in the continued presence of 0.3 μM OIT. Thus, the concentration-dependent change in the mean intensity of FluoZin-3 fluorescence was examined 60 min after treatment with OIT (0.03–3 μM). Treatment with 1–3 μM OIT significantly increased the mean fluorescence intensity of FluoZin-3 (Fig. 2B).

Fig. 1 OIT-induced shift of the FluoZin-3 fluorescence histogram. The shift of the histogram to a higher intensity indicates an elevation in the intracellular Zn²⁺ level. It is noted that the abscissa is displayed using a log scale. Each histogram was obtained from 2500 cells. The histograms were obtained 5 min (left panel) and 30 min (right panel) after the start of OIT application.

Fig. 2 Time- and concentration-dependent changes in the intensity of FluoZin-3 fluorescence by OIT. (A) Time-dependent change after treatment with 3 μM OIT. This experiment was repeated twice. (B) Concentration-dependent change following OIT application for 60 min at concentrations ranging from 0.03 μM to 3 μM. This experiment was repeated three times. The columns and bars indicate the mean and standard deviation of four samples. Asterisks (**) indicate significant difference (p < 0.01) between the control and test groups.

Effects of DTPA and TPEN on the OIT-induced change in FluoZin-3 fluorescence

The effect of OIT was examined under nominally Zn²⁺-free conditions to identify the source(s) of Zn²⁺ responsible for the OIT-induced change in FluoZin-3 fluorescence. The increase in the fluorescence intensity of FluoZin-3 by 3 μM OIT was completely abolished in the presence of 10 μM DTPA or 10 μM TPEN, membrane-impermeable and permeable Zn²⁺ chelators, respectively. Thus, the augmentation of FluoZin-3 fluorescence by OIT was lost when extracellular Zn²⁺ was chelated (Fig. 3).

Fig. 3 Changes in the fluorescence intensity of FluoZin-3 by 3 μM OIT in the presence of DTPA or TPEN. DTPA or TPEN was added to the cell suspension 10 min before the application of OIT. Open column; control. Filled column; 3 μM OIT. The columns and bars indicate the mean and standard deviation of four samples. Asterisks (**) indicate a significant difference in the pair indicated with an arrow. These experiments were repeated three times.

Changes in Fluo-3 and Oxonol fluorescence by OIT

To test the possibility that OIT non-specifically increases membrane ionic permeability to augment FluoZin-3 fluorescence, the effects of OIT on Fluo-3 and Oxonol fluorescence were examined. If OIT non-specifically increased membrane ionic permeability under normal Ca²⁺ conditions, OIT would also augment Fluo-3 fluorescence via Ca²⁺ influx by transmembrane Ca²⁺ gradient and Oxonol fluorescence via membrane depolarization. In brief, non-specific increases in membrane ionic permeability can theoretically cause membrane depolarization according to the Nernst equation.

As shown in Fig. 4A, 3 μM OIT slightly increased the intensity of Fluo-3 fluorescence. Addition of 2 mM MnCl₂, which quenches Fluo-3 fluorescence, slightly attenuated the Fluo-3 fluorescence intensity that was augmented by 3 μM OIT. Under extracellular Ca²⁺-free conditions, the increase in Fluo-3 fluorescence by 3 μM OIT was attenuated. Additionally, 3 μM OIT slightly decreased the intensity of Oxonol fluorescence (Fig. 4B). A23187, a Ca²⁺ ionophore, greatly decreased the intensity of Oxonol fluorescence through the activation of Ca²⁺-dependent K⁺ channels. Thus, it is unlikely that 3 μM OIT non-specifically increases membrane permeability, although OIT may slightly increase Ca²⁺ membrane permeability.

Fig. 4 Effect of OIT on calcium influx and membrane permeability. (A) Changes in OIT- or A23187-induced augmentation of Fluo-3 fluorescence by 2 mM MnCl₂. The increase in Fluo-3 fluorescence by A23187 was greatly attenuated by MnCl₂. The columns and bars indicate the mean and standard deviation of four samples. The experiments were repeated twice. (B) Changes in Oxonol fluorescence by 3 μM OIT or 100 nM A23187. A shift toward a increased or decreased fluorescence intensity corresponds to depolarization and hyperpolarization of the membrane potential, respectively. The columns and bars indicate the mean and standard deviation of four samples.

OIT-induced changes in FluoZin-3 fluorescence under low temperature conditions

At low temperatures (3–4 °C), increases in intracellular Zn²⁺ levels by externally applied Zn²⁺ were significantly attenuated.²³ Thus, the membranes of rat thymocytes seem to possess a temperature-sensitive Zn²⁺ pathway.²³ To determine whether the temperature-sensitive pathway is involved in OIT-induced increases in FluoZin-3 fluorescence, the effect of OIT was tested at low temperatures (2–4 °C).

As shown in Fig. 5, the increase in FluoZin-3 intensity by 3 μM OIT was significantly attenuated under low temperature conditions. Interestingly, warming to 36–37 °C greatly augmented the FluoZin-3 fluorescence in OIT-treated cells, but not in control cells.

Fig. 5 Changes in the fluorescence intensity of FluoZin-3 following 3 μM OIT treatment at cold temperatures (2–4 °C). The augmentation of FluoZin-3 fluorescence by OIT was attenuated under cold conditions. The columns and bars indicate the mean and standard deviation of four samples. The experiments were repeated three times.

Directionality of the OIT-activated Zn²⁺ pathway

The increase in FluoZin-3 fluorescence intensity following OIT treatment occurred in the presence of extracellular Zn²⁺ (Fig. 1–3). To determine whether the pathway carrying Zn²⁺ functioned in a bi-directional manner, the change in FluoZin-3 fluorescence by 3 μM OIT was studied using cells pre-loaded with Zn²⁺ and assayed in the absence of extracellular Zn²⁺.

As shown in Fig. 6, cells initially incubated with 3 μM ZnCl₂ were treated with 10 μM DTPA to create extracellular Zn²⁺-free conditions. OIT at 3 μM decreased the intensity of FluoZin-3 fluorescence in the presence of DTPA (Fig. 6).

Fig. 6 Dependence of OIT actions on a transmembrane Zn²⁺ gradient. (A) OIT-induced changes in FluoZin-3 fluorescence under control and external Zn²⁺-free conditions (the absence and presence of DTPA). Cell suspension contains 200–230 nM zinc that is derived during the cell preparation, although Tyrode's solution does not contain zinc salts.¹⁹ (B) Effect of increasing the transmembrane Zn²⁺ gradient using 3 μM ZnCl₂ on OIT-induced changes in FluoZin-3 fluorescence. This experiment was performed in the presence or absence of DTPA. The columns and bars indicate the mean and standard deviation of four samples. The experiments were repeated three times.

Discussion

Effect of OIT on the intracellular Zn²⁺ level

The increase and decrease in the fluorescence intensity of FluoZin-3 corresponds to the increase and decrease in intracellular Zn²⁺ levels.²⁰Fig. 1 and 2 suggest that 1–3 μM OIT greatly increases intracellular Zn²⁺ levels in rat thymocytes. The OIT-induced increase in intracellular Zn²⁺ levels is dependent upon extracellular Zn²⁺ because the removal of extracellular Zn²⁺ by DTPA completely inhibited the augmentation of FluoZin-3 fluorescence by OIT (Fig. 3). It is likely that Zn²⁺ influx is mediated via a temperature-sensitive Zn²⁺ pathway, since the OIT-induced increase in the FluoZin-3 fluorescence was suppressed at low temperatures (Fig. 5). Furthermore, we determined that the directionality of Zn²⁺ flux within the temperature-sensitive Zn²⁺ pathway is dependent on a transmembrane Zn²⁺ gradient, because OIT attenuated the FluoZin-3 fluorescence in the presence of DTPA (Fig. 3 and 6). Thus, we concluded that OIT increases membrane Zn²⁺ permeability via a pathway that is inhibited under cold conditions. In further studies, the hypothesis that rat thymocyte membranes possess a temperature-sensitive Zn²⁺ pathway activated by OIT will be examined.

There are many membrane zinc transporters.^24,25 The Zn²⁺ pathway activated by OIT may be different from ZnT (SLC30A) and Zip (SLC39A), because the OIT-activated pathway was bi-directional. The change in FluoZin-3 fluorescence by OIT was dependent on a transmembrane Zn²⁺ gradient (Fig. 6). However, the possibility cannot be ruled out that OIT simultaneously activates zinc transporters regulating Zn²⁺ influx and efflux. Unfortunately, these pathways could not be further examined, as there are no specific chemical antagonists of membrane Zn²⁺ transporters.

Effect of OIT on intracellular Ca²⁺ levels

The OIT-induced augmentation of Fluo-3 fluorescence suggests that OIT treatment increases intracellular Ca²⁺ levels. Nevertheless, the mechanism by which this occurs remains to be elucidated. MnCl₂ slightly, but significantly, attenuated the OIT-induced augmentation of Fluo-3 fluorescence. Mn²⁺ is known to quench Fluo-3 fluorescence by replacing the Ca²⁺ bound to Fluo-3. Thus, a small amount of Mn²⁺ may pass through the membrane after OIT treatment, resulting in the attenuation of Fluo-3 fluorescence.

Toxicological implication

The effects of OIT on cells may be dependent on its concentration in the blood. However, the blood concentration of OIT has not yet been determined. If the blood concentration of OIT reaches 0.3 μM or more, OIT would likely affect intracellular Zn²⁺ levels in some cell types. For example, in the case of the local anesthetic lidocaine, the agent equally increased FluoZin-3 fluorescence in thymocytes and cerebellar neurons.^26,27 The susceptibility of thymocytes to OIT may be similar to that in cerebellar neurons.

Zn²⁺ has physiological and pathological roles in cellular functions and signaling.^15,16 Therefore, OIT-induced increases in intracellular Zn²⁺ levels may disturb intracellular Zn²⁺ homeostasis, leading to cellular dysfunction. In thymocytes, the increase in intracellular Zn²⁺ levels may promote apoptosis.²⁸ Furthermore, the increase in intracellular Zn²⁺ levels by ZnCl₂ potentiates the cytotoxicity of hydrogen peroxide, suggesting that Zn²⁺ potentiates oxidative stress.¹³ Thus, micromolar level OIT may potentiate oxidative stress by increasing intracellular Zn²⁺ levels. Some PVA towels were reported to contain 208–531 μg OIT per g wet weight before washing and 9.9–281 μg OIT per g wet weight after washing.⁵ Therefore, it is likely that people who use OIT-containing PVA towels come in direct contact with OIT. When the ability of OIT to penetrate the skin was investigated using skin isolated from adult rats, 87% of the OIT from 30 mg L⁻¹ aqueous solution entered the skin 3 h after application.⁶ Combined, these data suggest that OIT may be absorbed through human skin. Information regarding the distribution (including blood–brain barrier penetration), metabolism, or excretion of OIT is lacking. Nevertheless, the rats exposed to OIT were reported to show signs of CNS depression such as ataxia, prostration, and apathy, as well as polyuria, diarrhea, and swollen, red-tinged noses.⁶ Furthermore, the OIT-induced action may be temperature sensitive, because the effect of OIT was abolished at cold temperatures (Fig. 5). Thus, the Zn²⁺-related toxicity of OIT may be partly dependent on skin and body temperatures, which vary due to changes in environmental temperature.

Conclusion

To increase intracellular Zn²⁺ levels in rat thymocytes, approximately 300 nM (64 ng mL⁻¹) of OIT was necessary (Fig. 2B). Since it is likely that this threshold level of OIT is much lower than the OIT content in PVA towels,⁵ the adverse effects of OIT are of great concern.

Acknowledgements

This study was supported by a Grant-in-Aid for Scientific Research (C26340039) from the Japan Society for the Promotion of Science.

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Footnote

† Present address: Medical Co. LTA Kyushu Clinical Pharmacology Research Clinic, Fukuoka 810-0064, Japan.

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