Toxicology 262 (2009) 245–249

Contents lists available at ScienceDirect

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

Tri-n-butyltin increases intracellular Zn2+ concentration by decreasing cellular

thiol content in rat thymocytes
Toshihisa B. Oyama 1 , Keisuke Oyama 2 , Takuya Kawanai, Tomohiro M. Oyama 3 , Erika Hashimoto,
Masaya Satoh, Yasuo Oyama ∗
Laboratory of Cellular Signaling, Faculty of Integrated Arts and Sciences, The University of Tokushima, Tokushima 770-8502, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Effect of tri-n-butyltin (TBT), an environmental pollutant, on intracellular Zn2+ concentration was tested
Received 10 May 2009 in rat thymocytes to reveal one of cytotoxic profiles of TBT at nanomolar concentrations using a flow
Received in revised form 11 June 2009 cytometer and appropriate fluorescent probes. TBT at concentrations of 30 nM or more (up to 300 nM)
Accepted 17 June 2009
significantly increased the intensity of FluoZin-3 fluorescence, an indicator for intracellular Zn2+ concen-
Available online 25 June 2009
tration, under external Ca2+ - and Zn2+ -free condition. Chelating intracellular Zn2+ completely attenuated
the TBT-induced augmentation of FluoZin-3 fluorescence. Result suggests that nanomolar TBT releases
Keywords:
Zn2+ from intracellular store site. Oxidative stress induced by hydrogen peroxide also increased the
Tri-n-butyltin
Intracellular zinc
FluoZin-3 fluorescence intensity. The effects of TBT and hydrogen peroxide on the fluorescence were
Intracellular thiol additive. TBT-induced changes in the fluorescence of FluoZin-3 and 5-chloromethylfluorescein, an indi-
cator for cellular thiol content, were correlated with a coefficient of −0.962. Result suggests that the
intracellular Zn2+ release by TBT is associated with TBT-induced reduction of cellular thiol content. How-
ever, chelating intracellular Zn2+ potentiated the cytotoxicity of TBT. Therefore, the TBT-induced increase
in intracellular Zn2+ concentration may be a type of stress responses to protect the cells.
© 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Quesada et al., 1996). Therefore, it is reminiscent of a possibility

Tri-n-butyltin (TBT), an environmental pollutant in edible mol-
lusks (Yamamoto, 1994; Kannan et al., 1996; Inoue et al., 2006; Choi
et al., 2009), exerts a variety of cytotoxic actions at environmentally
relevant nanomolar concentrations (Kannan et al., 1998; Nakata et
al., 1999; Whalen et al., 1999; Okada et al., 2000). TBT is immuno-
toxic and induces apoptosis of thymocytes (Aw et al., 1990; Raffray
et al., 1993), leading to thymus atrophy in rats (Raffray and Cohen,
1993).
The toxic action of TBT has been partly explained by TBT-induced
decrease or depletion of cellular thiol content (Cima and Ballarin,
2004; Powell et al., 2008; Tada-Oikawa et al., 2008). Cellular thiols
such as glutathione and metallothionein are complexed with Zn2+
(Diaz-Cruz et al., 1998; Jacob et al., 1998; Maret and Vallee, 1998;
Gelinsky et al., 2003). Thus, oxidative stress releases Zn2+ from
thiols via interchange between thiol and disulfide (Maret, 1994;
∗ Corresponding author. Tel.: +81 88 656 7256; fax: +81 88 656 7256.
E-mail address: oyama@ias.tokushima-u.ac.jp (Y. Oyama).
1
Present address: Faculty of Engineering, Okayama University, Okayama 700-
8530, Japan.
2
Present address: Faculty of Medicine, Saga University, Saga 849-8501, Japan.
3
Present address: Faculty of Pharmaceutical Sciences, Tokushima Bunri Univer-
sity, Tokushima 770-8512, Japan.
that TBT increases intracellular Zn2+ concentration via decreasing
cellular thiol content.
To test the possibility, the effect of TBT on FluoZin-3 fluo-
rescence, a fluorescent indicator for intracellular Zn2+ , has been
examined in rat thymocytes because of following reasons. Zn2+ is
the second most prevalent trace element and it is involved in the
structure and function of over 300 enzymes (Prasad, 1995). Zn2+
stimulates the activity of approximately 100 enzymes (Sandstead,
1994). Therefore, an abnormal increase in intracellular Zn2+ con-
centration by TBT may cause cytotoxic phenomena.
2. Materials and methods
2.1. Chemicals
Tri-n-butyltin (TBT) chloride was purchased from Tokyo Kasei Co. (Tokyo,
Japan). Chelators for Zn2+ , diethylenetriamine-N,N,N ,N ,N -pentaacetic acid (DTPA),
N,N,N ,N -tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), and ethylenediamine-
N,N,N ,N -tetraacetic acid (EDTA), were obtained from Dojin Chemical Laboratory
(Kumamoto, Japan). Fluorescent probes, propidium iodide, FluoZin-3 acetoxymethyl
ester (FluoZin-3-AM), and 5-chloromethylfluorescein diacetate (5-CMF-DA), were
products of Molecular Probes Inc. (Eugene, Oregon, USA). NaCl, MgCl2 , KCl, glucose,
HEPES, NaOH, and ZnCl2 were also obtained from Woko Pure Chemicals. Hydro-
gen peroxide (H2 O2 ) was purchased from Sumitomo Chemical Industry (Osaka,
Japan). Dimethyl sulfoxide (DMSO) was purchased from Wako Pure Chemicals
(Osaka, Japan). Final concentration of DMSO as a solvent for TPEN, FluoZin-3-
AM, 5-CMF-DA, and TBT in cell suspension was 0.3% or less. The incubation with

0300-483X/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.tox.2009.06.016
246 T.B. Oyama et al. / Toxicology 262 (2009) 245–249

DMSO at 0.3% or less did not affect the viability of rat thymocytes during experi-
ments.

2.2. Animals and cell preparation

This study was approved by the Committee for Animal Experiments in the
University of Tokushima (No. 05279 for Y. Oyama). The procedure to prepare cell
suspension was similar to that previously reported (Chikahisa and Oyama, 1992;
Chikahisa et al., 1996). In brief, thymus glands dissected from ether-anaesthetized
rats (Wistar strain) were sliced at a thickness of 400–500 ␮m with razor under an
ice-cold condition (1–4 ◦ C). The slices were triturated by gently shaking in chilled
normal Tyrode’s solution (in mM: NaCl 150, KCl 5, CaCl2 2, MgCl2 1, glucose 5, HEPES
5, with an appropriate amount of NaOH to adjust pH to 7.3–7.4) or chilled Ca2+ -free
Tyrode’s solution (in mM: NaCl 150, KCl 5, MgCl2 3, glucose 5, HEPES 5, with an
appropriate amount of NaOH to adjust pH to 7.3–7.4) to dissociate thymocytes. The
purities of chemicals for preparing Ca2+ -free Tyrode’s solution were greater than
99.999%. Thereafter, both Tyrode’s solutions containing the cells were respectively Fig. 1. TBT-induced change in histogram of FluoZin-3 fluorescence. Effect of TBT
passed through a mesh (a diameter of 10 ␮m) to prepare cell suspension (about was tested at 1 h after application. Each histogram was constructed with 2000 cells.
5 × 105 cells/ml). The beaker containing the cell suspension was water-bathed at The shift of histogram to a direction of higher intensity of FluoZin-3 fluorescence
36 ◦ C for 1 h before the start of experiment. indicates an increase in intracellular Zn2+ concentration.

2.3. Fluorescence measurements of cellular and membrane parameters

The methods for measurements of cellular and membrane parameters using a
flow cytometer equipped with an argon laser (CytoACE-150, JASCO, Tokyo, Japan) and
fluorescent probes were similar to those previously described (Chikahisa and Oyama,
1992; Chikahisa et al., 1996; Matsui et al., 2008). The fluorescence was analyzed by
JASCO software (Ver.3XX, JASCO). As to chemicals used in this study, there was no
fluorescence detected under our experimental condition.
To estimate cell size for identifying shrunken cells, the intensity of forward scat-
ter obtained from each cells in cytogram (forward scatter versus side scatter) was
measured. Reduction of forward scatter intensity indicates the decrease in cell size.
To assess cell lethality, propidium iodide was added to cell suspension to achieve
a final concentration of 5 ␮M. Since propidium stains dead cells, the measurement
of propidium fluorescence from cells provides a clue to estimate the lethality. The
fluorescence was measured at 2 min after the application of propidium iodide by a
flow cytometer. Excitation wavelength for propidium was 488 nm and emission was
detected at 600 ± 20 nm.
FluoZin-3-AM (Gee et al., 2002) is used as an indicator for intracellular Zn2+ . The
cells were incubated with 500 nM FluoZin-3-AM for 60 min before any fluorescence
measurements. FluoZin-3 fluorescence was measured from the cells that were not
stained with 5 ␮M propidium iodide to estimate the change in intracellular Zn2+ con-
centration of rat thymocytes with intact membranes (Matsui et al., 2008). Excitation
wavelength for FluoZin-3 was 488 nm and emission was detected at 530 ± 15 nm.
5-CMF-DA was used to monitor the change in cellular content of nonprotein thi-
ols (Chikahisa et al., 1996). The cells were incubated with 1 ␮M 5-CMF-DA for 30 min
before any fluorescence measurements. 5-CMF fluorescence was measured from the
cells that were not stained with 5 ␮M propidium iodide. Excitation wavelength for
5-CMF was 488 nm and emission was detected at 530 ± 15 nm.
2.4. Statistics
Values were expressed as the mean ± standard deviation of four experiments.
Statistical analysis was performed by using Tukey’s multivariate analysis. A P value
of <0.05 was considered significant.
3. Results
3.1. Increase in the intensity of FluoZin-3 fluorescence by TBT
As shown in Fig. 1, the histogram of FluoZin-3 fluorescence
monitored from rat thymocytes incubated with normal Tyrode’s
solution was shifted to a direction of higher intensity by 10–30 nM
TBT. The effect of TBT on FluoZin-3 fluorescence seemed to reach
a steady state level within 1 h because the further change in mean
intensity was less than 5% during next 0.5–1 h incubation. There-
fore, the effect of TBT on FluoZin-3 fluorescence was tested at 1 h
after the application of TBT in the experiments described below.
The TBT-induced augmentation of FluoZin-3 fluorescence was not
observed in the presence of TPEN, a chelator for extracellular and
intracellular Zn2+ . The results indicate the increase in intracellular
Zn2+ concentration by TBT.
Nanomolar TBT increases membrane permeability of Ca2+
(Chikahisa and Oyama, 1992; Chow et al., 1992). To rule out the
possibility that external Ca2+ and Zn2+ contribute to TBT-induced
increase in FluoZin-3 fluorescence intensity, the effect of TBT was
examined under external Ca2+ - and Zn2+ -free condition. The cells
were incubated with Ca2+ -free Tyrode’s solution containing 10 ␮M
DTPA. The threshold concentration of TBT to increase mean inten-
sity of FluoZin-3 fluorescence was 1–10 nM. As shown in Fig. 2, the
increases were statistically significant when the concentration of
TBT was 30 nM or more (up to 300 nM). Thus, it is suggested that
nanomolar TBT induces the release of intracellular Zn2+ , resulting in

Fig. 2. Concentration-dependent change in mean intensity of FluoZin-3 fluorescence by TBT. Column and bar respectively indicate average and standard deviation of four
experiments. Asterisks (* and **) show significant increase (P < 0.05 and P < 0.01, respectively) in comparison with control.
 T.B. Oyama et al. / Toxicology 262 (2009) 245–249 247

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

the increase in intracellular Zn2+ concentration. The concentration-
dependent change in FluoZin-3 fluorescence by TBT under external
Ca2+ - and Zn2+ -free condition was similar to that under normal
condition (not shown).
3.2. Decrease in the intensity of 5-CMF fluorescence by TBT
Since it has been reported that intracellular Zn2+ concentra-
tion is modulated via the interchange between thiol and disulfide
(Maret, 1994; Quesada et al., 1996), the effect of simultaneous appli-
cation of H2 O2 and TBT was tested on FluoZin-3 fluorescence. H2 O2
was used to decrease cellular thiol content, resulting in an increase
of cellular disulfide content. H2 O2 at 30 ␮M attenuated the 5-CMF
fluorescence by less than 40% without affecting the cell viability of
rat thymocytes (Chikahisa et al., 1996). The concentration of TBT
was 10–30 nM to observe further augmentation of the FluoZin-3
fluorescence by 30 ␮M H2 O2 . As shown in Fig. 3, H2 O2 signifi-
cantly increased the intensity of FluoZin-3 fluorescence. However,
the effects of H2 O2 and TBT seemed to be additive.
TBT decreases cellular content of thiols under normal condition
(Okada et al., 2000). The effect of TBT on intensity of 5-CMF fluo-
rescence was tested under external Ca2+ - and Zn2+ -free condition
to reveal a correlation between TBT-induced increase in intracellu-
lar Zn2+ concentration (Fig. 2) and TBT-induced change in cellular
thiol content. As shown in Fig. 4, TBT at concentrations ranging from
30 nM to 300 nM significantly decreased mean intensity of 5-CMF
fluorescence. The result of Fig. 4 was compared with those of Fig. 2
to see if there is a correlation between them. The correlation coef-
ficient was −0.962 (Fig. 5). In the presence of 10 ␮M TPEN (under
external and internal Zn2+ -free condition), TBT at 300 nM also sig-
nificantly decreased the intensity of 5-CMF fluorescence. Thus, TBT
is supposed to decrease cellular thiol content, being independent
from Zn2+ .
3.3. Effect of TPEN on the cells treated with TBT under normal
condition
The result of Fig. 5 suggests that TBT increases intracellular Zn2+
concentration by decreasing cellular thiol content. To see if the TBT-
induced increase in intracellular Zn2+ concentration contributes to
the TBT cytotoxicity, the effect of 300 nM TBT on rat thymocytes
incubated with normal Tyrode’s solution was examined in absence
and presence of 10 ␮M TPEN. The cells were incubated with and

Fig. 4. Concentration-dependent change in mean intensity of 5-CMF fluorescence

by TBT. Column and bar respectively indicate average and standard deviation of
four experiments. Asterisks (* and **) show significant increase (P < 0.05 and P < 0.01, Fig. 5. Correlation between the TBT-induced changes in FluoZin-3 and 5-CMF fluo-
respectively). rescence. Results are obtained from Figs. 2 and 4.
248 T.B. Oyama et al. / Toxicology 262 (2009) 245–249
Fig. 6. Effect of TPEN on TBT-induced cytotoxicity. Cell lethality was examined at 1 h after respective application. Asterisks (* and **) show significant increase (P < 0.05 and
P < 0.01, respectively) in comparison with CONTROL. Symbol (# ) indicates significant increase in comparison with both groups of cells incubated with TBT alone and TPEN
alone.
without TBT and/or TPEN for 1 h. Although the incubation with
300 nM TBT or 10 ␮M TPEN for 1 h did not significantly increase cell
lethality, the simultaneous application of TBT with TPEN induced
significant increase in cell lethality (Fig. 6). Thus, the removal of
intracellular Zn2+ is supposed to augment the cytotoxicity of TBT.
Cell shrinkage is one of characteristics during an early stage of
apoptosis (Klassen et al., 1993; Beauvais et al., 1995; Bortner and
Cidlowski, 1998). As to the population of shrunken cells, 300 nM
TBT significantly increased it under normal condition while it was
not the case under external Ca2+ -free condition (Fig. 6). The combi-
nation of TBT and TPEN further increased the population although
TPEN alone did not so (Fig. 6). This increase is statistically signifi-
cant in comparison with the control group and the group of cells
treated with 300 nM TBT.
4. Discussion
It is likely that TBT releases Zn2+ from intracellular stores,
resulting in the increase in intracellular Zn2+ concentration, via
decreasing cellular thiol content because of following reasons. First,
the TBT-induced increase in FluoZin-3 fluorescence intensity was
observed under external Ca2+ - and Zn2+ -free condition. The aug-
mentation of FluoZin-3 fluorescence by TBT was not observed in
the presence of TPEN. These results suggest that TBT increases
intracellular Zn2+ concentration by releasing intracellular Zn2+ from
intracellular stores. Second, the TBT-induced changes in fluores-
cence of FluoZin-3, an indicator for intracellular Zn2+ concentration,
and 5-CMF, an indicator for cellular thiol content, are correlated
with a coefficient of −0.962. Zn2+ makes a complex with thiol group
of protein and nonprotein such as metallothionein and glutathione
(Diaz-Cruz et al., 1998; Jacob et al., 1998; Maret and Vallee, 1998;
Gelinsky et al., 2003). The modification from thiol to disulfide has
been reported to release Zn2+ (Maret, 1994; Quesada et al., 1996). In
fact, N-ethylmaleimide, inducing a chemical depletion of intracellu-
lar thiol, decreased intensity of 5-CMF fluorescence and increased
that of FluoZin-3 fluorescence (Hashimoto et al., in press). These
results suggest that TBT increase in intracellular Zn2+ concentration
from intracellular stores by decreasing cellular thiol content.
TBT exerts an immunotoxic action on mammals (Snoeij
et al., 1987). Nanomolar TBT induces some toxic actions on
immune system cells under in vitro condition. TBT inhibits tumor-
killing capacity of human and murine natural killer lymphocytes
(Ghoneum et al., 1990; Whalen et al., 1999), elevates intracellu-
lar Ca2+ concentration (Chikahisa and Oyama, 1992; Chow et al.,
1992; Oyama et al., 1994) and increases the population of apop-
totic cells in murine thymocytes (Nakata et al., 1999). In addition,
TBT reduces the cellular content of GSH in rat thymocytes (Okada
et al., 2000). However, the increase in intracellular Zn2+ concen-
tration by nanomolar TBT may be not toxic since the removal of
intracellular Zn2+ by TPEN augmented the cytotoxicity of TBT. Intra-
cellular Zn2+ partly attenuates Ca2+ -dependent cell death induced
by A23187, a calcium ionophore, in rat thymocytes (Sakanashi et al.,
2009). Thus, the removal of intracellular Zn2+ by TPEN may poten-
tiate Ca2+ -dependent cell death induced by TBT since TBT increases
intracellular Ca2+ concentration (Chikahisa and Oyama, 1992; Chow
et al., 1992; Oyama et al., 1994). The increase in population of
 T.B. Oyama et al. / Toxicology 262 (2009) 245–249
shrunken cells by TBT was reduced under external Ca2+ -free condi-
tion, suggesting the involvement of Ca2+ in the cell shrinkage. The
combination of TBT and TPEN further increased the population of
shrunken cells. Therefore, the TBT-induced increase in intracellular
Zn2+ concentration in this study may be a type of stress responses
to protect the cells. In fact, TBT delays the process of cell death
induced by H2 O2 in rat thymocytes (Sakai et al., 2001). Both TBT
and H2 O2 increased intracellular Zn2+ concentration in this study.
The cell death induced by H2 O2 is dependent on Ca2+ (Sakanashi
et al., 2008). Cellular Zn2+ partly attenuates Ca2+ -dependent cell
death (Sakanashi et al., 2009). Thus, the TBT-induced increase in
intracellular Zn2+ concentration may delay the cell death induced
by H2 O2 .
Zinc pyrithione at nanomolar concentrations induces apoptosis
and the addition of zinc chelator protects the cells against apoptosis
induced by zinc pyrithione in murine thymic and splenic lympho-
cytes and human Ramos B and Jurkat T cells (Mann and Fraker,
2005). However, the chelation of intracellular zinc triggers apop-
tosis in murine and human mature thymocytes (McCabe et al.,
1993). Thus, the role of intracellular Zn2+ itself in cell death may
vary from cell to cell or may be complex. In addition, intracellular
Zn2+ affects the process of Ca2+ -dependent cell death in rat thymo-
cytes (Sakanashi et al., 2009). Therefore, the TBT-induced increase
in intracellular Zn2+ concentration may induce various effects on
the process of cell death, being dependent on the type of cells.
Conflict of interest statement
All authors declare that there are no conflicts of interest in this
study.
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