540

Journal of Health Science, 57(6) 540–546 (2011)— Research Letter —Elevation of Intracellular Ca2+
Level by Triclosan in Rat Thymic
Lymphocytes: Increase in
Membrane Ca2+ Permeability andInduction of Intracellular Ca2+
Releasesity still increased slightly after triclosan treatment,even under conditions free from external Ca2+ . Additionally, triclosan did not increase the intensity of
Fluo-3 fluorescence when Ca2+ was depleted from intracellular Ca2+ stores by A23187 under the external Ca2+ -free condition. Taken together, these datasuggest that micromolar concentrations of triclosan
affect intracellular Ca2+ homeostasis in thymocytes,
possibly resulting in cellular malfunction.

Ikumi Tamura, Minoru Saito,
Yumiko Nishimura, Masaya Satoh,
Hiroshi Yamamoto, and Yasuo Oyama∗

Key words —— triclosan, Ca2+ , Fluo-3, thymocyte

Division of Environmental Symbiosis Studies, Graduate Schoolof Integrated Arts and Sciences, The University of Tokushima,
1–1 Minamijosanjima, Tokushima 770–8502, Japan(Received July 30, 2011; Accepted September 22, 2011;
Published online September 28, 2011)

Triclosan is an antibacterial agent used in household items and personal care products. Because wild
animals and humans can harbor this compound intheir systems, the toxic effects of triclosan are a possibility and are suspected. Therefore, we examined theeffects of triclosan on intracellular Ca2+ concentration in rat thymocytes by cytometric techniques using fluorescent probes. Triclosan doses of 1–10 µM
significantly increased the intensity of Ca2+ -detecting
Fluo-3 fluorescence, indicating an increase in intracellular Ca2+ concentrations. The augmentation of
Fluo-3 fluorescence became more profound in a dosedependent manner after the addition of an externalsource of Ca2+ . Conversely, the removal of external
Ca2+ greatly attenuated the triclosan-induced augmentation of Fluo-3 fluorescence. These results suggest that triclosan treatment allows external Ca2+ topass through cell membranes. This phenomenon was
not specific for Ca2+ because external Mn2+ quenched
the triclosan-induced augmentation of Fluo-3 fluorescence, indicating that triclosan can also mediate Mn2+ permeation across membranes. Therefore,
these results suggest that triclosan increases membrane permeability to divalent metal cations. Furthermore, triclosan induces Ca2+ release from intracellular stores because the Fluo-3 fluorescence inten∗

To whom correspondence should be addressed: Division ofEnvironmental Symbiosis Studies, Graduate School of Integrated Arts and Sciences, The University of Tokushima, 1–1
Minamijosanjima, Tokushima 770–8502, Japan. Tel. & Fax:
+81-88-656-7256; E-mail: oyama@ias.tokushima-u.ac.jp

INTRODUCTION
Triclosan (2,4,4 -trichloro-2 -hydroxydiphenyl
ether) is used as an antibacterial agent in household items and personal care products.1–3) Since
triclosan-containing health care products are disposed of in residential drains,4, 5) triclosan is often
detected in terrestrial and aquatic environments.6– 8)
This agent is also found in the bodies of wild
animals as well as humans,9, 10) including maternal milk3, 11) and blood.12) Triclosan concentrations
in blood plasma of human mothers were in therange of 0.4–38 µg/l.3) Because human plasma concentrations of triclosan seem to be submicromolar(< 0.131 µM), it is unlikely that it affects the intracellular Ca2+ concentration of the cells present in
the bloodstream. However, triclosan is a lipophiliccompound (log Kow = 4.8). Indeed, the concentrations of triclosan in lipophilic biological substances, including mammalian maternal milk andthe bile of wild fishes, were measured at 60–
300 µg/kg lipid weight (0.207–1.036 µmol/kg lipid
weight) and 0.24–0.9 mg/kg fresh weight (0.828–
3.108 µmol/kg fresh weight), respectively.11) Therefore, the adverse effects of triclosan on these substances or on the cells contained in these substances
are a topic of concern and are currently unclear.
The previously published in vitro experiments
on triclosan toxicity were carried out with human gingival cells,13, 14) breast cancer cells,15) natural killer cells,16) and murine skeletal myotubes.17)
These studies found that micromolar triclosan concentrations induce various effects on the cells. Studies on murine myotubes showed that triclosan affected intracellular Ca2+ homeostasis such that itelevates intracellular Ca2+ level by releasing Ca2+
from the sarcoplasmic reticulum.17) Since Ca2+ isC 2011 The Pharmaceutical Society of Japan

541

No. 6

recognized to have many physiological roles as asecond messenger,18, 19) the disruption of intracellular Ca2+ homeostasis by chemical compounds is assumed to lead to cytotoxicity. In an epidemiologicalstudy, higher levels of triclosan in young people correlated with increased diagnosis of allergies or hay
fever, indicating that overexposure to triclosan may
influence the human immune system.20) Therefore,
it is of interest to study the effect of triclosan on
intracellular Ca2+ homeostasis of mammalian lymphocytes. In this study, we examined the characteristics of triclosan-induced increase in intracellular Ca2+ concentration in rat thymocytes (containing
thymic lymphocytes) as measured by flow cytometric detection of fluorescent probes specific for cell
death and Ca2+ flux, propidium iodide, and Fluo3-pentaacetoxymethyl ester (Fluo-3-AM). Rat thymocytes were used in this study for the following
2 reasons. First, thymocyte cell membranes remain
intact throughout the course of preparation because
a single cell suspension can be prepared without an
enzymatic treatment that might cause weakening orpermeabilization of the cell membrane. This is important for this study because an intact cell membrane is necessary to study the effects of chemical treatment on intracellular Ca2+ concentrations
due to a large transmembrane Ca2+ gradient. Second, the process of cell death is extensively studiedin murine thymocytes; additionally, there are several types of hormones, biological compounds, andchemicals that have been identified to induce apoptosis and can be studied to determine whether theyplay a role in the functional outcome of triclosaninduced Ca2+ flux.21, 22)

MATERIALS AND METHODS
Chemicals —— Triclosan was purchased from
Wako Pure Chemicals (Osaka, Japan). Fluo-3-AMand ethylendiaminetetraacetic acid (EDTA) werepurchased from Dojin Chemical Laboratory (Kumamoto, Japan). Propidium iodide and A23187
were obtained from Sigma Chemical Company (St.
Louis, MO, U.S.A.). Thapsigargin was obtained
from Biomol International (Enzo Life Sciences Inc.,
Farmingdale, NY, U.S.A.). Other chemicals (NaCl,
CaCl2 , MgCl2 , KCl, glucose, and NaOH) were purchased from Wako Pure Chemicals. The buffer
for Tyrode’s solution was 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES, Nacalai
Tesque, Kyoto, Japan).

Animals and Cell Preparation —— This study
was approved by the Committee for Animal Experiments at the University of Tokushima (No. 05279).
Male rats (Wistar strain, Charles River Laboratories Japan Inc., Yokohama, Japan) were provided
with automatic water and a commercial diet (MF,
Oriental Yeast, Tokyo, Japan) ad libitum. The
animal room was maintained at a temperature of
23 ± 2◦ C and a relative humidity of 55 ± 5%, andit was artificially illuminated with fluorescent lighton a 12-hr light/dark cycle (0800–2000). Cell suspension was prepared similar to that previously reported.23, 24) In brief, thymus glands dissected fromether-anesthetized rats were sliced to 400–500 µm
thickness with a razor under cold conditions (3–4◦ C). The slices were triturated by gently shaking them in chilled Ca2+ -free Tyrode’s solution(150 mM NaCl, 5 mM KCl, 1 mM MgCl2 , 5 mM
glucose, 5 mM HEPES adjusted to a pH of 7.3–7.4)
to dissociate thymocytes. Thereafter, the Tyrode’s
solution containing the cells was passed through a
10-µm mesh to prepare a single-cell cell suspension.
The cell suspension was maintained at 36–37◦ C ina water bath 1 hr before the experiment.
Fluorescence Measurements of Cellular andMembrane Parameters —— The methods formeasurements of cellular and membrane parameters
by using a flow cytometer equipped with an argon
laser (CytoACE-150, JASCO, Tokyo, Japan) andfluorescent probes were similar to those previously
described.23, 24) The fluorescence was analyzed using the JASCO software (Ver. 3.06).
Fluo-3-AM was used as a fluorescent indicator
for intracellular Ca2+ .25) Rat thymocytes with intact membranes were incubated with 500 nM Fluo3-AM for 60 min. After incubation, fluorescencemeasurements were made to estimate the change inintracellular Ca2+ concentration. The normal Ca2+
condition was prepared by adding 2 mM CaCl2 tothe cell suspension. Adding 2 mM MgCl2 and 1 mM
EDTA to chelate the free Ca2+ ions in the cell suspension was used to create the external Ca2+ -free
condition. Fluo-3 fluorescence was measured fromthe cells that were not stained with 5 µM propidium
iodide.24) Since propidium stains dead cells, measuring the propidium fluorescence from the experimental cells enables the monitoring of Fluo-3 fluorescence from cells with intact membranes that represent only the living cells. Cell viability was estimated from the population of living cells that were
not stained with propidium iodide. The excitation
wavelength for Fluo-3 and propidium was 488 nm.

542Vol. 57 (2011)

Emission was detected at 530 ± 20 nm for Fluo-3
fluorescence and 600 ± 20 nm for propidium fluorescence.
Statistics —— Values were expressed as themean ± standard deviation of 3 experiments unlessotherwise mentioned. Statistical analysis was
performed with Tukey multivariate analysis. A pvalue of less than 0.05 was considered significant.

RESULTSTime- and Concentration-dependent Changes in
the Intensity of Fluo-3 Fluorescence by Triclosan
The application of 3 µM triclosan to thymocytes shifted the Fluo-3 histogram toward the direction of higher fluorescence intensity as compared to control treated cells (Fig. 1A), indicatingthat triclosan increased intracellular the Ca2+ concentration. The effect of 3 µM triclosan on Fluo3 fluorescence reached a steady-state level within
1–3 min after drug application (Fig. 1B). Increasing the dose of triclosan to 10 µM further shifted
the histogram toward higher fluorescence intensity(Fig. 1A), demonstrating that the effect of 10 µM triclosan on intracellular Ca2+ concentration was more
profound than 3 µM and indicates a dose-response
effect of triclosan. This dose-response relationshipbetween triclosan treatment and the induction of
Ca2+ , as measured by Fluo-3 fluorescence, is summarized in Fig. 1C. On the lower end of the dose response curve, a significant increase in the intensityof Fluo-3 fluorescence over control cells was observed, beginning at the 1 µM dose of triclosan. Although the thymocyte cell viability in the presenceof 10 µM triclosan decreased in a time-dependent
manner, it did not significantly change within 10–
15 min after the application of 10 µM triclosan; thecell viability under control conditions was 94.7 ±
0.5% and remained at 93.7 ± 0.9% at 15 min after the application of 10 µM triclosan. Triclosan atthe 3-µM dose or below did not significantly affectcell viability during the 3-hr incubation (data notshown). Therefore, the subsequent experiments, described below, were mainly performed with 3 µM
triclosan.
Effect on Membrane Ca2+ Permeability
To determine if the increase in membrane permeability to Ca2+ can explain the triclosan-induced
increase in Fluo-3 fluorescence intensity, the effectof 3 µM triclosan on Fluo-3 fluorescence was stud-

Fig. 1. Triclosan-induced Change of Fluo-3 Fluorescence inThymocytes(A) Histograms of Fluo-3 fluorescence. The effects of triclosanwere examined 5 min after application. Each histogram was constructed with 2000 cells. (B) Time-dependent change in the intensity of
Fluo-3 fluorescence. The time “0” indicates the control before the application of 3 µM triclosan. The symbol (∗∗) shows significant change(p < 0.01) between the control group and the group of cells treatedwith triclosan. (C) Concentration-dependent changes in the Fluo-3 fluorescence intensity in response to triclosan. The effect was examined
5 min after the start of triclosan application. The column and bar indicate mean and standard deviation, respectively, of 3 experiments. The
symbol (∗∗) indicates a significant change (p < 0.01) between the control group and the group of cells treated with triclosan.

ied when the concentration of CaCl2 in the cell suspension was increased from 0.67 mM to 6.7 mM.
The cells were incubated with CaCl2 at the respective concentrations for 10–15 min before the application of triclosan. Dose-dependence on external
Ca2+ was observed because as the concentrationof CaCl2 increased, the Fluo-3 fluorescence by thesame dose of triclosan also increased and became
more profound (Fig. 2A).

No. 6543

can be assessed because Mn2+ can replace the Ca2+that is bound to Fluo-3, resulting in fluorescencequenching.25) As shown in Fig. 2B, the applicationof 2 mM MnCl2 to control thymocytes also treatedwith 2 mM CaCl2 in the absence of triclosan did not
significantly change the intensity of Fluo-3 fluorescence, indicating that divalent cation influx into thecell is well-regulated at baseline. A23187 was usedin this study as a positive control reference agent
because it nonspecifically increases membrane permeability of divalent metal cations.26) As predicted,
treatment with A23187 drastically increased the intensity of Fluo-3 fluorescence, while the further addition of 2 mM MnCl2 significantly quenched theFluo-3 fluorescence (Fig. 2C) because of the increased permeability. Similar to treatment with thepermeabilization positive control, MnCl2 at 2 mM
also quenched the Fluo-3 fluorescence augmented
by 3–10 µM triclosan (Fig. 2C). The results indicatethat triclosan increased membrane permeability to
not only Ca2+ but also other divalent cations like
Mn2+ .

Fig. 2. Increase in Membrane Permeability to Ca2+ by 3 µM
Triclosan(A) Triclosan-induced augmentation of Fluo-3 fluorescence at different concentrations of external Ca2+ . The effects were examined
5 min after the start of triclosan drug application. The column and bar
indicate mean and standard deviation, respectively, of 3 experiments.
Symbols (∗∗) show significant change (p < 0.01) between the drugtreated groups and respective control groups. Symbol (#) indicatessignificant difference (p < 0.05) between paired groups. (B) Effectof MnCl2 on Fluo-3 fluorescence of untreated control cells. The concentration of CaCl2 used in this experiment was 2 mM. (C) Effects of
MnCl2 on Fluo-3 fluorescence of the cells treated with A23187 or triclosan. Notably, 100 nM A23187 increased the intensity of Fluo-3 fluorescence to 625.2 ± 117.0. The effect of MnCl2 was examined 5 minafter the start of its application. Before the application of MnCl2 , thecells were treated with triclosan or A23187 for 5 min. Symbols (∗, ∗∗)show significant change (p < 0.05, p < 0.01, respectively) between the
drug-treated groups with and without MnCl2 . Dotted line indicates theintensity of Fluo-3 fluorescence of A23187-treated cells after MnCl2 induced quenching.

To test if the increase in membrane permeability by triclosan is specific for Ca2+ , the effect ofanother divalent cation, Mn2+ , on the triclosaninduced increase in the Fluo-3 fluorescence intensity was examined by treatment with 2 mM MnCl2
at 5 min after triclosan application. Permeability

Intracellular Source of Increased Ca2+As described above, external Ca2+ participatedin the triclosan-induced augmentation of Fluo-3 fluorescence. Therefore, the effect of triclosan on
Fluo-3 fluorescence was examined under conditions
free from external Ca2+ to determine if triclosan
still had any effect. Triclosan at the 3 µM dosestill increased the Fluo-3 fluorescence intensity under this external Ca2+ -free condition (Fig. 3A). A
steady state of Fluo-3 fluorescence after triclosan
treatment was achieved within 5 min after triclosan
application (Fig. 3B). This increase by triclosan in
the external Ca2+ -free condition was less than that
under the normal Ca2+ concentration condition. The
dose-response relationship between triclosan treatment and the augmentation of Fluo-3 fluorescence
under the external Ca2+ -free condition is shown in
Fig. 3C. The significant increase in the Fluo-3 fluorescence intensity by triclosan under the external
Ca2+ -free condition was also observed at doses of
1 µM or more.
To see if Ca2+ released from intracellular storesis also involved in the triclosan-induced increasein Fluo-3 fluorescence, the effect of 3 µM triclosan was tested on the Fluo-3 fluorescence ofcells treated with 100 nM A23187 under conditions
free from external Ca2+ . Under the external Ca2+ free condition, A23187 depletes Ca2+ in intracellular Ca2+ stores.27, 28) Although A23187 increased

544Vol. 57 (2011)even lower within 10 min in the continued presenceof A23187. Triclosan treatment of the 3 µM dose
did not change the intensity of Fluo-3 fluorescencein the cells treated with A23187 (Fig. 3D). Similarly, in cells treated with 300 nM thapsigargin, an
inhibitor for endoplasmic reticulum Ca2+ -ATPase,no change is observed in Fluo-3 fluorescence intensity (Fig. 3E). These results indicate that the increased Ca2+ levels by triclosan treatment under theexternal Ca2+ -free condition was derived from theendoplasmic reticulum stores.

DISCUSSION

Fig. 3. Increase in Intracellular Ca2+ Release by Triclosan(A) Effect of 3 µM triclosan on Fluo-3 fluorescence under the external Ca2+ -free condition. The effect was examined 5 min after thestart of triclosan drug application. The column and bar indicate meanand standard deviation, respectively, of 3 experiments. The symbol (∗∗)
shows significant change (p < 0.01) between the control group and thegroup of cells treated with triclosan. (B) Time-dependent change in theintensity of Fluo-3 fluorescence under the external Ca2+ -free condition.
The time “0” indicates the control before the application of 3 µM triclosan. Symbols (∗, ∗∗) show significant change (p < 0.05, p < 0.01,
respectively) between the control group and the group of cells treatedwith triclosan. (C) Concentration-dependent changes in the intensityof Fluo-3 fluorescence under the external Ca2+ -free condition. The effect was examined at 5 min after the start of drug application. Symbols(∗, ∗∗) show significant change (p < 0.05, p < 0.01, respectively) between the control group and the group of cells treated with triclosan.
(D) Effect of triclosan on Fluo-3 fluorescence of the cells treated with
A23187 under the external Ca2+ -free condition. The effect of triclosan
was examined at 5 min after the start of triclosan drug application. Before the application of triclosan, the cells were treated with A23187 for
10 min. Symbol (∗) shows significant change (p < 0.05) between thecontrol group and the group of cells treated with A23187 or triclosan.
(E) Effect of triclosan on Fluo-3 fluorescence of the cells treated withthapsigargin under external Ca2+ -free condition. The effect of triclosan
was examined 5 min after the start of triclosan drug application. Before
the application of triclosan, the cells were treated with thapsigargin for
10 min. The symbol (∗) shows significant change (p < 0.05) between
the control group and the group of cells treated with thapsigargin or
triclosan.

the Fluo-3 fluorescence intensity in thymocytes, thefluorescence intensity returned to control levels or

Effect of Triclosan
Because of the following evidence, it is likelythat triclosan at doses of 1 µM or more increases intracellular Ca2+ concentration by increasing membrane Ca2+ permeability and releasing Ca2+ fromintracellular store(s) in rat thymocytes. The augmentation of Fluo-3 fluorescence by triclosan became more profound as the membrane gradient of
Ca2+ increased by increasing external Ca2+ concentration (Fig. 2A), whereas the removal of external
Ca2+ greatly attenuated the augmentation of Fluo3 fluorescence by triclosan (Fig. 3A). Thus, it islikely that triclosan increases the external Ca2+ thatpasses through the cell membranes. The application of Mn2+ quenched the Ca2+ -dependent Fluo-3
fluorescence augmented by triclosan (Fig. 2C), indicating that Mn2+ passed across the membranes.
Taken together, the data suggest that triclosan can
increase membrane permeability to divalent metal
cations. Triclosan increased the intensity of Fluo3 fluorescence under conditions free from external
Ca2+ (Fig. 3A). However, triclosan did not increase
the Fluo-3 fluorescence intensity under the external Ca2+ -free condition when Ca2+ was depleted
from intracellular Ca2+ stores by A23187 (Fig. 3D).
Therefore, the data suggests that triclosan can release Ca2+ from intracellular stores.
In the study by Ahn et al.,17) a micromolar triclosan dose was shown to enhance the binding of
ryanodine to its receptor and augment the fluorescence of Fura-2, an indicator for intracellular Ca2+ ,in mouse skeletal myotubes. The results suggested
that triclosan induced release of Ca2+ from intracellular sarcoplasmic reticulum stores. As shownin Fig. 3, micromolar dose of triclosan induced theaugmentation of Fluo-3 fluorescence in rat thymocytes, also suggesting the release of Ca2+ from in-

545

No. 6

tracellular stores. Therefore, the intracellular Ca2+
release may be one of the common cytotoxic effectsof triclosan. If triclosan depletes Ca2+ from endoplasmic reticulum, it would activate an intracellularstore-operated Ca2+ influx.29–32) The introductionof MnCl2 quenched the Fluo-3 fluorescence augmented by triclosan (Fig. 2C), indicating that Mn2+
was able to permeate across the membranes. This
Mn2+ influx provides a qualitative evidence for theactivation of store-operated Ca2+ entry.31, 32) Therefore, triclosan may activate the intracellular-storeoperated Ca2+ influx by depleting Ca2+ from the endoplasmic reticulum of rat thymocytes.
Implications
Since intracellular Ca2+ plays a physiologicalrole as a second messenger,18, 19) the increase in
intracellular Ca2+ concentration by triclosan may
disturb the carefully regulated intracellular Ca2+
homeostasis and lead to cellular malfunctions. The
primary function of thymocytes is to generate T
lymphocytes (T cells). The function of T cells
is to recognize foreign antigens, and this is mediated by the T cell receptor. The thymocyte cells
that fail to produce a functional receptor are eliminated by apoptosis. Apoptosis is the fate of mostthymocytes.33) It has become clear that the cellular Ca2+ overload or perturbation of the intracellular Ca2+ compartmentalization can cause apoptotic
cell death.34) Thus, triclosan may affect the processof apoptosis in thymocytes, resulting in a change
in the competency of the immune system. Therefore, the main concern of the data presented here
is whether the concentrations of triclosan detected
in humans and wild animals reach a level that canincrease intracellular Ca2+ concentrations. Since
human plasma concentrations of triclosan3) seemto be in the submicromolar (< 0.131 µM) range, it
is unlikely that this triclosan concentration could
affect intracellular Ca2+ concentrations. However,the concentration of triclosan found in the maternal milk and bile of wild living fishes were 60–
300 µg/kg lipid weight (0.207–1.036 µmol/kg lipid
weight) and 0.24–0.9 mg/kg fresh weight (0.828–
3.108 µmol/kg fresh weight), respectively, indicating that triclosan may be accumulating in certain
lipophilic tissues.11) The possibility that triclosan
can increase intracellular Ca2+ concentration cannot be ruled out when triclosan accumulates in thetissues of humans and wild animals. At present,
however, there is not sufficient evidence for adverse
effects of triclosan on wild animals and humans.

Therefore, further studies should be performed todetermine whether adverse effects of triclosan exposure occur in nature.
Acknowledgement This study was supported by
Grant-in-Aid for Scientific Research (C23510078)
from Japan Society for the Promotion of Science.

REFERENCES
1) Perencevich, E. N., Wong, M. T. and Harris, A. D.
(2001) National and regional assessment of the antibacterial soap market: a step toward determining
the impact of prevalent antibacterial soaps. Am. J.
Infect. Control, 29, 281–283.
2) Schweizer, H. P. (2001) Triclosan: a widely usedbiocide and its link to antibiotics. FEMS Microbiol.
Lett., 202, 1–7.
3) Allmyr, M., Adolfsson-Erici, M., McLachlan, M.
S. and Sandborgh-Englund, G. (2006) Triclosan inplasma and milk from Swedish nursing mothers andtheir exposure via personal care products. Sci. Total
Environ., 372, 87–93.
4) Reiss, R., Mackay, N., Habig, C. and Griffin, J.
(2002) An ecological risk assessment for triclosanin lotic systems following discharge from wastewater treatment plants in the United States. Environ.
Toxicol. Chem., 21, 2483–2492.
5) Heidler, J. and Halden, R. U. (2008) Meta-analysis
of mass balances examining chemical fate during
wastewater treatment. Environ. Sci. Technol., 42,6324–6332.
6) Singer, H., Müller, S., Tixier, C. and Pillonel, L.
(2002) Triclosan: occurrence and fate of a widelyused biocide in the aquatic environment: field measurements in wastewater treatment plants, surface
waters, and lake sediments. Environ. Sci. Technol.,36, 4998–5004.
7) Zhao, J. L., Ying, G. G., Liu, Y. S., Chen, F., Yang,
J. F. and Wang, L. (2010) Occurrence and risks oftriclosan and triclocarban in the Pearl River system,
South China: from source to the receiving environment. J. Hazard. Mater., 179, 215–222.
8) Kookana, R. S., Ying, G. G. and Waller, N. J. (2011)
Triclosan: its occurrence, fate and effects in the Australian environment. Water Sci. Technol., 63, 598–
604.
9) Valters, K., Li, H., Alaee, M., D’Sa, I., Marsh,
G., Bergman, A. and Letcher, R. J. (2005) Polybrominated diphenyl ethers and hydroxylated andmethoxylated brominated and chlorinated analogues
in the plasma of fish from the Detroit River. Environ.
Sci. Technol., 39, 5612–5619.

546

10) Fair, P. A., Lee, H. B., Adams, J., Darling, C.,
Pacepavicius, G., Alaee, M., Bossart, G. D., Henry,N. and Muir, D. (2009) Occurrence of triclosan inplasma of wild Atlantic bottlenose dolphins (Tursiops truncatus) and in their environment. Environ.
Pollut., 157, 2248–2254.
11) Adolfsson-Erici, M., Petterson, M., Parkkonen, J.
and Sturve, J. (2002) Triclosan, a commonly usedbactericide found in human milk and in the aquatic
environment in Sweden. Chemosphere, 46, 1485–
1489.
12) Allmyr, M., Harden, F., Toms, L. M., Mueller,
J. F., McLachlan, M. S., Adolfsson-Erici, M. andSandborgh-Englund, G. (2008) The influence of age
and gender on triclosan concentrations in Australianhuman blood serum. Sci. Total Environ., 393, 162–
167.
13) Babich, H. and Babich, J. P. (1997) Sodium laurylsulfate and triclosan: in vitro cytotoxicity studies
with gingival cells. Toxicol. Lett., 91, 189–196.
14) Zuckerbraun, H. L., Babich, H., May, R. and
Sinensky, M. C. (1998) Triclosan: cytotoxicity,mode of action, and induction of apoptosis in human gingival cells in vitro. Eur. J. Oral Sci., 106,628–636.
15) Liu, B., Wang, Y., Fillgrove, K. L. and Anderson,
V. E. (2002) Triclosan inhibits enoyl-reductase oftype I fatty acid synthase in vitro and is cytotoxic
to MCF-7 and SKBr-3 breast cancer cells. Cancer
Chemother. Pharmacol., 49, 187–193.
16) Udoji, F., Martin, T., Etherton, R. and Whalen, M.
M. (2010) Immunosuppressive effects of triclosan,
nonylphenol, and DDT on human natural killer cells
in vitro. J. Immunotoxicol., 7, 205–212.
17) Ahn, K. C., Zhao, B., Chen, J., Cherednichenko, G.,
Sanmarti, E., Denison, M. S., Lasley, B., Pessah,
I. N., Kültz, D., Chang, D. P. Y., Gee, S. J. andHammock, B. D. (2008) In vitro biologic activitiesof the antimicrobials triclocarban, its analogs, andtriclosan in bioassay screens: receptor-based bioassay screens. Environ. Health Perspect., 116, 1203–
1210.
18) Clapham, D. E. (2007) Calcium signaling. Cell.,
131, 1047–1058.
19) Feske, S. (2007) Calcium signalling in lymphocyte
activation and disease. Nat. Rev. Immunol., 7, 690–
702.
20) Clayton, E. M. R., Todd, M., Dowd, J. B. and Aiello,A. E. (2011) The impact of bisphenol A and triclosan on immune parameters in the U.S. population, NHANES 2003–2006. Environ. Health Perspect., 119, 390–396.
21) McConkey, D. J., Jondal, M. and Orrenius, S. (1994)

Vol. 57 (2011)

The regulation of apoptosis in thymocytes. Biochem.
Soc. Trans., 22, 606–610.
22) Quaglino, D. and Ronchetti, I. P. (2001) Cell deathin the rat thymus: a minireview. Apoptosis, 6, 389–
401.
23) Chikahisa, L. and Oyama, Y. (1992) Tri-n-butyltin
increases intracellular Ca2+ in mouse thymocytes:
a flow-cytometric study using fluorescent dyes formembrane potential and intracellular Ca2+ . Pharmacol. Toxicol., 71, 190–195.
24) Chikahisa, L., Oyama, Y., Okazaki, E. and Noda,
K. (1996) Fluorescent estimation of H2 O2 -inducedchanges in cell viability and cellular nonprotein thiollevel of dissociated rat thymocytes. Jpn. J. Pharmacol., 71, 299–305.
25) Kao, J. P., Harootunian, A. T. and Tsien, R. Y.
(1989) Photochemically generated cytosolic calcium pulses and their detection by fluo-3. J. Biol.
Chem., 264, 8179–8184.
26) Erdahl, W. L., Chapman, C. J., Wang, E., Taylor,
R. W. and Pfeiffer, D. R. (1996) Ionophore 4BrA23187 transports Zn2+ and Mn2+ with high selectivity over Ca2+ . Biochemistry, 35, 13817–13825.
27) Itoh, T., Kanmura, Y. and Kuriyama, H. (1985)
A23187 increases calcium permeability of store
sites more than of surface membranes in the rabbit
mesenteric artery. J. Physiol. (Lond.), 359, 467–484.
28) Oyama, Y., Ueha, T., Hayashi, A. and Chikahisa, L.
(1994) Effect of tri-n-butyltin on intracellular Ca2+
concentration of mouse thymocytes under Ca2+ -free
condition. Eur. J. Pharmacol., 270, 137–142.
29) Mason, M. J., Mahaut-Smith, M. P. and Grinstein, S.
(1991) The role of intracellular Ca2+ in the regulation of the plasma membrane Ca2+ permeability of
unstimulated rat lymphocytes. J. Biol. Chem., 266,
10872–10879.
30) Mason, M. J. and Grinstein, S. (1993) Ionomycin
activates electrogenic Ca2+ influx in rat thymic lymphocytes. Biochem. J., 296, 33–39.
31) Parekh, A. B., Fleig, A. and Penner, R. (1997) The
store-operated calcium current ICRAC : nonlinear activation by InsP3 and dissociation from calcium release. Cell, 89, 973–980.
32) Kurebayashi, N. and Ogawa, Y. (2001) Depletion of
Ca2+ in the sarcoplasmic reticulum stimulates Ca2+
entry into mouse skeletal muscle fibres. J. Physiol.,533, 185–199.
33) Yang, Y. and Ashwell, J. D. (1999) Thymocyte
apoptosis. J. Clin. Immunol., 19, 337–349.
34) Orrenius, S., Zhivotovsky, B. and Nicotera, P.
(2003) Regulation of cell death: the calciumapoptosis link. Nat. Rev. Mol. Cell Biol., 4, 552–565.