Chemosphere 93 (2013) 353358

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Chemosphere
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Synergistic increase in cell lethality by dieldrin and H2O2 in rat

thymocytes: Effect of dieldrin on the cells exposed to oxidative stress
Tsolmon Chimeddorj 1, Tomoko Suzuki 1, Kazuhiro Murakane 1, Miyuki Inai 1, Masaya Satoh,
Yasuo Oyama
Division of Environmental Symbiosis Studies, Graduate School of Integrated Arts and Sciences, University of Tokushima, Tokushima 770-8502, Japan

h i g h l i g h t s
 Dieldrin is one of the most persistent pesticides.
 Synergistic increase in cell lethality by dieldrin and H2O2 was observed.
 Zn

2+

chelator signicantly attenuated the synergistic increase.

 Dieldrin accelerated the process of cell death induced by H2O2.

 Dieldrin may exhibit potent cytotoxicity on cells exposed to oxidative stress.

a r t i c l e

i n f o

Article history:
Received 27 December 2012
Received in revised form 4 March 2013
Accepted 27 April 2013
Available online 29 May 2013
Keywords:
Dieldrin
Oxidative stress
Hydrogen peroxide
Intracellular Zn2+
Nonprotein thiol
Cytotoxicity

a b s t r a c t
Dieldrin, one of persistent pesticides, is highly resistant to biotic and abiotic degradation. It is accumulated in organisms. Recent studies suggest that dieldrin exerts a potent cytotoxic action on cells exposed
to oxidative stress. In this study, the effect of dieldrin on rat thymocytes exposed to hydrogen peroxide
(H2O2)-induced oxidative stress was examined. Dieldrin at 5 lM and H2O2 at 300 lM slightly increased
cell lethality from a control value of 5.4 0.5% (mean standard deviation of four experiments) to
7.8 1.3% and 9.0 0.3%, respectively. Simultaneous application of dieldrin and H2O2 signicantly
increased cell lethality to 46.2 1.8%. The synergistic increase in cell lethality was dependent on dieldrin
concentration (0.35 lM) but not on H2O2 concentration (30300 lM). Dieldrin accelerated H2O2induced cell death, which was estimated with the help of annexin V-FITC and propidium iodide. Presence
of either dieldrin or H2O2 decreased the cellular content of nonprotein thiol and increased intracellular
Zn2+ concentration. The combination of dieldrin and H2O2 further pronounced these effects. TPEN, a
chelator of intracellular Zn2+, signicantly attenuated the synergistic increase in cell lethality induced
by dieldrin and H2O2. It is, therefore, suggested that dieldrin augments the cytotoxicity of H2O2 in a
Zn2+-dependent manner.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
Dieldrin is one of the most persistent pesticides, and is highly
resistant to biodegradation and abiotic degradation (US Environmental Protection Agency, 2000). This pesticide is toxic to aquatic
organisms, birds, and mammals (Clement Associates, 1985; Agency
for Toxic Substances and Diseases Registry, 2002). Because of concerns about damages to the environment and potentially to the human health, the US Environmental Protection Agency banned all
uses of dieldrin in USA since 1987. However, dieldrin is commonly
Corresponding author. Tel./fax: +81 88 656 7256.
E-mail address: oyama@ias.tokushima-u.ac.jp (Y. Oyama).
These authors (graduate students) contributed equally to this work. Experiments
and manuscript preparation were carried out during the graduate classes of
Environmental Symbiosis Studies.
1

0045-6535/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.chemosphere.2013.04.092

detected in human blood samples in USA and other countries

(Patterson et al., 2009; Mustafa et al., 2010; Azmi and Naqvi,
2011; Kaushik et al., 2012).
Recent studies suggest that dieldrin induces oxidative stress,
which possibly leads to adverse effects on mammals (Kitazawa
et al., 2001; Hatcher et al., 2007; Mao and Liu, 2008; Slotkin and
Seidler, 2009; Martyniuk et al., 2010; Sharma et al., 2010). Normal
metabolic processes produce a variety of reactive oxygen species,
which have a potential to cause oxidative damage to proteins, lipids, and DNA (Balaban et al., 2005). However, almost all aerobic
organisms are capable of quenching reactive oxygen species and
subsequently controlling their damaging actions (Halliwell, 1999;
Benzie, 2000). Therefore, the toxicity of dieldrin is supposed to
be obvious on the cells under oxidative stress since synergistic increase in the generation of reactive oxygen species was induced by

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T. Chimeddorj et al. / Chemosphere 93 (2013) 353358

the combination of dieldrin and lindane (Mao and Liu, 2008) and
lindane by itself induced oxidative stress (Barros et al., 1988; Junqueira et al., 1988). Dieldrin exerts cytotoxic actions on dopaminergic neurons (Sanchez-Ramos et al., 1998; Kitazawa et al.,
2001; Kanthasamy et al., 2005). Hydrogen peroxide (H2O2) and
other reactive oxygen species generated during the oxidative
metabolism of dopamine (Graham, 1978) possibly expose the cells
to chronic oxidative stress. Therefore, it may be one of common
features in dieldrin-related toxicity. In this study, the effect of dieldrin on rat thymocytes exposed to H2O2-induced oxidative stress
was examined to see if dieldrin exerts a potent cytotoxicity on cells
under oxidative stress. We also revealed that Zn2+ was involved in
the cytotoxicity induced by the combination of dieldrin and H2O2.
2. Materials and methods
2.1. Chemicals
Dieldrin was purchased from Kanto Chemical Co. Inc. (Tokyo, Japan). Annexin V-FITC, 5-chloromethyluorescein diacetate (5CMF-DA), FluoZin-3-AM, and propidium iodide were purchased
from Molecular Probes Inc. (Eugene, Oregon, USA). The chelator
of intracellular Zn2+, N,N,N0 ,N0 -tetrakis[2-pyridylmethyl]ethylenediamine (TPEN), was obtained from Dojin Chemical Laboratory
(Kumamoto, Japan). Other chemicals (NaCl, CaCl2, MgCl2, KCl, glucose, H2O2, and NaOH) were purchased from Wako Pure Chemicals
Industries, Ltd. (Osaka, Japan). The buffer used was 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Nacalai Tesque,
Kyoto, Japan).

for propidium was 488 nm, and emission was detected at

600 20 nm.
Exposure of phosphatidylserine on the outer surface of thymocyte membranes was detected using annexin V-FITC (Koopman
et al., 1994; Nakata et al., 1999; Oyama et al., 1999). The cells were
incubated with annexin V-FITC (10 lL mL1) for 30 min and with
5 lM propidium iodide for 2 min before the measurement. Excitation wavelength for FITC was 488 nm, while emission of FITC (annexin V binding to membranes) was detected at 530 20 nm.
5-CMF-DA was used to monitor changes in the cellular content
of nonprotein thiol (Chikahisa et al., 1996). The cells were incubated with 1 lM 5-CMF-DA for 30 min before any uorescence
measurements. 5-CMF uorescence was measured in the cells that
were not stained with 5 lM propidium iodide. The excitation
wavelength used for 5-CMF was 488 nm and emission was detected at 530 15 nm.
FluoZin-3-AM (Gee et al., 2002) was used as an indicator of
intracellular Zn2+ concentration. The cells were incubated with
500 nM FluoZin-3-AM for 60 min before any uorescence measurements were taken. FluoZin-3 uorescence was measured in
cells that were not stained with 5 lM propidium iodide (Matsui
et al., 2008). The excitation wavelength used for FluoZin-3 was
488 nm and emission was detected at 530 20 nm.
2.4. Statistics
Values were expressed as mean standard deviation of four
experiments. Statistical analysis was performed with Tukeys multivariate analysis. P < 0.05 was considered signicant.

2.2. Animals and cell preparation

3. Results

The study was approved by the Committee for Animal Experiments of the University of Tokushima (Registration No. 05279).
The cell suspension of rat thymocytes was prepared in a similar
manner to that previously reported (Chikahisa et al., 1996). In brief,
thymus glands dissected out from ether-anesthetized rats were
sliced at a thickness of 400500 lm by using a razor blade under
cold conditions (34 C). The slices were triturated with gentle
shaking in chilled Tyrodes solution (NaCl 150 mM, KCl 5 mM,
CaCl2 2 mM, MgCl2 1 mM, glucose 5 mM, and HEPES 5 mM, with
an appropriate amount of NaOH to adjust the pH to 7.37.4) to dissociate the thymocytes. Thereafter, the Tyrodes solution containing the cells was passed through a mesh (size: 10 lm) to prepare
the cell suspension. Beaker containing the cell suspension was
incubated in a water bath at 3637 C for 1 h before the experiment. Although Tyrodes solution did not contain ZnCl2, the cell
suspension contained 200230 nM zinc, derived from the cell
preparation (Sakanashi et al., 2009).

3.1. Dieldrin-induced increase in cell lethality

2.3. Fluorescence measurements of cellular and membrane parameters

Cellular and membrane parameters were measured using a ow
cytometer equipped with argon laser (CytoACE-150, JASCO, Tokyo,
Japan) and uorescent probes as described previously (Chikahisa
et al., 1996; Matsui et al., 2008). Fluorescence was analyzed using
JASCO software (Version 3XX, JASCO). Under the experimental conditions in the present study, no reagent showed uorescence, except the probes.
To assess cell lethality, propidium iodide was added to the cell
suspension to achieve a nal concentration of 5 lM. Since
propidium selectively stains dead cells, measurement of propidium
uorescence provides an estimate of cell lethality. Two minutes
following the application of propidium iodide, uorescence was
measured with a ow cytometer. The excitation wavelength used

As shown in Fig. 1A, incubation of rat thymocytes for 3 h with

5 lM dieldrin increased the population of cells showing propidium
uorescence signicantly, albeit slightly. Such an increase in propidium uorescence implies an increase in cell lethality due to dieldrin. However, dieldrin did not increase cell lethality at
concentrations 63 lM. Time-dependent and concentration-dependent changes in the effects of dieldrin are summarized in Fig. 1B.
3.2. Synergistic increase in cell lethality by dieldrin and H2O2
Effect of dieldrin on the cells under oxidative stress was examined. Simultaneous incubation of 5 lM dieldrin and 300 lM H2O2
for 3 h greatly increased the population of cells showing propidium
uorescence (Fig. 2A). As shown in Fig. 2B, 5 lM dieldrin and
300 lM H2O2 slightly, but signicantly increased cell lethality from
5.4 0.5% to 7.8 1.3% and 9.0 0.3%, respectively. Simultaneous
incubation of dieldrin and H2O2 signicantly increased cell lethality to 46.2 1.8% (Fig. 2B).
Incubation with dieldrin at concentrations ranging from 1 lM
to 5 lM for 3 h augmented the cytotoxicity of 300 lM H2O2 in a
concentration-dependent manner (Fig. 3A). H2O2 at 30300 lM
potentiated the cytotoxicity of 5 lM dieldrin (Fig. 3B). However,
the augmentation of dieldrin cytotoxicity by 100 lM H2O2 was
similar to that by 300 lM H2O2.
3.3. Effect of dieldrin on the process of cell death induced by H2O2
Effect of 5 lM dieldrin on the process of cell death induced by
300 lM H2O2 was examined using propidium iodide and annexin
V-FITC. As shown in Fig. 4, the cells were classied into the following four population groups on the basis of FITC and propidium

T. Chimeddorj et al. / Chemosphere 93 (2013) 353358

Fig. 1. Change in cell lethality induced by dieldrin. (A) Change in histogram of

propidium uorescence by dieldrin. Each histogram consisted of 2000 cells. The bar
under histogram indicates the population of cells showing propidium uorescence,
presumably the dead cells. (B) Concentration-dependent and time-dependent
changes in the population of cells stained with propidium (cell lethality) by
dieldrin. Column and bar respectively show mean and standard deviation of four
samples. Asterisk () indicates signicant difference (P < 0.05) between the control
groups (CONTROL, 0.1% DMSO) and the dieldrin treated group. The experiments
were repeated three times. The results of representative experiment are shown.

uorescence: intact living cells (area N); living cells with exposed
phosphatidylserine (area A); and dead cells (areas P and AP).
Fig. 4A shows the changes in cell populations following 1 h
incubation with dieldrin, H2O2, or both. The incubation with
5 lM dieldrin did not signicantly affect the cells. H2O2 at
300 lM increased the population of living cells with exposed phosphatidylserine. The combination of dieldrin and H2O2 increased
both the population of living cells with exposed phosphatidylserine and dead cells. The results are summarized in Fig. 5A. Fig. 4B
shows the changes in cell populations following 2 h incubation
with dieldrin, H2O2, or both. Incubation with H2O2 signicantly increased the population of living cells with exposed phosphatidylserine. The combination of H2O2 and dieldrin further accelerated
the conversion of living cells with exposed phosphatidylserine to
dead cells. Results are summarized in Fig. 5B.

355

Fig. 2. Synergistic increase in cell lethality by the combination of dieldrin and H2O2.
(A) Changes in the histogram of propidium uorescence by H2O2 alone and the
combination of dieldrin and H2O2. Each histogram consisted of 2000 cells. The bars
show the population of cells showing propidium uorescence. (B) Changes in cell
lethality by dieldrin alone, H2O2 alone, and their combination. Column and bar
respectively show mean and standard deviation of four samples. Asterisks (, )
indicate signicant difference (P < 0.05, P < 0.01) between the control groups
(CONTROL, 0.1% DMSO) and the group(s) of cells treated with chemical(s). Symbol
(##) shows signicant difference (P < 0.01) between the groups arrowed. The
experiments were repeated three times. The results of representative experiment
are shown.

3.5. Changes in intracellular Zn2+ concentration and cellular content of

nonprotein thiol induced by dieldrin, H2O2, and both
As seen in Fig. 6, it is likely that Zn2+ is involved in the synergistic increase in cell lethality by the combination of dieldrin and
H2O2. Both 3 lM dieldrin and 30 lM H2O2 signicantly increased
the intensity of FluoZin-3 uorescence, an indicator of intracellular
Zn2+ (Fig. 7A). A further augmentation of FluoZin-3 uorescence by
the combination of dieldrin and H2O2 was observed (Fig. 7A).
Effects of dieldrin, H2O2, and their combination on 5-CMF uorescence were examined to see if the combination synergistically
decreases cellular content of nonprotein thiol. H2O2 at 10 lM and
30 lM signicantly decreased the intensity of 5-CMF uorescence.
Similarly, the intensity decreased with 3 lM dieldrin. A further
attenuation of 5-CMF uorescence by the combination of dieldrin
and H2O2 was also observed (Fig. 7B).

3.4. Effect of TPEN on the synergistic increase in cell lethality by

dieldrin and H2O2
4. Discussion
Zn2+ has a critical role in cytotoxicity of H2O2 (Matsui et al.,
2010). Effect of TPEN on the increase in cell lethality induced by
the combination of H2O2 and dieldrin was examined. The combination increased cell lethality even in the presence of 10 lM TPEN,
however, the change was much smaller than that seen in the absence of TPEN (Fig. 6).

4.1. Effect of dieldrin on cells under oxidative stress

It is likely that dieldrin potentiates the cytotoxicity of H2O2 because of following observations. First, the increase in cell lethality
by simultaneous application of dieldrin and H2O2 was dependent

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T. Chimeddorj et al. / Chemosphere 93 (2013) 353358

Fig. 3. Concentration dependent changes in cell lethality by several combinations

of dieldrin and H2O2. (A) Concentration dependent changes in cell lethality by
dieldrin in the presence of 300 lM H2O2. Column and bar respectively show mean
and standard deviation of four experiments. Asterisks (, ) indicate signicant
difference (P < 0.05, P < 0.01) between the control (0.1% DMSO) and the group of
cells treated with dieldrin and H2O2. (B) Concentration dependent changes in cell
lethality by H2O2 in simultaneous presence of 5 lM dieldrin. Column and bar
respectively show mean and standard deviation of four samples. Asterisks (, )
indicate signicant difference (P < 0.05, P < 0.01) between the control (0.1% DMSO)
and the group of cells treated with dieldrin and H2O2. The experiments were
repeated three times. The results of representative experiment are shown.

on the concentration of dieldrin rather than on H2O2 (Fig. 3). Cell

lethality in the simultaneous presence of dieldrin and H2O2 increased as a function of dieldrin concentration (15 lM). On the

other hand, the increase in cell lethality by 5 lM dieldrin and

100 lM H2O2 was similar to that by 5 lM dieldrin and 300 lM
H2O2 (Fig. 3B). Second, as shown in Figs. 4 and 5, 300 lM H2O2 signicantly decreased the population of intact living cells and significantly increased the population of living cells with exposed
phosphatidylserine in a time-dependent manner, although the
change was not apparent with 5 lM dieldrin. Simultaneous application of dieldrin and H2O2 accelerated the process of conversion of
intact living cells to living cells with exposed phosphatidylserine,
in addition to accelerating the conversion to dead cells. Thus, dieldrin seems to accelerate the changes induced by H2O2.
The cytotoxicity of H2O2 alone was dependent on the increase in
intracellular Zn2+ concentration (Matsui et al., 2010). Dieldrin,
H2O2, and their combination increased the intensity of FluoZin-3
uorescence in living cells, suggesting an increase in intracellular
Zn2+ concentration (Fig. 7). Zn2+ seems to be involved in cell death
induced by the combination since TPEN, a chelator for intracellular
Zn2+, signicantly attenuated the increase in cell lethality by dieldrin and H2O2 (Fig. 6). The results of Figs. 6 and 7 are presumable
since an excessive elevation of intracellular Zn2+ concentration exerts cytotoxic action (for a review, Maret, 2008). The mechanism of
dieldrin toxicity was explained by dieldrin-induced oxidative
stress (Bachowski et al., 1998; Kitazawa et al., 2001). It is likely
that oxidative stress induced by dieldrin elicits an increase in intracellular Zn2+ concentration because an intracellular Zn2+ is known
to form a complex with the thiol group of protein and non-protein
(Jacob et al., 1998) and modication from thiol to disulde by oxidation releases Zn2+ (Maret, 1994). In fact, the cellular content of
nonprotein thiol was reduced by dieldrin alone, H2O2 alone, and
their combination (Fig. 7B).
The application of H2O2 increased the population of cells with
exposed phosphatidylserine (Figs. 4 and 5). Thus, phosphatidylserine that is normally localized to inner membrane surface is transferred to outer surface under severe oxidative stress. Disruption of
asymmetric sequence of the lipid bilayer due to oxidative stress
may change its physicochemical property. Since dieldrin is highly
lipophilic, it is likely that dieldrin easily penetrates into the membranes, leading to an enhanced perturbation of the membrane
integrity, such as membrane permeability. Nonionic surfactants

Fig. 4. Changes in cytograms following incubation with dieldrin alone, H2O2 alone, and their combination for 1 h (A) and 2 h (B). Fluorescence cytogram consisted of
propidium uorescence (abscissa) and FITC uorescence (ordinate). Each cytogram was constructed with 2000 cells after respective incubation.

T. Chimeddorj et al. / Chemosphere 93 (2013) 353358

Fig. 5. Changes in cell populations following incubation with dieldrin alone, H2O2
alone, and their combination for 1 h (A) and 2 h (B). Changes in cell populations
were classied on the basis of propidium and FITC uorescence, following
treatment with dieldrin alone, H2O2 alone, and their combination. Column and
bar show mean and standard deviation of four samples. Symbol (##) indicates
signicant difference (P < 0.01) between the control group (CONTROL) and the
group of cells treated with chemical(s).

357

Fig. 7. Changes in the intensity of FluoZin-3 uorescence (A) and 5-CMF uorescence (B) by dieldrin alone, H2O2 alone, and their combination. Column and bar
respectively indicate mean and standard deviation of four samples. Asterisk ()
indicates signicant difference (P < 0.01) between the control group and the group
of cells treated with chemical(s). Symbol (##) also shows signicant difference
(P < 0.01) between the groups arrowed.

such as cremophor EL and polysorbate 80 increase the vulnerability of cells suffering from oxidative stress induced by H2O2 (Iwase
et al., 2004; Tatsuishi et al., 2005). Therefore, the synergistic increase in cell lethality by dieldrin and H2O2 may also be dependent
on membrane perturbation.

4.2. Implications

Fig. 6. Effect of TPEN on synergistic increase in cell lethality by simultaneous

application of dieldrin and H2O2. Column and bar show mean and standard
deviation of four samples. Symbol (##) indicates signicant difference (P < 0.01)
between the groups arrowed.

Dieldrin is resistant to biotic and abiotic degradations. This

insecticide is commonly found in sera and tissues of humans and
wild animals (Naqvi and Jahan, 1999; Mustafa et al., 2010; for a review, Azmi and Naqvi, 2011). Analysis of maternal and cord blood
samples in the age group of 2529 years in the Guru Teg Bahadur
Hospital (Delhi, India) from August 2008 to March 2009 showed
the concentrations of dieldrin to be 2.87 0.48 lg L1 and
2.11 1.42 lg L1, respectively (Mustafa et al., 2010). In human
blood samples collected via venipuncture during August 2002 from
hospitals of Haryana (India), the mean concentration was
17.9 lg L1 (Kaushik et al., 2012). Higher dieldrin concentrations
were observed in the sera samples obtained from chlorination
plant of Pakistan Agriculture Research Center at Karachi (Azmi
and Naqvi, 2011). Molecular weight of dieldrin is 380.91.

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T. Chimeddorj et al. / Chemosphere 93 (2013) 353358

Therefore, the blood concentrations of dieldrin are supposed to be

in nanomolar range. Despite log Kow of dieldrin being 5.37, dieldrin
would not exert synergistic toxicity on the cells suffering from oxidative stress, unless it is accumulated in human tissues.
There is no evidence for adverse actions of dieldrin on human
thymocytes under oxidative stress at present. Primary function of
thymocytes is the generation of T lymphocytes. The function of T
lymphocytes to recognize foreign antigens is mediated by T cell
receptor. If dieldrin decreases the population of intact thymocytes
under oxidative stress, it would disturb normal development and
functioning of cells mediating immunity.
Acknowledgements
This study was supported by a Grant-in-Aid for Scientic Research (C23510078) from the Japan Society for the Promotion of
Science and by a grant from the Ofce of the Dean, Institute of Socio-Arts and Sciences, The University of Tokushima).
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