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RES E AR C H A R T I C L E Open Access
Immunomodulatory role of Emblica officinalis in
arsenic induced oxidative damage and apoptosis
in thymocytes of mice
Manish K Singh, Suraj S Yadav, Vineeta Gupta and Sanjay Khattri
*
Abstract
Background: Arsenic is widely distributed in the environment and has been found to be associated with the
various health related problems including skin lesions, cancer, cardiovascular and immunological disorders. The fruit
extract of Emblica officinalis (amla) has been shown to have anti-oxidative and immunomodulatory properties. In
view of increasing health risk of arsenic, the present study has been carri ed out to investigate the protective effect
of amla against arsenic induced oxidative stress and apoptosis in thymocytes of mice.
Methods: Mice were exposed to arsenic (sodium arsenite 3 mg/kg body weight p.o.) or amla (500 mg/kg body
weight p.o.) or simultaneously with arsenic and amla for 28 days. The antioxidant enzyme assays were carried out
using spectrophotometer and generation of ROS, apoptotic parameters, change in cell cycle were carried out using
flow cytomete r following the standard protocols.
Results: Arsenic exposure to mice caused a significant increase in the lipid peroxidation, ROS production and
decreased cell viability, levels of reduced glutathione, the activity of superoxide dismutase, catalase, cytochrome c
oxidase and mitochondrial membrane potential in the thymus as compared to controls. Increased activity of
caspase-3 linked with apoptosis assessed by the cell cycle analysis and annexin V/PI binding was also observed in
mice exposed to arsenic as compared to controls. Co-treatment with arsenic and amla d ecreased the levels of
lipid pe roxidati on, ROS production, activity of caspase- 3, apoptosis and increased cell viability, levels of
antiox idant enzymes, cytochrome c o xidase and mitochondrial membrane pot ential as compared to mice treated
with arsenic alone.
Conclusions: The results of the present study exhibits that arsenic induced oxidative stress and apoptosis
significantly protected by co-treatment with amla that could be d ue to its strong antioxidant potential.
Keywords: Arsenic, Emblica officinalis, Oxidative stress, Apoptosis, Thymocytes
Background
Arsenic is considered as an environmental contaminant
and widely distributed in the environment due to its natural
existence and anthropogenic applications [1,2]. It has long
been used in pharmaceuticals, glass industries, manufac-
turing of sheep-dips, leather preservatives, poisonous baits,
pesticides and semiconductor devices [3-5]. Exposure to
arsenic in human could occur through air, soil and other
occupational sources [6]. Human exposure to arsenic
through contaminated food materials is quite common
in the area having high levels of arsenic in ground water
[6-8]. High levels of arsenic in ground water in India
and many other regions of the world have been found to
be associated with various health related problems including
arsenicosis, skin lesions, cardiovascular diseases, reproduct-
ive problems, psychological, neurological and immunotoxic
responses [2,9]. In view of increasing risk of arsenic toxicity,
World Health Organization lowered the limit of arsenic in
ground water from 50 μg/l to 10 μg/l [6].
Epidemiological studies have suggested that exposure
to arsenic in humans may attributed to various immune
related disorders [10-13]. In utero exposure to arsenic
has been shown to suppress the immune mediated cells
* Correspondence: drskhattri01@gmail.com
Department of Pharmacology, King George Medical University, Lucknow,
Chowk 226 003, India
© 2013 Singh et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Singh et al. BMC Complementary and Alternative Medicine 2013, 13:193
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and impaired child thymic development associated with
increased morbidity in children [14]. Exposure to arsenic
during pregnancy has been found to lower thymic index
suggested poor development of thymus in infants [15].
Increased placental inflammatory response, reduced pla-
cental T cells and altered levels of cord blood cytokines
linked with fetal death, impaired infant health associated
with enhanced oxidative stress have also been reported
following exposur e to arsenic [16,17]. Andrew et al. [10]
observed that arsenic exposure may stimulate the over
expression of genes involved in the defense system, im-
mune function, cell growth, apoptosis, regulation of cell
cycle, T cell receptor signaling pathway and diabetes.
Association of arsenic intoxication with cancer, black foot
disease and diabetes has a lso been reported [18,19]. A r-
senic exposure in mice has been found to be associated
with the increased free radical ge neration that affect s
the primary electron rich sites within the cells and cause
DNA damage in human lymphocytes [20-22], breaking
of DNA strand, DNA base modifications, protein crosslink,
structural carbohydrates and lipids [23-25]. Arsenic in-
duced cell death has been strongly linked to the induc-
tion of autophagy in human lymphoblastoid cell lines
to impart its immunotoxic effects [11,12]. Numerous
studies have reported that exposure t o inorganic arsenic
increased the frequency of micronuclei, chromosome ab-
errations and sister chromatid exchanges both in humans
and experimental animals [26,27].
Recently, plant derived natural compounds and their
active constituents have received great attention as a poten-
tial antioxidant against arsenic induced toxicity [21,28,29].
The fruit extract of Emblica officinalis (amla) with a history
of medicinal value, long been used in Chinese and Indian
traditional system of medicine and ha s s hown anti-
oxidative and immunomodulatory properties [30-33].
Amla contains a wide variety of phenolics including
anthocyanins, flavonols, ellagic acid and its derivatives
which protects against the harmful action of ROS and
exhibits a wide range of biological effects including anti-
oxidant, anti-tumour, anti-inflammatory, anti-bacterial and
hepato-protective [32-34]. The dose of arsenic selected in
the present study is quite low and based on the earlier
studies [35,36]. Although a number of studies have been
carried out to understand the protective efficacy of herbal
agents against arsenic induced toxicity, not much is known
about its mechanism involved in immunotoxicity and
protective management. In recent years , the intake of
dietary polyphenols ha s rece ived a great attention of
health scientists to use them in the therapeutic manage-
ment of various disease conditions . The prese nt study
has therefore been focused to investigate the immuno-
modulatory role of the fruit extract of amla in arsenic
induced oxidative damage including lipid peroxida-
tion, status of antioxidant enzymes and mitochondrial
membrane potential and apoptosis and necrosis in thymo-
cytes of mice.
Methods
Chemicals
Sodium arsenite, RNase A, 2′,7′-dichlorofluorescein diace-
tate (DCFH-DA), 3-(4,5-dimethyl-2-yl)- 2,5-diphenyl tetrazo-
lium bromide (MTT), 7-amino-4-trifluoro methylcoumarin
(AFC), DPPH (1,1-diphenyl-2-2′-picrylhydrayl) and all
other chemicals were purchased from Sigma–Aldrich,
USA. Rhodamine 123 (Rh 123) and from Molecular Probes,
propidium iodide ( PI) from Calbiochem, and Annexin
V-FITC were purc hased fro m Biovision.
Plant material
Studies have been reported that phenolics and flavonoi ds
including gallic acid, ellagic acid, isocorilagin, chebulanin
and chebulagic acid are the maj or constituents present
in ethyl acetate extract of amla [37,38]. To investigate
the combined effect of theses constituents, ethyl acetate
extract of amla has been selected for the present study.
Briefly, the fresh fruits of amla were collected from authen-
tic source and fruit powder was extracted three times using
95% ethanol (Plant Identification No. - 13394 obtained by
the Herbarium of Birbal Sahni Institute of Palaeobotany,
Lucknow, India). The combined extracts were filtered and
evaporated to dryness with a rotary e vaporator under
reduced pressure and the residue was suspended in water
and extracted successively with diethyl ether and ethyl
acetate. The extract was evaporated under reduced pres-
sure to get powdered form of ethyl acetate fraction.
Animals and treatment
The Balb/c male mice (15 ± 2 g) were obtained from the
animal breeding colony of CSIR-Indian Institute of Toxi-
cology Research, Lucknow used for the study. Mice were
housed in an air-conditioned room at 25 ± 2°C with a 12 h
light/dark cycle under standard hygiene conditions and
had free access to pellet diet and water ad libitum.The
study was approved by the institutional animal ethics
committee of King George Medical University, Lucknow
(No. 121 IAH/Pharma-11) and all experiments were car-
ried out in accordance with the guidelines laid down by
the committee for the purpose of control and supervi-
sion of experiments on animals (CPCSEA), Ministry of
Environment and Forests (Government of India), New Delhi,
India. The animals were randomly divided into four groups
contained ten animals in each group as follows
Group I. Mice were treated with vehicle (2% gum acacia)
for the duration of the treatment to serve as controls.
Group II. Mice were treated with arsenic a s sodium
arsenite (dissolved in distilled water 3 mg/kg body
weight p.o., daily for 30 days).
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Group III. Mice were treated with fruit extract of amla
(500 mg/kg body weight, suspended in 2% gum
acacia, p.o., daily for 30 days).
Group IV. Mice were co-treated with arsenic and fruit
extract of amla identically as in group II and III.
After the last dose of treatment, animals were sacrificed
and thymus of all the animals was i solated. Out of all
isolated thymus three of each set, put into the phosphate
buffer s aline (PBS) and processed for the measurement
of apoptotic parameters. While remaining thymus were
immediately placed in an ice cold saline solution (0.15M),
blotted on filter paper, quickly weighed and processed for
enzymatic and non-enzymatic antioxidants assays.
Preparation of thymocyte suspension
Thymus was dissected from mice and single cell suspen-
sion was prepared under aseptic condition. The suspen-
sion was passed through stainless steel mesh centrifuged
at 200 × g at 4°C for 10 min and resuspended in complete
cell culture medium (RPMI-1640 containing HEPES and
2mM glutamine, supplemented with 10% FBS and 1%
antibiotic antimycotic solution) the cell density were ad-
justed to 1.5 × 10
6
cell/ml.
Measurement of antioxidant activity
Assay of DPPH radical scavenging activity
The free radical scavenging activity of ethyl acetate ex-
tract amla wa s measured by the scavenging of the DPPH
radical using the standard method with slight modifica-
tions [39]. Amla extract at different concentrations (1.0,
2.5, 5, 10, 20 and 40 μg/ml) in ethanol (2ml) was mixed
with DPPH (2 ml, 1mM in ethanol) and incubated for
30 min in the dark. The absorbance of DPPH radical was
read at 517 nm using a spectrophotometer. The DPPH
radical scavenging activity was calculated using the follow-
ing equation
Scavenging activity %ðÞ¼ A
0
−A
1
ðÞ=A
0
fg
100
where A
0
is the absorbance of the control reactions and A
1
is the absorbance in the presence of the test compound.
Biochemical parameters
Assessment of cell viability
The cell viability was measured by the MTT reduction
method following the standard procedure [40]. Cells were
seeded at a density of 1.0 × 10
4
in a 96 well plate. 10 μlof
MTT (5 mg/ml PBS) was added to the each well and incu-
bated for 4 h at 37°C in a CO
2
incubator. The plate was
centrifuged at 1200 × g for 10 min and 100 μlofDMSO
was added after aspirating supernatant to dissolve the
formazan formed in the wells. After 5 min the absorbance
was read on a microplate reader (Synergy HT of BIO-TEK
International, USA) at 530 nm.
Assay of lipid peroxidation
Lipid peroxidation as a measure of thiobarbituric acid
reactive substances was measured following the standard
procedure [41]. Malondialdehyde (MDA) forms as an inter-
mediate product of the peroxidation of lipids and serves as
an index of the intensity of oxidative stress. The intensity of
pink color formed during the reaction was read on a spec-
trophotometer at 532 nm.
Assay of reduced glutathione levels
Levels of reduced glutathione (GSH) in the thymus hom-
ogenate were estimated following the standard protocol
[42]. The assay involves the reaction of GSH with 5, 5′-
dithiobis-2 nitrobenzoic acid (DTNB) that forms yellow
color and its absorbance was taken by spectrophotometer
at 412 nm. The result has been expressed at μgGSH/mg
protein.
Assay of superoxide dismutase activity
Activity of superoxide dismutase in thymus homogenate
was assayed according to the method of Marklund and
Marklund [43]. The unit of enzyme activity is defined as
the enzyme required for 50% inhibition of pyrogallol auto-
oxidation. The results have been expressed as unit/min/
mg protein.
Assay of catalase activity
Activity of catalase in thymus homogenate was assayed
following the standard protocol [44] using hydrogen perox-
ide (H
2
O
2
) as substrate. The activity of catalase was mea-
sured on a spectrophotometer and has been expressed in
μmole/min/mg protein.
Assay of caspase-3 activity
Activity of caspase-3 in thymocytes was measured fol-
lowing the standard procedure described by Pathak and
Khandelwal [45]. Briefly, the cells (3.0 × 10
6
/ml) were lysed
on ice for 10 min with the help of lysis buffer. Further,
the reaction buffer (10 mM Tris–HCl, 1 mM EDTA, 10 mM
DTT, 5% glycerol) and DEVD-AFC substrate (50 μM) were
added and incubated at 37°C in dark for 2 h. AFC was used
as standard and fluorescence was measured at excitation
and emission wavelengths of 400 nm/505 nm, respectively,
onamicroplatereader.Theenzymeactivityisexpressedas
nmoles AFC/60 min.
Assay of cytochrome c oxidase activity
Cytochrome c oxidase activity was a ssayed through the
colorimetric assay kit purcha sed from Sigma-Aldrich
ChemicalCo.(St.Louis,MO,USA).Absorbancewas
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measured on a spe ctrophotometer at 550 nm and values
are expressed as U/ml.
Assay of ROS generation
The generation of ROS was measured using DCFH-DA
by flow cytometry as described previously [46]. Single cell
suspension (1 × 10
6
/ml) of thymus from control and treated
mice was suspended in PBS and incubated with DCFH-DA
at 37°C for 1 h. Hydrolysis of DCFH-DA leads to formation
of fluorescence DCFH that was measured by the fluores-
cence intensity (FL-1, 530 nm).
Assay of mitochondrial membrane potential
The detection of mitochondrial membrane potential was
assessed by flow cytometry following the standard proced-
ure [47]. Single cell suspension (1 × 10
6
/ml) was incubated
with Rh-123 for 60 min in dark at 37°C. The mitochon-
drial membrane potential was measured using FL-1 filter
at fluorescence intensity of 530 nm.
Analysis of apoptotic DNA
The assay of apoptotic DNA was carried out using the
standard procedure [48]. Briefly, cell suspension of thy-
mocytes at a density of 1 × 10
6
cells from control and
treated mice was prepared for the detection of cell cycle
analysis. Cells were washed with PBS and fixed by 70%
ethanol. The fixed cells were again washed with PBS and
added phosphate citrate buffer (200 μl, pH 7.8) and incu-
bated for 60 min at room temperature. After centrifugation
the cells were resuspended in 0.5 ml propidium iodide (PI)
and 0.5 ml RNAse (50 μg/ml) and further incubated for
30 min in the dark. The PI fluorescence was measured
through a FL-2 filter (585 nm) and a total of 10,000 events
were acquired.
Assessment of apoptotic and necrotic cell
The apoptotic and necrotic cell distribution was analyzed
through Annexin V binding and PI uptake following the
procedure of Vermes et al. [49]. Briefly, thymocytes were
suspended in 1 ml binding buffer (1×), an aliquot of
100 μl was incubated with 5 μlAnnexinV-FITCand10μl
PI for 15 min in dark at room temperature and 400 μlbind-
ing buffer (1×) was added to each sample, the FITC and PI
fluorescence will be measured through FL-1 (530 nm) and
FL-2 filters (585 nm) respectively.
Protein estimation
Protein concentration in thymus homogenates was mea-
sured following the standard procedure [50] using bovine
serum albumin as the reference standard.
Statistical analysis
The statistical analysis wa s carried out by GraphPad
Prism 3.02 using one way analysis of variance followed
by Newman–Keuls test for multiple pair wise compari-
sons among the groups. All values have been expressed
as mean ± SEM. P value <0.05 has been considered
significant.
Results
Effect on DPPH free radical scavenging activity
The different concentration of ethyl acetate extract of
amla (1.0, 2.5, 5 10, 20 and 40 μg/ml) in ethanol (2 ml) was
used for the DPPH radical scavenging activity and results
were presented in Figure 1. The 50% inhibitory concentra-
tion (IC
50
) of fruit extract was found to be 8.32 μg/ml and
it become saturated over 20 μg/ml concentration where the
activity was more than 90%. The results showed that ethyl
acetate extract of amla has strong free radical scavenging
activity associated with its antioxidant potential.
Effect on body weight and thymus weight in m ice
Effect of arsenic and co-treatment of arsenic and amla on
mice has been presented in Table 1. Exposure to arsenic
in mice caused a significant decrease in body weight (25%,
p < 0.01) and thymus weight (34%, p < 0.001) as compared
to controls suggesting the general toxic effe ct of the ar-
senic and could be a ssociated with decreased food con-
sumption and water intake. Co-treatment with arsenic
and amla increased the body weight (21%, p < 0.05) and
thymus weight (26%, p < 0.05) as compared to mice treated
with arsenic alone. No significant effect on body weight
and thymus weight was observed in mice treated with amla
alone as compared to controls (Table 1).
Effect on cell viability in thymus of mice
Effect of arsenic and co-treatment of arsenic and amla
on cell viability in thymus has been presented in Figure 2.
Mice exposed to arsenic exhibited a significant decrease
in cell viability (34%, p < 0.001) as compared to controls.
Co-treatment with arsenic and amla increased the cell
viability (21%, p < 0.01) in thymus as compared to those
Figure 1 DPPH free radical scavenging activity of fruit extract
of Emblica officinalis. The concentration of extract above 20 μg/ml
showed a saturation in the plot indicating the radical scavenging
activity of more than 90%.
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treated with arsenic alone. No significant effect on cell
viability was observed in mice treated with amla alone as
compared to controls (Figure 2).
Effect on the lipid peroxidation in thymus of mice
To assess the level of oxidative damage to the biological
membrane, effect of arsenic and co-treatment of arsenic
and amla on lipid peroxidation in thymus has been car-
ried out and presented in Figure 3. A significant increase
in lipid peroxidation (52%, p < 0.001) in thymus wa s ob-
served in mice exposed to arsenic as compared to controls.
Co-treatment with arsenic and amla decreased in the level
of lipid peroxidation (36%, p < 0.001) in thymus as com-
pared to those treated with arsenic alone. No significant ef-
fect on the level of lipid peroxidation was observed in mice
treated with amla alone as compared to controls (Figure 3).
Effect on the reduced glutathione levels in thymus of mice
Arsenic has high affinity to GSH and thus enhances vul-
nerability towards oxidative stress. Figure 4 indicates the
effect of arsenic and co-treatment of arsenic and amla
on reduced glutathione levels in thymus of mice . Expos-
ure to arsenic in mice caused a significantly decreased in
the levels of reduced glutathione (34%, p < 0.001) in thy-
mus as compared to controls. Co-treatment with arsenic
and amla increase the levels of reduced glutathione (49%,
p < 0.001) in thymus of mice as compared to those treated
with arsenic alone. No significant effect on the levels of
reduced glutathione was observed in the thymus of mice
treated with amla alone as compared to controls (Figure 4).
Effect on the activity of superoxide dismutase in thymus
of mice
Effect of arsenic and co-treatment of arsenic and amla
on the activity of superoxide dismuta se in thymus has
been presented in Figure 5. The activity of superoxide
dismutase, an enzyme involved in the dismutation of
superoxide radicals, was found to be significant decreased
(31%, p < 0.01) in thymus of mice exposed to arsenic as
compared to controls. Co-treatment w ith arsenic and
amla increased the activity of superoxide dismutase (36%,
p < 0.05) in thymus a s compared to those treated with
arsenic alone. No significant effe ct on the activity o f
superoxide dismuta se wa s o bserved in the t hymus of
mice treated with amla alone as compa red to controls
(Figure 5).
Effect on the activity of catalase in thymus of mice
Effect of arsenic and co-treatment of arsenic and amla
on the activity of catalase in thymus has been presented
in Figure 6. Exposure to arsenic in mice caused a signifi-
cant decrease the activity of catalase (35%, p < 0.05) in
thymus of mice as compared to controls. Co-treatment
with arsenic and amla increased the activity of catalase
in thymus (32%, p < 0.05) as compared to those treated
Table 1 Effect on body, thymus weight and thymus cellularity in mice exposed to arsenic, amla and their co-treatment
for 30 days
Parameters Control Treatment groups
Arsenic Amla Amla
(3 mg/kg) (500 mg/kg) + Arsenic
Body weight (g) 19.40 ± 1.12 15.40 ± 0.97
*a
19.80 ± 0.80 18.60 ± 1.16
*b
Thymus weight (mg) 79.2 ± 3.73 52.2 ± 3.26
*a
77.6 ± 3.29 66.2 ± 4.54
*b
Thymus cellularity (×10)
-6
32.3 ± 6.60 26.2 ± 5.30
*a
33.1 ± 5.20 30.6 ± 4.80
*b
Values are mean ± SEM of five animals in each group.
*a-compared to control gro up; *b-compared to arsenic treated group.
*Significantly differs (p < 0.05).
Figure 2 Effect of arsenic, amia and their co-treatment on cell
viability in thymus of mice. Values are mean ± SEM of five animals
in each group a-compared to control group, b-compared to arsenic
treated group *Significantly differs (p < 0.05).
Figure 3 Effect of arsenic, amla and their co-treatment on the levels
of lipid peroxidation in thymus of mice. Values are mean ± SEM of
five animals in each group a-compared to control group, b-compared to
arsenic treated group *Significantly differs (p < 0.05).
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with arsenic alone suggesting a protective effect of amla
against oxidative insult. No significant effect on the activ-
ity of catalase was observed in the thymus of mice treated
with amla alone as compared to controls (Figure 6).
Effect on caspase-3 activity in thymus of mice
Figure 7 demonstrate the effect of arsenic and co-treatment
of arsenic and amla on the activity of caspase-3 in thymus
of mice. Exposure to arsenic caused a significant increased
in the caspase activity (2.42 fold, p < 0.001) in thymus of
mice as compared to controls. Co-treatment with arsenic
and amla decrease the activity of caspase (0.3 fold, p < 0.01)
in thymus as compared to mice treated with arsenic alone
suggesting a trend of recovery. No significant effect on the
caspase activity was observed in mice treated with amla
alone as compared to controls (Figure 7).
Effect on cytochrome c oxidase activity in thymus of mice
Effect of arsenic and co-treatment of arsenic and amla
on the cytochrome c oxidase activity in thymus has been
presented in Figure 8. Exposure of arsenic in mice showed
a decreased cytochrome c oxidase activity (43%, p < 0.001)
in thymus as compared to controls. Co-treatment with
arsenic and amla increa sed the cytochrome c oxidase
activity (62%, p < 0.001) in thymus as compared to mice
treated with arsenic alone. No significant effe ct on the
cytochrome c oxidase activity was observed in mice treated
with amla alone as compared to controls (Figure 8).
Effect on the generation of ROS in thymus of mice
Arsenic has been found to be associated with the increased
generation of ROS. Eff e ct of arsenic and co-treatment of
arsenic and amla on ROS generation in thymus ha s been
presented in Figure 9. Exposure of arsenic to mice caused
an increased generation of ROS (90%, p < 0.01) in thymus
as compared to controls. Co-treatment with arsenic and
amla de crea sed the ROS generation (52%, p < 0.01) in
thymus as compared to mice treated with arsenic alone
suggested the antioxidant a nd free radical scavenging
activity of amla. No significant effe ct on the production
of ROS wa s observed in mice treated with amla alone as
compared to controls (Figure 9).
Effect on the mitochondrial membrane depolarization in
thymus of mice
Effect of arsenic and co-treatment of arsenic and amla on
mitochondrial membrane depolarization in thymus has
been presented in Figure 10. Exposure of arsenic in mice
showed decrea se mitochondrial membrane depolariza-
tion (53%, p < 0.001) in thymus a s compared to con-
trols. Co-treatment with arsenic and amla increased
Figure 4 Effect of arsenic, amla and their co-treatment on the
levels of reduced glutathione levels in thymus of mice. Values
are mean ± SEM of five animals in each group a-compared to
control group, b-compared to arsenic treated group *Significantly
differs (p < 0.05).
Figure 5 Effect of arsenic, amla and their co-treatment on the
activity of superoxide dismutase in thymus of mice. Values are
mean ± SEM of five animals in each group a-compared to control group,
b-compared to arsenic treated group *Significantly differs (p < 0.05).
Figure 6 Effect of arsenic, amla and their co-treatment on the
catalase activity in thymus of mice. Values are mean ± SEM of
five animals in each group a-compared to control group, b-compared
to arsenic treated group *Significantly differs (p < 0.05).
Figure 7 Effect of arsenic, amla and their co-treatment on the
catalase-3 activity in thymus of mice. Values are mean ± SEM of
five animals in each group a-compared to control group, b-compared
to arsenic treated group *Significantly differs (p < 0.05).
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the mitochondrial membrane depolarization (43%, p <
0.05) in thymus as compared to mice treated with ar-
senic a lone. No significant effect on the mitochondrial
membrane depolarization was observed in m ice treated
with amla alone as c ompared to controls (Figure 10).
Effect on the cell cycle in thymus of mice
Cell cycle represents the DNA damage in cells in sub G1
peak. Figure 11 demonstrate the effect of arsenic and co-
treatment of arsenic and amla on the DNA damage as ob-
served by a sub G1 peak in thymus. Exposure of arsenic to
mice caused an increased number of sub G1 peak indicating
the DNA damage (21.37%, p < 0.01) in thymus as compared
to controls. Co-treatment with arsenic and amla reduced
the number of cells in sub G1 peak (11.67%, p < 0.01) in thy-
mus as compared to mice treated with arsenic alone. No sig-
nificant effect on the cell cycle was observed in mice treated
with amla alone as compared to controls (Figure 11).
Effect on the annexin V/PI binding assay in thymus of mice
Annexin binding assay has been used to measure the num-
ber of apoptosis and necrotic cells. Effect of arsenic and co-
treatment of arsenic and amla on the apoptosis in thymus
has been presented in Figure 12. Exposure of arsenic in
mice caused an increased number of necrotic (9%) and
apoptotic cells (11.52%) in thymus as compared to con-
trols. Co-treatment with arsenic and amla decreased the
number of necrotic (2.03%) and apoptotic cells (3.34%) in
thymus as compared to mice treated with arsenic alone.
No significant effect of apoptosis and necrosis in cells of
the thymus was observed in mice treated with amla alone
as compared to controls (Figure 12).
Discussion
Enhanced oxidative stress has been found to play a crucial
role in the induction of apoptosis under both pathological
Figure 8 Effect of arsenic, amla and their co-treatment on the
cytochrome c oxidase activity in thymus of mice. Values are
mean ± SEM of five animals in each group a-compared to control group,
b-compared to arsenic treated group *Significantly differs (p < 0.05).
Figure 9 Effect of arsenic, amla and their co-treatment on the generation of reactive oxygen species in thymocytes of mice. Cells were
incubated with DCFH-DA and fluorescence was measured using a flow cytometer with FL-1 filter. Values are presented as mean ± SEM of three
assays performed independently a-compared to control group, b-compared to arsenic treated group *Significantly differs (p < 0.05).
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and physiological conditions. Increased generation of free
radicals associated with enhanced oxidative stress has been
found to be implicated in arsenic induced thymic atrophy
[20,21]. Numerous studies have reported that exposure to
arsenic increased the production of free radical generation
and cause oxidative damage to the biological membrane
through increa sed levels of lipid peroxidation, protein
carbonyl contents followed by decreased antioxidant defense
system [20,21,28]. GSH is an important biomolecule in-
volve in the antioxidant defense system against toxicants.
The decrease in the levels of GSH following exposure to
arsenic has been found to be associated with high affinity
of arsenic with GSH [51]. Chronic arsenic exposure has
largely been associated with the apoptosis in the lympho-
cytes and involve in immunotoxic response. Viability of
cells has been found to be decreased in thymus of mice
following exposure to arsenic as observed in the present
study suggested the immunotoxicity of arsenic. Increased
production of ROS associated with enhanced lipid peroxi-
dation and decreased levels of reduced glutathione, the
activity of superoxide dismutase and catalase as observed
in the present study consistent with the earlier findings
[20,21]. Most of the toxic chemicals directly act on the
mitochondria, disrupt its phospholipids membrane and
cause mitochondrial dysfunctions [2,52,53]. Arsenic com-
pounds have been shown to be strong inducers of apop-
tosis in normal and transformed cells through production
of ROS [54], decreased mitochondrial membrane potential
[52], activation of caspases [25,55], increased fragmenta-
tion of DNA [55], decreased expression of anti-apoptotic
proteins (Bcl-2, Bcl-XL) and increased expression of pro-
apoptotic proteins [55]. Martin-Chouly et al. [56] reported
that inorganic arsenic directly acts on human T-cells and
impaired their activity via up-regulation of se veral im-
mune and stress response genes. However, the inhib-
ition of T cell proliferation wa s independently of heme
oxygenase −1 expression and monocyte related accessory
signals [56]. Exposure to arsenic through semiconductor
elements including indium arsenide and gallium arsenide
could induce alterations in gene expression and immune
response as sociated with increased production of ROS
which might be involved in the apoptosis and ne crosis
in T-lymphocytes and neuronal cells [57,58]. A decreased
in the activity of cytochrome c oxidase, mitochondrial
membrane potential and increased activity of mitochon-
drial caspase-3, number of cells in sub G1 peak and
Figure 10 Effect of arsenic, amla and their co-treatment on mitochondrial membrane potential of thymocytes of mice. Cells were
incubated with Rh 123 and fluorescence was measured using flow cytometer with FL-1 filter. Values are presented as mean ± SEM of three assays
performed independently a-compared to control group, b-compared to arsenic treated group *Significantly differs (p < 0.05).
Singh et al. BMC Complementary and Alternative Medicine 2013, 13:193 Page 8 of 13
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number of apoptotic and necrotic cells following exposure
to arsenic in mice as obser ved in the present study is
consistent with the earlier reports.
Experimental studies have reported that weigh t of or-
gans (thymus, spleen, adrenals) responsible for immune
response wa s decreased following exposure to a rsenic
[20,59]. Subc hronic low-le vel ex posure to arsenic may
affect immune responses [59]. Decrease body weight in
experimental animals exposed to arsenic has been reported
[59-61]. Decrease in the body weight indicates the general
toxic effect of the chemical. A decrease in the body weight
and thymus weight as observed in the present study could
be due to immunotoxic response of arsenic that have
been found to be recovered by co-treatment with amla
and arsenic in mice.
Various pharmacological preparations and plant extracts
are reported to have strong antioxidant potential and used
against arsenic induced oxidative damage to investigate
their protective efficacy [2,21,30]. Phyto constituents
including flavanoids found in the plant extracts are effect-
ive as radical scavengers and inhibitors of lipid peroxida-
tion [61-63]. Amla contains a wide variety of phenolics
and its derivatives a ssociated with it s strong antioxidant
potential. Amla is widely accepted immune booster among
the people since it possesses multiple pharmacological
and immunomodulatory properties [21,30-33]. It ha s
been reported that amla protect s against the harmful ac-
tion of free radicals and exhibit it s ameliorating effects
in biological system [32,33 ]. Sharma et al. [21] reported
that the antioxidant potential of amla may be due to the
presence of the many phyto-constitue nts, which p rovide
maximum conjugation with free radical species , thus
reducing the number of free radicals available and the
extent of cellular damage. They further suggested that
pre and post supplementation of fruit extract of amla
significantly reduce arsenic induced oxidative stress in
the liver as a result serum transaminases and MDA content
become lowered in the liver and also increa sed activity
Figure 11 Effect of arsenic, amla and their co-treatment on the cell cycle progression of thymocytes of mice. Control and amla treated
cells showed no sub-G1 peak while arsenic showed high numbers of cells in sub-G1 peak and group co-treated with of arsenic and amla showed reduce
number of cells in sub-G1 peaks as compared to arsenic. Cells were incubated with propidium iodide and flourescence was measured using flow cutometer
with FL-2 filter. Values are presented in the historam as mean ± SEM of three assays performed independently representing sub-G1 population of cells.
Singh et al. BMC Complementary and Alternative Medicine 2013, 13:193 Page 9 of 13
http://www.biomedcentral.com/1472-6882/13/193
of superoxide dismutase, catalase, glutathione S trans-
ferase and serum alkaline phosphatase activities [21].
Reddy et al. [64] reported that alcohol-induced oxidative
stress in plasma of rats could b e ameliorated through
the amla that could be due to the combined effect of
phytophenols present in i t. In another study, Kumar
et al. [65] found that amla significantly protects against
lead induced toxicity by decrease generation of free radi-
cals in one day old male broiler chicks. Haque et al. [66]
suggested that amla extract was very effective in reducing
cyclophosphamide induced suppression of humoral im-
munity in mice. They also reported that amla extract
treatment in no rmal animals could modulate levels of
certain antioxidants of kidney and liver resulted in restor-
ation of antioxidant enzymes in cyclophosphamide treated
animals and further suggested that amla or its m edi-
cinal preparations may be useful in combination therapy
in cancer patients. The supplementation with Triphala
(Terminalia chebula, Terminalia beler ica and Embl ica
officinalis) prevent s the noise-stress induced changes
in the antioxidant as well as cell-mediated immune re-
sponse in rat s [67]. Enhanced lipid peroxidation and
decreased levels of reduced glutathione, activity of super-
oxide dismutase and catalase following exposure to arsenic
has been found to be protected following simultaneous
treatment with arsenic and amla in mice suggesting pro-
tectiveefficacyofamla.
In the present study, treatment with amla alone had
no significant effect on cell viability, lipid peroxidation,
levels of reduced glutathione, the activity of superoxide
dismutase, Catalase and other apoptotic markers includ-
ing the activity of caspase–3 and cytochrome c oxidase.
However, decrea sed cell viability has been found to be
protected following co-treatment with arsenic and amla in
thymus of mice. Also, increased levels of lipid peroxdation,
activity of caspase-3 and decreased levels of reduced gluta-
thione, activity of superoxide dismutase, catalase and activ-
ity of cytochrome c oxidase following arsenic exposure
Figure 12 Effect of arsenic, amla and their co-treatment on the annexin-V/PI dual staining of thymocytes of mice. Cells in the upper
right (UR) portion are late apoptotic cells, upper left (UL) are necrotic cells whereas cells in the lower left (LL) and lower right (LR) portion are
viable and early apoptotic cells respectively. Values are presented as mean ± SEM of three assays performed independently.
Singh et al. BMC Complementary and Alternative Medicine 2013, 13:193 Page 10 of 13
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were protected in mice co-treated with arsenic and amla.
Such an immunoprotective effect of amla may attribute
to it s antioxidant potential to counteract free radicals
and prevented from enhanced oxidative stress. The fruit
extract of amla has been reported to enhance cyto-
protection, decrease apoptosis and DNA fragmentation
[68,69]. It has also been found to protect against heavy
metals induced clastogenicity [70]. Chromium (VI) in-
duced free radical generation a ssociated with enhanced
oxidative stress has been found t o protected following
treatment with amla [30]. Further, chromium ( VI) in-
duced apoptosis, DNA fragmentation and immunosup-
pressive effect s on lymphocyte proliferation ha s been
ameliorated following treatment with amla and it also
restored the altered levels IL-2 and γ-IFN [30]. Decreased
activity of cytochrome c oxidase, mitochondrial mem-
brane potential and increa sed activity of mitochondrial
caspase-3, number of cells in sub G1 peak and number
of apoptotic and ne crotic cells in arsenic exposed mice
have also been found to be protected following sim-
ultaneous treatment with arsenic and amla in the
present study.
Conclusions
In conclusion, the results of the present study clearly in-
dicated that ars enic induced free radical generation and
enhance oxidative stress leading to apoptosis in thymo-
cytes of mice . Arsenic induced decrease mitochondrial
membrane potential has been found to increased follow-
ing co-treatment with arsenic and amla. Further , increased
number of apoptosis, necrotic cells and DNA damage fol-
lowing exposure to arsenic has also been found to be de-
creased following co-treatment with arsenic and amla
indicatestheanti-apoptoticpropertyofamlathatcouldbe
due to its strong antioxidative potential. Although t he
results of the present study exhibit immunomodulatory
effects of amla through its antioxidant properties, further
studies are required to understand the detailed mechanism
of immunoprotection.
Competing interests
The authors declare that they have no financial or personnel competing
interests.
Authors’ contributions
MS: designed the experiment; MS and SY conducted research and drafting
of the manuscript; VS: acquisition of data; analysis and interpretation of data;
statistical analysis; SK: review of the manuscript; analysis and interpretation of
data; obtained funding; administrative support; study supervision. All authors
read and approved the final manuscript.
Acknowledgement
The authors thank to Head, Department of Pharmacology, King George
Medical University, Lucknow for his interest in the study. The authors are also
thankful to Dr. P.C. Cho udhury, Professor, Government Ayurvedic College
and Hospital, Lucknow. Manish Kumar Singh is grateful to the Indian Council
of Medical Research, New Delhi for the award of research fellowship. The
technical support by Mr. Veerendra Kumar Saini is also acknowledged.
Received: 6 March 2013 Accepted: 25 July 2013
Published: 27 July 2013
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doi:10.1186/1472-6882-13-193
Cite this article as: Singh et al.: Immunomodulat ory role of Emblica
officinalis in arsenic induced oxidative damage and apoptosis in
thymocytes of mice. BMC Complementary and Alternat ive Medicine
2013 13:193.
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