Cardiovasc Toxicol (2013) 13:123–137

DOI 10.1007/s12012-012-9191-x

Arsenic Trioxide Toxicity in H9c2 Myoblasts—Damage to Cell

Organelles and Possible Amelioration with Boerhavia diffusa
V. P. Vineetha • A. Prathapan • R. S. Soumya •

K. G. Raghu

Published online: 19 November 2012

Ó Springer Science+Business Media New York 2012

Abstract Arsenic trioxide (ATO) has been long used as a Keywords Arsenic trioxide  Boerhavia diffusa  H9c2 
chemotherapeutic agent because of its significant anticancer Intracellular calcium  Mitochondria  Oxidative stress
property. Unfortunately, the use of ATO is limited due to its
cardiotoxic effects. The present study evaluates the pro- Abbreviations
tective property of ethanolic extract of Boerhavia diffusa APD20 Action potential duration at 20 % of
(BDE) against ATO-induced toxicity on various cell repolarization
organelles in H9c2 cardiomyocytes. The effects of different APL Acute promyelocytic leukaemia
concentrations of ATO (5, 7.5 and 10 lM) on cell organ- ATO Arsenic trioxide
elles like mitochondria, endoplasmic reticulum (ER), BDE Ethanolic extract of Boerhavia diffusa
lysosome and actin, generation of reactive oxygen species, CAT Catalase
antioxidant enzyme status and intracellular calcium over- ECG Electrocardiograph
load were evaluated. ATO significantly (P B 0.05) altered ER Endoplasmic reticulum
mitochondrial transmembrane potential, intracellular cal- LDH Lactate dehydrogenase
cium level, ER, lysosomal activity and F-actin network in NR Neutral red
addition to induction of oxidative stress. Co-treatment with ROS Reactive oxygen species
BDE protected the cardiomyocytes from the adverse effects SOD Superoxide dismutase
of ATO, especially at 5 lM concentration, which was evi-
dent from decreased activity of lactate dehydrogenase
(5 lM ATO ? 20 lg/mL BDE: 6.61 ± 1.97 lU/mL,
respective control group: 16.15 ± 1.92 lU/mL), reduced
oxidative stress, calcium influx and organelle damage. Introduction
Results obtained from the present study allow for a better
characterization of the effects of ATO on H9c2 myoblasts. Arsenic trioxide (ATO) is a very potent antitumor agent
In conclusion, our data suggest that cell organelles are also used to treat acute promylocytic leukaemia (APL) patients
the targets of ATO-induced cardiotoxicity in addition to who acquire resistance to all-trans retinoic acid therapy [1,
other reported targets like ion channels, and BDE has the 2]. Clinical use of ATO is restricted due to its toxicity
potential to protect the cardiotoxicity induced by ATO. profile that includes cardiotoxicity [3] and certain other
organ toxicity [4]. Cardiotoxicity associated with ATO
ranges from electrocardiographical (ECG) changes and
V. P. Vineetha  A. Prathapan  R. S. Soumya  temporary left ventricular ejection fraction decline to
K. G. Raghu (&) congestive heart failure [3]. The major ECG lesions include
Agroprocessing and Natural Products Division, Council of QT prolongation and torsedes de pointes. The mechanisms
Scientific and Industrial Research-National Institute for
of anticancer activity of ATO include induction of apop-
Interdisciplinary Science and Technology (CSIR-NIIST),
Thiruvananthapuram 695019, Kerala, India tosis mediated by reactive oxygen species (ROS) and
e-mail: raghukgopal2009@gmail.com excessive intracellular calcium ([Ca2?]i) influx [5–7].

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124
Our previous studies showed that ATO prolonged car-
diac action potential in a dose- and duration-dependent
manner, as well as biochemical alterations in cardiomyo-
cytes [8]. It has been reported that administration of anti-
oxidant phytochemicals such as curcumin [9], resveratrol
[10] and a-lipoic acid [11] attenuated the ATO-induced
cardiotoxicity, and the reported mechanisms for protection
against cardiotoxicity was via their antioxidant-related
effects. Medicinal plants are reported to be a good source
of potent, safe and affordable bioactive for cardioprotec-
tion, and recently, there is a high demand for the same in
the pharmaceutical sector [12].
Boerhavia diffusa (Family: Nyctaginaceae), a well-
known indigenous medicinal plant, also known as pun-
arnava, has pleiotropic medicinal properties [13–15] and
is also a main ingredient of many ayurvedic formula-
tions for the treatment of jaundice, inflammation,
oedema, hypertension etc. This plant is also being used
as a green leafy vegetable in different parts of Asia and
Africa due to its nutraceutical properties. It is a rich
source of minerals, vitamins and carbohydrates. It con-
tains large number of compounds such as alkaloids
(punarnavine), rotenoids (boeravinones A-F), flavonoids,
amino acids, lignans, saponins, b-sitosterols and tetra-
cosanoic, eicosanoic, stearic and ursolic acids [16]. Our
preliminary investigation on the properties of ethanolic
extract of Boerhavia diffusa (BDE) revealed that they
are potent antioxidants [15] and rich resources of bio-
logically active polyphenols. The present study aims to
evaluate the toxic effects of ATO on various cell
organelles like mitochondria, endoplasmic reticulum
(ER), lysosomes and contractile protein F-actin network
in H9c2 cells and the possible amelioration with BDE
against toxicity.
Methods
Chemicals
Arsenic trioxide (ATO), 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide (MTT), dimethyl
sulfoxide (DMSO), 20 ,70 dichlorodihydrofluorescein
diacetate (DCFH-DA), acridine orange (AO), ethidium
bromide (EB), phalloidin and 40 ,6-diamidino-2-pheny-
lindole (DAPI) were purchased from Sigma (St. Louis,
MO, USA). Lysosome–green fluorescent protein (GFP)
and endoplasmic reticulum red fluorescent protein (ER-
RFP) were purchased from Invitrogen, USA. All sol-
vents were of HPLC grade. Dulbecco’s modified Eagle’s
medium (DMEM), foetal bovine serum (FBS) and sup-
plements were purchased from Himedia Pvt Ltd
(Mumbai, India).
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Cardiovasc Toxicol (2013) 13:123–137
Plant Material
Boerhavia diffusa (BD) was collected locally in the month
of May as per the advice of traditional practitioner to avoid
batch to batch and season to season variations and
authenticated by taxonomist from the Department of Bot-
any, University of Kerala. A voucher specimen was kept in
our herbarium for future reference. All the experiments
were finished with the same lot of plant material.
Preparation of BDE
The fresh whole plant was air-dried, defatted with hexane
and extracted with ethanol at low temperature (24 ± 1 °C)
under stirring for 6 h, and the extraction process was
repeated until the solvent became colourless. The solvent
fractions were filtered through Whatman No. 1 filter paper
and concentrated in vacuum under reduced pressure in
rotavapour (Hiedolph, Germany) followed by lyophiliza-
tion. The lyophilized extract was stored at 4 °C until
analysis.
Cell Culture and Treatment
H9c2 cells derived from rat embryonic cardiomyocytes
were obtained from National Centre for Cell Science
(NCCS), Pune, India. Cells were cultured in DMEM sup-
plemented with FBS, 100 U penicillin/mL, and 100 lg
streptomycin/mL and cultured in 5 % CO2 at 37 °C. Cells
were subcultured to 80 % confluence before the experi-
ments. The experimental group consist of (a) control cells;
(b) cells treated with BDE alone (20 lg/mL) for 24 h;
(c) cells treated with various concentration of ATO (5, 7.5
and 10 lM) for 24 h; and (d) cells co-treated with ATO (5,
7.5 and 10 lM) and BDE (20 lg/mL) for 24 h.
In Vitro Cytotoxicity Assay
Morphological Analysis
H9c2 cells in the exponential growth phase were trypsini-
zed and re-suspended in the medium and 5 9 104 cells
were seeded in 24-well plate. After 48 h, cells from all
experimental groups were checked for morphological
alterations under the phase-contrast microscope (Nikon
Eclipse TS100, Japan) at 409 magnification.
MTT Assay
Cells in exponential growth phase were plated at 5 9 104
cells per well in 24-well plate. After 48 h of incubation at
37 °C, cells were subjected to various treatments. 350 lL
of MTT solution (5 mg/mL) was added to each well and
Cardiovasc Toxicol (2013) 13:123–137
incubated for 4 h at 37 °C. The formazan crystals thus
formed were dissolved in DMSO. Then the plates were
read after 45 min in a microplate reader (Biotek Synergy 4,
US) at 570 nm.
Lactate Dehydrogenase Release
Lactate dehydrogenase (LDH) is a stable cytosolic enzyme
which is released into the culture medium due to the disruption
of plasma membrane. This was determined by cytotoxicity
detection kit (Cayman chemical company, USA).
Neutral Red (NR) Uptake Assay
The NR uptake assay provides a quantitative estimation of
the number of viable cells in culture. It is based on the
ability of viable cells to incorporate and bind to the supra-
vital dye NR in the lysosomes. The cells are incubated for
4 h with a medium containing NR (0.33 % of NR dye in
ethanol acetic acid solution). Then cells were washed, the
dye was extracted from each well and the absorbance was
read using a multiwell plate reader at 540 nm.
DNA Integrity
Briefly, the cells from all experimental groups were
labelled with AO/EB to detect apoptosis and processed for
fluorescent imaging to see alteration with various treat-
ments. The working stain (100 lg/mL of AO and 100
lg/mL EB in phosphate buffered saline) was added to cells
and then examined under spinning disc fluorescent micro-
scope (BD PathwayTM Bioimager system, USA).
Detection of Intracellular Reactive Oxygen Species
(ROS)
Intracellular ROS content was determined by oxidative
conversion of cell-permeable DCFH-DA to fluorescent
20 ,70 dichlorofluorescein (DCF). H9c2 cells were seeded in
96-well plate at a density of 5,000 cells per well. DCFH-
DA stain in serum-free medium was added and co-
incubated with H9c2 cells at 37 °C for 20 min. After three
washes, DCF fluorescence was measured by fluorimetry
(570 nm) in multiwell plate reader and fluorescent imaging
was done to detect the difference in the intensity of fluo-
rescence emitted.
Activities of Antioxidant Enzymes
Activities of antioxidant enzymes like superoxide dismu-
tase (SOD) and catalase (CAT) were assayed according to
the method of Misra and Fridovich [17] and Cohen et al.
[18], respectively.
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Mitochondrial Transmembrane Potential Change
The cells were seeded in 96-well plate at a density of 5,000
cells per well in 200 lL of culture medium. The experiment
was done as per the protocol provided with the kit (JC1 kit,
Sigma) and visualized under spinning disc microscope and
fluorescence was measured in multiwell plate reader.
[Ca2?]i Overload
[Ca2?]i overload was detected by staining the various
experimental groups with Fura-2AM for 20 min at 37 °C and
visualized under spinning disc fluorescent microscope. The
fluorescence intensity was measured in multiwell plate reader.
Analysis of Lysosome and Endoplasmic Reticulum
For fluorescent imaging of lysosomes, the cells were
stained with 1 lM lysosome-GFP in serum-free medium
for 16 h at 37 °C and for ER examination, 1 lM ER-RFP
in serum-free medium was added and incubated for 16 h at
37 °C, after 24 h of exposure to various treatments. The
stains were washed off with PBS and examined.
Studies on cell Integrity
The cells from experimental groups were washed with PBS.
Then cells were fixed with 4 % paraformaldehyde in PBS for
10 min, permeabilised and dehydrated with cold 100 %
acetone for 3–5 min. Phalloidin stain (in PBS) was added
and kept at room temperature for 30 min. Nucleus was
counterstained with DAPI and visualized. The fluorescence
emission of actin was read in multiwell plate reader.
Statistical Analysis
All experiments were performed in sextuplicates (n = 6).
Data are reported as mean ± CV of control and treated
cells. The data were subjected to one-way analysis of
variance (ANOVA) and the significance of differences
between means were calculated by Duncan’s multiple
range test using SPSS for windows, standard version 7.5.1,
and the significance accepted at P B 0.05.
Results
Effects of BDE on ATO-Induced Cytotoxicity
The morphological observation showed that cells had
undergone marked changes such as shrinkage, rounding up
(Fig. 1c, e), detachment from the plate and membrane
blebbing (Fig. 1g) with ATO. BDE was effective in
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126 Cardiovasc Toxicol (2013) 13:123–137

maintaining the normal cell morphology, especially at a
concentration of 5 & 7.5 lM (Fig. 1d, f) of ATO, but not at
higher concentration.
The MTT results showed that ATO reduced cell via-
bility in a dose-dependent manner (Table 1). 5, 7.5 and
10 lM of ATO caused 9.96 ± 1.03, 22.93 ± 1.08 and
34.81 ± 3.49 % of cell death, respectively, which were
statistically significant (P B 0.05) compared to control. Co-
treatment with BDE on 5, 7.5 and 10 lM of ATO-chal-
lenged cells reduced the cell death up to 0.94 ± 0.82,
6.29 ± 2.37 and 23.59 ± 2.02 %.
In order to evaluate membrane integrity, LDH leakage
of various experimental groups was measured. 5, 7.5 and
10 lM of ATO caused a significant increase (P B 0.05) in
LDH leakage while BDE co-treatment reduced LDH
leakage caused by ATO (Table 1).
As illustrated in Table 1, a decline in cell viability was
observed after 24 h of ATO treatment, based on the NR
uptake by the cells. ATO treatment showed 18.92 ± 0.61,
12.40 ± 1.76 and 10.06 ± 0.70 % of dye uptake with 5,
7.5 and 10 lM, respectively, indicating dose-dependent
reduction in cell viability. In this case, BDE was effective

Fig. 1 Morphological
observation of cells with ATO.
Images of H9c2 cells from
different experimental groups
under phase-contrast
microscope (Original
magnification 910). a Control
cells; b Cells treated with BDE
alone; c, e, g cells treated with
5, 7.5 and 10 lM ATO,
respectively; d, f, h cells treated
with 5, 7.5 and 10 lM ATO and
BDE, respectively; High dose
(10 lM) caused conspicuous
morphological alteration. BDE
was protective at low and
medium doses but not at high
dose of ATO

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Cardiovasc Toxicol (2013) 13:123–137 127

Table 1 Viability of H9c2 cardiac myoblast cells treated with ATO

Control 20 lg/mL 5 lM ATO 7.5 lM ATO 10 lM ATO
BDE alone
Without With 20 lg/ Without With 20 lg/ Without With 20 lg/
BDE mL BDE BDE mL BDE BDE mL BDE

MTT assay 0 0.28 ± 0.45 9.96 ± 0.10 0.94 ± 0.87a 22.93 ± 0.04 6.29 ± 0.37a 34.81 ± 0.10 23.59 ± 0.08
(% toxicity)
LDH leakage 5.38 ± 0.27 6.15 ± 0.19 16.15 ± 0.11 6.61 ± 0.29a 24.72 ± 0.10 12.76 ± 0.13a 31.61 ± 0.03 29.53 ± 0.03a
(lU/mL)
Neutral red 25.17 ± 0.02 24.43 ± 0.03 18.92 ± 0.03 23.15 ± 0.04 12.40 ± 0.14 14.83 ± 0.04 10.06 ± 0.06 11.44 ± 0.03
uptake
assay (%)
Each value represents the mean ± CV (n = 6)
a
Significant (P B 0.05) changes in value compared with their respective control groups

(P B 0.05) in protecting the cells from the toxic effects Alteration in Transmembrane Potential (Wm)
only at 5 lM concentration of ATO. of Mitochondria

Effects of BDE on ATO-Induced Apoptosis
AO/EB double staining showed that ATO induced lesions
of apoptosis, such as cell shrinkage, nuclear condensation
and fragmentation of DNA (Fig. 2c, e, g). Morphological
features of cells exposed to high dose showed apoptosis
even with BDE co-treatment (Fig. 2h). As in previous case,
BDE was effective (P B 0.05) to protect the cells from
apoptosis only at low dose, that is, 5 lM (Fig. 2d), but not
much effective at other two doses (Fig. 2f, h).
ATO-Induced Intracellular ROS Generation in Cardiac
Myocytes
Figure 3 shows the intracellular ROS generation in control
and ATO-treated cells. Higher DCF fluorescence in cells
exposed with different doses of ATO indicates increased
generation of intracellular ROS level (Fig. 3i). BDE was
found to be significantly (P B 0.05) reducing the ROS
generation in lower dose of ATO (5 lM) (Fig. 3d).
Activity of SOD and CAT in ATO-treated Cells
Activity of SOD was found to be inhibited significantly
(P B 0.05) by 15.5, 24.8 and 56.8 % in 5, 7.5 and 10 lM,
respectively, in ATO-treated cells when compared to
untreated control cells (Table 2). Co-treatment with BDE
reduced the inhibition of SOD activity to 4.7, 11.9 and
34.5 % in 5, 7.5 and 10 lM, respectively. Likewise, CAT
activity was also inhibited significantly (P B 0.05) by
ATO. Co-treatment with BDE in these cells also reduced
CAT inhibition from 22.4, 38.7 and 65.2 % to 4.7, 12.6 and
48.5 % in 5, 7.5 and 10 lM of ATO-treated cells, respec-
tively (Table 2).
Exposure of H9c2 cells to ATO for 24 h caused significant
alteration of the mitochondrial Wm, as measured with the
JC-1 probe, with an average ratio of red:green fluorescence
of 243:110, 182:192 and 55:203 in 5, 7.5 and 10 lM,
respectively. The untreated cells had a red: green ratio of
368:33 (Fig. 4a). ATO caused an increase in the intensity
of green fluorescence when compared to control H9c2
cells. Valinomycin was used as positive control (Fig. 4b).
ATO treatment showed dose-dependent alteration in
mitochondrial membrane potential which was supported by
fluorimetric analysis data (Fig. 4i). BDE was able to
maintain the transmembrane potential significantly
(P B 0.05) intact at 5 and 7.5 lM (Fig. 4d, f), but was
ineffective at 10 lM (Fig. 4h).
Effects of BDE on ATO-Induced [Ca2?]i Influx
in H9c2 Cells
As shown in Fig. 5c, e, g, ATO increased [Ca2?]i influx in
a dose-dependent manner. Fluorescence intensity (Fig. 5i)
increased around 53.7 % in 10-lM-ATO-treated group
when compared to the control group (Fig. 5i). Co-treatment
with BDE, partially inhibited the increase in intracellu-
lar calcium in 5-lM-ATO-treated groups (Fig. 5d), but
not effective in 7.5- and 10-lM-ATO-treated groups
(Fig. 5f, h).
ATO-induced Alteration in the Lysosomes
Staining of the lysosomes with lysosome-GRP probe
revealed a number of aberrations in the lysosomes of cells
with ATO (Fig. 6). An increase in the number and size of
the lysosomes was noted in H9c2 cells exposed to all doses
of ATO in a dose-dependent manner. BDE treatment was

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Fig. 2 Alteration in DNA

integrity with ATO.
Photomicrographs showing
apoptosis of H9c2 cells under
AO/EB staining (Original
magnification 920). a Control
H9c2 cells; b H9c2 cells treated
with BDE alone; viable cells
were uniformly green, showing
the normal structure c, e H9c2
cells treated with 5 and 7.5 lM
ATO, early-apoptotic cell,
showing the nuclear shrinkage
and chromatin condensation;
g H9c2 cells treated with 10 lM
ATO, late-apoptotic cell that
had lost its selective
permeability, allowing EB to
intercalate into DNA, produced
a red colour; d, f and h H9c2
cells treated with 5, 7.5 and
10 lM ATO and BDE. BDE
was effective in maintaining the
integrity of DNA at lower doses

able to retain size as well as number of lysosome more or
less same to that of control except at high dose (10 lM).
Effect of BDE against ATO-Induced ER Stress
The ER of H9c2 cells treated with ATO (Fig. 7c, e, g)
differed in their appearance from that of the control cells
(Fig. 7a). The ER of ATO-treated groups were less com-
pact than those of the control group and appeared more
diffused (Fig. 7c, e, g) and even collapsed. It was also
found that ER had accumulated around the nuclei (Fig. 7e,
g) in ATO-treated groups. At high dose, ER appeared to be
denser. BDE was effective only at low dose of ATO and
was almost ineffective at medium and high concentrations.
BDE Against ATO-induced Alterations
in the Contractile Protein
Staining of F-actin with phalloidin revealed the alteration
of contractile protein (Fig. 8c, e, g, i) in ATO-treated
groups. The cytoskeleton of control cells had an intact
filamentous network (Fig. 8a), while cells treated with

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Cardiovasc Toxicol (2013) 13:123–137 129

Fig. 3 Generation of ROS.

Fluorescent microscopic images
of H9c2 cells stained with
DCF-DH (Original
magnification 9 20). a Control
cells; b cells treated with BDE
alone; c, e, g cells treated with
5, 7.5 and 10 lM ATO,
respectively; d, f, h cells treated
with 5, 7.5 and 10 lM ATO and
BDE, respectively; Dose-
dependent increase in
fluorescence had been
visualized with various doses of
ATO indicating ROS
generation. BDE was protective
at low and medium doses, but
not at high dose of ATO. A
decrease in DCF fluorescence
was seen in the case of lower
and medium dose of ATO when
co-treated with BDE; i The
fluorometric analysis supported
the microscopic data

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Table 2 Effect of ATO on superoxide dismutase (SOD) and catalase (CAT) activity
Control 20 lg/mL BDE 5 lM ATO
alone
Without BDE
SOD (Units/mg 0.139 ± 0.020 0.139 ± 0.010 0.118 ± 0.018
protein)
CAT (Units/mg 0.255 ± 0.047 0.251 ± 0.073 0.198 ± 0.082
protein)
Each value represent the mean ± CV (n = 6)
a
Significant (P B 0.05) change in value compared with their respective control group
ATO showed disruption of filamentous network in a dose-
dependent manner. BDE was significantly effective
(P B 0.05) in holding intact the mesh-like architecture of
the cells at 5 and 7.5 lM (Fig. 8d, f), but not significant at
10 lM (Fig. 8h) concentration.
Discussion
Although ATO is a widely-used drug for the treatment of
APL, the clinical utility of this drug is limited due to its
cardiotoxic effects mediated through cardiac ion channels
mainly potassium channel human ether-a-go-go-related
gene [19] and L-type calcium channel [20]. It also causes
histopathological and ultrastructural changes in cardiac
cells [21]. In this study, our goal was to investigate the
cardiotoxicity caused by ATO on various cell organelles in
H9c2 cells. The H9c2 clonal myoblastic cell line, derived
from embryonic rat heart, has many features of adult car-
diomyocyte and thus is used as an experimental model to
investigate the molecular mechanism of cardiomyocyte
pathophysiologies [22].
BDE is a pharmacologically well-characterized medicinal
plant [23]. It has been reported that BDE contains bioactives
like eupalitin 3-O-b-D-galactopyranoside (5,40 -dihydroxy
6,7-dimethoxy-flavonal-3-O-b-D-galactopyranoside), eupal-
itin [24], eupalitin 3-O-b-D-galactopyranoside (1 [ 2)-b-D-
glucopyranoside [25], eupalitin 3-O-b-D-galactopyranosyl-
(100 [ 200 )-O-b-D-galactopyranoside, 3,30 ,5-trihydroxy-7-
0 0
methoxyflavone 4 ,7-dihydroxy-3 -methylflavone, 4-dime-
thoxyphenyl-1-O-b-D-apiofuranosyl -(100 [ 30 )-O-b-D-gluco-
pyranoside [26] etc. These compounds have several prop-
erties including cardioprotection, hepatoprotection, immu-
nomodulation antihypertension etc. Thus, our study focuses
on the protective efficacy of ethanolic extract of BDE
against ATO-induced toxicity.
MTT, LDH and NR assays were done to evaluate the
effects of ATO on cytotoxicity in H9c2 cells. In addition to
these, the morphological appearances and the DNA integ-
rity of the cells of various experimental groups were also
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Cardiovasc Toxicol (2013) 13:123–137
7.5 lM ATO 10 lM ATO
With 20 lg/mL Without BDE With 20 lg/mL Without BDE With 20 lg/mL
BDE BDE BDE
0.133 ± 0.037 0.105 ± 0.033 0.123 ± 0.040 0.060 ± 0.140 0.091 ± 0.093
0.243 ± 0.037 0.156 ± 0.054 0.223 ± 0.022a 0.089 ± 0.039 0.131a ± 0.043
observed. We found typical apoptotic way of cell death like
vacuolation, blebbing and granulation in all the cases, and
BDE was found to be effective against cytotoxicity except
10 lM concentration of ATO. It was notable that the
mitochondrial DNA was red in colour in AO/EB staining at
lower concentrations of ATO rather than the nuclear DNA.
Thus, it can be conferred that mitochondrial DNA is
affected prior to nuclear DNA in ATO-induced toxicity.
Generation of ROS causes altered cellular signal trans-
duction such as activation of transcription factors and
changes in gene expression and contributes to damage in
DNA, lipids and proteins resulting in apoptotic death [27,
28]. Co-incubation with BDE almost suppressed both ROS
generation as well as apoptosis. The evaluation of innate
antioxidant enzymes like SOD and CAT in ATO-treated
cells found a dose-dependent inhibition with ATO though
not significant. BDE was able to protect the enzymes from
depletion by ATO mainly due its antioxidant property.
ATO exerted a rapid noxious effect on the mitochondria
of H9c2 cells. The mitochondrion is the target of multiple
pro-apoptotic signals and contains a number of effecter
proteins that can promote cell death. Cardiac myocytes are
endowed with high content of mitochondria and maintain
an elevated rate of ATP synthesis to satisfy the energy
demand of the heart. These make the cells highly vulner-
able to damage. In normal cells, due to the electrochemical
potential gradient, the JC1 dye concentrates in the mito-
chondrial matrix, where it forms red fluorescent aggre-
gates. Any event that dissipates a change in the
mitochondrial membrane potential prevents the accumu-
lation of the JC-1 dye in the mitochondria, and thus, the
dye is dispersed throughout the entire cell leading to a shift
from red to green fluorescence (JC-1 monomers). ATO
caused depolarization of the transmembrane potential, as
JC1 monomer was dispersed throughout the cytoplasm due
to decrease in negative charge in ATO-treated cells. The
effects on mitochondria evident from this study reveal that
mitochondria are one of the target sites of action of ATO
on cardiomyocyte. BDE was able to keep transmembrane
potential intact except for 10 lM concentration of ATO.
Cardiovasc Toxicol (2013) 13:123–137 131

Fig. 4 Mitochondrial
transmembrane potential change
with ATO. Fluorescent
microscopic merged images of
H9c2 cells stained with JC1 for
mitochondrial study (Original
magnification 920). a Control
cells; b cells treated with BDE
alone; c, e, g cells treated with
5, 7.5 and 10 lM ATO,
respectively; d, f, h cells treated
with 5, 7.5 and 10 lM ATO and
BDE, respectively; A shift in
fluorescence from red to green
was seen in the case of high
ATO concentration due to a
dissipation in the
transmembrane potential. BDE
was effective at lower and
medium dose of ATO in
maintaining the normal
membrane potential; i Relative
fluorescence also showed a
similar trend

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132 Cardiovasc Toxicol (2013) 13:123–137

Fig. 5 Intracellular calcium

overload with ATO. Fluorescent
microscopic images of H9c2
cells stained with Fura-2AM
(Original magnification 920).
a Control cells; b cells treated
with BDE alone; c, e, g cells
treated with 5, 7.5 and 10 lM
ATO, respectively; d, f, h cells
treated with 5, 7.5 and 10 lM
ATO and BDE, respectively;
Dose-dependent increase in
fluorescence was observed.
BDE was protective at low and
medium doses, but not at high
dose of ATO. A decrease in
fluorescence was seen in the
case of lower and medium dose
of ATO when co-treated with
BDE. i The fluorometric
analysis supported the
microscopic data

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Cardiovasc Toxicol (2013) 13:123–137 133

Fig. 6 Changes in lysosome

with ATO. Fluorescent
microscopic images of H9c2
cells stained with lysosome-
GFP (Original magnification
920). a control cells; b cells
treated with BDE alone; c, e,
g cells treated with 5, 7.5 and
10 lM ATO respectively; d, f,
h cells treated with 5, 7.5 and
10 lM ATO and BDE
respectively; Dose-dependent
alteration in lysosome had been
observed. BDE was protective
at low and medium doses but
not at high dose of ATO

Nominal calcium is very much essential for function of
cardiomyocyte. ATO causes calcium overload both in pri-
mary cardiac myocyte [29] and H9c2 cells [30]. Calcium
overload through enhanced L-type calcium channel activity
is possible as ATO activate L-type calcium channel activity
and prolong action potential duration at 20 % repolarization
(APD20) [8]. Oxidative stress inhibits Ca2?-ATPases that
leads to the alteration of calcium levels and then cell death.
Free radical overproduction also increases the cytosolic
calcium contributing to the activation of endonucleases that
degrade DNA, leading to cell death. The calcium overload
by ATO also acts as a catalyst to mitochondrial damage due
to its action on various proteins on inner mitochondrial
membrane. BDE prevented accumulation of calcium in low
and medium doses of ATO-treated cells, but not at high
dose. Alteration in ER with ATO has significant contribu-
tion in cardiotoxicity as ER is the centre of protein folding
for synthesis of functional protein [31]. The visual evalua-
tion of ER in ATO-treated group showed signs of initiation
of ER stress. There are reports that ER chaperons during ER
stress-induced apoptosis. Based on this, we speculate that
this organelle may partially contribute in apoptosis.

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134 Cardiovasc Toxicol (2013) 13:123–137

Fig. 7 Endoplasmic reticulum

stress with ATO. Fluorescent
images of H9c2 cells stained
with ER-RFP for labelling ER
(Original magnification 920).
a Untreated control cells; b cells
treated with BDE alone; c, e,
f cells treated with 5, 7.5 and
10 lM ATO, respectively; d, f,
h cells treated with 5, 7.5 and
10 lM ATO and BDE,
respectively; Dose-dependent
alteration in ER had been
observed. BDE was protective
at low and medium doses, but
not at high dose of ATO

Disruption of cytoskeletal assembly is one of the early
effects of any stress that can ultimately lead to cell death.
Stabilization of cytoskeleton assembly, therefore, is a
critical event that regulates cell survival under stress. ATO
caused disorganization of actin into fragmented and fragile
form. This disorganization affects the contraction effi-
ciency of heart by reducing the force of contraction of
cardiac myocytes [8]. In addition, we found an increase in
size as well as number of lysosome in ATO-treated cells
which were more conspicuous at high dose. This enlarge-
ment is mainly due to loss of integrity of lysosomal
membrane and efflux of lysosomal enzymes [32]. Overall
result indicates that co-treatment of H9c2 cells with ATO
and BDE reduced cytotoxicity significantly at low and
medium doses. This may be due to irreversible death of
cells at high dose of ATO. Apoptosis offers target for
therapeutic intervention in ATO-induced cardiotoxicity. A
detailed investigation on apoptosis is being carried on that
shall provide further understanding of the mechanism of
this anticancer drug–induced toxicity at molecular level
and provide new insights into novel cardioprotective
strategies in vivo.

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Cardiovasc Toxicol (2013) 13:123–137 135

Fig. 8 Alteration in integrity of

cytoskeleton with ATO.
Fluorescent microscopic images
of H9c2 cells stained with
phalloidin (Original
magnification 920). a Control
cells; b cells treated with BDE
alone; c, e, g cells treated with
5, 7.5 and 10 lM ATO,
respectively; d, f, h cells treated
with 5, 7.5 and 10 lM ATO and
BDE, respectively; Dose-
dependent alteration in
cytoskeleton was observed.
BDE was protective at low and
medium doses, but not at high
dose of ATO; i The fluorometric
analysis supported the
microscopic data

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136
To conclude, ATO causes alterations in mitochondria,
ER, lysosomes, antioxidant enzymes and cytoskeleton in a
dose-dependent manner providing a proof that cell organ-
elles are also target of ATO-induced cardiotoxicity in
addition to the reported targets like ion channels. Although
BDE supplementation failed to reverse ATO-induced cel-
lular damage at high concentration, its efficacy at low and
medium doses confirms the presence of cardiotonic phy-
tochemicals in BDE. Since the plant is edible, these effects
may explain the use of BDE as a potent cardioprotectant
with possible health benefits and can serve as an ideal
nutraceutical.
Acknowledgments This research is supported by ICMR grants-in-
aid (59/39/2009 BMS/TRM) from the Ministry of Health and Family
welfare, Govt of India. We thank the Director, CSIR-NIIST and Head,
Agroprocessing and Natural Products Division, CSIR–NIIST for
providing all the necessary facilities for conducting the experiments.
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