IJEB 52(4) 323-331

Indian Journal of Experimental Biology
Vol. 52, April 2014, pp. 323-331
Protective effect of Azadirachta indica A. Juss against doxorubicin-induced
cardiac toxicity in tumour bearing mice
Ashwani Koul*, Renu Goyal & Sanjay Bharati
Department of Biophysics, Basic Medical Sciences Block, Panjab University, Chandigarh 160 014, India
Received 14 June 2013; revised 13 January 2014
Doxorubicin (DOX) treatment (12 µg/g body weight, once a week for 2 weeks) resulted in a significant decrease in the
heart rate along with an increase in QRS, ST, and QT intervals. Histopathological studies showed cardiomyocyte
degeneration, cytoplasmic vacuolation and macrophage infiltration in cardiac tissue. A marked increase in the rate of
apoptosis was also observed. An increased oxidative stress was evidenced by significantly higher levels of lipid peroxidation
(LPO) and depletion of reduced glutathione. A decrease in the activity of cellular antioxidant defence enzymes was also
observed. The decrease in the heart rate and ECG alterations were prevented significantly by AAILE (100 µg/g body
weight, po) co-treatment, started two weeks prior to DOX treatment and continued till the termination of the experiment.
The cardioprotection was also evident from histopathology and decrease in the rate of apoptosis in cardiomyocytes. AAILE
co-treatment also prevented DOX-induced increase in LPO and decrease in antioxidant defence enzymes. The results
suggest that AAILE administration prevents DOX-induced cardiotoxicity.
Keywords: Azadirachta indica, Cardiac toxicity, Doxorubicin, Oxidative stress, Tumour
Doxorubicin (DOX) has been established as an
effective anticancer drug for the treatment of wide
range of malignant conditions. However, DOX
treatment leads to severe cardiotoxicity which
remains a major concern for its use in cancer
patients1. The risk of cardiotoxicity is dose dependent
and increases with an increase in the amount of dose.
Cardiotoxicity can be acute, sub-acute, or chronic.
Acute cardiotoxicity develops within minutes of
intravenous administration of DOX and is usually
reversible but early onset of cardiac toxicity has been
shown to be predictive of future heart failure.
Sub-acute and chronic forms of DOX-mediated
cardiotoxicity manifest as a permanent and
irreversible damage to the cardiac tissue2.
The exact mechanism(s) of DOX-induced
cardiotoxicity is yet to be fully elucidated. A number
of proposed mechanisms include: the generation of
reactive oxygen species (ROS), apoptosis, DNA
interference, and metabolic alterations etc3-5. DOX on
reaction
with
molecular
oxygen
generates
significantly large amount of free radicals which is
the major source of oxidative stress mediated cellular
——————
*Correspondent author
Telephone: +91-0172-2534124
E-mail: [email protected]; [email protected]
damage6. The role of iron has also been investigated
by several authors in DOX-induced cardiotoxicity and
it is proposed that free intracellular iron increases
oxidative stress of the cell3,7. These events contribute
to death of cardiomyocytes and are considered the
primary reason of DOX-induced cardiac side effects.
Since DOX is an effective antineoplastic drug,
considerable efforts have been made to explore
strategies effective in reducing DOX-induced cardiac
dysfunction. However, there is no effective cure
against DOX-induced cardiotoxicity1. Pharmaceutical
agents in the form of supplements have been tested in
experimental animal studies to reduce the risk of
DOX-induced cardiotoxicity8. Antioxidants such as
mercaptopropionyl glycine (MPG), selenium,
superoxide dismutase and dexrazoxane have been
reported to decrease DOX-induced cardiotoxicity9-12.
The use of antioxidants, iron chelators, amino acid
supplements etc. have limited success and in some
cases additional side effects13. Dexrazoxane, drug
known for its iron-chelating effects is in clinical use
but usage is limited because of its myelosuppressive
side effects14. Other strategies such as slow infusion
of DOX and reduction in the cumulative dose have
decreased the incidence of acute cardiotoxicity but do
not completely eliminate the risk of chronic
cardiotoxicity15. Therefore, further research is
324
INDIAN J EXP BIOL, APRIL 2014
required to develop interventions to prevent the
cardiotoxicity arising due to DOX treatment.
Azadirachta indica A. Juss is one of the most
versatile medicinal plants having wide spectrum of
biological activity. It has been shown to be effective
against coronary artery disease, hypertension and
arrhythmias16,17. Peer et al.18 have reported
cardioprotective potential of A. indica against
isoprenaline induced myocardial infarction in rats.
More than 135 compounds have been isolated from
different parts of the plant. The presence of wide
range of biologically active molecules makes
A. indica effective against various health aliments19,20.
In addition, it is also known to be a strong
antioxidant, iron chelator and modulator of immune
response as well as antioxidant defence system of the
body21,22. Elevation of antioxidant defence enzyme
levels have been reported after treatment of A. indica
to mice23,24. In some cases antioxidant mediated
effects of A. indica are reported to have better potency
in comparison to known antioxidants like vitamin E,
ascorbate, and melatonin25.
Considering the multifaceted etiology of
DOX-induced cardiotoxicity and cardioprotective
potential of A. indica in experimental animal models,
the present study has been designed to investigate
the possibility of use of A. indica to eliminate
DOX-induced cardiac toxicity.
Materials and Methods
Chemicals
and
reagents—7,12dimethylbenz(a)anthracene
(DMBA),
reduced
glutathione (GSH), oxidised glutathione (GSSH),
nitroblue tetrazolium (NBT) and reduced nicotinamide
adenine dinucleotide phosphate (NADPH) were
purchased from Sigma Chemical Company, St. Louis,
USA. Doxorubicin (DOX), bovine serum albumin
(BSA), 2-thiobarbituric acid (TBA), 5-5’–dithiobis
(2-thiobarbiotic acid) (DTNB), hydrogen peroxide
(H2O2) and glutathione reductase (GR) were
purchased from local Indian firms and were of highest
purity grade. TUNEL Assay kit was purchased from
Trevigen, Inc, 8405 Helgerman Ct. Gaithersburg.
Preparation of aqueous Azadirachta indica leaf
extract—A. Indica leaves were collected after proper
identification by botanist from Panjab University
campus, Chandigarh, India. The leaf extract was
prepared as describe earlier26. Briefly, fresh leaves of
A. indica were obtained and washed properly with
distilled water. The aqueous extract was obtained by
grinding the leaves in a mixer and filtering the
aqueous portion of mixture. The fraction obtained was
then centrifuged and supernatant was collected.
The supernatant was then freeze dried to obtain
aqueous A. indica leaf extract (AAILE).
Treatment of animals—Male Balb/c mice in body
weight range 25-30 g were procured from Central
Animal House, Panjab University, Chandigarh (India)
and housed in the animal room of Department of
Biophysics, Panjab University, Chandigarh (India).
The animal room was well-ventilated and maintained
at 21±2 °C, and 50-60 % RH.
The animals had free access to drinking water and
fed with standard pellet diet. The experimental
animals were acclimatized to the experimental
conditions for one week before starting the experiment.
All experimental procedures were approved by
institutional ethics committee and conducted according
to the guidelines of Indian National Science Academy
for the use and care of experimental animals.
The skin cancer model was developed as described
earlier26. Briefly, animals received topical application
of DMBA (500 nmol/100 µL of acetone) for two weeks,
twice a week followed by TPA (1.7 nmol/100 µL of
acetone) for 18 weeks, twice a week.
After 22 weeks of first DMBA application, the
animals were segregated into following five groups of
9-10 animals each: (I) normal, (II) tumour, (III)
tumour+AAILE, (IV) tumour+DOX and (V)
tumour+DOX+AAILE. DOX was administered
intravenously to group IV and group V animals twice
at an interval of two weeks (cumulative dose 12 µg/g
body weight). AAILE was administered by oral route
to group III and group V animals thrice a week on
alternate days for four weeks (100 µg/g body weight).
The treatment of AAILE was started 2 weeks prior to
the administration of DOX. Group I and group II
animals did not receive any special treatment.
Electrocardiographic
assessment—ECG
was
recorded in animals at different time intervals as
described previously27. Elastic cotton jacket
containing two circular platinum leads (radius: 3 mm)
was wrapped around the thoracic cavity of mouse.
Lead II electrodes were placed below right clavicle
and 11th intercostal space left to sternum and right
lower abdomen respectively. Prior to ECG, the ventral
thoracic region of each animal was carefully shaved
and a conductive ECG gel was applied over each
electrode. For each ECG record, the most stable 3 min
KOUL et al.: AZADIRACHTA INDICA PROTECTS AGAINST DOXORUBICIN INDUCED TOXICITY
continuous segment was chosen for study. The heart
segment analysis of ECG was done with the help of
computer software as described previously28.
Histopathological investigations—After 24 h of
second DOX administration, animals were sacrificed
by cervical dislocation under mild ether anaesthesia
and left ventricle portions of cardiac tissue of mouse
from each group were fixed in neutral formalin for
24 h. Tissues were then dehydrated in grades of
alcohol and embedded in paraffin wax. Section (5 µm
thick) were cut and placed on slides and stained with
Haematoxylin and Eosin stains. The stained sections
were visualized under light microscope (LEICA DM
3000).
Cell death assay (TUNEL assay)—TUNEL assay
was performed on deparaffinised and rehydrated liver
sections for the detection of DNA fragmentation with
in situ apoptosis detection kit following the
manufacturer’s
specifications
with
minor
modifications. The DNA fragmentation in apoptotic
cells was visualized by the detection of brominated
nucleotide (BrdU), incorporated by terminal
deoxynucleotidyltransferase (TdT) onto the free 3’OH
residue of the DNA fragment. To make DNA
accessible to the labelling enzymes, the membranes
were permeabilized by incubating with proteinase K
for 30 min. Quenching of endogenous peroxidase
activity was done by incubating in H2O2. The TdT
incorporated brominated nucleotides were revealed by
incubation with biotin labelled anti-BrdU antibody.
Biotin labelled fragments were visualized by
incubation with streptavidin horse radish peroxidase
and subsequently with 3’,3-diamino benzidine (DAB).
The slides were counter stained with methyl-green for
better background details and observed under light
microscope.
Biochemical parameters—
29
Myocardial
lipid
peroxidation :
Tissue
homogenates were incubated in 150 mM KCl/TrisHCl buffer, pH 7.4 containing 0.3 µmoles NADPH in
a metabolic shaker at 37 ºC for 60 min. The reaction
was stopped by addition of cold TCA-HCl mixture.
Samples were centrifuged for 10 min at 3000 g and
supernatants were boiled in boiling water bath after
addition of 1% TBA. The pink coloured MDA-TBA
complex was measured at 535 nm and the LPO levels
were expressed as nanomole of MDA-TBA complex
formed/mg protein.
325
Myocardial
reduced
glutathione30:
Tissue
homogenates were precipitated using cold TCA. The
non-protein sulfhydryl groups were assayed with
DTNB solution to yield yellow coloured complex
which was read at 412 nm. GSH was used as a
standard and GSH content of the sample was
expressed as nanomole GSH/mg protein.
Myocardial
superoxide
dismutase31:
The
superoxide scavenging activity of superoxide
dismutase (SOD) was assessed by measuring the
inhibition of nitroblue tetrazolium reduction to blue
colour formazon at 560 nm by superoxide anions
generated by photo-oxidation of hydroxylamine
hydrochloride. The activity was expressed as IU/mg
protein, where 1 IU is defined as the amount of
enzyme inhibiting the increase in optical density by
50%.
Myocardial catalase32: The catalase activity was
measured as the rate of reduction in intensity of H2O2
at 240 nm. The rate of change in OD was obtained
and the activity of enzyme was expressed as IU/mg
protein.
Myocardial glutathione peroxidase33: The activity
was assessed by measuring the rate of oxidation of
NADPH at 340 nm in the presence of reduced
glutathione and H2O2. Enzyme activity was expressed
as nanomole of NADPH consumed/min protein.
Myocardial glutathione reductase34: The activity of
glutathione reductase was assessed as rate of
oxidation of NADPH by GR in presence of GSSG at
340 nm. Enzyme activity was expressed as nanomole
of NADPH oxidized/min/mg protein.
Protein estimation: Protein content of samples was
estimated by the method of Lowry et al.35 using
bovine serum albumin (BSA) as a standard.
Statistical analysis—The data were expressed as
mean±SD and analysed by one-way ANOVA
followed by post hoc test. P value less than or equal
to 0.05 was considered to be statistically significant.
Results
Electrocardiographic assessment—A significant
decrease in the heart rate (bradycardia) was noticed
after 2 weeks of DOX treatment to tumour bearing
mice (Gr. IV) (Fig. 1). However, administration of
AAILE to tumour bearing-DOX treated animals
(Gr. V) had significantly improved heart rate
INDIAN J EXP BIOL, APRIL 2014
326
compared to tumour bearing-DOX treated animals
(Gr. IV). ST segment, QT interval and QRS complex
which were significantly increased in Gr. IV animals
compared to Gr. I and Gr. II got normalized after
AAILE co-administration in Gr. V animals (Table 1).
Heart rate and duration of cardiac intervals were not
affected in Gr. II and III animals compared to normal
animals (Gr. I).
Histopathology—Histopathological analysis of
cardiac tissue was conducted at the end of the
treatment period in all groups. Normal myocardial
histoarchitecture was observed in Gr. I, II and III
animals (Fig. 2a, 2b). The administration of DOX to
tumour bearing mice (Gr. IV) resulted in myocardial
Fig. 1—Effect of AAILE on ECG after DOX treatment. Sweep:
100 mm/s; Gain: 2.00 cm/mV.
degradation, which is characterized by partial or total
loss of cytoplasm. In some myocytes the myofibrils
were completely disorganised by large, clear space
indicative of intracellular edema (Fig. 2c, 2d).
In addition to cardiomyocytes injury, DOX treatment
(Gr. IV) produced vacuolation and induction of fibrosis.
A significant improvement was observed upon AAILE
co-administration to DOX treated animals (Gr. V).
There was a reduction in the overall vacuolation and
degree of myofibrillar loss (Fig. 2e, 2f).
Cell death analysis—Figure 3 showed left
ventricular TUNEL stained sections of mice heart
from different treatment groups. Apoptotic cells in
addition for being TUNEL positive (i.e. nucleus
stained brown) also displayed certain features such as;
chromatin material was confined to nucleus,
chromatin
condensation
and
chromatin
marginalization etc. The overall percentage of
apoptotic cells in the myocardium of normal, tumour
bearing and AAILE treated animals (Gr. I, II and III)
were very low (Fig. 4). DOX treatment
(Gr. IV) significantly increased number of apoptotic
cells in myocardium compared to normal animals
(Gr. I). A significant reduction was noted upon
AAILE co-treatment in group V compared to group
IV mice. However, the percentage of apoptotic cells
remained significantly higher compared to normal
animals (Gr. I).
Lipid peroxidation (LPO)—The lipid peroxidation
level as assessed in terms of Thiobarbituric acid
reactive substances (TBARS) was significantly
increased after DOX treatment to tumour bearing
mice (Gr. IV) compared to other treatment groups.
A significant decrease was observed in LPO levels
upon AAILE co-administration to tumour bearingDOX treated mice (Gr. V) compared to tumour
bearing-DOX treated animals (Gr. IV) (Table 2).
Gr. III mice had significantly decreased LPO
compared to Gr. II. Statistically non-significant
alterations were observed in other treatment groups.
Table 1—Effects of AAILE on various ECG parameters after DOX treatment
[Values are mean±SD from 9-10 animals in each group]
Parameters
Normal (Gr. I)
Tumour (Gr. II)
Tumour+AAILE (Gr. III)
Tumour+DOX (Gr. IV)
Tumour+DOX+AAILE
(Gr. V)
QRS (ms)
QT (ms)
ST (ms)
R-R (ms)
34.6±3.28
68.6±5.57
32.3±2.77
93.0±3.40
35.6±3.78
69.8±4.54
32.0±2.43
90.9±4.04
37.2±5.25
70.8±3.24
33.1±2.23
91.8±5.97
49.2±1.64ab
83.6±6.83ab
48.2±4.82ab
116.4±7.59ab
38.3±2.86d
75.6±4.21d
37.3±3.94d
101.3±4.0abcd
One way ANOVA followed by post hoc test. P values: ≤0.05 with reference to aGroup I, bGroup II, cGroup III, dGroup IV
KOUL et al.: AZADIRACHTA INDICA PROTECTS AGAINST DOXORUBICIN INDUCED TOXICITY
327
Fig. 2—Effect of AAILE on histoarchitecture of heart after DOX treatment (a) Group I; left ventricle myocardium showing
cardiomyocytes arranged in regular pattern (100X). (b) normal myocardium (Group I) at a higher magnification (400X). (c) Group IV;
DOX treated mice heart showing deranged myocardium and inflammatory cells (arrow) (100X). (d) DOX treated heart (Group IV)
(400X) showing extensive cytoplasmic vacuolation (arrows) and myocardial degeneration e) Group V; mice heart showing mild
myocardial derangement (Magnification: 100X). (f) (400X) showing cytoplasmic vacuolation (arrows).
Reduced glutathione (GSH)—A significant
depletion of GSH was observed in tumour
bearing-DOX treated animals (Gr. IV) compared to
Gr. I and II. AAILE co-administration (Gr. V)
increased the levels of GSH compared to tumour
bearing-DOX treated mice (Gr. IV). No significant
change was observed in myocardial GSH level of
tumour bearing animals (Gr. II) compared to normal
animals (Gr. I). However, AAILE administration in
tumour bearing mice (Gr. II) had significantly
increased level of GSH compared to other treatment
groups. (Table 2).
Superoxide dismutase (SOD)—Cardiac SOD
activity decreased significantly after DOX treatment
to tumour bearing mice (Gr. IV) compared to other
treatment groups. AAILE administration to tumour
bearing-DOX treated mice (Gr. V) elevated the SOD
activity compared to tumour bearing-DOX treated
328
INDIAN J EXP BIOL, APRIL 2014
Fig. 3—TUNEL assay of cardiac tissue in various treatment groups (a) showing TUNEL negative, normal cardiomyocytes (400X) b)
DOX plus AAILE treated (Group V) (400X) (c) DOX treated (Group IV) showing brown coloured TUNEL positive apoptotic cells
brown colour confined to nucleus, with mild chromatin condensation (400X). Arrows indicate TUNEL positive cells.
mice (Gr. IV). Tumour bearing mice (Gr. II) showed
significant depression in cardiac SOD activity
compared to normal mice (Table 2).
Catalase—A significant increase in myocardial
catalase was observed in tumour bearing-AAILE
treated (Gr. III), tumour bearing-DOX treated
(Gr. IV) and tumour bearing-DOX + AAILE
co-treated (Gr. V) mice compared to normal (Gr. I)
and tumour bearing (Gr. II) mice. However, no
significant change was noticed in tumour
bearing-DOX + AAILE co-treated (Gr. V) mice
compared to tumour bearing-DOX treated (Gr. IV)
mice (Table 2).
Glutathione peroxidase (GPx)—DOX treatment to
tumour bearing mice (Gr. IV) significantly decreased
myocardial GPx activity compared to normal (Gr. I)
and tumour bearing mice (Gr. II). AAILE
supplementation to tumour bearing-DOX treated mice
(Gr. V) significantly increased the activity of GPx
Fig. 4—Effect of AAILE on apoptotic index in candiac tissue
after DOX treatment [Values are mean±SD (n=9-10). P values:
≤0.05 with reference to aGroup I, bGroup II, cGroup III,
d
Group IV].
KOUL et al.: AZADIRACHTA INDICA PROTECTS AGAINST DOXORUBICIN INDUCED TOXICITY
329
Table 2—Effects of AAILE on antioxidant defence system of myocardial tissue after DOX treatment
[Values are mean±SD from 9-10 animals in each group]
Parameters
Lipid peroxidation
Reduced glutathione
Superoxide dismutase
Catalase
Glutathione
peroxidase
Glutathione reductase
Normal (Gr. I)
Tumour (Gr. II)
Tumour+AAILE
(Gr. III)
Tumour+DOX
(Gr. IV)
Tumour+DOX+AAILE
(Gr. V)
51.1± 4.50
1.12 ±0.08
12.4 ± 0.52
0.55 ± 0.05
25.7 ± 1.46
59.2 ± 6.90
1.26 ± 0.07
10.7 ± 1.31a
0.52 ± 0.07
27.1 ± 1.82
42.5 ± 4.30b
1.61 ± 0.14ab
12.2 ± 0.69b
0.64 ± 0.05ab
29.3 ± 0.65a
82.7 ± 5.20ab
0.68 ± 0.09ab
7.15 ± 0.40ab
0.60 ± 0.02ab
20.9 ± 2.04ab
67.0± 6.60cd
0.95 ± 0.09abd
9.59 ± 1.05acd
0.62 ± 0.06ab
23.9 ± 0.94bcd
4.03 ± 0.46
3.70 ± 0.24
3.53 ± 0.41
2.78 ± 0.42ab
3.51 ± 0.57d
One way ANOVA followed by post hoc test. P values: ≤0.05 with reference to aGroup I; bGroup II; cGroup III; dGroup IV. Units: Lipid
peroxidation: nanomole TBA-MDA chromophore/mg protein; Reduced glutathione: nanomole GSH/mg protein; Superoxide dismutase:
IU/mg protein; Catalase: IU/mg protein; Glutathione peroxidase: nanomole of NADH oxidized/min/mg protein; Glutathione reductase:
nanomole of NADH oxidized/min/mg protein.
compared to tumour bearing-DOX treated mice
(Gr. IV). GPx activity did not differ significantly in
normal (Gr. I) and tumour bearing mice (Gr. II).
AAILE treatment to tumour bearing mice (Gr. III)
significantly increased the activity of GPx compared
to normal mice (Gr. II) (Table 2).
Glutathione reductase (GR)—GR activity was
significantly decreased after DOX treatment to
tumour bearing mice (Gr. IV) compared to normal
(Gr. I) and tumour bearing mice (Gr. II). The activity
was significantly increased in tumour bearing-DOX +
AAILE co-treated group (Gr. V) compared to tumour
bearing-DOX treated animals (Gr. IV). No significant
alterations were observed in other treatment groups
(Table 2).
Discussion
Azadirachta indica has tremendous medicinal
potential. The extracts of various parts of the plant
have been used against variety of ailments and have
been demonstrated to be effective against various
types of cancers19,36. The plant not only offers
protection against cancer but also reduces the side
effects of certain chemotherapeutic drugs when given
as an adjunct to standard chemotherapy. Pre-treatment
of Swiss mice with A. indica reduced leucopenia
and neutropenia induced by cyclophosamide37 and
cisplatin plus 5-Flurouracil38. Therefore, it is
legitimate to explore cardioprotective potential of
A. indica against DOX-induced cardiotoxicity.
The development of cancer model prior to the
induction of DOX-induced cardiotoxicity was
considered important because studies suggested that
during carcinogenesis physiology of the whole body
gets affected39. In the present study, treatment of
DOX to skin tumour bearing mice resulted in
significant cardiotoxicity as revealed by changes in
ECG and myocardium morphology. The ECG
alterations such as elongation of QT interval,
widening of ST segment, and bradycardia observed
in ECG of DOX treated animals are considered
as one of the most reliable predictor of
DOX-induced cardiotoxicity40. The QRS, ST
elongation and PR widening after DOX treatment
has also been demonstrated41,42. QT elongation is
attributed to the disturbance in the ion flux across
the membrane of myocytes. This is further related to
the structural damage incurred to these cells by
DOX43. The histopathological studies revealed that
left ventricular tissue of animals treated with
DOX had significant structural and organisational
alterations in terms of presence of myocardial
degeneration, cytoplasmic vacuolization, and
intracellular edema which correlated well with
ECG changes in DOX-induced cardiotoxicity.
However, the QT segment elongation was
significantly
prevented
by
AAILE
and
histopathology of animals co-treated with AAILE
was improved signifying the cardioprotection.
The bradycardia observed after DOX administration
could be correlated to the free radical accumulation
and cellular damage. AAILE co-treatment returned
heart rate of animals to normal, indicating that it
has reduced the deteriorating effect of DOX on
cardiac tissue. The DOX-induced cardiac cell
death and cardiac failure are the fields of major
concern and extensive research44. The apoptotic
index assessed in accordance to the morphological
criteria of apoptosis along with TUNEL staining
characteristics45 demonstrated a decrease in apoptosis
after pre-treatment of AAILE to DOX administered
INDIAN J EXP BIOL, APRIL 2014
330
mice. This observation indicated that AAILE played
an important role in reducing the extent of damage
produced by DOX treatment to cardiomyocytes.
The damage produced by free radicals manifests as
lipid peroxidation (LPO) at the cell membrane. LPO
is not just an indicator of free radical damage but also
induces further tissue damage by production of wide
range of other free radicals which are far more potent
and damaging46. The cardiac tissue is susceptible to
DOX-induced damage due to the higher number of
mitochondria which are source of extensive ROS
generation. The profound amount of oxygen present
in the cardiac tissue makes it a major site for the
metabolism of DOX5. DOX metabolism is mediated
through the use of molecular oxygen, whose
metabolism products act as important contributor to
toxicity 5,47. The superoxide free radicals produced in
the process are converted to less reactive H2O2 by
action of SOD. H2O2 is further decomposed to water
by the action of catalase or GPx. The activity of
myocardial catalase is very low and the conversion of
H2O2 or related reactive molecules is carried out
primarily by myocardial GPx. Therefore, activity of
myocardial GPx is thought to play a significant role in
DOX-induced cardiotoxicity48. The increased activity
of SOD and GPx in AAILE co-treated group might
indicate the protective role played by AAILE in
DOX-induced cardiotoxicity. The results were further
supported by decreased levels of LPO in cardiac
tissue of this group.
Conclusion
It may be concluded that AAILE pre-treatment to
tumour bearing mice has provided protection against
cardiotoxicity induced by DOX. The modulatory
effect of AAILE on antioxidant defence system might
have played a significant role in the reduction of
DOX-induced cardiotoxicity.
4
5
6
7
8
9
10
11
12
13
14
15
16
References
1
2
3
Minotti G, Menna P, Salvatorelli E, Cairo G & Gianni L,
Anthracyclines: Molecular advances and pharmacologie
developments in antitumour activity and cardiotoxicity,
Pharmacol Rev, 56 (2004) 185.
Bernstein D, Fajardo G, Zhao M, Urashima T, Powers J,
Berry G & Kobilka B K, Differential cardioprotective/cardiotoxic
effects mediated by beta adrenergic receptor subtypes, Am
J Physiol Heart Circ Physiol, 289 (2005) H 2441.
Ghibu S, Delemasure S, Richard C, Guilland J C, Martin L,
Gambert S, Rochette L & Vergely C, General oxidative
stress during doxorubicin-induced cardiotoxicity in rats:
Absence of cardioprotection and low antioxidant efficiency
of alpha-lipoic acid, Biochimie, 94 (2012) 932.
17
18
19
20
Doroshow J H & Davies K J A, Redox cycling of
anthracyclines by cardiac mitochondria. II. Formation of
superoxide anion, hydrogen peroxide, and hydroxyl radical,
J Biol Chem, 261 (1986) 3068.
Chen B, Peng X, Pentassuglia L, Lim C C & Sawyer D B,
Molecular and cellular mechanisms of anthracycline
cardiotoxicity, Cardiovasc Toxicol, 7 (2007) 114.
Grankvist K, Stendahl U & Henriksson R, Comparative
Study of Demethoxydaunorubicin with other anthracyclines
on generation of oxygen radicals and clonogenic survival of
fibroblasts, Pharmacol Toxicol, 65 (1989) 40.
Castellani R J, Moreira P I, Perry G & Zhu X, The role of
iron as a mediator of oxidative stress in Alzheimer disease,
Biofactors, 38 (2012) 133.
Singal P K, Iliskovic N, Li T & Kumar D, Adriamycin
cardiomyopathy: pathophysiology and prevention, FASEB J,
12 (1997) 931.
El-Missiry M A, Othman AI, Amer M A & Abd El-Aziz M
A, Attenuation of the acute Adriamycin-induced cardiac and
hepatic oxidative toxicity by N-(2-mercaptopropionyl)
glycine in rats, Free Radic Res, 35 (2001) 575.
Lipshultz S E, Rifai N, Dalton V M, Levy D E, Silverman L
B, Lipsitz S R, Colan W S D, Asselin B L, Clavell L A,
Hurwitz C A, Moghrabi R D, Samson Y, Schorin M A,
Gelber R D & Sallan S E, The effect of dexrazoxane on
myocardial injury in doxorubicin-treated children with acute
lymphoblastic leukemia, N Engl J Med, 351 (2004) 145.
Singal P K, Siveski-Iliskovic N, Hill M, Thomas P T, & Li T,
Combination therapy with probucol prevents Adriamycininduced cardiomyopathy, J Mol Cell Cardiol, 27 (1995) 1055.
Berthiaume J M, Oliveira P J, Fariss M W, & Wallace K B,
Dietary vitamin E decreases doxorubicin-induced oxidative
stress without preventing mitochondrial dysfunction, Cardiovasc
Toxicol, 3 (2005) 257.
Van Vleet J F, Ferrans V J & Weirich W E, Cardiac disease
induced by chronic adriamycin administration in dogs and an
evaluation of vitamin E and selenium as cardioprotectants,
Am J Pathol, 99 (1980) 13.
Seifert C F & Nesser M E, Thompson D F, Dexrazoxane in
the prevention of doxorubicin-induced cardiotoxicity, Ann
Pharmacother, 28 (1994) 1063.
Kruger A & Wojnowski L, Cardiotoxicity of anthracyclines
– an Unsolved problem, Dtsch Arztebl Int, 37 (2006) A-2393.
Obiefuna I & Young R, Concurrent administration of
aqueous Azadirachta indica (neem) leaf extract with
DOCA-salt prevents the development of hypertension and
accompanying electrocardiogram changes in the rat,
Phytother Res, 19 (2005) 792.
Koley K M & Lal J, Pharmacological effects of Azadirachta
indica (neem) leaf extract on the ECG and blood pressure of
rat, Indian J Physiol Pharmacol, 38 (1994) 223.
Peer P A, Trivedi P C, Nigade P B, Ghaisas M M &
Deshpande A D, Cardioprotective effect of Azadirachta
indica A. Juss. on isoprenaline induced myocardial infarction
in rats, Int J Cardiol, 126 (2008) 123.
Imam H, Hussain A & Ajij A, Neem (Azadirachta indica A.
Juss) - A Nature's drugstore: An overview, I. Res. J. Biological
Sci, 1 (2012) 76.
Das B K, Mukherjee S C, Sahu B B & Murjani G, Neem
(Azadirachta indica) extract as an antibacterial agent against
fish pathogenic bacteria, Indian J Exp Biol, 37 (1999) 1097.
KOUL et al.: AZADIRACHTA INDICA PROTECTS AGAINST DOXORUBICIN INDUCED TOXICITY
21 Biswas K, Chattopadhyay I, Banerjee R K &
Bandyopadhyay U, Biological activities and medicinal
properties of neem (Azadirachta indica), Curr Sci, 11
(2002) 1336.
22 Ray A, Banerjee B D & Sen P, Modulation of humoral and
cell-mediated immune response by Azadirachta indica
(Neem) in mice, Indian J Exp Biol, 34 (1996) 698.
23 Koul A, Binepal G & Gangar S C, Impediment of
diethylnitrosamine induced hepatotoxicity in male Balb/c
mice by pretreatment with aqueous Azadirachta indica leaf
extract, Indian J Exp Biol,4 (2007) 359.
24 Koul A, Kapoor N & Bharati S, Histopathological,
enzymatic, and molecular alterations induced by cigarette
smoke inhalation in the pulmonary tissue of mice and its
amelioration by aqueous Azadirachta indica leaf extract,
J Environl Pathol Toxicol Oncol,31 (2012) 7.
25 Bandyopadhyay U, Biswas K, Chatterjee R, Bandyopadhyay
D, Chattopadhyay I, Ganguly C K, Chakraborty T,
Bhattacharya K & Banerjee R K, Gastroprotective effect of
Neem (Azadirachta indica) bark extract: Possible
involvement of H+-K+-ATPase inhibition and scavenging of
hydroxyl radical, Life Sci, 71 (2002) 2845.
26 Arora N, Koul A & Bansal M P, Chemopreventive activity of
Azadirachta indica on two-stage skin carcinogenesis in
murine model, Phytother Res, 25 (2011) 408.
27 Pereira-Junior P P, Marocolo M, Rodrigues F P, Medei E &
Nascimento J H M, Noninvasive method for electrocardiogram
recording in conscious rats: Feasibility for heart rate variability
analysis, An Acad Bras Cienc, 82 (2010) 431.
28 Van Acker S A, Kramer K, Grimbergen J A, Van den Berg D J,
Van der Vijgh W J & Bast A, Monohydroxyethylrutoside as
protector against chronic doxorubicin-induced cardiotoxicity, Br
J Pharmacol, 115 (1995) 1260.
29 Trush M A, Mimnaugh E G, Ginsburg E & Gram T E, In
vitro stimulation by paraquat of reactive oxygen-mediated
lipid peroxidation in rat lung microsomes, Toxicol Appl
Pharmacol, 60 (1981) 279.
30 Moron M S, Depierre J W & Mannervik B, Levels of
glutathione, glutathione reductase and glutathione-Stransferase activities in rat lung and liver, Biochim et Biophy
Acta, 582 (1979) 67.
31 Kono Y, Generation of superoxide radical during autooxidation of hydroxylamine and assay for superoxide
Dismutase, Arch Biochem Biophys, 186 (1978) 189.
32 Luck H, Catalase, in Methods of enzymatic analysis,
edited by HU Bergmeyer, (Academic Press, New York)
1963, 885.
33 Paglia D E & Valentine W N, Studies on the quantitative and
qualitative characterization of erythrocyte glutathione
peroxidase, J Lab Clin Med, 70 (1967) 158.
34 Carlberg I & Mannervik B, Purification and characterization
of the flavo enzyme glutathione reductase from rat liver,
J Biol Chem, 250 (1975) 5475.
331
35 Lowry O H, Rosebrough N J, Farr A L & Randell R J,
Protein measurement with Folin phenol reagent, J Biol
Chem, 193 (1951) 256.
36 Bharati S, Rishi P & Koul A, Azadirachta indica exhibits
chemopreventive action against hepatic cancer: Studies on
associated histopathological and ultrastructural changes,
Microsc Res Tech, 75 (2012) 586.
37 Ghosh D, Bose A, Haque E & Baral R, Pretreatment with
neem (Azadirachta indica) leaf preparation in Swiss mice
diminishes leukopenia and enhances the antitumour activity
of cyclophosphamide, Phytother Res, 20 (2006) 814.
38 Ghosh D, Bose A, Haque E & Baral R, Neem (Azadirachta
indica) leaf preparation prevents leukocyte apoptosis
mediated by cisplatin plus 5-fluorouracil treatment in Swiss
mice, Chemotherapy, 55 (2009) 137.
39 Koul A, Ghara A R & Gangar S C, Chemomodulatory effects
of Azadirachta indica on the hepatic status of skin tumour
bearing mice, Phytother Res, 20 (2006) 169.
40 Hazari M S, Haykal-Coates N, Winsett D W, Costa D L &
Farraj A K, Continuous electrocardiogram reveals
differences in the short-term cardiotoxic response of WistarKyoto and spontaneously hypertensive rats to doxorubicin,
Toxicol Sci, 110 (2009) 224.
41 Xin Y, Zhang S, Gu L, Liu S, Gao H, You Z, Zhou G, Wen
L, Yu J & Xuan Y, Electrocardiographic and biochemical
evidence for the cardioprotective effect of antioxidants in
acute doxorubicin-induced cardiotoxicity in the beagle dogs,
Biol Pharm Bull, 34 (2011) 1523.
42 Kelishomi R B, Ejtemaeemehr S, Tavangar S M, Rahimian
R, Mobarakeh J I & Dehpour A R, Morphine is protective
against doxorubicin induced cardiotoxicity in rats,
Toxicology, 243 (2008) 96.
43 Puri A, Maulik S K, Ray R & Bhatnagar V,
Electrocardiographic and biochemical evidence for the
cardioprotective effect of vitamin E in doxorubicin-induced
acute cardiotoxicity in rats, Eur J Pediatr Surg, 15 (2005) 387.
44 Kalyanaraman B, Joseph J, Kalivendi S, Wang S, Konorev E
& Kotamraju S, Doxorubicin-induced apoptosis: Implications
in cardiotoxicity, Mol Cell Biochem, 234 (2002) 119.
45 Gujral J S, Bucci T J, Farhood A & Jaeschke H, Mechanism
of cell death during warm hepatic ischemia-reperfusion in
rats: Apoptosis or necrosis?, Hepatology, 33 (2001) 397.
46 Negre-Salvayre A, Coatrieux C, Ingueneau C & Salvayre R,
Advanced lipid peroxidation end products in oxidative
damage to proteins, Potential role in diseases and therapeutic
prospects for the inhibitors, Br J Pharmacol, 153 (2008) 6.
47 Nohl H & Jordan W, OH • generation by adriamycin
semiquinone and H2O2; an explanation for the cardiotoxicity
of anthracycline antibiotics, Biochem Biophy Res Commun,
114 (1983) 197.
48 Matés J M, Pérez-Gómez C & Núñez de Castro I,
Antioxidant enzymes and human diseases, Clin Biochem, 32
(1999) 595.