Food and Chemical Toxicology 48 (2010) 11781184

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Food and Chemical Toxicology

journal homepage: www.elsevier.com/locate/foodchemtox

Cranberry (Vaccinium macrocarpon) protects against doxorubicin-induced

cardiotoxicity in rats
Ahmed A. Elberry a, Ashraf B. Abdel-Naim b, Essam A. Abdel-Sattar c, Ayman A. Nagy d, Hisham A. Mosli e,
Ahmed M. Mohamadin f, Osama M. Ashour b,*
a

Department of Clinical Pharmacy, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia
Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia
Department of Natural Products, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia
d
Department of Pathology and Forensic Medicine, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
e
Department of Urology, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
f
Department of Clinical Biochemistry, Faculty of Medicine, Taibah University, Madinah, Saudi Arabia
b
c

a r t i c l e

i n f o

Article history:
Received 8 November 2009
Accepted 3 February 2010

Keywords:
Cranberry
Doxorubicin
Cardiotoxicity
Antioxidation

a b s t r a c t
Doxorubicin (DOX) is a widely used cancer chemotherapeutic agent. However, it generates free oxygen
radicals that result in serious dose-limiting cardiotoxicity. Supplementations with berries were proven
effective in reducing oxidative stress associated with several ailments. The aim of the current study
was to investigate the potential protective effect of cranberry extract (CRAN) against DOX-induced cardiotoxicity in rats. CRAN was given orally to rats (100 mg/kg/day for 10 consecutive days) and DOX
(15 mg/kg; i.p.) was administered on the seventh day. CRAN protected against DOX-induced increased
mortality and ECG changes. It signicantly inhibited DOX-provoked glutathione (GSH) depletion and
accumulation of oxidized glutathione (GSSG), malondialdehyde (MDA), and protein carbonyls in cardiac
tissues. The reductions of cardiac activities of catalase (CAT), superoxide dismutase (SOD), glutathione
peroxidase (GSH-Px) and glutathione reductase (GR) were signicantly mitigated. Elevation of cardiac
myeloperoxidase (MPO) activity in response to DOX treatment was signicantly hampered. Pretreatment
of CRAN signicantly guarded against DOX-induced rise of serum lactate dehydrogenase (LDH), creatine
phosphokinase (CK), creatine kinase-MB (CK-MB) as well as troponin I level. CRAN alleviated histopathological changes in rats hearts treated with DOX. In conclusion, CRAN protects against DOX-induced cardiotoxicity in rats. This can be attributed, at least in part, to CRANs antioxidant activity.
2010 Elsevier Ltd. All rights reserved.

1. Introduction
Cranberries are small, dark red fruits that are widely consumed
as juice and sauce. They come from a shrub, Vaccinium macrocarpon
Aiton [Ericaceae], native to eastern North America (Cunningham
et al., 2004). Active constituents of cranberries include several
avonols and avonoids as proanthocyanidins and anthocyanins
(Ariga, 2004). Cranberry juice has long been consumed for the prevention of urinary tract infections (Foo et al., 2000). Studies have
shown that supplementations with berries were effective in reducing oxidative stress associated with aging. Further, cranberries have
been reported to possess anti-inammatory and anti-mutagenic
properties and provide cardioprotection (Bagchi et al., 2004).
Doxorubicin (DOX) is one of the most effective antitumor antibiotics belonging to the class of anthracyclines. However, its use is
* Corresponding author. Tel.: +966 56 2134533; fax: +966 2 6951696.
E-mail address: ashour032000@yahoo.com (O.M. Ashour).
0278-6915/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2010.02.008

limited by high incidence of a dose-dependent cardiotoxicity that

can vary from transient electrocardiographic abnormalities to cardiomyopathy and heart failure (Buzdar et al., 1985). With the
increasing use of this anthracycline antibiotic, an acute cardiotoxicity has been recognized as a severe complication of DOX chemotherapy (Doroshow, 1991). The mechanism by which DOX causes
myocardial injury is not fully understood. Several explanations account for the DOX cardiotoxicity, e.g., free radical production, calcium overloading, mitochondrial dysfunction and peroxynitrite
formation have been proposed (Olson and Mushlin, 1990; De Beer
et al., 2001; Shuai et al., 2007). Nonetheless, the oxidative stress
hypothesis of DOX toxicity remains the cornerstone. Following entry into cardiomyocytes, DOX generates reactive oxygen species
(ROS) via several mechanisms (Zweier et al., 1986; Malisza and
Hasinoff, 1995). The role of ROS in DOX-induced cardiac toxicity
is supported by the ndings that treatment of animals with a variety of antioxidants protects heart against the toxicity of DOX (Nazeyrollas et al., 1999; Liu et al., 2002). Furthermore, overexpression

A.A. Elberry et al. / Food and Chemical Toxicology 48 (2010) 11781184

of antioxidant enzymes such as manganese superoxide dismutase,

catalase, or glutathione peroxidase in cardiomyocytes of transgenic
mice greatly attenuates DOX-induced cardiac injury (Kang et al.,
1996; Yen et al., 1996; Xiong et al., 2006). In spite of the effectiveness of some antioxidants such as vitamin E and N-acetylcysteine,
they failed to eliminate oxygen radicals clinically (Peng et al.,
2005). Natural products have long gained wide acceptance among
the public and scientic community (Bauer, 2000). Therefore, the
present study was designed to explore the potential protective effects of the alcoholic extract of cranberry V. macrocarpon against
DOX-induced cardiotoxicity in rats.
2. Material and methods
2.1. Chemicals
DOX was obtained as doxorubicin hydrochloride (2 mg/ml) from EBWE Pharma,
A-4866 Unterach, Austria. 4-Aminoantipyrine, bovine serum albumin, carboxymethylcellulose (CMC), Ellmans reagent, Folin reagent, glutathione reduced form
(GSH), glutathione reductase (GR), hydrogen peroxide (H2O2), nitroblue-tetrazolium, nicotinamide adenine dinucleotide phosphate reduced form (NADPH), oxidized glutathione (GSSG), phenol, 1,1,3,3-tetraethoxypropane and trichloroacetic
acid were purchased from SigmaAldrich Chemical Company (St. Louis, MO, USA).
All other chemicals were of the highest grade commercially available.

2.2. Extraction of cranberry

Cranberry (V. macrocarpon) capsules were purchased from GNC products (GNC
Natures Fingerprint Cranberry 100 Capsules, USA). Each capsule contained
500 mg of lyophilized powdered fruits. The powdered fruit content of 100 capsules
(equivalent to 50 g) was extracted with 90% MeOH (3  150 ml) by using Ultraturrax T25 homogeniser (Janke and Kunkel, IKA Labortechnik, Stauten, Germany) at a
temperature not exceeding 25 C, using ice bath. The extract was evaporated under
reduced pressure, lyophilized and protected from light at 4 C until use. The extract was analyzed by thin layer chromatography as ngerprint using chromatographic system reported by Camag (http://www.camag.com, last visited 7/11/
2009).

1179

2.5. Cardiac biochemical assays

2.5.1. Reduced glutathione (GSH) and oxidized glutathione (GSSG)
Cardiac GSH content was determined according to the method of Adams et al.
(1983). GSSG level was assessed according to the method of Hissin and Hilf
(1976) and values are expressed as nmol/mg protein.
2.5.2. Lipid peroxidation
Lipid peroxidation products were determined by measuring malondialdehyde
(MDA) content in tissue homogenates according to the method of Uchiyama and
Mihara (1978). The MDA content was measured spectrophotometrically at
532 nm. The MDA content was calculated based on a standard curve using
1,1,3,3-tetraethoxypropane as a standard. Values are expressed as nmol/g protein.
2.5.3. Protein carbonyl (PC) content
Cardiac PC content was determined spectrophotometrically by a method based
on the reaction of the carbonyl group with 2,4-dinitrophenylhydrazine to form 2,4dinitrophenylhydrazone (Levine et al., 1990) and values are expressed as nanomoles carbonyl/mg protein.
2.5.4. Catalase (CAT) activity
Cardiac CAT activity was determined according to the method described by Aebi
(1984) based on determination of the H2O2 decomposition rate at 240 nm and values are expressed as U/mg protein.
2.5.5. Superoxide dismutase (SOD) activity
Cardiac SOD activity was determined according to the method of Sun et al.
(1988). The principle of the method is based on the inhibition of nitroblue-tetrazolium reduction by the xanthinexanthine oxidase system as a superoxide generator.
Values are expressed as U/mg protein.
2.5.6. Glutathione peroxidase (GSH-Px) activity
Cardiac GSH-Px activity was assessed spectrophotometrically according to the
method of Paglia and Valentine (1967) and expressed as U/mg protein.
2.5.7. Glutathione reductase (GR) activity
Cardiac GR activity was determined according to the method described by Staal
et al. (1969). The principle of the assay is based on the determination of the amount
of NADPH utilized to convert GSSG to the reduced form. Values are expressed as U/g
protein.

2.3. Animals and experimental protocol

Male Wister rats, weighing 250300 g were used in the study in accordance
with the guidelines of the Biochemical and Research Ethical Committee at King
Abdulaziz University, Jeddah, Saudi Arabia. Animals were housed in a well-ventilated, temperature-controlled room at 22 3 C with 12 h lightdark cycle. Food
consisted of normal rat chow and water was provided ad libitum. Care was taken
to avoid stressful conditions. All experimental procedures were performed between
8 and 10 a.m.
Rats were randomly assigned to four groups (18 animals in DOX group and 12
animals in the rest of the groups). Group I received carboxymethylcellulose (CMC;
0.5%) (1 ml/200 g body weight/day) orally for 10 consecutive days. Group II received
CRAN alone suspended in 0.5% CMC (100 mg/kg; orally once daily for 10 consecutive days). Group III received CMC orally for 10 consecutive days and a single dose
of DOX (15 mg/kg, i.p.) on day 7. Group IV received both CRAN and DOX in the previously mentioned doses; CRAN was administered for 10 consecutive days and DOX
was administered once on day 7. Dosing volume was 0.5 ml/100 g body weight. The
chosen doses and regimens were consistent with those in the literature and pilot
studies (Fadillioglu et al., 2004).
Twenty-four hours after the last CRAN or CMC treatment (day 11), rats were
anesthetized with thiopentone (35 mg/kg; i.p.) and subjected to ECG recording.
Blood samples were collected by orbital puncture in serum separating tubes. The
blood was centrifuged at 3000g for 15 min to separate the sera that were kept
at 70 C for biochemical analyses. Abdomen of each rat was opened and hearts
were rapidly dissected out, washed in ice-cold isotonic saline and blotted between
two lter papers. Four hearts from each group were xed in 10% formalin for histopathological examination and the remaining hearts from each group were homogenized in ice-cold 0.1 M potassium phosphate puffer (pH 7.4) and stored at 70 C
for subsequent analyses.

2.4. General toxicity study

For determination of mortality, 12 animals were used in each group. Mortality
and general condition of the animals were observed daily throughout the study.
Fluid accumulation in the abdominal cavity was determined at the end of the study
after abdominal opening and scored according to a graded scale of 0 to 3+; 0, non;
1+, mild; 2+, moderate and 3+, severe (Kelishomi et al., 2008).

2.5.8. Myeloperoxidase (MPO) activity

Cardiac MPO activity was determined by the method of Wei and Frenkel (1993).
The principle of the assay is based on using 4-aminoantipyrine/phenol solution as
the substrate for MPO-mediated oxidation by H2O2 and recording the changes in
absorbance at 510 nm. Values are expressed as mU/g protein.
2.5.9. Determination of protein content
The protein content of cardiac tissue homogenates was determined by the Lowry
protein assay using bovine serum albumin as the standard (Lowry et al., 1951).
2.6. Serum biochemical assays
Creatine phosphokinase (CPK), creatine kinase isoenzyme-MB (CK-MB) and lactate dehydrogenase (LDH) activities were determined according to standard methods using diagnostic kits from BioSystems S.A. (Barcelona, Spain). Assessment of
serum troponin I was carried out by enzyme-linked immunosorbent assay (ELISA)
using kit purchased from DRG International Inc. (Mountainside, NJ, USA).
2.7. Electrocardiography (ECG)
ECG was recorded in thiopentone anesthetized rats using Bioscience ECG recorder (Bioscience, Washington, USA). Needle electrodes were inserted under the skin
for the limb lead at position II.
2.8. Histopathological study
Hearts were cut at 0.5 lm, mounted on slides, stained with hematoxylin and
eosin (H&E) and examined under light microscope (Olympus BX-50 Olympus Corporation, Tokyo, Japan).
2.9. Statistical analysis
Survival and effusion intensity score were analyzed using X2 test. All other data
are expressed as means SEM. Assessment of these results was performed using
one-way ANOVA procedure followed by TukeyKramer multiple comparisons tests

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A.A. Elberry et al. / Food and Chemical Toxicology 48 (2010) 11781184

using Software GraphPad InStat, Version 4 (GraphPad Software Inc., La Jolla, CA,
USA). Statistical signicance was determined as p value below 0.05.

Table 2
The effect of cranberry extract (CRAN) on doxorubicin (DOX)-induced alterations in
heart rate, ST interval and QT interval.

3. Results
3.1. Evaluation of general toxicity
Four rats died in DOX-only-treated group (33.3%) two days after
DOX administration. However, no mortality was observed in all
other groups including the combined CRAN + DOX-treated group.
Rats in the DOX-only-treated group showed scruffy fur and developed a light yellow tinge. These animals showed, also, red exudates
around the eyes which appeared to sicker, weaker and lethargic as
compared to CRAN + DOX-treated group. Strikingly, these animals
developed ascites, as determined by a grossly distended abdomen
and later conrmed during necropsy. The hallmark gross pathologic changes in DOX-only-treated rats were excessive amounts
of pericardial, pleural and peritoneal uids. Effusion intensity score
was moderate in 25% and severe in 75% in DOX-only treated animals, compared with two rats only (16.7%) in the CRAN + DOX
group, where the intensity score was severe (Table 1).

Control
CRAN
DOX
CRAN + DOX

Heart rate (beat/min)

ST interval (ms)

QT interval (ms)

360 15.0
355 12.0
260 12.0a
360 14.0b

100 6.00
90.0 8.00
360 9.00a
110 4.00b

500 40.0
480 20.0
1280 104a
460 42.0b

CRAN was given orally to rats (100 mg/kg/day for 10 consecutive days) and DOX
(15 mg/kg; i.p.) was administered on the seventh day.
Data are the mean SEM of 12 rats.
a
p < 0.05 vs. corresponding control group.
b
p < 0.05 vs. corresponding DOX group.

3.2. Evaluation of ECG changes

ECG tracing showed normal cardiac activity in all rats in the
control and CRAN groups with a mean heart rate of 360 15 and
355 12 beat/min, respectively. Rats in the DOX-only-treated
group showed several ECG changes including bradycardia
(260 12 beat/min), ST segment depression and prolongation of
both ST and QT intervals. Such ECG abnormalities were obviously
improved in the CRAN + DOX group as evidenced by normalization
of heart rate, ST segment and both ST and QT intervals (Table 2 and
Fig. 1).
3.3. Evaluation of cardiac tissues
Cardiac content of GSH was reduced signicantly in the DOXonly-treated group as compared with control group. Animals
receiving combined CRAN + DOX showed signicantly higher levels of GSH as compared to DOX-treated animals. However, GSH level in the CRAN + DOX group was still signicantly lower than that
of control group. Cardiac levels of GSSG, MDA and protein carbonyl
contents were increased signicantly in the DOX-only-treated
group compared with control group. It was observed that CRAN
pretreatment could signicantly guard against the increases in
the latter parameters. Although not normalized, GSSG and MDA
levels were signicantly lower than the corresponding values of
the control group. Also, protein carbonyl content in CRAN + DOX

Table 1
The effect of cranberry extract (CRAN) on doxorubicin (DOX)-induced pleural,
pericardial and peritoneal effusion intensity score in surviving rats.
Group

Control
CRAN
DOX
CRAN + DOX

No. of
animals

12
12
8
12

Effusion intensity score

0

+1

+2

+3

No.

No.

No.

No.

12
12
0
10

100
100
0
83.3

0
0
0
0

0
0
0
0

0
0
2
0

0
0
25
0

0
0
6
2

0
0
75a
16.7b

CRAN was given orally to rats (100 mg/kg/day for 10 consecutive days) and DOX
(15 mg/kg; i.p.) was administered on the seventh day.
Score: (0) non, (1) mild, (2) moderate, (3) severe. Statistical analysis was done using
X2 test.
a
p < 0.05 vs. corresponding control group.
b
p < 0.05 vs. corresponding DOX group.

Fig. 1. Effect of cranberry extract (CRAN) on doxorubicin (DOX)-induced alterations

in ECG pattern. CRAN was given orally to rats (100 mg/kg/day for 10 consecutive
days) and DOX (15 mg/kg; i.p.) was administered on the seventh day. Control ECG
tracing (1a) shows control heart rate, ST segment and interval, and QT interval.
CRAN ECG tracing (1b) shows control heart rate, ST segment and interval, and QT
interval. DOX-treated group (1c) shows bradycardia, depressed long ST segment,
and long QT interval. CRAN + DOX group (1d) shows normal heart rate, ST segment
and QT interval.

group was signicantly lower than that of the DOX group and is
statistically non-signicant from the corresponding value of the
control group (Table 3).
Administration of DOX to rats signicantly reduced the cardiac
activities of CAT, SOD, GSH-Px and GR as compared to the control
activities. Catalase activity showed no alteration by pretreatment
with CRAN. Meanwhile, the activities of SOD and GR in the com-

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A.A. Elberry et al. / Food and Chemical Toxicology 48 (2010) 11781184

Table 3
The effect of cranberry extract (CRAN) on doxorubicin (DOX)-induced alterations in the cardiac reduced and oxidized glutathione (GSH and GSSG), malondialdehyde (MDA) and
protein carbonyl contents.

Control
CRAN
DOX
CRAN + DOX

GSH (lmol/g protein)

GSSG (lmol/g protein)

MDA (nmol/g protein)

Protein carbonyl content (nmol/mg protein)

4.61 0.21
4.85 0.11
2.01 0.08a
3.89 0.17a,b

0.87 0.10
0.92 0.08
1.61 0.07a
0.98 0.02b

60.6 3.20
55.2 2.40
129.3 6.20a
79.8 3.80a.b

0.182 0.03
0.179 0.04
0.386 0.05a
0.189 0.07b

CRAN was given orally to rats (100 mg/kg/day for 10 consecutive days) and DOX (15 mg/kg; i.p.) was administered on the seventh day.
Data are the mean SEM of 6 rats.
a
p < 0.05 vs. corresponding control group.
b
p < 0.05 vs. corresponding DOX group.

Table 4
The effect of cranberry extract (CRAN) on doxorubicin (DOX)-induced alterations in the cardiac activities of catalase (CAT), superoxide dismutase (SOD) glutathione peroxidase
(GSH-Px) and glutathione reductase (GR).

Control
CRAN
DOX
CRAN + DOX

CAT (U/mg protein)

SOD (U/mg protein)

GSH-Px (U/mg protein)

GR (U/mg protein)

8.13 0.27
8.05 0.23
3.46 0.17a
3.92 0.26a

33.1 1.42
34.6 1.71
18.6 1.21a
29.1 1.16b

5.12 0.12
5.03 0.17
1.82 0.10a
4.86 0.18a,b

5.82 0.22
5.53 0.24
3.10 0.20a
5.36 0.32b

CRAN was given orally to rats (100 mg/kg/day for 10 consecutive days) and DOX (15 mg/kg; i.p.) was administered on the seventh day.
Data are the mean SEM of 6 rats.
a
p < 0.05 vs. corresponding control group.
b
p < 0.05 vs. corresponding DOX group.

bined CRAN + DOX group were signicantly elevated almost to the

control values. Further, GSH-Px activity was elevated in the combined CRAN + DOX group. However, it was signicantly lower than
that of the control group (Table 4).
Assessment of the MPO activity in cardiac tissues indicated a
signicant increase in the DOX-only-treated group. However, pretreatment with CRAN exerted a signicant reduction compared to
DOX-only-treated group that was still signicantly higher than the
control group (Fig. 2).

3.5. Histopathological examination

Cardiotoxicity induced by DOX was further assessed using of
hematoxylin and eosin stained sections. Hearts from control and
CRAN groups showed regular cell distribution and normal myocardium architecture (Fig. 3a and b). Histological examination of the
rat hearts from DOX-only treated animals revealed cytoplasmic
vacuole formation, interstitial edema and brotic bands (Fig. 3c).
Myocardial lesions were signicantly reduced in animals from
CRAN + DOX group (Fig. 3d).

3.4. Evaluation of serum markers of cardiac injury

4. Discussion

The serum markers indicating myocardial injury; LDH, CPK, CKMB and troponin I, were signicantly (p < 0.05) elevated in the
DOX-only-treated group compared with control and CRAN-onlytreated group. Pretreatment with CRAN in DOX + CRAN group signicantly reduced their levels as compared with DOX-only-treated
group. However, none of the assessed parameters was returned to
corresponding control values (Table 5).

CRAN ranks high among fruits in both antioxidant quality and

quantity because of its substantial avonoid content, including
proanthocyanidins, anthocyanins, and avonols, and a wealth of
phenolic acids (Vinson et al., 2001). DOX continues to be an effective and widely used broad spectrum chemotherapeutic agent.
However, its clinical use is limited because of its serious dosedependent cardiotoxicity (Singal and Iliskovic, 1998). Clinical and
experimental investigations suggested that increased oxidative
stress plays a critical role in subsequent cardiomyopathy and heart
failure associated with DOX treatment (Siveski-Iliskovic et al.,
1995; Mihm et al., 2002). The present study was designed to investigate the potential protective effects of the methanolic extract of
CRAN against DOX-induced cardiotoxicity in rats.
In the present study, DOX administration was accompanied by a
high mortality as compared to control animals. Live animals
showed excessive degree of pericardial, pleural and peritoneal
effusion. These ndings are in line with those observed by previous
investigators (Li and Singal, 2000; Hayward and Hydock, 2007).
Ascites has been reported to be a characteristic of DOX-induced
heart failure (Kim et al., 2005). Some researchers have dismissed
the accumulation of ascitic uid as evidence of DOX-mediated
heart failure (Jensen et al., 1984), whereas others have assessed
the degree of heart failure according to the volume of the ascitic
uid (Siveski-Iliskovic et al., 1994). The existence of mortality
and effusions are explained on the basis of the development of

MPO (mU/g protein)

4
3

a,b

Control
CRAN
DOX
CRAN+DOX

2
1
0

Fig. 2. The effect of cranberry extract (CRAN) on doxorubicin (DOX)-induced

alterations in cardiac myeloperoxidase (MPO) activity. CRAN was given orally to
rats (100 mg/kg/day for 10 consecutive days) and DOX (15 mg/kg; i.p.) was
administered on the seventh day. Data are the mean SEM of four rats in DOX
group and eight rats in the other groups. ap < 0.05 vs. corresponding control group.
b
p < 0.05 vs. corresponding DOX group.

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A.A. Elberry et al. / Food and Chemical Toxicology 48 (2010) 11781184

Table 5
The effect of cranberry extract (CRAN) on doxorubicin (DOX)-induced alterations in serum biomarkers of cardiac injury; lactate dehydrogenase (LDH), creatine phosphokinase
(CPK), creatine phosphokinase isoenzyme-MB (CK-MB) and troponin I.

Control
CRAN
DOX
CRAN + DOX

LDH (IU/L)

CPK (IU/L)

CK-MB (IU/L)

Troponin I (ng/ml)

122 9.80
126 8.30
211 10.3a
166 9.80a,b

246 13.0
252 11.0
780 28.0a
363 15.0a,b

93.0 6.70
90.0 7.20
189 10.0a
127 9.20a,b

9.62 0.13
10.3 0.23
17.2 1.13a
13.9 0.30a,b

CRAN was given orally to rats (100 mg/kg/day for 10 consecutive days) and DOX (15 mg/kg; i.p.) was administered on the seventh day.
Data are the mean SEM of 12 rats.
a
p < 0.05 vs. corresponding control group.
b
p < 0.05 vs. corresponding DOX group.

Fig. 3. Effect of cranberry extract (CRAN) on doxorubicin (DOX)-induced histopathological alterations in cardiac tissues. CRAN was given orally to rats (100 mg/kg/day for 10
consecutive days) and DOX (15 mg/kg; i.p.) was administered on the seventh day. Cardiac tissues from control and cranberry extract (CRAN) groups show normal histological
pattern (a and b, respectively). Cardiac tissues from doxorubicin (DOX)-treated group show swollen vacuolated cardiomyocytes (V), interstitial edema (E) and brosed bands
(F) (c). Cardiac tissues from DOX + CRAN treated rats show some vacuolated cardiomyocytes (V) and minimal interstitial edema (E) with no brosed bands (d) (H&E X125).

heart failure. The ability of CRAN to protect against DOX-induced

high mortality and effusion intensity score was considered an early
sign of cardioprotection. This was conrmed by ECG tracing. Rats
in the DOX group showed a reduction of cardiac rate, ST segment
depression and prolongation of both ST and QT intervals. These
changes reected arrhythmias, conduction abnormalities and
attenuation of left ventricular function. Similar changes in ECG
tracing have been reported by other studies (Puri et al., 2005;
Kelishomi et al., 2008; Li et al., 2009). A positive correlation between DOX-induced cardiotoxicity and ST-interval duration has
been demonstrated by previous studies (den Hartog et al., 2004).
The ECG tracings collected from the combined CRAN + DOX veried a guardian role for CRAN.
Since oxidative stress is a cornerstone in DOX-induced cardiotoxicity (Takemura and Fujiwara, 2007), it was reasonable to investigate the oxidant/antioxidant status of the rats. Current data show
that cardiac levels of oxidized glutathione (GSSG), MDA and protein carbonyl were signicantly elevated while those of GSH were

signicantly reduced following DOX administration as compared

to control group. Such data clearly indicate an overt oxidative
stress. These data are in accordance with those reported by previous investigators (Kim et al., 2003; Choi et al., 2008; Ibrahim et al.,
2009). The observed GSH deciency and the rise of the level of
GSSG caused by DOX might be due to GSH consumption in the
interactions of DOX-induced free radicals with bio-membrane
and the subsequent lipid peroxidation. Pretreatment of rats with
CRAN signicantly guarded against the oxidative stress observed
in the DOX group. Further, the activity of the cardiac antioxidant
enzymes CAT, SOD, GSH-Px and GR were signicantly reduced
while that of MPO was signicantly elevated in response to DOX
administration as compared to control rats. These data are in
accordance with those reported by many investigators (Dbrowska
et al., 2008; Tatlidede et al., 2009). The observed decrease in the
antioxidant enzyme activities can be explained on the basis of their
exhaustion in combating the previously observed oxidative stress.
Our results indicated that CRAN mitigated the decrease of the

A.A. Elberry et al. / Food and Chemical Toxicology 48 (2010) 11781184

activity of the antioxidant enzymes. This explains and conrms the

previously observed GSH depletion and GSSG, MDA and protein
carbonyls accumulation in cardiac tissues. This can be explained
on the basis of its high content of several antioxidants including
avonoids and phenolic acids (Kresty et al., 2001). CRAN rich in
these compounds were reported to inhibit oxidative processes in
other tissues (Ofek et al., 1991; Willett, 1995; Harborne and Williams, 2001). Specically, most of CRANs protective properties
may be attributed to its proanthocyanidins content. They have
been shown to scavenge 1,1-Diphenyl-2-picrylhydrazyl (DPPH)
and superoxide effectively (Chang et al., 2007) and improve the ferric-reducing antioxidant power in plasma (Busserolles et al., 2006).
Further, they were shown to suppress the DNA damage induced by
DOX (de Rezende et al., 2009). Moreover, Li and co-workers (2009)
reported that procyanidins from grape seeds protected from DOXinduced myocardial damage and cardiac dysfunction by scavenging the free radical in rats. The nding that cardiac MPO activity
was increased in DOX group is important because it denotes leukocyte accumulation in cardiac tissue. This is in line with previous reports implicating inammation in DOX cardiotoxicity (Riad et al.,
2009). Neutrophils have a role in oxidant injury via many mechanisms (Arnhold et al., 2001; Hamza et al., 2008). Based on our data,
it may be suggested that inhibition of CAT and GSH-Px activities
would channel the produced H2O2 to the MPO pathway with the
resultant cardiac injury. The protective effects of CRAN are supported by the work of Aruoma (1994) who reported that CRAN prevents oxidative and inammatory damage to the vascular
endothelium. This is in line with our histological nding that CRAN
mitigates neutrophil inltration.
DOX administration to rats signicantly elevated serum LDH
activity and CPK, CK-MB and troponin I levels; which are released from damaged myocytes and sensitive indicators of cardiac injury (Herman et al., 1971). Similar observations were
previously reported (Shi et al., 2007; Iqbal et al., 2008). The increase in CPK, CK-MB and troponin I levels in rats administered
DOX is in line with the published data (Venkatesan, 1998;
Ahmed et al., 2005; Shi et al., 2007). CRAN signicantly inhibited
DOX-induced elevations in serum activity of LDH, and CK-MB as
well as troponin I levels. The aforementioned biochemical data
and ECG abnormalities were further strengthened by histopathological examination of rats hearts. They showed cardiac injury in
the form of cytoplasmic vacuole formation, interstitial edema and
brotic bands typically present in acute DOX-induced cardiotoxicity in rats (Morishima et al., 1998; Saad et al., 2001; Mukherjee
et al., 2003) and mice (Xin et al., 2007). The severity of the histological changes was much less in sections from animals pretreated with CRAN. Thus, the observed maintenance of the
cardiomyocyte integrity would lead to decreased leakage of cardiac markers.
In conclusion, our data indicate that CRAN protects against
DOX-induced cardiotoxicity in rats as evidenced by improved
mortality and effusion scores, mitigation of ECG abnormalities,
improved cardiac injury markers and restoration of the oxidant/antioxidant status as well as lessening histopathological
changes. This can be attributed, at least in part, to its antioxidant
activity.
Conict of interest statement
The authors declare that there are no conicts of interest.
Acknowledgement
This work was supported by Grant # 429/004-11 offered by the
Deanship of Scientic Research, King Abdulaziz University, Jeddah,
Saudi Arabia.

1183

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