Cranberry (Vaccinium macrocarpon) protects against doxorubicin-induced cardiotoxicity in rats

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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
b
Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia
c
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
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 car-
diotoxicity 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 significantly 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 significantly mitigated. Elevation of cardiac
myeloperoxidase (MPO) activity in response to DOX treatment was significantly hampered. Pretreatment
of CRAN significantly 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 histopathol-
ogical changes in rats’ hearts treated with DOX. In conclusion, CRAN protects against DOX-induced car-
diotoxicity in rats. This can be attributed, at least in part, to CRAN’s antioxidant activity.
Ó 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
flavonols and flavonoids as proanthocyanidins and anthocyanins
(Ariga, 2004). Cranberry juice has long been consumed for the pre-
vention of urinary tract infections (Foo et al., 2000). Studies have
shown that supplementations with berries were effective in reduc-
ing oxidative stress associated with aging. Further, cranberries have
been reported to possess anti-inflammatory and anti-mutagenic
properties and provide cardioprotection (Bagchi et al., 2004).
Doxorubicin (DOX) is one of the most effective antitumor anti-
biotics belonging to the class of anthracyclines. However, its use is
limited by high incidence of a dose-dependent cardiotoxicity that
can vary from transient electrocardiographic abnormalities to car-
diomyopathy and heart failure (Buzdar et al., 1985). With the
increasing use of this anthracycline antibiotic, an acute cardiotox-
icity has been recognized as a severe complication of DOX chemo-
therapy (Doroshow, 1991). The mechanism by which DOX causes
myocardial injury is not fully understood. Several explanations ac-
count for the DOX cardiotoxicity, e.g., free radical production, cal-
cium 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 en-
try 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 findings that treatment of animals with a vari-
ety of antioxidants protects heart against the toxicity of DOX (Naz-
eyrollas et al., 1999; Liu et al., 2002). Furthermore, overexpression
0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2010.02.008
* Corresponding author. Tel.: +966 56 2134533; fax: +966 2 6951696.
E-mail address: ashour032000@yahoo.com (O.M. Ashour).
Food and Chemical Toxicology 48 (2010) 1178–1184
Contents lists available at ScienceDirect
Food and Chemical Toxicology
journal homepage: www.elsevier.com/locate/foodchemtox

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 effective-
ness 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 scientific community (Bauer, 2000). Therefore, the
present study was designed to explore the potential protective ef-
fects 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, carboxy-
methylcellulose (CMC), Ellman’s reagent, Folin reagent, glutathione reduced form
(GSH), glutathione reductase (GR), hydrogen peroxide (H2O2), nitroblue-tetrazo-
lium, nicotinamide adenine dinucleotide phosphate reduced form (NADPH), oxi-
dized glutathione (GSSG), phenol, 1,1,3,3-tetraethoxypropane and trichloroacetic
acid were purchased from Sigma–Aldrich 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
Nature’s FingerprintÒ
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 Â 150 ml) by using Ultratur-
rax 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 À4 °C until use. The ex-
tract was analyzed by thin layer chromatography as fingerprint using chromato-
graphic system reported by Camag (http://www.camag.com, last visited 7/11/
2009).
2.3. Animals and experimental protocol
Male Wister rats, weighing 250–300 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-venti-
lated, temperature-controlled room at 22 ± 3 °C with 12 h light–dark 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 consecu-
tive 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 pre-
viously 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 3000Âg for 15 min to separate the sera that were kept
at À70 °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 filter papers. Four hearts from each group were fixed in 10% formalin for histo-
pathological examination and the remaining hearts from each group were homog-
enized in ice-cold 0.1 M potassium phosphate puffer (pH 7.4) and stored at À70 °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. 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 2,4-
dinitrophenylhydrazone (Levine et al., 1990) and values are expressed as nano-
moles 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 val-
ues 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-tetrazo-
lium reduction by the xanthine–xanthine 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.
2.5.8. 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 lac-
tate dehydrogenase (LDH) activities were determined according to standard meth-
ods 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 recor-
der (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 Cor-
poration, 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 Tukey–Kramer multiple comparisons tests
A.A. Elberry et al. / Food and Chemical Toxicology 48 (2010) 1178–1184 1179

using Software GraphPad InStat, Version 4 (GraphPad Software Inc., La Jolla, CA,
USA). Statistical significance was determined as p value below 0.05.
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 devel-
oped 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 confirmed during necropsy. The hallmark gross patho-
logic changes in DOX-only-treated rats were excessive amounts
of pericardial, pleural and peritoneal fluids. Effusion intensity score
was moderate in 25% and severe in 75% in DOX-only treated ani-
mals, compared with two rats only (16.7%) in the CRAN + DOX
group, where the intensity score was severe (Table 1).
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 significantly in the DOX-
only-treated group as compared with control group. Animals
receiving combined CRAN + DOX showed significantly higher lev-
els of GSH as compared to DOX-treated animals. However, GSH le-
vel in the CRAN + DOX group was still significantly lower than that
of control group. Cardiac levels of GSSG, MDA and protein carbonyl
contents were increased significantly in the DOX-only-treated
group compared with control group. It was observed that CRAN
pretreatment could significantly guard against the increases in
the latter parameters. Although not normalized, GSSG and MDA
levels were significantly lower than the corresponding values of
the control group. Also, protein carbonyl content in CRAN + DOX
group was significantly lower than that of the DOX group and is
statistically non-significant from the corresponding value of the
control group (Table 3).
Administration of DOX to rats significantly 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-
Table 1
The effect of cranberry extract (CRAN) on doxorubicin (DOX)-induced pleural,
pericardial and peritoneal effusion intensity score in surviving rats.
Group No. of
animals
Effusion intensity score
0 +1 +2 +3
No. % No. % No. % No. %
Control 12 12 100 0 0 0 0 0 0
CRAN 12 12 100 0 0 0 0 0 0
DOX 8 0 0 0 0 2 25 6 75a
CRAN + DOX 12 10 83.3 0 0 0 0 2 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.
Table 2
The effect of cranberry extract (CRAN) on doxorubicin (DOX)-induced alterations in
heart rate, ST interval and QT interval.
Heart rate (beat/min) ST interval (ms) QT interval (ms)
Control 360 ± 15.0 100 ± 6.00 500 ± 40.0
CRAN 355 ± 12.0 90.0 ± 8.00 480 ± 20.0
DOX 260 ± 12.0a
360 ± 9.00a
1280 ± 104a
CRAN + DOX 360 ± 14.0b
110 ± 4.00b
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
b
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.
1180 A.A. Elberry et al. / Food and Chemical Toxicology 48 (2010) 1178–1184

bined CRAN + DOX group were significantly elevated almost to the
control values. Further, GSH-Px activity was elevated in the com-
bined CRAN + DOX group. However, it was significantly lower than
that of the control group (Table 4).
Assessment of the MPO activity in cardiac tissues indicated a
significant increase in the DOX-only-treated group. However, pre-
treatment with CRAN exerted a significant reduction compared to
DOX-only-treated group that was still significantly higher than the
control group (Fig. 2).
3.4. Evaluation of serum markers of cardiac injury
The serum markers indicating myocardial injury; LDH, CPK, CK-
MB and troponin I, were significantly (p < 0.05) elevated in the
DOX-only-treated group compared with control and CRAN-only-
treated group. Pretreatment with CRAN in DOX + CRAN group sig-
nificantly reduced their levels as compared with DOX-only-treated
group. However, none of the assessed parameters was returned to
corresponding control values (Table 5).
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 myocar-
dium architecture (Fig. 3a and b). Histological examination of the
rat hearts from DOX-only treated animals revealed cytoplasmic
vacuole formation, interstitial edema and fibrotic bands (Fig. 3c).
Myocardial lesions were significantly reduced in animals from
CRAN + DOX group (Fig. 3d).
4. Discussion
CRAN ranks high among fruits in both antioxidant quality and
quantity because of its substantial flavonoid content, including
proanthocyanidins, anthocyanins, and flavonols, and a wealth of
phenolic acids (Vinson et al., 2001). DOX continues to be an effec-
tive and widely used broad spectrum chemotherapeutic agent.
However, its clinical use is limited because of its serious dose-
dependent 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 inves-
tigate 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 findings 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 fluid 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
fluid (Siveski-Iliskovic et al., 1994). The existence of mortality
and effusions are explained on the basis of the development of
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.
GSH (lmol/g protein) GSSG (lmol/g protein) MDA (nmol/g protein) Protein carbonyl content (nmol/mg protein)
Control 4.61 ± 0.21 0.87 ± 0.10 60.6 ± 3.20 0.182 ± 0.03
CRAN 4.85 ± 0.11 0.92 ± 0.08 55.2 ± 2.40 0.179 ± 0.04
DOX 2.01 ± 0.08a
1.61 ± 0.07a
129.3 ± 6.20a
0.386 ± 0.05a
CRAN + DOX 3.89 ± 0.17a,b
0.98 ± 0.02b
79.8 ± 3.80a.b
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.
a
b
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).
CAT (U/mg protein) SOD (U/mg protein) GSH-Px (U/mg protein) GR (U/mg protein)
Control 8.13 ± 0.27 33.1 ± 1.42 5.12 ± 0.12 5.82 ± 0.22
CRAN 8.05 ± 0.23 34.6 ± 1.71 5.03 ± 0.17 5.53 ± 0.24
DOX 3.46 ± 0.17a
18.6 ± 1.21a
1.82 ± 0.10a
3.10 ± 0.20a
CRAN + DOX 3.92 ± 0.26a
29.1 ± 1.16b
4.86 ± 0.18a,b
5.36 ± 0.32b
a
b
0
1
2
3
4
5
CRAN
DOX
CRAN+DOX
Control
a
a,b
MPO(mU/gprotein)
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. a
b

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 confirmed 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 reflected 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 be-
tween 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 veri-
fied a guardian role for CRAN.
Since oxidative stress is a cornerstone in DOX-induced cardio-
toxicity (Takemura and Fujiwara, 2007), it was reasonable to inves-
tigate the oxidant/antioxidant status of the rats. Current data show
that cardiac levels of oxidized glutathione (GSSG), MDA and pro-
tein carbonyl were significantly elevated while those of GSH were
significantly 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 previ-
ous investigators (Kim et al., 2003; Choi et al., 2008; Ibrahim et al.,
2009). The observed GSH deficiency 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 significantly 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 significantly reduced
while that of MPO was significantly 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
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.
LDH (IU/L) CPK (IU/L) CK-MB (IU/L) Troponin I (ng/ml)
Control 122 ± 9.80 246 ± 13.0 93.0 ± 6.70 9.62 ± 0.13
CRAN 126 ± 8.30 252 ± 11.0 90.0 ± 7.20 10.3 ± 0.23
DOX 211 ± 10.3a
780 ± 28.0a
189 ± 10.0a
17.2 ± 1.13a
CRAN + DOX 166 ± 9.80a,b
363 ± 15.0a,b
127 ± 9.20a,b
13.9 ± 0.30a,b
a
b
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 fibrosed bands
(F) (c). Cardiac tissues from DOX + CRAN treated rats show some vacuolated cardiomyocytes (V) and minimal interstitial edema (E) with no fibrosed bands (d) (H&E X125).

activity of the antioxidant enzymes. This explains and confirms 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
flavonoids 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 Wil-
liams, 2001). Specifically, most of CRAN’s 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 fer-
ric-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 DOX-
induced myocardial damage and cardiac dysfunction by scaveng-
ing the free radical in rats. The finding that cardiac MPO activity
was increased in DOX group is important because it denotes leuko-
cyte accumulation in cardiac tissue. This is in line with previous re-
ports implicating inflammation in DOX cardiotoxicity (Riad et al.,
2009). Neutrophils have a role in oxidant injury via many mecha-
nisms (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 sup-
ported by the work of Aruoma (1994) who reported that CRAN pre-
vents oxidative and inflammatory damage to the vascular
endothelium. This is in line with our histological finding that CRAN
mitigates neutrophil infiltration.
DOX administration to rats significantly elevated serum LDH
activity and CPK, CK-MB and troponin I levels; which are re-
leased from damaged myocytes and sensitive indicators of car-
diac injury (Herman et al., 1971). Similar observations were
previously reported (Shi et al., 2007; Iqbal et al., 2008). The in-
crease 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 significantly 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 histopathol-
ogical examination of rats’ hearts. They showed cardiac injury in
the form of cytoplasmic vacuole formation, interstitial edema and
fibrotic bands typically present in acute DOX-induced cardiotox-
icity in rats (Morishima et al., 1998; Saad et al., 2001; Mukherjee
et al., 2003) and mice (Xin et al., 2007). The severity of the his-
tological changes was much less in sections from animals pre-
treated with CRAN. Thus, the observed maintenance of the
cardiomyocyte integrity would lead to decreased leakage of car-
diac 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 oxi-
dant/antioxidant status as well as lessening histopathological
changes. This can be attributed, at least in part, to its antioxidant
activity.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgement
This work was supported by Grant # 429/004-11 offered by the
Deanship of Scientific Research, King Abdulaziz University, Jeddah,
Saudi Arabia.
References
Adams, J., Lauterburg, B., Mitchell, J., 1983. Plasma glutathione and glutathione
disulfide in the rat. Regulation and response to oxidative stress. J. Pharmacol.
Exp. Ther. 227, 749–754.
Aebi, H., 1984. Catalase in vitro. In: Packer, L., Orlando, F.L. (Eds.), Methods in
Enzymology, vol. 105. Academic Press, New York, pp. 121–126.
Ahmed, H.H., Mannaa, F., Elmegeed, G.A., Doss, S.H., 2005. Cardioprotective activity
of melatonin and its novel synthesized derivatives on doxorubicin-induced
cardiotoxicity. Bioorg. Med. Chem. 13 (5), 1847–1857.
Ariga, T., 2004. The antioxidative function, preventive action on disease and
utilization of proanthocyanidins. Biofactors 21, 197–201.
Arnhold, J., Osipov, A.N., Spalteholz, H., Panasenko, O.M., Schiller, J., 2001. Effects of
hypochlorous acid on unsaturated phosphatidylcholines. Free Radical Biol. Med.
31 (9), 1111–1119.
Aruoma, O.I., 1994. Nutrition and health aspects of free radicals and antioxidants.
Food Chem. Toxicol. 32, 671–683.
Bagchi, D., Sen, C.K., Bagchi, M., Atalay, M., 2004. Anti-angiogenic, antioxidant, and
anti-carcinogenic properties of a novel anthocyanin-rich berry extract formula.
Biochemistry (Mosc) 69, 75–80.
Bauer, B.A., 2000. Herbal therapy: what a clinician needs to know to counsel
patients effectively. Mayo Clin. Proc. 75, 835–841.
Buzdar, A.U., Marcus, C., Smith, T.L., Blumenschein, G.R., 1985. Early and delayed
clinical cardiotoxicity of doxorubicin. Cancer 55 (12), 2761–2765.
Busserolles, J., Gueux, E., Balasin´ ska, B., Piriou, Y., Rock, E., Rayssiguier, Y., Mazur, A.,
2006. In vivo antioxidant activity of procyanidin-rich extracts from grape seed
and pine (Pinus maritima) bark in rats. Int. J. Vitam. Nutr. Res. 76, 22–27.
Chang, W.T., Shao, Z.H., Yin, J.J., Mehendale, S., Wang, C.Z., Qin, Y., Li, J., Chen, W.J.,
Chien, C.T., Becker, L.B., Vanden-Hoek, T.L., Yuan, C.S., 2007. Comparative effects
of flavonoids on oxidant scavenging and ischemia-reperfusion injury in
cardiomyocytes. Eur. J. Pharmacol. 566, 58–66.
Choi, E.H., Lee, N., Kim, H.J., Kim, M.K., Chi, S., Kwon, D.Y., Chun, H.S., 2008.
Schisandra fructus extract ameliorates doxorubicin-induce cytotoxicity in
cardiomyocytes: altered gene expression for detoxification enzymes. Genes
Nutr. 2, 337–345.
Cunningham, D.G., Vannozzi, S.A., Turk, R., Roderick, R., O’Shea, E., Brilliant, K., 2004.
Cranberry phytochemicals and their health benefits. In: Shahidi, F.,
Weerasinghe, D.K. (Eds.), Nutraceutical Beverages: Chemistry, Nutrition, and
Health Effects, ACS Symposium Series 871. American Chemical Society,
Washington, DC, pp. 35–51.
Dbrowska, K., Stuss, M., Gromadzin´ ska, J., Wasowicz, W., Sewerynek, E., 2008. The
effects of melatonin on glutathione peroxidase activity in serum and
erythrocytes after adriamycin in normal and pinealectomised rats.
Endokrinol. Pol. 59 (3), 200–206.
De Beer, E.L., Bottone, A.E., Voest, E.E., 2001. Doxorubicin and mechanical
performance of cardiac trabeculae after acute and chronic treatment: a
review. Eur. J. Pharmacol. 415, 1–11.
de Rezende, A.A., Graf, U., Guterres Zda, R., Kerr, W.E., Spanó, M.A., 2009. Protective
effects of proanthocyanidins of grape (Vitis vinifera L.) seeds on DNA damage
induced by doxorubicin in somatic cells of Drosophila melanogaster. Food Chem.
Toxicol. 47, 1466–1472.
den Hartog, G.J., Haenen, G.R., Boven, E., wan der Vijgh, W.J., Bast, A., 2004.
Lecithinized copper, zinc–superoxide dismutase as a protector against
doxorubicin-induced cardiotoxicity in mice. Toxicol. Appl. Pharmacol. 194,
180–188.
Doroshow, J.H., 1991. Doxorubicin-induced cardiotoxicity. N. Engl. J. Med. 324, 843–
845.
Fadillioglu, E., Oztas, E., Erdogan, H., Yagmurca, M., Sogut, S., Ucar, M., Irmak, M.K.,
2004. Protective effects of caffeic acid phenethyl ester on doxorubicin-induced
cardiotoxicity in rats. J. Appl. Toxicol. 24, 47–52.
Foo, L.Y., Lu, Y., Howell, A.B., Vorsa, N., 2000. A-type proanthocyanidin trimers from
cranberry that inhibit adherence of uropathogenic P-fimbriated Escherichia coli.
J. Nat. Prod. 63, 1225–1228.
Hamza, A., Amin, A., Daoud, S., 2008. The protective effect of a purified extract of
Withania somnifera against doxorubicin-induced cardiac toxicity in rats. Cell
Biol. Toxicol. 24 (1), 63–73.
Harborne, J.B., Williams, C.A., 2001. Anthocyanins and other flavonoids. Nat. Prod.
Rep. 18, 310–333.
Hayward, R., Hydock, D.S., 2007. Doxorubicin cardiotoxicity in the rat: an in vivo
characterization. J. Am. Assoc. Lab. Anim. Sci. 46 (4), 20–32.
Herman, E., Mhatre, R., Lee, I.P., Vick, J., Waravdekar, V.S., 1971. A comparison of the
cardiovascular actions of daunomycin, adriamycin and N-acetyldaunomycin in
hamsters and monkeys. Pharmacology 6, 230–241.
Hissin, P.J., Hilf, R.A., 1976. Afluorometric method for determination of oxidized and
reduced glutathione in tissues. Anal. Biochem. 74, 214–226.
Ibrahim, M.A., Ashour, O.M., Ibrahim, Y.F., El-Bitar, H.I., Gomaa, W., Abdel-Rahim,
S.R., 2009. Angiotensin-converting enzyme inhibition and angiotensin AT(1)-
receptor antagonism equally improve doxorubicin-induced cardiotoxicity and
nephrotoxicity. Pharmacol. Res. 60 (5), 373–381.
Iqbal, M., Dubey, K., Anwer, T., Ashish, A., Pillai, K.K., 2008. Protective effects of
telmisartan against acute doxorubicin-induced cardiotoxicity in rats.
Pharmacol. Rep. 60 (3), 382–390.
Jensen, R.A., Acton, E.M., Peters, J.H., 1984. Doxorubicin cardiotoxicity in the rat:
comparison of electrocardiogram, transmembrane potential, and structural
effects. J. Cardiovasc. Pharmacol. 6, 186–200.

Kang, Y.J., Chen, Y., Epstein, P.N., 1996. Suppression of doxorubicin cardiotoxicity by
overexpression of catalase in the heart of transgenic mice. J. Biol. Chem. 271,
12610–12616.
Kelishomi, R.B., Ejtemaeemehr, S., Tvangar, S.M., Rahimian, R., Mobarakeh, J.I.,
Dehpour, A.R., 2008. Morphine is protective against doxorubicin-induced
cardiotoxicity in rat. Toxicology 243, 96–104.
Kim, C., Kim, N., Joo, H., Youm, J.B., Park, W.S., Cuong, D.V., Park, Y.S., Kim, E., Min,
C.K., Han, J., 2005. Modulation by melatonin of the cardiotoxic and antitumor
activities of adriamycin. J. Cardiovasc. Pharmacol. 46 (2), 200–210.
Kim, Y., Ma, A.G., Kitta, K., Fitch, S.N., Ikeda, T., Ihara, Y., Simon, A.R., Evans, T.,
Suzuki, Y.J., 2003. Anthracycline-induced suppression of GATA-4 transcription
factor: implication in the regulation of cardiac myocyte apoptosis. Mol.
Pharmacol. 63, 368–377.
Kresty, L.A., Morse, M.A., Morgan, C., Carlton, P.S., Lu, J., Gupta, A., Blackwood, M.,
Stoner, G.D., 2001. Chemoprevention of esophageal tumorigenesis by dietary
administration of lyophilized black raspberries. Cancer Res. 61, 6112–6119.
Levine, R.L., Garland, D., Oliver, C.N., Amici, A., Climent, I., Lenz, A.G., Ahn, B.W.,
Shalteil, S., Stantman, E.R., 1990. Determination of carbonyl content in
oxidatively modified proteins. In: Packer, L., Glazer, A.N. (Eds.), Methods in
Enzymology, V 186, Oxygen Radicals in Biological Systems. Academic Press,
New York, pp. 464–478.
Li, T., Singal, P.K., 2000. Adriamycin-induced early changes in myocardial
antioxidant enzymes and their modulation by probucol. Circulation 102,
2105–2110.
Li, W., Xu, B., Xu, J., Wu, X.L., 2009. Procyanidins produce significant attenuation of
doxorubicin-induced cardiotoxicity via suppression of oxidative stress. Basic
Clin. Pharmacol. Toxicol. 104, 192–197.
Liu, X., Chen, Z., Chua, C.C., Ma, Y.S., Youngberg, G.A., Hamdy, R., Chua, B.H., 2002.
Melatonin as an effective protector against doxorubicin-induced cardiotoxicity.
Am. J. Physiol. Heart Circ. Physiol. 283, H254–H263.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement
with the Folin phenol reagent. J. Biol. Chem. 193, 265–275.
Malisza, K.L., Hasinoff, B.B., 1995. Production of hydroxyl radical by iron(III)–
anthraquinone complexes through self-reduction and through reductive
activation by the xanthine oxidase/hypoxanthine system. Arch. Biochem.
Biophys. 321, 51–60.
Mihm, M.J., Yu, F., Weinstein, D.M., Reiser, P.J., Bauer, J.A., 2002. Intracellular
distribution of peroxynitrite during doxorubicin cardiomyopathy: evidence for
selective impairment of myofibrillar creatine kinase. Br. J. Pharmacol. 135, 581–
588.
Morishima, I., Matsui, H., Mukawa, H., Hayashi, K., Yukio, T., Okumura, K., Ito, T.,
Hayukawa, T., 1998. Melatonin, a pineal hormone with antioxidant property,
protects against adriamycin cardiomyopathy in rats. Life Sci. 63, 511–521.
Mukherjee, S., Banerjee, S.K., Maulik, M., Dinda, A.K., Talwar, K.K., Maulik, S.K., 2003.
Protection against acute adriamycin-induced cardiotoxicity by garlic: role of
endogenous antioxidants and inhibition of TNF-a expression. BMC Pharmacol.
3, 16.
Nazeyrollas, P., Prévost, A., Baccard, N., Manot, L., Devillier, P., Millart, H., 1999.
Effects of amifostine on perfused isolated rat heart and on acute doxorubicin-
induced cardiotoxicity. Cancer Chemother. Pharmacol. 43, 227–232.
Ofek, I., Goldhar, J., Zafriri, D., Lis, H., Adar, R., Sharon, N., 1991. Anti-Escherichia coli
adhesin activity of cranberry and blueberry juices. N. Engl. J. Med. 324 (22),
1599.
Olson, R.D., Mushlin, P.S., 1990. Doxorubicin cardiotoxicity: analysis of prevailing
hypotheses. FASEB J. 4, 3076–3086.
Paglia, D.E., Valentine, W.N., 1967. Studies on the quantitative and qualitative
characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70,
158–169.
Peng, X., Chen, B., Lim, C.C., Sawyer, D.B., 2005. The cardiotoxicology of
anthracycline chemotherapeutics: translating molecular mechanism into
preventative medicine. Mol. Interv. 5 (3), 163–171.
Puri, A., Maulik, S.K., Ray, R., Bhatnagar, V., 2005. Electrocardiographic and
biochemical evidence for the cardioprotective effect of vitamin E in
doxorubicin-induced acute cardiotoxicity in rats. Eur. J. Pediatr. Surg. 15,
387–391.
Riad, A., Bien, S., Westermann, D., Becher, P.M., Loya, K., Landmesser, U.,
Kroemer, H.K., Schultheiss, H.P., Tschöpe, C., 2009. Pretreatment with statin
attenuates the cardiotoxicity of doxorubicin in mice. Cancer Res. 69 (2),
695–699.
Saad, S.Y., Najjar, T.A., Ai-Rikabi, A.C., 2001. The preventive role of deferoxamine
against acute doxorubicin-induced cardiac, renal and hepatic toxicity in rats.
Pharmacol. Res. 43, 211–218.
Shi, R., Liu, L., Huo, Y., Cheng, Y.Y., 2007. Study on protective effects of Panax
notoginseng saponins on doxorubicin-induced myocardial damage. Zhongguo
Zhong Yao Za Zhi 32 (24), 2632–2635.
Shuai, Y.I., Guo, I.B., Peng, S.-G., Zhang, L.-S., Guo, J., Han, G., Shen Dong, Y., 2007.
Metallothionein protects against doxorubicin-induced cardiomyopathy through
inhibition of superoxide generation and related nitrosative impairment. Toxicol.
Lett. 170 (11), 66–74.
Singal, P.K., Iliskovic, N., 1998. Doxorubicin-induced cardiomyopathy. N. Engl. J.
Med. 339, 900–905.
Siveski-Iliskovic, N., Kaul, N., Singal, P.K., 1994. Probucol promotes endogenous
antioxidants and provides protection against adriamycin-induced
cardiomyopathy in rats. Circulation 89, 2829–2835.
Siveski-Iliskovic, N., Hill, M., Chow, D.A., Singal, P.K., 1995. Probucol protects against
adriamycin cardiomyopathy without interfering with its antitumor effect.
Circulation 91, 10–15.
Staal, G.E., Visser, J., Veeger, C., 1969. Purification and properties of glutathione
reductase of human erythrocytes. Biochim. Biophys. Acta 185, 39–48.
Sun, Y., Oberley, L.W., Li, Y., 1988. A simple method for clinical assay of superoxide
dismutase. Clin. Chem. 34, 497–500.
Takemura, G., Fujiwara, H., 2007. Doxorubicin-induced cardiomyopathy from
Cardiotoxic mechanisms to management. Prog. Cardiovasc. Dis. 49, 330–
352.
Tatlidede, E., Sehirli, O., Veliog˘lu-Og˘ünc, A., Cetinel, S., Yeg˘en, B.C., Yarat, A.,
Süleymanog˘lu, S., Sener, G., 2009. Resveratrol treatment protects against
doxorubicin-induced cardiotoxicity by alleviating oxidative damage. Free
Radical Res. 43, 195–205.
Uchiyama, M., Mihara, M., 1978. Determination of malonaldehyde precursor in
tissues by thiobarbituric acid test. Anal. Biochem. 86, 271–278.
Venkatesan, N., 1998. Curcumin attenuation of acute adriamycin myocardial
toxicity in rats. Br. J. Pharmacol. 124, 425–427.
Vinson, J.A., Su, X., Zubik, L., Bose, P., 2001. Phenol antioxidant quantity and quality
in foods: fruits. J. Agric. Food Chem. 49 (11), 5315–5321.
Wei, H., Frenkel, K., 1993. Relationship of oxidative events and DNA oxidation in
SENCAR mice to in vivo promoting activity of phorbol ester-type tumor
promoters. Carcinogenesis 14, 1195–1201.
Willett, W.C., 1995. Diet, nutrition, and avoidable cancer. Environ. Health Perspect.
103 (l8), 165–170.
Xin, Y.F., Zhou, G.L., Shen, M., Chen, Y.X., Liu, S.P., Chen, G.C., Chen, H., You,
Z.Q., Xuan, Y.X., 2007. Angelica sinensis: a novel adjunct to prevent
doxorubicin-induced chronic cardiotoxicity. Basic Clin. Pharmacol. Toxicol.
101, 421–426.
Xiong, Y., Liu, X., Lee, C.P., Chua, B.H., Ho, Y.S., 2006. Attenuation of doxorubicin-
induced contractile and mitochondrial dysfunction in mouse heart by cellular
glutathione peroxidase. Free Radical Biol. Med. 41, 46–55.
Yen, H.C., Oberley, T.D., Vichitbandha, S., Ho, Y.S., St Clair, D.K., 1996. The protective
role of manganese superoxide dismutase against adriamycin-induced acute
cardiac toxicity in transgenic mice. J. Clin. Invest. 98, 1253–1260.
Zweier, J.L., Gianni, L., Muindi, J., Myers, C.E., 1986. Differences in O2 reduction by
the iron complexes of adriamycin and daunomycin: the importance of the side
chain hydroxyl group. Biochim. Biophys. Acta 884, 326–336.

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