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3698
Journal of Applied Sciences Research, 9(6): 3698-3707, 2013
ISSN 1819-544X
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLES
Corresponding Author: Naglaa K. Madkour, Department of Zoology, Girls’ College for Arts, Science and Education, Ain
Shams University, Egypt
E-mail: naglaamadkour@hotmail.com
Amelioration of amiodarone-induced lung fibrosis in rats by grape seed extract
Naglaa K. Madkour and Mona Ahmed
Department of Zoology, Girls’ College for Arts, Science and Education, Ain Shams University, Egypt
ABSTRACT
Amiodarone (AM) hydrochloride is indicated for the treatment of serious cardiac arrhythmias.
Unfortunately it is associated with pulmonary toxicity, sometimes with fatal sequelae. The purpose of this study
was to elucidate the antioxidant and anti-fibrotic capacity of grape seed extract (GSE) against AM-induced lung
injury in rats. Twenty four adult male albino rats were divided into four groups: group I (normal control), group
II (rats given GSE), group III (rats given AM) and group IV (rats given GSE and AM). Oral administration of
AM (30 mg/kg) daily for 8 weeks increased oxidative stress markers [thiobarbituric acid reactive substances
(TBARS) and 8-hydroxy-2′-deoxyguanosine (8-OHdG)], myloperoxidase (MPO) activity and hydroxyproline
(HP) content in the lung homogenates compared with control rats. Transforming growth factor-β1 (TGF-β1) and
tumor necrosis factor-α (TNF-α) levels in serum were also markedly increased in AM-treated rats. Further,
histological alterations in the lung architecture were also observed in AM group, characterized by thickening of
interalveolar septa, cellular infiltration, vacuolar degeneration, congestion, inflammatory infiltration and focal
necrosis, compared to those of the control group. However, co-administration of GSE (150 mg/kg) ameliorated
oxidative and fibrotic damage in the lung of AM-treated rats. These findings showed that supplementation of
GSE could be useful in alleviating AM-induced lung injury.
Key words: Amiodarone, Grape seed extract, Fibrotic markers, Histopathology, Rat.
Introduction
Amiodarone (AM) is recognized as an orally effective antiarrhythmic drug (Connolly, 1999) that is widely
used throughout the world (Singh, 1996; Vassallo and Trohman, 2007). AM use has gained favor as a first-line
therapy for the treatment of acute ventricular tachycardia (Singh et al., 2005) and to reduce mortality in patients
with a high risk for arrhythmia (Doval et al., 1994). However, AM use has been associated with a variety of
adverse effects. The most serious of which is pulmonary toxicity, which manifests as pneumonitis and can
progress to potentially life-threatening pulmonary fibrosis (Oyama et al., 2005; Garg et al., 2012) and drug
withdrawal is often needed. In one third of cases, AM pulmonary toxicity presents within weeks of
commencement of the therapy (Iskandar et al., 2006). Range et al. (2013) reported that a subacute iatrogenic
AM overdose causes fatal interstitial pneumonitis within one month after initiation of treatment.
Nikaido et al. (2010) speculated that AM-induced pulmonary toxicity (AIPT) is related to lung alveolar
epithelial cell apoptosis. AM in vitro and in vivo has been shown to generate free radicals that may be involved
in the pathogenesis of its toxicity (Card et al., 1999; Ray et al., 2000). Alteration of membrane properties and
activation of alveolar macrophages and cytokine release are the other proposed mechanism of AM toxicity
(Punithavathi et al., 2003).
AM extensively distributes to body tissues due to its lipophilic structure, lending itself to a very high
volume of distribution (Hosaka et al., 2002). In humans AM also has moderate to low bioavailability, a low
hepatic extraction ratio, and a long elimination half-life, likely due to its extensive distribution to tissues
(Shayeganpour et al., 2007). The rat shares many of the pharmacokinetic characteristics of AM with human,
except that its hepatic extraction ratio is higher (Shayeganpour et al., 2005 and 2007). In both species AM is
extensively metabolized, with the prevalent metabolite being desethylamiodarone (DEA), which is a product of
mono-deethylation of AM by cytochrome (Shayeganpour et al., 2006).
Fruits and vegetables contain a vast array of antioxidant components, mainly polyphenols and flavonoids
(Potter, 1997). Flavonoids have the ability to protect against free radical attack in both aqueous and lipid
environments because of their hydrophilic or relatively lipophilic properties and may interact with plasma
proteins as well as the polar surface region of phospholipid bilayers in lipoproteins and cell membranes (Terao
et al., 1994).
Grape seed extract (GSE), a well-known dietary supplement, contains important vitamins, minerals, and
polyphenols including flavonoids, proanthocyanidins and procyanidins (Weber et al., 2007). Proanthocyanidins
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J. Appl. Sci. Res., 9(6): 3698-3707, 2013
are the most abundant phenolic compounds in grape seeds and are high molecular weight polymers comprised
of dimmers or trimers of catechin and epicatechin (Bagchi et al., 2001).
It has become clear that GSE exhibits chemoprotective properties against reactive oxygen species (ROS)
(Ashtiyani et al., 2013), anti-inflammatory (Terra et al., 2009), anti-bacterial (Mayer et al., 2008), anti-cancer
(Kaur et al., 2006), anti-ulcer (Abbas and Sakr, 2013) and anti-diabetic activities (Pinent et al., 2004). Ray et al.
(2000) demonstrated that GSE provided significant cellular protection against AM-induced lung toxicity.
The aim of the present study was to determine the possible role of GSE in preventing or minimizing AM-
induced lung injury.
Materials And Methods
Chemicals:
AM was obtained from Sanofi-Aventis, Montpellier, France (Commercially found in the form of
cordarone). Grape seed extract was obtained from Arab Company for Pharmaceuticals and Medicinal Plants
(MEPACO), Egypt.
GSE was administered two weeks before AM in a daily dose of 150 mg/kg orally (Hemmati et al., 2006)
and this dose was continued till the end of the experiment. Rats were given AM (30 mg/kg) orally (Agelaki et
al., 2007) daily for eight weeks using gastric tube.
Animals and experimental design:
Twenty four adult male albino rats (Rattus norvegicus) (150–180 g) were obtained from the National
Research Centre, Dokki, Egypt, and were kept at a constant temperature (25±2ºC) with 12-h light and dark
cycles. Rats were acclimatized to laboratory conditions for 7 days before commencement of the experiment and
free access to food and water was allowed at all times.
The animals were divided into the following four groups:
Group I (control group): Rats were orally treated with saline.
Group II (GSE group): Rats received GSE for 10 weeks.
Group III: Rats received AM for 8 weeks.
Group IV: (GSE and AM group): Rats were orally given GSE daily for 10 consecutive weeks and AM was
administered orally for 8 weeks starting 2 weeks after the commencement of the first dose of GSE.
At the end of the experimental duration rats were sacrificed after being fasted over night; blood was
collected in centrifuge tubes and centrifuged at 3000 rpm for 20 min. Lung was quickly removed and cleaned
with physiological saline. The superior lobe of the left lung was kept in 10% formaldehyde for histological
investigation. Both serum and lung tissues were immediately stored at -80°C until assayed.
Biochemical assays:
Determination of malondialdehyde (MDA):
MDA levels in the lung tissue were determined as an indicator of lipid peroxidation and as a reliable marker
of oxidative tissue injury. Lung tissues were homogenized in ice-cold Tris-HCl buffer (pH 7.4) and centrifuged
for 10 minutes at 1000× g. MDA levels were measured using the thiobarbituric acid reactive substance
(TBARS) method according to Ohkawa et al. (1979).
Measurement of 8-hydroxy-2′-deoxyguanosine (8-OHdG) level:
8-OHdG, an indicator of oxidative DNA damage, was estimated by by Enzyme-Linked Immunosorbent
Assay (ELISA) kit (BIOXYTECH® OXIS Health Products, Inc. USA) according to the manufacturer’s
instructions.
Determination of lung myeloperoxidase (MPO) activity:
The lung MPO activity was determined using a 4-aminoantipyrine/phenol solution as the substrate for
MPO-mediated oxidation by H2O2 and changes in absorbance at 460 nm were recorded (Manktelow and Meyer,
1986).
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J. Appl. Sci. Res., 9(6): 3698-3707, 2013
Detection of lung hydroxyproline content:
As an index of collagen, hydroxyproline content was estimated in the lung homogenate according to the
method of Woessner (1961). It depends on the acid digestion of collagen, and the resultant hydroxyproline was
then oxidized and converted to a colored product that was measured spectrophotometrically at 550 nm.
Measurement of TGF-β1 and TNF-α levels:
TGF-β1 and TNF-α were analyzed by ELISA technique using Quantikine® kit, supplied by R & D systems,
Inc. Minneapolis, USA.
Histological observation:
Small pieces of the left lung were immediately fixed in 10% neutral buffered formalin solution, dehydrated
in graded series of alcohol, embedded in paraffin wax, sectioned at 4 µm and stained with hematoxylin and
eosin. The stained sections were examined and photographed under a light microscope (Drury and Wallington,
1980).
Statistical analysis:
Data are presented as means ± SE and were analyzed by ANOVA using the SPSS version 13 statistical
program. Hypothesis testing methods included one-way analysis of variance (ANOVA) followed by least
significant difference (LSD) The significance level was tested at P < 0.05.
Results:
Effect of GSE on AM-induced pulmonary oxidative injury:
MDA and 8-OHdG of lungs in the AM-treated group were elevated compared to normal rats. The
improvement of MDA and 8-OHdG was shown after administration of GSE to AM-treated rats (Table 1) and no
differences were observed between both GSE and control groups.
Table 1: Effect of GSE on pulmonary MDA and 8-OHdG contents in AM-induced lung injury.
MDA (nmol/ mg)
M ± SE
8-OHdG (ng/mg DNA)
M ± SE
Control 3.64 ± 0.46 59.35 ± 1.85
GSE 3.60 ± 0.37
b
60.68 ± 2.09
b
AM 7.70 ± 0.51 a 81.99 ± 1.42 a
GSE and AM 4.99 ± 0.16 a/b 66.44 ± 2.23 a/b
a Significant change at p < 0.05 in comparison with control group. b Significant change at p < 0.05 in comparison with AM-treated group.
GSE: grape seed extract; AM: amiodarone; MDA: malondialdehyde; 8-OHdG: 8-hydroxy-2′-deoxyguanosine.
Effect of GSE and/or AM on pulmonary MPO activity:
Table 2 provides the description of pulmonary tissue activity of MPO in different treatment groups. AM-
treated group exhibited significant elevation in MPO activity, compared to normal control group. However, co-
treatment with GSE significantly attenuated the increase in MPO activity in comparison with the AM-treated
group.
Effect of GSE and/or AM on pulmonary hydroxyproline content:
Hydroxyproline content is an important indicator of lung fibrosis. A significant increase in hydroxyproline
content of the lung tissue was observed in the group that received AM compared to control group. Treatment
with GSE plus AM effectively reduced the hydroxyproline content in the lung (Table 2).
Effect of GSE and/or AM on serum levels of TGF-β and IFN-α:
The levels of the TGF-β1 and TNF-α were determined in serum. As shown in Table 3, treatment of rats
with AM resulted in a significantly marked increase in these cytokines compared with those in the control
group. Administration of GSE (150 mg/kg) led to a significant decrease in TGF-β and TNF-α as compared to
the AM group.
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J. Appl. Sci. Res., 9(6): 3698-3707, 2013
Table 2: Effect of GSE on pulmonary MPO and HPO contents in AM-induced lung injury.
MPO (U/g tisuue)
M ± SE
HPO (μg/g tissue)
M ± SE
Control 2.30 ± 0.12 9.01 ± 0.43
GSE 2.43 ± 0.19
b
9.43 ± 0.25
b
AM 6.19 ± 0.30 a 17.03 ± 0.81 a
GSE and AM 3.10 ± 0.11 a/b 10.79 ± 0.53 a/b
a Significant change at p < 0.05 in comparison with control group. b Significant change at p < 0.05 in comparison with AM-treated group.
GSE: grape seed extract; AM: amiodarone; MPO: myloperoxidase; HPO: hydroxyproline.
Table 3: Effect of GSE on serum TGF-β1 and TNF-α levels in AM-induced lung injury.
TGF-β1 (pg/ml)
M ± SE
TNF-α (pg/ml)
M ± SE
Control 41.71 ± 0.95 21.52 ± 1.23
GSE 40.31 ± 1.30
b
19.05 ± 1.83
b
AM 77.61 ± 1.22a 46.52 ± 0.93
a
GSE and AM 47.91 ± 1.21 a/b 28.10 ± 1.44
a/b
a Significant change at p < 0.05 in comparison with control group. b Significant change at p < 0.05 in comparison with AM-treated group.
GSE: grape seed extract; AM: amiodarone; TGF-β1: transforming growth factor-β1; TNF-α: tumor necrosis factor-α.
Effect of GSE on AM-induced pulmonary histological changes:
Examination of sections stained with H and E from the control group (group I) showed normal histological
structure of the lung (Fig. 1). Histological evaluation showed no structural changes in lung histology between
the control and GSE (group II) group. In the AM-treated group (group III), the lungs showed focal interstitial
pneumonia (Fig. 2), focal interstitial leukocyte aggregations associated with fibrous tissue (Fig. 3),
peribronchial leukocyte infiltration and focal pulmonary haemorrhage (Fig. 4), marked necrosis and
desquamation of bronchial epithelium (Fig. 5), congestion of pulmonary blood vessels (Fig. 6) and focal
pulmonary emphysema (Fig. 7). Administration of GSE before and with AM (group IV) showed mild thickened
interalveolar septa and mild cellular infiltration (Fig. 8) indicating that GSE treatment showed marked
improvement of histological architecture of the lung.
Fig. 1: Lung section from control group showing normal lung architecture. H and E ×100.
Fig. 2: Lung section from AM-treated group showing focal interstitial pneumonia (↑). H and E ×400.
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Fig. 3: Lung section from AM-treated group showing focal interstitial leukocyte cells aggregation (*) associated
with fibrous tissue (↑). H and E ×400.
Fig. 4: Lung section from AM-treated group showing peribronchial leukocyte cells infiltration (↑) and focal
pulmonary haemorrhage (*). H and E ×400.
Fig. 5: Lung section from AM-treated group showing marked necrosis of bronchial epithelium (↑). H and E
×400.
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J. Appl. Sci. Res., 9(6): 3698-3707, 2013
Fig. 6: Lung section from AM-treated group showing congestion of pulmonary blood vessel (↑). H and E ×400.
Fig. 7: Lung section from AM-treated group showing focal pulmonary emphysema (↑). H and E ×200.
Fig. 8: Lung section from GSE and AM-treated group showing mild thickened interalveolar septa (↑) and mild
cellular infiltration. H and E ×200.
Discussion:
Plant material in the human diet contains a large number of natural compounds, which may be of benefit in
protecting the body against the development of several diseases. One of the constituents reputed to possess
protective properties was GSE (Clouatre and Kandaswami, 2005). The present investigation was directed to
study the possible protective effects of orally administered GSE against AM-induced lung injury.
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In this study the MDA and 8-OHdG levels in the lung tissue were markedly increased after AM
administration, verifying that AM induces excessive production of ROS that leads to serious oxidative damage.
Our findings are consistent with the findings of Taylor et al. (2000) and Punithavathi et al. (2003), who reported
increased cellular oxidant production in AM rats. Moreover MDA and 8-OHdG levels were significantly lower
in the intervention group than in the AM group, indicating the ability of GSE to remove toxic oxygen radicals
and therefore reduce oxidative damage caused by ROS to some extent.
Several mechanisms have been proposed by which AM results in pulmonary toxicity. AM impairs lipid
metabolism resulting in damage to the pulmonary endothelium. It can also produce toxic oxidants when exposed
to high oxygen concentrations (Kaushik et al., 2001). There is accumulating evidence that oxidative stress may
play a major role in the pathogenesis of pulmonary fibrosis. Due to their powerful oxidizing capability, ROS can
lead to generation of advanced oxidation molecular products and induce damage to cellular and subcellular
structures within the lung, including DNA, proteins, cell membranes, and mitochondria (Kinnula et al., 2005).
Some data suggested the role of oxidative stress in AM-induced lung fibrosis. In the ventilated perfused rabbit
lung system, AM increased the levels of ROS and oxidized glutathione (Kennedy et al., 1988). Another study
revealed that AM is metabolized to an aryl radical that may give rise to other ROS (Nicolescu et al., 2007).
GSE has been shown to attenuate AM-induced oxidative stress in rats. The possible reason may be that the
GSE functions as an in vivo antioxidant by virtue of its ability to directly scavenge ROS as was reported when
administered before whole-body irradiation in rats (Enginar et al., 2007). Also, GSE prevented DNA oxidative
damage in various tissues induced by many agents (Llopiz et al., 2004) and this action may be due to
detoxification of cytotoxic radicals and presumed contribution to DNA repair (Ray et al., 2000) along with its
ability to protect against both water- and fat-soluble free radicals provides incredible protection to the cells
(Bagchi et al., 2001).
In the lung, ROS production may result from increasing MPO levels (Kinnula et al., 2005). Winterbourn
and Kettle (2000) suggested that reactive oxygen metabolites (ROM) play a role in the recruitment of
neutrophils into damaged tissue, but activated neutrophils are also a potential source of ROM and it is not
certain if neutrophil accumulation and activation are the causes or the result of injury. In the current study,
increased lung MPO activity due to AM administration indicated that tissue injury involves the contribution of
neutrophil infiltration and GSE treatment along with its antioxidant activity suppressed neutrophil accumulation.
It may thus be suggested that AM-induced oxidative injury in the lung tissue is neutrophil dependent and is
improved by GSE treatment. This observation was in agreement with Dulundu et al. (2007), where the levels of
MPO were increased in experimental biliary obstruction and GSE treatment abolished this increase.
Oxidative stress, in particular lipid peroxidation, induces collagen synthesis (Muriel and Moreno, 2004). In
this study, increases in lipid peroxidation induced increases in fibrotic activity, as assessed by lung
hydroxyproline content, while this effect was also reduced by GSE treatment. These findings suggested that
GSE has an additional protective effect on oxidant-induced production and deposition of collagen which
resulted in lung fibrosis. Hemmati et al. (2008) suggested that GSE could exert antifibrotic effect by scavenging
ROS in a rat model of silica-induced pulmonary fibrosis and delayed the process of silicosis. Also, Terra et al.
(2009) reported that GSE may be useful in the treatment of rheumatoid arthritis by attenuating collagen-induced
arthritis in mice.
A complex cytokine network involving TGF-β1 and TNF-α, that is initiated immediately after AM
administration has been shown to be the major etiological factor in pulmonary injury (Punithavathi et al., 2003).
TGF-β1 is a key profibrotic cytokine that stimulates collagen accumulation in several ways. It induces the
differentiation of fibroblasts into myofibroblasts, increases collagen synthesis by these cells and reduces
collagenase activity (Lasky and Brody, 2000). Expression of TGF-β1 is upregulated in several rodent models of
pulmonary fibrosis, including a rat model of AM-induced pulmonary fibrosis (Yi et al., 1996; Iyer et al., 1999;
Chung et al., 2001). Also, accumulation of TNF-α in increased amount would modulate fibroblast functions and
synthesis of collagen (Postlethwaite and Seyer, 1990). In the present study, TGF-β1 and TNF-α levels were
increased after AM treatment, while the GSE inhibited this overproduction. Because induction of TGF-β1 and
TNF-α levels is possibly through oxidant-induced damage giving rise to high levels of inflammation, our results
suggested that the anti-pulmonary fibrosis effect of GSE might be ascribed to both anti-inflammatory activity
and downregulation of TGF-β1 and TNF-α. These results are concomitant with studies that have been
performed on the anti-inflammatory effect of GSE, e.g., Hemmati et al. (2006, 2008) who investigated the
protective effect of GSE against the fibrogenic effect of bleomycin and silica in rat lung.
In this study, histological examination of the lung of the AM-treated group showed tissue injury in the form
of inflammation characterized by leukocyte infiltration and injury to alveolar epithelial cells; focal pulmonary
emphysema and congestion of pulmonary blood vessels; focal interstitial leukocyte aggregations associated with
fibroblasts proliferation; perivascular oedema associated with mononuclear cells infiltration and thickening of
interstitial tissue with mononuclear cells. Several mechanisms of AM adverse pulmonary effects have been
proposed, including direct cellular damage, induction of phospholipidosis, and immune mediated mechanisms
such as the activation of natural killer cell activity (Taylor et al., 2003)
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J. Appl. Sci. Res., 9(6): 3698-3707, 2013
It was reported that AM induces phospholipidosis in humans and animals because of the inhibition of
lysosomal phospholipases resulting in an abnormal degradation of phospholipids promoting its intracytoplasmic
accumulation and permitting phagocytic cells to accumulate large quantities of lipids leading to the appearance
of vacuolated pneumocytes type II and foamy macrophages (Mortuza et al., 2003). The present study showed
thickening of interalveolar septa, which could be explained by the increased interstitial fiber deposition and
marked cellular infiltration with lymphocytes, neutrophils, eosinophils, and macrophages. This finding was
previously reported by Stankiewicz et al. (2002) who stated that alveolar macrophages could release many
mediators such as tumor necrotizing factor, which augment the inflammatory response of airways and alveoli. In
addition, alveolar macrophages release a chemotactic substance specific for neutrophils, which in turn release
proteases and toxic ROS that increase the destruction of tissue and maintain alveolitis (Nagata et al., 1997).
Congested blood vessels in our study was explained to be due to direct vasodilator effects of AM by blocking
alpha receptors and calcium channels inhibitory effects, as AM increased production of free oxygen radicals
(Oyama et al., 2005), compatible with emphysematous lung and similar findings described by other researchers
(Looney et al., 2009). Co-administration of GSE with AM caused a reduction in severity of lung damages. GSE,
a potent antioxidant, can exert anti-fibrotic effects by scavenging ROS; although it cannot suppress the
progression of the disease, it probably delays the injury induced by AM (Hemmati et al., 2008).
In conclusion, the present study showed that GSE, an anti-inflammatory and antioxidant compound,
mitigates AM-induced lung injury.
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