Grape (Vitis vinifera ) Seed Extract Inhibits the Cytotoxicity and Oxidative Stress in Liver of Rats Treated with Carbon Tetrachloride

Abstract

The present study examined the curative action of Grape seed extract (GSE) on experimentally
induced hepatic damage in rats by Carbon tetrachloride (CCl4). Rats were divided into six groups (8 rats/ group)
treated for 21 days and included the control group, Group 2 the group treated orally twice a week with CCl4
(1.0 mg/kg b.w) in corn oil, Groups 3 and 4 treated orally with GSE at low dose (100 mg/kg b.w) or high dose
(200 mg/kg b.w) and Groups 5 and 6 treated with CCl4 plus GSE at the two tested doses. At the end of
experimental period, blood and liver samples were collected from all groups for the biochemical, histological,
histochemical and cytogenetic analysis. The results indicated that CCl4 induced hepatic damage in the rats
as evidenced by a significant increase in serum AST, ALT, ALP, triglycerides, MDA, nitric oxide, LDH, CEA,
total lipid, cholesterol, DNA fragmentation and NO accompanied with a significant decrease in total proteins,
GPX, SOD, DNA and RNA content in the liver and Fas and TNF gene expression in the liver. Treatment with
GSE reversed the values of the biochemical parameters to near normal values and improved the content of
nucleic acids in hepatic tissues, the gene expression and the histopathological and histochemical picture of
the liver. It could be concluded that GSE may be used in the protection against and/or the treatment of liver
disease.

Full text

Global Journal of Pharmacology 7 (3): 258-269, 2013
ISSN 1992-0075
© IDOSI Publications, 2013
DOI: 10.5829/idosi.gjp.2013.7.3.7637
Corresponding Author: Mosaad A. Abdel-Wahhab, Department of Food Toxicology and Contaminants,
National Research Center, Cairo, Egypt. Tel: 202-2283-1943,
Fax: 202-3337-0931.
258
Grape (Vitis vinifera ) Seed Extract Inhibits the Cytotoxicity and
Oxidative Stress in Liver of Rats Treated with Carbon Tetrachloride
Ghada M.A. Ragab, Ezzeldeen S. El-Denshary,
1 1,2
Aziza M. Hassan, Sekena H. Abdel-Azeim, Nabila S. Hassan,
3,4 3 5
Fathia A. Mannaa and Mosaad A. Abdel-Wahhab
67
Department of Pharmacology and Toxicology, Faculty of Pharmacy,
1
Misr University for Science and Technology, Cairo, Egypt
Department of Pharmacology and Toxicology,
2
Faculty of Pharmacy, Cairo University, Cairo, Egypt
Cell Biology Department, National Research Center, Dokki, Cairo, Egypt
3
Biotechnology Department, Faculty of Science, Taif University-KSA
4
Pathology Department, National Research Center, Dokki, Cairo, Egypt
5
Medical Physiology Department, National Research Center, Dokki, Cairo, Egypt
6
Department of Food Toxicology and Food Contaminants, National Research Center, Cairo, Egypt
7
Abstract: The present study examined the curative action of Grape seed extract (GSE) on experimentally
induced hepatic damage in rats by Carbon tetrachloride (CCl ). Rats were divided into six groups (8 rats/ group)
4
treated for 21 days and included the control group, Group 2 the group treated orally twice a week with CCl4
(1.0 mg/kg b.w) in corn oil, Groups 3 and 4 treated orally with GSE at low dose (100 mg/kg b.w) or high dose
(200 mg/kg b.w) and Groups 5 and 6 treated with CCl plus GSE at the two tested doses. At the end of
4
experimental period, blood and liver samples were collected from all groups for the biochemical, histological,
histochemical and cytogenetic analysis. The results indicated that CCl induced hepatic damage in the rats
4
as evidenced by a significant increase in serum AST, ALT, ALP, triglycerides, MDA, nitric oxide, LDH, CEA,
total lipid, cholesterol, DNA fragmentation and NO accompanied with a significant decrease in total proteins,
GPX, SOD, DNA and RNA content in the liver and Fas and TNF gene expression in the liver. Treatment with
GSE reversed the values of the biochemical parameters to near normal values and improved the content of
nucleic acids in hepatic tissues, the gene expression and the histopathological and histochemical picture of
the liver. It could be concluded that GSE may be used in the protection against and/or the treatment of liver
disease.
Key words: Liver Grape seed extract Cytogenetic Gene expression Oxidative stress Antioxidant
INTRODUCTION with more than 500.000 cases diagnosed annually. The
Liver diseases are amongst the most serious increasing in Egypt with a doubling in the incidence rate
health problems in the world today and their in the past 10 years [2]. Egypt has possibly the highest
prevention and treatment options still remain limited hepatitis C virus (HCV) prevalence worldwide [3],
despite tremendous advances in modern medicine. In estimated among the general population to be around
Egypt, liver cancer is the second cause of deaths from 14% [4]. Moreover, hepatitis B virus (HBV) accounts
cancer after breast cancer and it is third frequent for 10-30% of chronic liver diseases and there is likewise
occurring cancer after bladder and breast cancer [1]. a large occult reservoir of HCV caused chronic liver
Hepatocellular carcinoma (HCC) is a major health problem, disease.
burden of hepatocellular carcinoma (HCC) has been

Global J. Pharmacol., 7 (3): 258-269, 2013
259
Current liver mortality, including liver cirrhosis and important role in controlling of some liver diseases, high
cancer, is over 40,000/year and is increasing annually. blood pressure and anemia. Also fibers and fruit acids in
This represents more than 10% of total mortality. grape have vital role in cleaning blood functions of
The conventional or synthetic drugs used in the treatment digestive system and kidney [15]. Flavonoid component
of liver diseases are inadequate and sometimes can have of grape seed extract, especially proanthocyanidin has
serious side effects [5]. In view of severe undesirable side antioxidant activity and it used in treatment of liver
effects of synthetic agents, there is growing focus to disease [16]. The aims of the current study were to
follow systematic research methodology and to evaluate evaluate the protective role of aqueous extract of grape
scientific basis for the traditional herbal medicines that seed against the cytotoxicity, DNA damage and oxidative
are claimed to possess hepatoprotective activity [6]. stress in liver tissue in rats treated with CCl .
Most liver disorders cause some degree of hepatocellular
injury and necrosis, resulting in various abnormal MATERIAL AND METHODS
laboratory testes results and sometimes symptoms.
Symptoms may be due to liver disease itself (e.g, jaundice Chemicals and Kits: Alanine aminotransferase (ALT)
due to Hepatitis) or to complications of liver disease.
As the liver is the first organ to metabolize all
foreign compounds so it is susceptible to almost as
many different diseases. Some are rare but there are a few,
including hepatitis, cirrhosis, alcohol related disorders
and liver cancer. A major cause of these disorders is due
to exposure to different environmental pollutants and
xenobiotics e.g., paracetamol, carbon tetrachloride,
thioacetamide, alcohol, etc. These toxicants mainly
damage liver by producing reactive oxygen species
(ROS). Reactive oxygen free radicals have been
known to produce tissue injury through covalent
binding and lipid peroxidation and have been shown
to augment fibrosis as seen from increased collagen
synthesis [7]. Scavenging of free radicals by antioxidants
could reduce the fibrosis process in the tissues also free
radicals may act as a contributory factor in a progressive
decline in the function of immune system [8]. Cooperative
defense systems that protect the body from free radical
damage include the antioxidant nutrients and enzymes
[9-11].
Plant origin polyphenolic compounds are intensely
studied in the recent years thank to their potent
antioxidant, anti-inflammatory and immunomodulatory
properties [12]. Grapes (Vitis vinifera), which are one of
the most widely consumed fruits in the world have
enormous health benefits. They contain a great variety of
polyphenolic antioxidants with preventive and also
therapeutic effects in several cancers, 60-70% of their
content being represented by proanthocyanidins,
composed mainly of dimers, trimers, tetramers and
oligomers of monomeric catechins [13]. Grape seed extract
can be considered among the most powerful natural
nutrients efficient in the protection of body health,
proanthocyanidins from grape seeds being stronger
antioxidants and free radical scavengers than ascorbic
acid or vitamin E [14]. It has been reported that grape has
4
and Aspartate aminotransferase (AST) were purchased
from Spectrum-diagnostics Co. (Cairo, Egypt).
Triglycerides, total proteins, glutathione peroxidase (GPx),
superoxide dismutase (SOD), lipid peroxidase, alkaline
phosphatase, nitric oxide, lactic dehydrogenase (LDH),
carcinoembrionic antigen (CEA), total lipid and
cholesterol kits were purchased from Biodiagnostic Co.
(Giza, Egypt). All other chemicals used throughout the
experiments were of the highest analytical grade available.
Plant Extract: Grape seed extract (GSE) was obtained from
Mepaco Arabian Pharmaceutical Company Cairo, Egypt).
Experimental Animals: Three-month old female
Sprague-Dawley rats (100-150g) purchased from Animal
House Colony, National Research Centre Dokki, Giza,
Egypt. Animals were maintained on the specified diet
and housed in filter-top polycarbonate cages in a room
free from any source of chemical contamination, artificially
illuminated (12h dark/light cycle) and thermally controlled
(25 ±1°C) at the Animal House Lab., National Research
Centre. All animals were received humane care in
compliance with the guidelines of the Animal Care and
Use Committee of the National Research Centre.
Experimental Design: Animals were divided into six
groups (8 rats/ group) and were maintained on their
respective diet for 3 weeks as follow: group 1, normal
control animals which fed on basal diet and water without
any treatment; Group 2, animals treated orally with CCl4
(1.0 mg/ kg b.w) suspended in corn oil twice a week [17];
Group 3, animals treated orally with low dose of GSE
(100 mg/kg) [18]; Group 4, animals treated orally with high
dose of GSE (200 mg/kg), Group 5, animals treated orally
with low dose of GSE plus CCl , rats treated orally with
4
the low dose of GSE plus CCL and Group 6,
4
animals treated orally with high dose of GSE plus CCl .
4

Global J. Pharmacol., 7 (3): 258-269, 2013
260
The animals were observed daily for signs of toxicity separated; one-half the volume was used for gel
during the experimental period. At the end of the electrophoresis and the other half together with the pellet
treatment period (i.e. day 21) all animals were fasted for 12 containing large pieces of DNA were used for
h, then blood samples were collected from the retro-orbital quantification of fragmented DNA by the Diphenyl amine
venous plexus under diethyl ether anesthesia. Sera were (DPA) assay. The samples were treated with equal
separated using cooling centrifugation and stored at-20°C volumes of absolute isopropyl alcohol and NaCl to
until analysis. The sera were used for the determination precipitate DNA. Extracted DNA was electrophoresed
of ALT, AST, ALP, LDH, NO, CEA, total protein, on 1% agarose gels containing 0.71 µg/ml ethidium
cholesterol, triglycerides, total lipid, uric acid and bromide. At the end of the runs, gels were examined
createnine according to the kits instructions. using UV transillumination. The Diphenyl amine (DPA)
After the collections of blood samples, animals were assay reaction suggested earlier [20, 23]. The colorimetric
sacrificed and samples of the liver of each animal were reaction was measured spectrophotometrically at 575 nm
dissected, weighed and was homogenized in phosphate and the percentage of DNA fragmentation was calculated.
buffer (pH 7.4) to give 20% w/v homogenate [19].
This homogenate was centrifuged at 1700 rpm and 4 °C for RNA Isolation: Hepatic tissue cells were ground in
10 min; the supernatant was stored at-70°C until analysis. liquid nitrogen and total RNA was extracted from all
This supernatant (20%) was used for the determination of groups of the experiment (five samples from each group).
hepatic lipid peroxidation and it was further diluted with The extraction of total RNA was performed using Biozol
phosphate buffer solution to give 2% and 0.5% dilutions reagent according to the manufacturer’s procedures.
for the determination of hepatic glutathione peroxidase The concentration and purity of RNA was measured at
(2%) and superoxide dismutase (0.5%) activities. 260/280 nm using ultraviolet spectrophotometer
Other samples of the liver from all animals were (ratios fell between 1.75 and 1.9, indicating very pure RNA
fixed in 10% neutral formalin and paraffin embedded. in all cases). Equal amounts of RNA isolated from
Sections (5µm thickness) were stained with hematoxylin individual rat of each group were prepared for the
and eosin (H&E) for the histological examination. semi-quantitative RT-PCR [24, 25].
Other sections from liver were stained with Bromophenol
blue for the determination of protein content in liver and Reverse Transcription-polymerase Chain Reaction
kidney tissue [20]. (RT-PCR): Reverse transcription-PCR was performed in
Cytogenetic Analysis: Determination of nucleic acids in total hepatic RNA, 200 U Superscript TM II reverse
hepatic tissue: Nucleic acids were determined in hepatic transcriptase (Life Technologies) at 42°C for 10 min
tissues according to the method described previously followed by 42°C for 1 h. In a total volume of 20 µl, the
[21]. In brief, the hepatic tissues were homogenized and PCR mixture contained 150 µM dNTPs, 1 µM antisense
the homogenate was suspended in ice-cold trichloroacetic and sense primers for Fas or TNF , 1 µl reverse-
acid (TCA). After centrifugation, the pellet was extracted transcribed cDNA and 2 U Taq polymerase (PE Applied
with ethanol. The levels of DNA were determined by Biosystems, Foster City, CA). The sequences of
treating the nucleic acid extract with diphenylamine oligonucleotide primers were: 5`-Fas, CGC CTA TGG TTG
reagent and reading the intensity of blue color at 600 nm. TTG ACC, 3`-Fas, CTC CAG ACA TTG TCC TTC,
For quantification of RNA, the nucleic acid extract was 5`-TNF , ACA GAA AGC ATG ATC CGC, 3`-TNF ,
treated with orcinol and the green color was read at 660 GTA GAC CTG CCC GGA CTC, 5'- -actin,
nm. Standard curves were used to determine the amounts C GTGA CATCA AAG A GAA G CTGT GC-3 '- -actin,
of nucleic acids present. CTCAGGAGGAGCAATGATCTTGAT-3'. The expected
DNA Fragmentation Assays for Apoptosis: Apoptotic TNF . Amplification conditions were (94°C 15 s, 54°C
changes in the liver were evaluated calorimetrically by
DNA fragmentation and by agarose gel electrophoresis
according to the procedure of the published method [22].
Liver samples were homogenized in 700 µl hypotonic
lysis buffer and centrifuged for 15 min at 11,000 rpm.
The supernatants containing small DNA fragments were
a 20-µl volume that contained 5 µM oligo dT12-18, 2 µg
amplicon lengths were 477 bp for Fas and 692 bp for
1 min, 72°C for 30 s) for 15–35 cycles. The expected
amplicon lengths were 477 bp for Fas and 692 bp for
TNF [26]. An aliquot of the RT-PCR reactants (10 µl)
was separated on a 1.2% agarose gel containing ethidium
bromide, visualized under UV light and analyzed using
NIH Image software.

Global J. Pharmacol., 7 (3): 258-269, 2013
261
Statistical Analysis: All data were statistically analyzed The results also indicated that rats treated with CCl
by analysis of Variance (ANOVA) using the General
Linear Model Procedure of the Statistical Analysis
System [27]. The significance of the differences among
treatment groups was determined by Waller-Duncan
k-ratio [28]. All statements of significance were based
on probability of P = 0.05.
RESULTS
The results of the current study revealed that
various liver enzymes were seriously affected with CCl4
treatment while upon treatment with grape seed extract
whether in low or high doses, a significant improvement
was achieved. Significant increases in AST, ALT, ALP,
LDH, total lipids, cholesterol, triglycerides, CEA and NO
accompanied with a significant decrease in total protein
were detected after CCl treatment (Table 1). Animals
4
received GSE at the two tested doses plus CCl showed a
4
significant improvement in all biochemical parameters
toward the normal value of the controls. Moreover
treatment with CCl resulted in a significant decrease in
4
GPX and SOD activities in liver accompanied with a
significant increase in MDA. Treatment with GSE
succeeded to induce a significant improvement in
antioxidant parameters and oxidative stress markers
(Table 2).
4
showed a significant cytotoxicity as indicated by the
depletion in DNA and RNA content in the hepatic tissue
(Table 3). The results also indicated that CCl -induced
4
DNA damage was evaluated by measuring the level of
fragmented DNA calorimetrically using Diphenylamine
(DPA) and by comparing DNA profiles on agarose gel
electrophoresis. These results showed that CCl caused
4
marked DNA fragmentation in the liver (39.40 %)
compared to control untreated rat (7.80) as indicated by
DPA assay (Table 4). Treatment with GSE significantly
brought down the percentage of the DNA damage to
15.93% and 10.15% at the low and high doses
respectively. The DNA fragmentation in response to
exposure to CCl and the other treatments was also
4
detected by gel electrophoresis as a DNA ladder
representing a series of fragments that are multiples of
180-200 bp (Fig. 1).
In the current study, bands produced from amplifying
cDNA of fatty acid synthesis (Fas), tumor necrosis factor
(TNF) and the house keeping gene -actin as a control
were analyzed and the results of gene expression was
based on quantifying the signal intensities in each band.
The results were expressed as the ratio between maximum
optical density (OD max) for each band of the target
amplification product and the corresponding OD max of
-actin (Fig. 2). These results indicated that exposure to
Table 1: Effect of grape seed extract (GSE) on serum biochemical parameters in rats treated with CCl4
Groups Parameters Control CCl LGSE HGSE CCl + LGSE CCl + HGSE
4 44
AST (U/L) 150.75 ± 5.89 230.93 ± 4.79 151.38 ± 1.98 156.81 ± 3.47 212.88 ± 2.15 157.35 ± 6.10
acaada
AST (U/L) 65.83 ±1.42 98.73 ± 1.76 57.88 ± 1.48 66.43 ± 1.51 65.75 ± 1.73 61.44 ± 1.98
a b c aaa
ALP (U/L) 160.95 ± 15.16 226.53 ± 13.29 161.42 ± 13.5 160.65 ± 10.98 176.09 ± 4.33 169.88 ± 4.59
a b a ac d
LDH (IU/L) 1973.70 ± 162.53 7867.18 ± 460.74 1842.38 ± 161.52 1954.42 ± 181.06 4157.26 ± 242.02 3441.66 ± 196.71
a b c ad e
TP (mg/dl) 5.96 ± 0.08 3.76 ± 0.27 7.09 ± 0.21 7.06 ± 0.25 5.71 ± 0.22 7.45 ± 0.20
a b c ca c
TL (mg/dl) 145.15 ± 6.06 181.71 ± 9.11 137.56 ± 8.25 134.27 ± 9.10 117.73 ± 9.88 117.73 ± 9.88
a b c cd d
ThiG (mg/dl) 33.68 ± 2.80 90.24 ± 2.28 30.07 ± 2.55 28.18 ± 2.30 49.27 ± 4.66 31.72 ± 2.16
a b a cd a
Cho (mg/dl) 78.42 ± 5.45 154.26 ± 3.96 74.37 ± 5.19 69.08 ± 8.47 73.28 ± 1.10 62.23 ± 7.10
a ba c a c
CEA (ng/ml) 4.99 ± 0.39 14.03 ± 0.18 4.49 ± 0.28 4.74 ± 0.31 5.17 ± 0.32 5.41 ± 0.56
a ba a c c
NO (µmol/L) 17.84 ± 0.61 28.73 ± 1.07 15.57 ± 0.53 12.41 ± 0.21 14.65 ± 0.97 14.52 ± 0.42
adbcbb
Within each row, means with different superscripts are significantly different at P<0.05.
Table 2: Effect of grape seed extract (GSE) on oxidative stress markers in live of rats treated with CCl4
Groups Parameters Control CCl LGSE HGSE CCl + LGSE CCl + HGSE
4 44
GPX (U/mg protein) 23.18 ± 0.72 7.91 ± 1.55 22.32 ± 1.32 24.94 ± 0.75 18.53 ± 1.41 21.8 ± 0.10
ad a b e f
SOD (U/mg protein) 2.81 ± 0.13 1.5 ±0.11 3.07 ± 0.14 2.81 ± 0.21 2.5 ±0.11 2.56 ± 0.16
ac b a a a
MDA (mol/mg protein) 11.31 ± 1.43 27.72 ± 0.38 11.24 ± 1.38 11.72 ± 0.84 12.72 ± 1.51 13.17 ± 1.37
aba a c c
Within each row, means with different superscripts are significantly different at P<0.05.

0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
Control
CCl4 LGSE HGSE CCl4 + LGSE CCl4 + HGSE
Relative Transcript Level
Fa s -O T NF- O
Global J. Pharmacol., 7 (3): 258-269, 2013
262
Table 3: Effects of Grape seed extract on nucleic acid contents in hepatic
tissues of rats treated with CCl4
Treatments DNA mg/gm RNA mg/gm
Control 0.356 ± 0.012 0.189 ± 0.003
a ab
CCl 0.170 ± 0.013 0.109 ± 0.006
4ee
LGSE 0.285 ± 0.013 0.126 ± 0.014
bc e
HGSE 0.279 ± 0.017 0.135 ± 0.016
c cd
CCl + LGSE 0.220 ± 0.011 0.141 ± 0.010
4d cd
CCl + HGSE 0.277 ± 0.012 0.190 ± 0.020
4c ab
Within each column, means with different superscripts are significantly
different at P<0.05.
Table 4: Effects of Grape seed extract on DNA fragmentation in liver of rat
treated with CCl4
Treatments DNA Fragmentation % Changes
Control 7.80 ± 1.43 --
de
CCl 39.40 ± 1.91 + 31.60
4a
LGSE 7.60 ± 0.89 - 0.20
e
HGSE 7.24 ± 0.80 - 0.56
e
CCl + LGSE 15.93 ± 0.96 + 8.13
4bc
CCl + HGSE 10.15 ± 0.71 + 2.35
4d
Within each column, means with different superscripts are significantly
different at P<0.05.
Fig. 1: Effects of GE on DNA fragmentation of hepatic
tissue in CCl -treated rats. Agarose gel
4
electrophoretic pattern of DNA isolated from liver
tissue of different groups. Lane M: phi x marker,
Lane 1: Control, Lane 2: low dose of GE, Lane 3:
high dose of GE, Lane 4: CCl , Lane 5: CCl + low
44
dose of GE and Lane 6: CCl + high dose of GE.
4
Fig. 2: RNA gene expression of Fas and TNF in the liver of control and treated rats. The results depicted are normalized
to levels of -actin gene. Data are mean of ratios of intensity for each gene divided by that for -actin.
Ccl resulted in a significant increased in mRNA liver sections in the control rats (Fig. 4a) or those treated
4
expression of the Fas gene accompanied with increased with GSE at the low or high dose (Fig. 4b,c) showed
in TNF gene expression in the hepatic tissues compared normal structure of hepatocytes with no cellular
to the other experimental groups. Treatment with GSE infiltration around the central vein, portal area or in
alone at the low and high doses induced insignificant between the hepatocytes. The microscopic examination
increase in the expression of Fas and TNF genes of liver sections from CCl -treated group showed
compared to the control group. On the other hand, vacoulation and mononuclear cellular infiltration in
treatment with GSE at high dose normalized the expression between the hepatocytes (Fig. 4d). The liver sections of
levels of Fas and TNF genes compared to the control level the animals treated with CCl plus the low dose of GSE
in liver tissues (Fig. 3). showed prominent improve in hepatocytes (Fig. 4e)
The biochemical results were confirmed by the however; those treated with CCl plus the high dose of
histological and histochemical examination of the liver GSE showed prominent improve in hepatocytes but the
and kidney tissues. The histological examination of the fibrous tissues are still present (Fig. 4f).
4
4
4

Global J. Pharmacol., 7 (3): 258-269, 2013
263
Fig. 3: Effects of GSE on transcript product of hepatic Fas & TNF genes in CCl -treated rats. Agarose gel
4
electrophoresis of Fas, TNF and -actin RT-PCR products of different groups. Lane M: phi x marker, Lane 1:
Control, Lane 2: LGSE, Lane 3: HGSE, Lane 4: CCl , Lane 5: CCl + LGSE and Lane 6: CCl + HGSE.
44 4
Fig. 4: Photomicrographs of liver sections from (a) control animal showing the normal structure of hepatocytes with no
cellular infiltration around the central vein, portal area or in between the hepatocytes, (b) animals treated with
low dose of GSE showing the normal structure of hepatocytes with no cellular infiltration around the central vein,
portal area or in between the hepatocytes, (c) animals treated with high dose of GSE showing the normal structure
of hepatocytes with no cellular infiltration around the central vein, portal area or in between the hepatocytes, (d)
animals treated with CCl showing vacoulation and mononuclear cellular infiltration in between the hepatocytes
4
(arrow), (e) rats received low dose of GSE plus CCl showing prominent improve in hepatocytes, (f) rats received
4
high dose of GSE plus CCl showing prominent improve in hepatocytes but the fibrous tissues are still present.
4
The histochemical examination of the liver The liver section of rats treated with Ccl showed
section of the control rats (Fig. 5a), low dose of a decrease in protein reaction in hepatocytes
GSE (Fig. 5b) and high dose of GSE (Fig. 5c) stained (Fig. 5d). The liver section of rats treated with CCl
with bromophenol blue stain for protein evaluation plus low dose of GSE (Fig. 5e) and high dose of
namely in the hepatocytes cytoplasm and nucleus GSE (Fig. 5f) showed obvious improvements in protein
membrane showing normal distribution of blue color. reaction.
4
4

Global J. Pharmacol., 7 (3): 258-269, 2013
264
Fig. 5: Hotomicrographs of liver sections from (a) control rats, (b) rat treated with low dose of GSE, (c) rats treated with
high dose of GSE showing normal distribution of blue color, (d) rats treated with CCl showing a decrease in
4
protein reaction in hepatocytes, (e) rats treated with CCl plus low dose of GSE and (f) tars treated with CCl plus
44
high dose of GSE showing obvious improvements in protein reaction. (Bromophenol blue stainX400)
DISCUSSION target organ for CCl consequently, the activities of
Ccl is one of the most extensively studied necrosis [31, 35-37].
4
hepatotoxicants. The mechanism by which CCl causes The elevated levels of ALT, AST, ALP, LDH,
4
hepatotoxicity is well documented in a series of reports triglycerides, cholesterol and total lipids reported herein
which indicated that its hepatotoxicity undergoes of indicated severe hepatic parenchymal cells injury [38-40].
two phases. The first results from its metabolic Whereas, the decrease in total protein indicated liver
conversion to free radical product CCl by Cyt P-450 [29]. necrosis and/or kidney dysfunction [39, 41]. These results
-
3
Once CCl has been formed it reacts very rapidly with O clearly showed that CCl has a harmful and stressful
-
32
to produce CCl OO, a much more reactive radical than CCl influence on the hepatic tissue consistent with those
3
-
[30]. These free radicals attack microsomal lipids leading reported earlier [10, 42].
to its peroxidation and also covalently bind to microsomal Alteration in the hepatic antioxidant status may
lipids and proteins. This results in the generation of therefore manifestation of oxidative stress caused by CCl
reactive oxygen species (ROS), which includes the and its metabolites. Both GPX and SOD are considered
super-oxide anion O , H O and the hydroxyl radical. enzymatic free-radical scavengers in cells. In the present
2 22
In the current study, the results indicated that study, GPX and SOD were found to decline significantly
CCl induced a severe toxicity to the animals and were in in rats treated with CCl . It is well known that SOD plays
4
agreement with the previous reports. CCl caused an important role in the elimination of ROS derived from
4
significant changes in serum biochemical parameters the peroxidative process in liver tissues [43]. Moreover,
typical to those reported in the literature [11, 26, 31, 32]. SOD removes superoxide by converting it to H O ,
Serum AST and ALT are the simple and well-accepted which can be rapidly converted to water by CAT [44].
biomarkers for hepatic dysfunction. Hepatic ALT only Taken together, the increased level of MDA and the
locates in cytoplasm, while AST distributes in both decreased activity of antioxidant enzymes GPX and SOD
cytosolic and mitochondrial fraction. AST release is an may be attributed to free radical formation which initiated
important index of relatively severe liver injuries, whereas chain reactions of direct and indirect bond formation with
ALT is more reliable (longer half-life than AST) and cellular molecules (nucleic acids, proteins, lipids and
liver-specific for acute and moderate liver injuries [33, 34]. carbohydrates) impairing crucial cellular processes that
Moreover, the liver is considered to be the principal may ultimately culminate.
4
transaminases are sensitive indicators of acute hepatic
4
4
4
22

Global J. Pharmacol., 7 (3): 258-269, 2013
265
Regarding the elevated serum level of tumor markers stress. Different forms of oxidative stress may give rise to
(CEA) in CCl administrated rats, previous reports different oxidation products and the biomarkers that are
4
indicated that CCl is a potent hepatotoxicity, enhances sensitive to other insults may differ from those that
4
reactive oxygen species (ROS) formation and causes were clearly elevated in this CCl model.
oxidative DNA damage, which may play a role in its In the current study, bands produced from amplifying
toxicity and carcinogenicity [26, 31]. CEA are considered cDNA of fatty acid synthesis (Fas), tumor necrosis factor
specific biomarkers for liver cancer and it is synthesized (TNF) and the house keeping gene -actin as a control
mainly in the fetal stage; practically no production of this were analyzed and the results of gene expression was
marker occurs in the normal adult. However, when some based on quantifying the signal intensities in each
adult cells are transformed to cancer cells, the synthesis band. These results were expressed as the ratio between
of CEA commences again. Therefore, the current study maximum optical density (OD max) for each band of the
affirmed that CCl can induce hepatotoxicity in rats as target amplification product and the corresponding OD
4
indicated by the elevation of CEA level in serum. max of -actin. The results indicated that exposure to
MDA, an end product of lipid peroxidation, is widely CCl resulted in a significant increased in mRNA
used as a marker of lipid peroxidation. Lipid peroxidation expression of the Fas gene accompanied with increased
(LP) is one of the main manifestations of oxidative damage in TNF gene expression in the hepatic tissues compared
and has been found that it plays an important role in to the other experimental groups. It is well documented
toxicity and carcinogenicity. It is well documented that that CCl significantly increased caspase 3 activity and a
CCl enhanced LP [31, 45] that is an indication of free pro-apoptotic gene (bax) expression as well as decreased
4
radical mediated toxicity. Free radicals are known to attack anti-apoptotic gene (Bcl-2) gene expression [32].
the highly unsaturated fatty acids of the cell membrane The significant high level of fatty acid synthesis
and induce lipid peroxidation that is considered a key (Fas) and tumor necrosis factor (TNF) expression found
process in many pathological events induced by oxidative in liver of CCl -treated rats indicated that these cells are
stress [46]. In the present study, MDA was found to be susceptible to apoptosis. In this concern, Masson et al.
significantly higher in the animals treated with CCl alone [53] reported that the proapoptotic proteins Bad and Bax
4
suggesting that this agent has a significant effect on LP were significantly higher in liver cirrhosis induced by
and supported the earlier findings [42, 47, 48]. CCl and apoptosis takes place in liver during CCl -
Nitric oxide (NO) is produced by macrophages and induced cirrhosis. Moreover, apoptosis lead to DNA
it plays an important role in tumor conditions [49]. damage as indicated by DNA fragmentation and comet
The generation of NO by the inducible nitric oxide formation reported in the current study since a 39.4 %
synthase (iNOS) plays a key role in the cytokine-mediated enhancement of DNA fragmentation in liver of rats
cell destruction [50]. In the current study, the ingestion treated with Ccl compared to the control group. Similar
of CCl significantly increased NO suggesting that CCl results were observed by Lee et al. [54] who reported that
44
preferentially affects macrophage functions. It is well CCl induced hepatocyte DNA fragmentation and
documented that excess ROS, a condition referred to as cytosolic caspase-3 and caspase-8 activity in rats.
oxidative stress, is considered to be a major contributor to Moreover, CCl induced DNA strand breaks in
cell injury, although many studies have shown that higher hepatocytes measured by single cell gel electrophoresis
levels of ROS can also activate specific genetic programs through the increase in comet tail length in CCl -treated
in various cells [51]. group compared to control group. Similar result noticed
In the current study, treatment with CCl resulted in by Vanitha et al. [55] who reported that CCl induced
4
a significant decrease in hepatic nucleic acid content. toxicity by comet formation in rats. Moreover, it was
It has been reported that cellular DNA damage occurs reported that CCl increased chromosomal aberrations and
due to free radicals generated under different conditions SCE’s in bone marrow which arise from DNA breaks and
and a number of techniques have been developed to reversion of broken fragments at almost homologous loci
measure the oxidatively modified nucleobases in DNA after their exchange between the two sister chromatids of
[52]. CCl induced damage in hepatic DNA (strand breaks) the same chromosome [56, 57] and hence their formation
4
and RNA. This demonstrates that oxidation products of is dependent on the S-phase of the cell cycle [58] or on
hepatic nucleic acids measured in the present study are DNA replication processes [59].
good biomarkers of oxidative liver damage after CCl The biochemical and cytogenetic results of the
4
exposure. CCl -mediated oxidative damage is just one current study were further confirmed by the
4
example of the more general phenomenon of oxidative histopathological and histochemical study. The
4
4
4
4
44
4
4
4
4
4
4

Global J. Pharmacol., 7 (3): 258-269, 2013
266
histological and histochemical results showed that liver properties of bioflavonoids, in terms of the availability of
tissue more or less have significant changes in the the phenolic hydrogens as hydrogen donating radical
histological and histochemical pictures. It is clear that scavengers and singlet oxygen quenchers, predicts their
animals treated with CCl showed severe histological antioxidant activity [66, 68]. Moreover, GSE has also been
4
changes in liver typical to those reported in the demonstrated as potent inhibitors of the enzymes
literature. In CCl -treated group, the liver showed phospholipase A2, cyclooxygenase and lipooxygenase
4
vacoulation and mononuclear cellular infiltration in [68].
between the hepatocytes. Some sections of the same The proanthocyanidins or polyphenolic
group showed coagulative necrosis in blood sinusoids bioflavonoids may act as antioxidants and/or by other
around the central vein. Moreover, other sections of the mechanisms, contributing to chemoprotective and health
animals treated with CCl showed evident fibrosis and bene?ts. It is well documented that the Bcl-2 gene is an
4
cellular infiltration around the portal area accompanied important antagonist of apoptosis, the programmed cell
with decrease in protein reaction in hepatocytes. death. Bcl-2 is highly expressed variety of tumor cells
These histological changes were similar to those including lymphomas leukemia and other solid tumors.
reported earlier [45, 60]. Similar observations of the liver These cells particularly the etastatic cells, are resistant to
tissues in rats treated with CCl were also reported apoptosis. One of the mechanisms of induction of
4
previously. resistance is via increased expression of Bcl-2 [69].
In this study, treatment with GSE at the two tested In this regard, the liver cells treated with GSE also had an
doses did not induce any significant effect on liver increased expression of Bcl-2 gene. The other apoptosis
function or the tumor marker (CEA). However, they related gene p53 is a pro-apoptosis gene [69]. Thus, one
improve the antioxidant capacity of the body and reduce of the cellular mechanisms of chemoprevention of
the oxidative stress as indicated by the reduction of GSE appears to be via up-regulation of Bcl-2 gene and
MDA and NO levels as well as DNA fragmentation and down-regulation of p53 gene. The present study
up-regulate gene expression. Treatment with GSE to the demonstrated that GSE signi?cantly normalize Fas and
intoxicated rats could markedly suppress the high serum TNF genes in a dose dependent manner. According to
level of ALT and exerted a resistant effect for the Joshi et al. [70], one of the possible mechanisms of the
increase of serum AST. These results suggest that the action of GSE is via up-regulation of anti-apoptotic gene
administration of GSE may exert protective effects Bcl-2 expression in these cells which makes the cells
for further deterioration of the mitochondrial resistant to apoptotic cell death.
membranes of the hepatocytes [64, 65]. Moreover,
these protectives succeeded to improve the histological CONCLUSION
and histochemical changes resulted in liver tissue in
CCl -treated rats. It could be concluded that treatment with CCl
4
The biological pharmacological and medicinal resulted in sever biochemical and histological changes
properties of bioflavonoids have been extensively accompanied with sever cytotoxic effect in the live.
reviewed [66]. Increasing interest in proanthocyanidins Treatment with GSE resulted in a significant improvement
and other polyphenolic compounds in GSE is based on a in all tested parameters in dose dependent manner. GSE it
variety of pharmacological, medicinal and therapeutic self was safe at the tested doses and it may be a candidate
potential including inhibition of DNA topoisomerase II, for the prevention as well as treatment of liver diseases.
modulation of protein kinase C, angiotensin-converting
enzyme and hyaluronidase enzyme activities [66]. REFERENCES
Proanthocyanidins has also been demonstrated to exhibit
antihypertensive effects [66], anti-peptic activity, 1. Ferlay, J., H.R. Shin, F. Bray, D. Forman, C. Mathers
monocyte stimulating ability and anti-hepatotoxic and D.M. Parkin, 2010. Estimates of worldwide
activity [67]. In the current study, animals treated with burden of cancer in 2008: GLOBOCAN 2008. Int. J.
CCl plus GSE at the two tested doses showed significant Cancer, 127(12): 1893-2917.
4
improvements in the antioxidant enzymes, oxidative stress 2. El-Shenawy, S.Z., M.M. El.Sabawi, N. Sheble,
markers, nucleic acids content and gene expression as M. Abd El-Raof, M.M. Allam and S.K. Fath Allah,
well as the significant improvement in the histological 2012. Diagnostic role of serum glypican-3 as a tumor
and histochemical picture in liver in dose dependent marker for hepatocellular carcinoma. J. Nat. Sci.,
fashion. Previous studies indicated that the chemical 10(4): 32-38.
4

Global J. Pharmacol., 7 (3): 258-269, 2013
267
3. WHO, 2012. Hepatitis C. World Health Organization, 14. Katiyar, S., 2008. Grape seed proanthocyanidins and
department of communicable diseases surveillance cancer prevention: inhibition of oxidative stress and
and response. protection of immune system. Mol. Nut. Food Res.,
4. Heintges, T. and J.R. Wands, 1997. Hepatitis C 52: 871-876.
virus: epidemiology and transmission. J. Hepatol, 15. Bhragual, D.D., N. Kumar, V.K. Garg and P.K. Sharma,
26: 521-526. 2010. Review on plants having hepatoprotective
5. Arun, K. and U. Balasubramanian, 2010. activity. J. Pharm. Res., 3(9): 2077-2082.
Comparative Study on hepatoprotective activity 16. Mirzaei, A., M. Mirzaei and N. Mirzaei, 2011.
of Phyllanthus amarus and Eclipta prostrata Hepatoprotectivity of grape seed and jaft extracts
against alcohol induced in albino rats. J. Environ. formulation in hepatoxic rats. J. Clin. Bioch.
Sci., 2(1): 361-379. Poster-[A-10-225-3].
6. Hemamalini, K., B. Preethi, A. Bhargav and 17. Feng, Y., K.Y. Siu, X. Ye, N. Wang, M.F. Yuen,
U. Vasireddy, 2012. Hepatoprotective activity of C.H. Leung, Y. Tong and S. Kobayashi, 2010.
Kigelia africana and Anogeissus accuminata against Hepatoprotective effects of berberine on carbon
paracetamol induced hepatotoxicity in rats. Int. J. tetrachloride-induced acute hepatotoxicity in rats.
Pharma. Biomed. Res., 3(3): 152-156. Chin. Med., 5: 33-38.
7. Singh, D. and R.S. Gupta, 2011. Hepatoprotective 18. Pallarès, V., A. Fern ndez-Iglesias, L. Cedó,
activity of methanol extract of Tecomella undulate A. Castell-Auví, M. Pinent, A. Ardévol, M.J. Salvadó,
against alcohol and paracetamol induced S. Garcia-Vallvé, M. Blay, 2013. Grape seed
hepatotoxicity in rats. Life Sci. Med. Res., 26: 1-8. procyanidin extract reduces the endotoxic effects
8. Haggag, M.H., 2011. Protective effect of Coriandrum induced by lipopolysaccharide in rats. Free Radic
sativum plant of hepatotoxicity and nephrotoxicity Biol Med., 60: 107-114.
induced by carbon tetrachloride in male albino 19. Lin, C.C., Y.F. Hsu, T.C. Lin, F.L. Hsu and H.Y. Hsu,
rats. In 'The 6 Arab and 3 International Annual 1998. Antioxidant and hepatoproductive activity
th rd
Scientific Conference on: Development of Higher of punicalagin and punicalin on carbon tetrachloride
Specific Education Programs in Egypt and the Arab induced liver damage in rats. J. Pharmacol,
World in the Light of Knowledge Era Requirements'. 50: 789-794.
pp: 2332-2348. 20. Drury, R., E. Wallington and R. Cancerson, 1976.
9. Sreelatha, S., P.R. Padma and M. Umadevi. Carleton's histological technique. 4 ed. Oxford
2009. Protective effects of Coriandrum sativum University Press, London.
extracts on carbon tetrachloride-induced 21. Bregman, A., 1983. Laboratory Investigation and
hepatotoxicity in rats. Food Chem. Toxicol, Cell Biology. John Wiley and Sons, New York,
47: 702-708. pp: 51-60.
10. Abdel-Wahhab, M.A., N.S. Hassan, A.A. El-Kady, 22. Perandones, C.E., V.A. Illera, D. Peckham, L.L. Stunz
Y.A. Mohamed, A.A. El-Nekeety, S.R. Mohamed, and R.F. Ashman, 1993. Regulation of apoptosis
H.A. Sharaf and F.A. Mannaa, 2010. Red ginseng in vitro in mature murine spleen T cells. J. Immunol.
extract protects against aflatoxin B and 151(7): 3521-3529.
1
Fumonisins-induced hepatic pre-cancerous lesions 23. K. Burton, K., 1956. A study of the conditions and
in rats. Food Chem. Toxicol, 48(2): 733-742. mechanisms of the diphenylamine reaction for the
11. El-Denshary, E.S., M.A. Al-Gahazali, F.A. Mannaa, estimation of deoxyribonucleic acid. Biochem. J.,
H.S. Salem, N.S. Hassan and M.A. Abdel-Wahhab, 62: 315-323.
2012. Dietary honey and ginseng protect against 24. Brun, M.E., S. Gasca, C. Girard, K. Bouton,
carbon tetrachloride-induced hepatonephrotoxicity B. De Massy and A. De Sario, 2006. Characterization
in rats. Exper. Toxicol. Pathol, 64: 753-760. and expression analysis during embryo development
12. Nichols, J.A. and S.K. Katiyar, 2010. of the mouse ortholog of MLL3. Gene, 371: 25-33.
Skin photoprotection by natural polyphenols: 25. Hassan, A.M., S.H. Abdel-Aziem and M.A. Abdel-
antiinflammatory, anti-oxidant and DNA repair Wahhab, 2012. Modulation of DNA damage and
mechanisms. Arch. Dermatol. Res., 302(2): 71-83. alteration of gene expression during aflatoxicosis via
13. Silva, R.C., J. Rigaud, V. Cheynier and A. Chemina, dietary supplementation of Spirulina (Arthrospira)
1991. Procyanidin dimers and trimers from grape and whey protein concentrate. Ecotoxicol. Environ.
seeds. Phytochem, 30: 1259-1264. Safety, 79: 294-300.
th

Global J. Pharmacol., 7 (3): 258-269, 2013
268
26. Abdel-Aziem, S.H., A.M. Hassan and M.A. Abdel- 39. Abdel-Wahab, M., N. El-Ghawalby, M. Mostafa,
Wahhab, 2011. Dietary supplementation with whey
protein and ginseng extract counteract the oxidative
stress and DNA damage in rats fed aflatoxins-
contaminated diet. Mutat. Res., 723: 65-71.
27. SAS Institute, Inc., 1982. SAS User’s Guide.
Statistics. SAS Institute, Cary, NC.
28. Waller, R.A. and D.B. Duncan, 1969. A Bayes rule
for the symmetric multiple comparison problems. J.
Am. Stat. Assoc, 64: 1484-1503.
29. Noguchi, T., K.L. Fong, E. Lai, S.S. Alexander,
M.M. King, L. Olson, J.L. Poyer and P.B. Mccay,
1982. Specificity of a phenobarbital induced
cytochrome P450 for metabolism of
carbontetrachloride to the trichloromethyl radical.
Biochem. Pharmacol, 31: 615-624.
30. Packer, J.E., T.F. Slater and R.L. Willson, 1978.
Reactions of the carbon tetrachlonde-related
peroxy free radical with amino acids: pulse radiolysis
evidence. Life Sci., 23: 2617-2620.
31. Sarhan, N.A., E.S. El-Denshary, N.S. Hassan,
F.M. Abou-Salem and M.A. Abdel-Wahhab, 2012.
Isoflavones-Enriched soy protein prevents CCL -
4
induced hepatotoxicity in rats. ISRN Pharmacol.
347930. Epub 2012 Mar.1.
32. Abdel-Aziem, S.H., A.M. Hassan, A.S. Salman,
A.I. Waly and M.A. Abdel-Wahhab, 2011.
Genetic alterations and gene expression profile in
male Balb/c mice treated with carbon tetrachloride
with or without carboxymethyl chitosan. J. Am. Sci.
7(6): 7-17.
33. Nyblom, H., U. Berggren, J. Balldin and R. Olsson,
2004. High AST/ALT ratio may indicate advanced
alcoholic liver disease rather than heavy drinking.
Alcohol Alcohol, 39: 336-339.
34. Ramaiah, S.K., 0000. A toxicologist guide to the
diagnostic interpretation of hepatic biochemical
parameters. Food Chem. Toxicol, 45: 1551-1557.
35. Kaplan, M.M., 1987. Primary biliary cirrhosis. New
Engl. J. Med., 316: 521-526.
36. Abdel-Wahhab, M.A. and S.E. Aly, 2003.
Antioxidants and radical scavenging properties of
vegetable extracts in rats fed aflatoxin-contaminated
diet. J. Agric. Food Chem., 51(8): 2409-2414.
37. Abdel-Wahhab, M.A. and S.E. Aly, 2005.
Antioxidant property of Nagilia Sativa (Black cumin)
and Syzygium Aromaticum (Clove) rats during
aflatoxicosis. J. Appl. Toxicol, 25: 218-223.
38. Abdel-Wahhab, M.A., S.A. Nada and M.S. Arbid,
1999. Ochratoxicosis: prevention of developmental
toxicity by L methionine in rats. J. Appl. Toxicol.
19(1): 7-12.
A. Sultan, M. El-Sadany, O. Fathy, T. Salah and
F. Ezzat, 2007. Epidemiology of hepatocellular
carcinoma in lower Egypt. Mansoura
Gastroenterology Center, Hepato-gastroenterol.
54(73): 157-162.
40. Barton, C.C., E.X. Barton, P.E. Ganey, S.L. Kunkel
and R.A. Roth, 2001. Bacterial lipopolysaccharide
enhances aflatoxin B hepatotoxicity in rats by a
1
mechanism that depends on tumor necrosis factor.
Hepatol, 33(1): 66-73.
41. Shyamal, S., P.G. Latha, S.R. Suja, V.J. Shine,
G.I. Anuja, S. Sini, S. Pradeep, P. Shikha and
S. Rajasekharan, 2010. Hepatoprotective effect of
three herbal extracts on aflatoxin B -intoxicated rat
1
liver. Singapore Med. J., 51: 326-331.
42. Bhattacharjee, R. and P.C. Sil, 2007. Protien isolate
from herb, Phyllanthus niruri L. (Euphorbiaceae),
plays hepatoprotective role against carbon
tetrachloride induced liver damage via its antioxidant
properties. Food Chem. Toxicol, 45: 817-826.
43. Abdel-Wahhab, M.A., H.H. Ahmed and
M.M. Hagazi, 2006, Prevention of aflatoxin
B - initiated hepatotoxicity in rat by marine algae
1
extracts. J. Appl. Toxicol, 26(3): 229-238.
44. Halliwell, B., 1999. Antioxidant defence mechanisms:
from the beginning to the end (of the beginning).
Free Rad. Res., 31(4): 261-272.
45. El-Denshary, E.S., M.A. Al-Gahazali, F.A. Mannaa,
H.A. Salem, N.S. Hassan and M.A. Abdel-Wahhab,
2011. Dietary honey and ginseng protect against
carbon tetrachloride-induced hepatonephrotoxicity
in rats. Exp. Toxicol. Pathol., 64: 753-760.
46. Schinella, G.R., H.A. Tournier, J.M. Prieto,
D.B.P. Mordujovich and J.L. Rios, 2002. Antioxidant
activity of anti-inflammatory plant extracts. Life Sci.
18: 1023-1033.
47. Abdel-Wahhab, M.A., A.A. Ibrahim, A.A. El-
Nekeety, N.S. Hassan and A.A. Mohamed, 2012.
Panax ginseng C.A. Meyer extract counteracts the
oxidative stress in rats fed multi-mycotoxins-
contaminated diet. Comunicata Scientiae,
3(3): 143-153.
48. Mansour, M.A., 2000. Protective effect of
thymoquinone and desferrioxamine against
hepatotoxicity of carbon tetrachloride in mice. Life
Sci., 66: 2583-2591.
49. Moon, E.Y. and S. Pyo, 2000. Aflatoxin B inhibits
1
CD14-mediated nitric oxide production in murine
peritoneal macrophages. Int. J. Immunopharmacol.
22(3): 237-246.

Global J. Pharmacol., 7 (3): 258-269, 2013
269
50. Azeredo-Martins, A.K., S. Lortz, S. Lenzen, R. Curi, 60. Song, J.Y., L. Li, J.B. Ahn, J.G. Park, J.S. Jo and
D.L. Eizirik and M. Tiedge, 2003. Improvement of D.H. Park, 2007. Acute liver toxicity by carbon
the mitochondrial antioxidant defense status tetrachloride in HSP70 knockout mice. Exp. Toxicol.
prevents cytokine induced nuclear factor-kappa B Pathol, 59(1): 29-34.
activation in insulin-producing cells. Diabetes, 61. Geerts, A.M., E. Vanheule, M. Praet, H. Van,
52: 93-101. M. DeVos and I. Colle, 2008. Comparison of three
51. Bartosz, G., 2009. Reactive oxygen species: research models of portal hypertension in mice:
destroyers or messengers? Biochem. Pharmacol. macroscopic, histological and portal pressure
77(8): 1303-1315. evaluation. Int. J. Exp. Pathol, 89(4): 251-63.
52. Rouzer, C.A., A.K. Chaudhary, M. Nokubo, 62. Kuriakose, G.C. and G.M. Kurup, 2008. Antioxidant
D.M. Ferguson, G.R. Reddy, I.A. Blair and activity of Aulosira fertilisima on CCl induced
L.J. Marnett, 1997. Analysis of the malondialdehyde- hepatotoxicity in rats. Ind. J. Exp. Biol., 46(1): 52-59.
2V-deoxyguanosine adduct pyrimidopurinone in 63. Bilgin, H.M., M. Atmaca, B.D. Obay, S. O¨zekinci,
human leukocyte DNA by gas chromatography/ E. Tas-demir and A. Ketani, 2011. Protective effects
electron capture/ negative chemical ionization/mass of coumarin and coumarin derivatives against
spectrometry. Chem. Res. Toxicol, 10: 181-188. carbon tetrachloride-induced acute hepatotoxicity in
53. Masson, S., M. Scotte, S. Garnier, A. Francois, rats. Exp. Toxicol. Pathol, 67(4): 569-576.
P. Teniere, J. Fallu, J. Salier and M. Daveau, 2000. 64. Wang, M.E., Y.C. Chen, I.S. Chen, S.C. Hsieh,
Differential expression of apoptosis-associated S.S. Chen and C.H. Chiu, 2012. Curcumin protects
genes post-hepatoectomy in cirrhotic vs. normal rats. against thioacetamide-induced hepatic fibrosis by
Apoptosis, 5(2): 173-179. attenuating the inflammatory response and inducing
54. Lee, T.Y., H.H. Chang, G.J. Wang, J.H. Chitu, apoptosis of damaged hepatocytes. J. Nutr. Biochem.
Y.Y. Yang and H.C. Lin, 2006. Water-soluble extract 23(10): 1352-366.
of Salvia miltiorrhiza ameliorates carbon 65. Li, J., J. Li, S. Li, B. He, Y. Mi, H. Cao, C. Zhang and
tetrachloride-mediated hepatic apoptosis in rats. J. L. Li, 2012. Ameliorative effect of grape seed
Pharm. Pharmacol, 58(5). 659-665. proanthocyanidin extract on thioacetamide-induced
55. Vanitha, A., K.N. Chidambara, V. Kumar, mouse hepatic fibrosis. Toxicol. Lett., 213: 353-360.
G. Sakthivelu and J.M. Veigas, 2007. Effect of the 66. Rice-Evans, C.A. and L. Packer, 1997. Flavonoids in
carotenoid-producing alga, Dunaliella bardawil, Health and Disease. Marcel Dekker, NY, USA.
on Ccl -induced toxicity in rats. Int. J. Toxicol, 67. Kolodziej, H., C. Haberl and, H.J. Woerdenbag
4
26: 159-167. and A.W.T. Konings, 1995. Moderate cytotoxicity
56. Latt, S.A., J.W. Allen, S.E. Bloom, A. Carrano, of proanthocyanidins to human tumor cell lines.
E. Flake, D. Kram, E. Schneider, R. Schreck, Phytoth. Res., 9(6): 410-415.
B. Whitefield and S. Wolf, 1981. Sister chromatid 68. Rice-Evans, C.A., N.J. Miller and G. Paganda, 1996.
exchanges: a report of Gen-Tox Program. Mutat. Res., Structure antioxidant activity relationships of
87: 17-62. ?avonoids and phenolic acids. Free Rad. Biol. Med.
57. Louis, H., J.L. Van, W. Wu, E. Quertinmont, 20(7): 933-956.
C. Degraef and K. Van, 1998. Interleukin-10 controls 69. Caligaris-Cappio, F. and T.J. Hamblin, 1999.
neutrophilic infiltration, hepatocyte proliferation B-cell chronic lymphocytic leukemia: a bird of a
and liver fibrosis induced by carbon tetrachloride different feather. J. Clin. Oncol, 17: 399-408.
in mice. Hepatol, 28: 1607-1615. 70. Joshi, S.S., C.A. Kuszynski, M. Bagchi and D. Bagchi,
58. Kato, H., 1977. Mechanisms for sister chromatid 2000. Chemopreventive effects of grape seed
exchanges and their relation to the production of proanthocyanidin extract on Chang liver cells.
chromosomal aberrations. Chromosoma, 59: 179-191. Toxicology, 155: 83-90.
59. Lasne, C., Z.W. Gu, W. Venegas and
I. Chouroulinkov, 1984. The in vitro micronucleus
assay for detection of cytogenetic effects induced by
mutagen carcinogens: Comparison with in vitro
sister chromatid exchanges assay. Mutat. Res.,
130: 273-282.
4