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Antioxidant and antigenotoxic effects of lycopenein obstructive jaundice
Sevtap Aydın, PhD,a Mehmet Tokac, MD, PhD,b Gokce Taner, MSc,c
Ata Turker Arıkok, MD, PhD,d Halit Ziya Dundar, MD, PhD,b
Alper Bilal Ozkardes‚, MD, PhD,b Mine Yavuz Tas‚lıpınar, MD, PhD,e
Mehmet Kılıc, MD, PhD,f Arif Ahmet Bas‚aran, PhD,g and Nurs‚en Bas‚aran, PhD
a,*aDepartment of Pharmaceutical Toxicology, Faculty of Pharmacy, University of Hacettepe, Ankara, TurkeybDepartment of Surgery, Ministry of Health Etlik _Ihtisas Education and Research Hospital, Ankara, TurkeycDepartment of Biology, Faculty of Science, University of Gazi, Ankara, TurkeydDepartment of Clinical Pathology, Ministry of Health Dıs‚kapı Yıldırım Beyazıt Education and Research Hospital, Ankara, TurkeyeDepartment of Clinical Biochemistry, Ministry of Health Etlik _Ihtisas Education and Research Hospital, Ankara, TurkeyfDepartment of Surgery, Faculty of Medicine, University of Yıldırım Beyazıt, Ankara, TurkeygDepartment of Pharmacognosy, Faculty of Pharmacy, University of Hacettepe, Ankara, Turkey
a r t i c l e i n f o
Article history:
Received 31 August 2012
Received in revised form
12 October 2012
Accepted 17 October 2012
Available online 7 November 2012
Keywords:
Lycopene
Cholestasis
Obstructive jaundice
Alkalinesingle cell gel electrophoresis
DNA damage
Oxidative stress
Peripheral lymphocytes
Liver
Kidney
* Corresponding author. Department of Toxic3052178; fax: þ90 312 3114777.
E-mail address: [email protected]/$ e see front matter ª 2013 Elsevhttp://dx.doi.org/10.1016/j.jss.2012.10.031
a b s t r a c t
Background: Obstructive jaundice, a frequently observed condition caused by obstruction of
the common bile duct or its flow and seen in many clinical situations, may end up with
serious complications like sepsis, immune depression, coagulopathy, wound breakdown,
gastrointestinal hemorrhage, and hepatic and renal failures. Intrahepatic accumulation of
reactive oxygen species is thought to be an important cause for the possible mechanisms of
the pathogenesis of cholestatic tissue injury from jaundice. Carotenoids have been well
described that are able to scavenge reactive oxygen species. Lycopene, a carotenoid present
in tomatoes, tomato products, and several fruits and vegetables, have been suggested to
have antioxidant activity, so may play a role in certain diseases related to the oxidative
stress. The aim of the present study was to determine the effects of lycopene on oxidative
stress and DNA damage induced by experimental biliary obstruction in Wistar albino rats.
Materials and methods: Daily doses of 100 mg/kg lycopene were given to the bile duct-
ligation (BDL) rats orally for 14 days. DNA damage was evaluated by an alkaline comet
assay. The levels of aspartate transferase, amino alanine transferase, gamma glutamyl
transferase, alkaline phosphatase, and direct bilirubin were analyzed in plasma for the
determination of liver functions. The levels of malondialdehyde, reduced glutathione,
nitric oxide, catalase, superoxide dismutase, and glutathione S transferase were deter-
mined in the liver and kidney tissues. Pro-inflammatory cytokine tumor necrosis factor-
alpha level was determined in the liver tissues. Histologic examinations of the liver and
kidney tissues were also performed.
Results: According to this study, lycopene significantly recovered the parameters of liver
functions in plasma, reduced malondialdehyde and nitric oxide levels, enhanced reduced
glutathione levels, as well as enhancing all antioxidant enzyme activity in all tissues ob-
tained from the BDL group. Moreover, the parameters of DNA damage in the liver and
ology, Faculty of Pharmacy, Hacettepe University, TR-06100, Ankara, Turkey. Tel.: þ90 312
.tr (N. Bas‚aran).ier Inc. All rights reserved.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 2 ( 2 0 1 3 ) 2 8 5e2 9 5286
kidney tissue cells, whole blood cells, and lymphocytes were significantly lower in the
lycopene-treated BDL group, compared with the BDL group.
Conclusions: Lycopene significantly reduced the DNA damage, and markedly recovered the
liver and kidney tissue injuries seen in rats with obstructive jaundice.
ª 2013 Elsevier Inc. All rights reserved.
1. Introduction communication, immune system, and metabolic pathways,
Obstructive jaundice (OJ), caused by the structural and func-
tional impairment of the hepatobiliary system, is seen in
many clinical situations, such as benign tumor or stricture of
the bile duct, gallstone complications of pancreatitis, or biliary
surgery. Thesemay end upwith serious complications such as
sepsis, immune depression, coagulopathy, wound break-
down, gastrointestinal hemorrhage, and cardiovascular,
hepatic, and renal failure [1]. The intrahepatic accumulation
of toxic bile salts is thought to be one of the important causes
in the pathogenesis of liver damage from OJ. In addition, the
increased productions of pro-inflammatory cytokines, such as
tumor necrosis factor alpha (TNF-a), interleukin-1 beta and
interleukin-6 [2,3], bacterial endotoxins [2,4], and oxidative
stress [4] are suggested to be responsible for liver damage in
OJ. Intracellular accumulation of bile acids, cholesterol and
bilirubin stimulate proinflammatory cytokines and apoptosis
enhancement leading hepatocellular damage [5,6]. Over-
production of reactive oxygen species, which take a pivotal
role of oxidative stress, may cause lipid peroxidation and
disturb the integrity of cellular membranes and promote of
hepatic injury in OJ [3,4,7]. The antioxidant defense system is
impaired by the decrease in glutathione (GSH) reductase and
in the activity of glutathione peroxidase in OJ [8,9]. Oxidative
stressmay lead to DNA base damage, altered gene expression,
and DNA strand breaks, thereby causing mutagenesis and
carcinogenesis [10e12].
Bile duct ligation (BDL) produces a well established exper-
imentalmodel of acute OJ, the progression of biliary fibrosis to
cirrhosis, and secondary biliary cirrhosis in rats [13]. In this
process, the accumulation of hydrophobic bile acids and
inflammatory cells in the liver tissue may cause increased
production of free radicals [1]. The natural antioxidants for
protection and prevention against the damage caused by free
radicals have emerged as a subject of great interest in recent
years, and many experimental studies have reported the
beneficial effects of antioxidants in OJ [14e17].
Lycopene, a natural carotenoid and an acyclic isomer of b-
carotene, has no provitamin A activity [18]. It is present in
tomatoes, tomato products, and several fruits and vegeta-
bles. The potentially beneficial properties of lycopene have
been attributed to its ability to scavenge free radicals [19] and
to physically quench singlet molecular oxygen [20]. It is one
of the most potent antioxidants [21] and has been suggested
to prevent carcinogenesis and atherosclerosis by protecting
biomolecules such as low-density lipoproteins, proteins, and
DNA [22e24]. Several studies have suggested that lycopene
may play a role in certain diseases related to oxidative stress
[21,25,26]. Although the antioxidant properties of lycopene
are thought to play a role in the protective effects, other
possible mechanisms, such as modulations of gap junction
may be involved [18,27e29]. The aim of this study was to
determine the protective effects of lycopene against the
DNA and oxidative damages induced by an experimental
obstructive jaundice in peripheral lymphocytes, whole blood
cells, and the liver and kidney tissues of Wistar albino rats.
2. Materials and methods
2.1. Experimental design
The chemicals used in the experiments were purchased from
the following suppliers: normalmelting point agarose and low
melting point agarose from Boehringer Mannheim (Man-
nheim, Germany). Sodium chloride and sodium hydroxide
from Merck Chemicals (Darmstadt, Germany), dimethyl sulf-
oxide, ethidium bromide, Triton X-100, phosphate-buffered
saline tablets, ethylenediamine tetra acetic acid disodium
salt dihydrate (EDTA), N-lauroyl sarcosinate, and Tris from
ICN Biomedicals Inc. (Aurora, OH), lycopene from Sigma (St.
Louis, MO), ketamine hydrochloride from Ketalar, Eczacıbas‚ ı-
Warner Lambert (_Istanbul, Turkey),and xylazine from Bayer
Rompun (_Istanbul, Turkey).
Twenty-four healthy adult male Wistar albino rats weigh-
ing 200e250 g, housed in stainless steel cages under controlled
temperature (22�C) and humidity (55%e60%) andwith 12-hour
light-dark cycle were used in this study. Standard laboratory
rat feed containing 21% protein and fresh drinking water were
given ad libitum before and after the operation. The animals
were treated humanely with regard for alleviation of suffering
and the study protocol was designed according to the ethical
standards for animal use and approved by the local committee
of animal use.
All surgical procedures were performed under anesthesia
by intraperitoneal injection of 80 mg/kg ketamine hydro-
chloride (Ketalar, Eczacıbas‚ ı-Warner Lambert, _Istanbul,
Turkey) plus 10 mg/kg xylazine (Rompun, Bayer, _Istanbul,
Turkey). A midline incision was made under sterile tech-
niques. After a midline laparotomy of 1e2 cm, the common
bile duct was identified, doubly ligated using 5-0 silk sutures,
and transected at the level 0.7e0.8 cm distal to the last
bifurcation [30,31].
The animals were randomized to three groups (eight in
each group). Group I (the shamgroup)was subjected to a sham
operation and treated once daily with 0.5 mL of maize oil
orally. Group II (the BDL-group) was subjected to BDL and was
treated once daily with 0.5mL ofmaize oil orally. Group III (the
BDL þ L-group) was subjected to BDL and treated once daily
with 100 mg/kg body weight of lycopene (Sigma, St Louis, MO)
in 0.5 mL of maize oil orally. All treatments started 3 d before
the operation and continued 14 d after operation and were
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 2 ( 2 0 1 3 ) 2 8 5e2 9 5 287
performed by gastric intubation. In the sham-operated group,
the silk sutures were passed through the extra-hepatic bile
duct without ligation and transaction. The re-laparotomy was
performed through the old incision on postoperative day 14
under anesthesia, and subjects were sacrificed.
Blood samples via cardiac puncture were collected in
preservative-free heparin tubes for biochemical evaluation
and DNA damage analysis. The liver and kidney tissues were
carefully dissected from their attachments and totally
excised. Excised tissues were divided into three parts for
histopathologic examination, DNA damage analysis, and
determination of antioxidant parameters. The samples were
kept in the dark at 4�C and processed within 6 h.
2.2. Biochemical analysis
Heparinized blood samples were centrifuged at 800 g for 15
min. Plasma was collected and examined for direct bilirubin
(D-Bil), aspartate transferase (AST), amino alanine trans-
ferase, gamma glutamyl transferase, and alkaline phospha-
tase (AP) by spectrophotometric analysis as indicative of
hepatic function using standard diagnostic kits (Roche Diag-
nostics, Mannheim, Germany) and a Roche modular P800
clinical chemistry analyzer.
2.3. Determination of malondialdehyde (MDA), GSH,nitric oxide (NO), superoxide dismutase (SOD), catalase(CAT), and glutathione S transferase (GST) levels
The liver and kidney tissues were extracted following
a homogenization and sonication procedure [32]. The levels of
MDA, GSH, NO, SOD, CAT, and GST in the tissue homogenates
were analyzed.
MDA levels, biomarker of lipid peroxidation, were deter-
mined spectrophotometrically by measuring thiobarbituric
acid-reactive substances [33]; 2.5 mL of 20% trichloroacetic
acid and 1.0 mL of 0.67% thiobarbituric acid were added to
0.5 mL of the 10% homogenates of the tissue samples. The
mixtures were incubated at 100�C for 30 min. 4 mL of n-
butanol was added after cooling and mixed vigorously. After
centrifugation, absorbance of the butanol layer was
measured at 535 nm. 1,1,3,3-Tetraethoxypropane was used
for standard curve. The results were expressed as pmol/mg
protein.
GSH levels were determined spectrophotometrically with
a GSH assay kit (Cayman Chemicals Co., Ann Arbor, MI) at 405
nm according to manufacturer’s introduction. NO levels were
determined using a nitrate/nitrite colorimetric assay kit
(Cayman Chemicals Co.) at 550 nm according to manufac-
turer’s introduction. Results were expressed as nmol/mg
tissue.
The determination of tissue CAT, SOD, and GST levels
were performed with a CAT colorimetric assay kit (Sigma-
Aldrich, St Louis, MO, USA) at 520 nm, a SOD assay kit
(Cayman Chemicals Co.) at 440 nm, and GST assay kit
(Cayman Chemicals Co.) at 340 nm, respectively, according to
manufacturer’s introduction. Results were expressed as U/
mg protein. Protein concentrations of the tissue homoge-
nates were determined using the method described by Lowry
et al. [34].
2.4. Determination of TNF-a level in liver tissue
TNF-a levels in liver tissues were determined using commer-
cial enzyme-linked immunosorbent assay kit (eBioscience,
Vienna, Austria) at 450 nm according to manufacturer’s
introduction. Results were expressed as pg/mg tissue.
2.5. Evaluation of DNA damage
The DNA damage evaluation was performed by single cell gel
electrophoresis (comet assay). The basic alkaline technique of
Singh et al. [35], as further described by Anderson et al. [36] and
Collins et al. [37] was followed. Lymphocytes from whole
heparinized blood were separated by Ficoll-Hypaque density
gradient and centrifugation [38]. Then the cells were washed
with phosphate-buffered saline buffer. A small piece of the
liver and kidney tissues were placed in 1 mL of cold Hank’s
balanced salt solution containing 20 mM EDTA/10% dimethyl
sulfoxide and is minced into fine pieces. After it was settled
the supernatant was used. Cell viability was determined by
trypan blue and was found to be higher than 85% in all cases.
The 1 � 104 cells in 25, 15, 5, and 50 mL of the liver and
kidney tissue cells, whole blood cells, and lymphocytes,
respectively, were suspended in 75 mL of 0.5% low melting
point agarose. The suspensionswere then embedded on slides
pre-coated with a layer of 1% normal melting point agarose
and the slides were allowed to solidify on ice for 5 min. The
coverslips were then removed. The slides were immersed in
cold lysing solution (2.5 M sodium chloride,100 mM EDTA, 100
mMTris,1% sodium sarcosinate pH 10) for aminimumof 1 h at
4�C, with 1% Triton X-100 and 10% dimethyl sulfoxide added
just before use. Then they were removed from the lysing
solution, drained, andwere left in the electrophoresis solution
(1mMsodiumEDTA and 300mM sodiumhydroxide, pH 13) for
20 min at 4�C to allow unwinding of the DNA and expression
of alkali-labile damage. Electrophoresis was also conducted at
a low temperature (4�C) for 20 min using 24 V and adjusting
the current to 300 mA by raising or lowering the buffer level.
The slides were neutralized by washing three times in 0.4 M
Tris-HCL (pH 7.5) for 5 min at room temperature. After
neutralization, the slides were incubated in 50%, 75%, and 98%
of alcohol for 5 min for each, successively.
The dried microscope slides were stained with ethidium
bromide (20 mg/mL in distilled water, 60 mL/slide), covered
with a cover-glass prior to analysis with a Leica (Wetzlar,
Germany) fluorescence microscope under green light. The
microscope was connected to a charge-coupled device
camera and a personal computer-based analysis system
(Comet Analysis Software, v. 3.0; Kinetic Imaging Ltd., Liv-
erpool, UK) to determine the extent of DNA damage after
electrophoretic migration of DNA fragments in the agarose
gel. In order to visualize DNA damage, slides were examined
at 40� magnification. Images of 100 cells from each of two
replicate slides were counted. Results were expressed as tail
length, tail intensity, and tail moment.
2.6. Histopathologic evaluation
Excised tissue specimens were fixed in 10% formalin pro-
cessed in graded alcohol, and xylene, and then embedded in
Table 1 e Serum biochemical measurements.
Group I Group II Group III
D-Bil (mg/dL) 0.09 � 0.02 9.3 � 1.8a 7.3 � 1.3a,b
AST (mg/dL) 117.0 � 19.7 782.0 � 119.2a 333.5 � 195.7a,b
ALT (mg/dL) 54.3 � 10.2 214.7 � 62.0a 125.6 � 43.2a,b
GGT (mg/dL) 1.6 � 1.9 129.1 � 40.3a 31.3 � 51.4a,b
AP (mg/dL) 440.6 � 192.8 903.6 � 498.9a 748.5 � 274.0a,b
The results are given as mean � SD.a P < 0.05, group I compared with group II and group III.b P < 0.05, group II compared with group III.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 2 ( 2 0 1 3 ) 2 8 5e2 9 5288
paraffin. Paraffin blocks were sliced into 5 mm sections. The
sections were stained with hematoxylin and eosin and Mas-
son’s Trichrome according to standard procedure for routine
histopathologic examination under a light microscope
(Olympus BX51, Hamburg, Germany). The histologic exami-
nation was performed by an experienced histologist who was
not aware of the treatment groups.
Portal inflammation, focal necrosis, and ductal prolifer-
ation for the liver histopathologies were examined accord-
ing to the modified histologic activity index score proposed
by Knodell et al. [39]. Histopathologic scoring for portal
inflammation was as follows: no inflammation, grade 0;
slight inflammation, grade 1; severe inflammation, grade 2.
Histologic scoring for focal necrosis was as follows: no focal
necrosis, grade 0; 1e2 areas of focal necrosis, focal necrosis,
grade 1; >2 areas of focal necrosis, widespread necrosis,
grade 2. Histopathologic scoring for bile ductal proliferation
was as follows: no proliferation in portal area, grade 0; 10%e
50% ductal proliferation in portal area, grade 1; >50% ductal
proliferation in portal area, grade 2; >50% ductal prolifera-
tion in portal area without expansion of the portal area,
grade 3; >50% ductal proliferation in portal area with
expansion of the portal area, grade 4; same as grade 4 plus
bridging portal tracts in <20% instances, grade 5; same as
grade 4 plus bridging portal tracts in >20% instances,
grade 6.
The scoring renal glomerular damage was as follows: no
damage, grade 0; <25% damage, grade 1; 25%e50% damage,
grade 2; >50% damage, grade 3 [40]. The scoring of renal
interstitial inflammation, hydropic degeneration, tubular
necrosis, tubular desquamation, and tubular dilatation was as
follows: no damage, grade 0; <25% damage, grade 1; 25%e50%
damage, grade 2; >50% damage, grade 3 [41].
2.7. Statistical analysis
Statistical analysis was performed by the computer program
SPSS for Windows 15.0 (SPSS Inc., Chicago, IL). Differences
between the means of data were compared by ANOVA test
and post hoc analysis of group differences was performed by
least significant difference test. The Kruskal-Wallis H test was
used in comparing parameters displaying abnormal distribu-
tion between groups. The results were given as the
mean � SD, and P values of less than .05 were considered as
statistically significant.
Table 2 e Hepatic MDA, GSH, NO, CAT, SOD, and GSTlevels.
Group I Group II Group III
MDA (pmol/mg
protein)
251.3 � 79.4 753.0 � 219.1a 266.4 � 38.2b
GSH (nmol/mg tissue 3.1 � 1.2 0.9 � 0.8a 2.5 � 0.9b
NO (nmol/mg tissue) 68.6 � 8.4 112.3 � 16.6a 73.0 � 3.4b
CAT (U/mg protein) 164.8 � 34.9 93.4 � 23.1a 174.9 � 38.5b
SOD (U/mg protein) 86.1 � 6.0 50.9 � 4.7a 77.5 � 7.8a,b
GST (U/mg protein) 0.4 � 0.1 0.2 � 0.1a 0.4 � 0.1b
The results are given as mean � SD.a P < 0.05, group I compared with group II and group III.b P < 0.05, group II compared with group III.
3. Results
3.1. Biochemical findings
The plasma biochemical parameters and the markers of
hepatocellular damage are shown in Table 1. D-Bil, AST,
amino alanine transferase (ALT), gamma glutamyl transferase
(GGT), and AP levels were found to be significantly higher in
both group II and group III than group I (P < 0.05). The levels of
AST, ALT, and GGT were found to significantly decrease in
group III compared with group II (P < 0.05). There was no
significant difference between group II and group III in the
levels of both D-Bil and AP.
3.2. Hepatic and renal MDA, GSH, NO, SOD, CAT, GSTlevels
Hepatic and renal MDA, GSH, NO, SOD, CAT, and GST levels
are shown in Table 2 and Table 3, respectively.
Hepatic MDA level in group II was found to be significantly
higher than group I (P < 0.05). The level in group III was found
to be significantly lower than group II (P < 0.05). There was no
significant difference in hepatic MDA level between group I
and group III (Table 2). Renal MDA levels in both group II and
group III were found to be significantly higher than group I (P<
0.05). But the level in group III was found to be significantly
lower than group II (P < 0.05) (Table 3).
Hepatic and renal NO levels in group II were found to be
significantly higher than group I (P < 0.05). These levels in
group III were found to be significantly lower than group II (P<
0.05). There was no significant difference in hepatic and renal
NO levels between group I and group III (Table 2 and Table 3).
Hepatic and renal GSH levels in group II were found to be
significantly lower than group I (P < 0.05). These levels in
group III were found to be significantly higher than group II (P
< 0.05). There was no significant difference in hepatic and
renal GSH levels between group I and group III (Table 2 and
Table 3).
Hepatic and renal SOD, CAT, and GST levels in group II
were found to be significantly lower than group I (P < 0.05).
SOD levels in group III were found to be significantly lower
than group I (P < 0.05). However, all of these enzyme levels in
group III were significantly higher than group II (P < 0.05).
There was no significant difference in hepatic and renal CAT
and GST levels between group I and group III (Table 2 and
Table 3).
Table 3 e Renal MDA, GSH, NO, CAT, SOD, and GST levels.
Group I Group II Group III
MDA (pmol/mg
protein)
12.3 � 5.3 52.0 � 17.0a 20.8 � 5.1a,b
GSH (nmol/mg tissue 1.7 � 0.5 0.5 � 0.2a 1.5 � 0.3b
NO (nmol/mg tissue) 117.4 � 63.5 178.4 � 65.0a 106.4 � 34.9b
CAT (U/mg protein) 75.8 � 9.6 51.4 � 12.3a 76.0 � 10.5b
SOD (U/mg protein) 68.1 � 6.7 43.5 � 11.3a 57.1 � 5.8a,b
GST (U/mg protein) 0.3 � 0.06 0.1 � 0.04a 0.2 � 0.06b
The results are given as mean � SD.aP < 0.05, group I compared with group II and group III.bP < 0.05, group II compared with group III.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 2 ( 2 0 1 3 ) 2 8 5e2 9 5 289
3.3. Hepatic TNF-a levels
Hepatic TNF-a level in group II was found to be significantly
higher than in group I (P < 0.05). The level in group III was
significantly lower than in group II (P < 0.05). There was no
significant difference on hepatic TNF-a level between group I
and group III (Fig. 1).
3.4. Determination of DNA damage
Bile duct ligation seemed to induce DNA damage in the whole
blood cells, lymphocytes, and hepatic and renal tissue cells
because all of the parameters studied were found to increase
in group II compared with group I (P < 0.05) (Fig. 2). Lycopene
seemed to significantly reduce DNA damage, since all the
parameters studied were found to decrease significantly in
group III compared with group II (P <0.05). There was no
significant difference in terms of DNA tail length between
group I and group III (Fig. 2). Even in whole blood cells,
lymphocytes, and renal tissue cells, the comet parameters
such as DNA tail intensity and DNA tail moment in group III
were found to be significantly lower than in group I (P < 0.05)
(Fig. 2A, B, D). There was no statistically significant difference
in hepatic cell DNA between group I and group III (Fig. 2C).
3.5. Histopathologic findings
The histologic structure of the liver was observed to be normal
in group I as seen in Figure 3A. Therewas portal inflammation,
Fig. 1 e Hepatic TNF-a level in the experimental groups.
Results were given as mean ± SD. a P < 0.05, group I
compared with group II and group III. b P < 0.05, group II
compared with group III.
parenchymal necrosis, and ductal proliferation including
bridging of the portal tracts in group II (Fig. 3B). Portal
inflammation and ductal proliferationmarkedly attenuated in
group III compared with group II (Fig. 3C).
There was no portal inflammation in 37.5% of group I.
Slight inflammation and severe inflammation in portal tracts
were observed in 50.0% and 12.5% of group I, respectively.
Slight inflammation and severe inflammation were seen in
25% and 75% of group II, respectively. Slight inflammationwas
found in 100% of group III. The mean grades of liver portal
inflammation were found to be 0.75 � 0.71, 1.75 � 0.38, and
1.00� 0.00 in group I, group II, and group III, respectively. Liver
portal inflammation was found to increase significantly in
group II compared with group I (P < 0.05). Lycopene treatment
seemed to reduce liver portal inflammation significantly in
group III compared with group II (P < 0.05). There was no
significant difference between group I and group III (Table 4).
There was no necrosis in group I. Focal necrosis of grade 1
and widespread necrosis of grade 2 were observed in 62.5%
and 37.5% of group II, respectively. There was no necrosis in
25% of group III. Focal necrosis of grade 1 and widespread
necrosis of grade 2 were seen in 50% and 25% of group III,
respectively. The mean grades of liver focal necrosis were
found to be 1.38� 0.53 and 1.00� 0.76 in group II and group III,
respectively. Liver focal necrosis was found to increase
significantly in both group II and group III compared with
group I (P < 0.05). However, focal necrosis was found to be
lower in group III than in group II, which was not significant
(Table 4).
There was no bile ductal proliferation in the liver of portal
area in group I. The scores of grade 4, grade 5, and grade 6were
observed in 12.5%, 62.5%, and 25.5% of group II, respectively.
The scores of grade 4 and grade 5 were observed in 50% and
50% of group III, respectively. The mean grades of ductal
proliferation in portal area were found to be 5.13 � 0.69 and
4.50 � 0.53 in group II and group III, respectively. The bile
ductal proliferations in the portal area were found to increase
significantly in both group II and group III compared with
group I (P < 0.05). However, it was found to decrease signifi-
cantly in group III compared with group II (Table 5).
As seen in Figure 4A, renal histologic structure was
observed to be normal in group I. There were glomerular and
tubular degenerations in group II. The epithelial cell desqua-
mation of the tubule and the congestion of blood vessel in the
interstitial tissue were observed (Fig. 4B). Renal degeneration
was markedly attenuated in group III compared with group II
(Fig. 4C).
There were no glomerular damages, tubular necrosis, and
tubular dilatation in both group I and group III. Glomerular
damage grade 1 ofwas observed in 87.5% of group II. Themean
grades of glomerular damage were found to be 0.88 � 0.35 in
group II. The glomerular damage was found to decrease
significantly in group III compared with group II (P < 0.05)
(Table 6).
Interstitial inflammation grade 1 was determined in 12.5%
and 100%of group I and group II, respectively. Themean grades
of interstitial inflammation were found to be 0.13 � 0.35 and
1.00 � 0.00 in group I and group II, respectively. The interstitial
inflammation was found to increase significantly in group II
compared with group I (P < 0.05). The interstitial inflammation
Aa
b
0,00
5,00
10,00
15,00
20,00
25,00
Group I
Group II
Group III
DNA
Tail
Leng
th
a
a,b
0,002,004,006,008,00
10,0012,00
Group I
Group II
Group III
DNA
Tail
Inte
nsity
a,b
a
0,000,200,400,600,801,001,201,401,60
Group I
Group II
Group III
DNA
Tail
Mom
ent
B
b
a
0,005,00
10,0015,0020,0025,0030,0035,0040,00
Group I
Group II
Group III
DNA
Tail
Leng
th
a,b
a
0,002,004,006,008,00
10,0012,0014,0016,00
Group I
Group II
Group III
DNA
Tail
Inte
nsity
a
a,b
0,00
0,50
1,00
1,50
2,00
2,50
Group I
Group II
Group III
DNA
Tail
Mom
ent
C a
b
0,005,00
10,0015,0020,0025,0030,0035,0040,00
Group I
Group II
Group III
DNA
Tail
Leng
th
b
a
0,00
5,00
10,00
15,00
20,00
25,00
Group I
Group II
Group III
DNA
Tail
Inte
nsity
b
a
0,001,00
2,003,00
4,005,00
Group I
Group II
Group III
DNA
Tail
Mom
ent
D
b
a
0,005,00
10,0015,0020,0025,0030,0035,00
Group I
Group II
Group III
DNA
Tail
Leng
th
a,b
a
0,00
5,00
10,00
15,00
20,00
Group I
Group II
Group III
DNA
Tail
Inte
nsity
a,b
a
0,000,501,001,502,002,503,003,50
Group I
Group II
Group III
DNA
Tail
Mom
ent
Fig. 2 e DNA damages expressed as DNA tail length, DNA tail intensity, and DNA tail moment in the whole blood cells (A),
lymphocytes (B), the liver (C), and renal (D) tissue cells of the experimental groups. aP< 0.05, group I compared with group II
and group III. bP < 0.05, group II compared with group III.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 2 ( 2 0 1 3 ) 2 8 5e2 9 5290
was found to decrease significantly in group III compared with
group II (P < 0.05). Moreover interstitial inflammation was not
observed in group III (Table 6).
Hydropic degeneration grade 1 was determined in 12.5% of
both group I and group III. Grade 1 and the grade 2 were
determined in 62.5% and 12.5% of group II, respectively. The
mean grades of hydropic degeneration were found to be
0.13 � 0.35, 1.75 � 1.04, and 0.13 � 0.35 in group I, group II, and
group III, respectively. Hydropic degeneration was found to
increase significantly in group II compared with group I (P <
0.05). Hydropic degeneration was found to decrease signifi-
cantly in group III comparedwith group II (P< 0.05). Moreover,
there was no significant difference between group I and group
III (Table 6).
4. Discussion
Oxygen-derived free radicals, produced in the course of
several biochemical reactions, are extremely reactive
Fig. 3 e Histopathology of hepatic tissue in group I (A), group II (B), and group III (C). Normal histological structure of liver
was observed in group I (hematoxylin and eosin [H and E], 320). There was portal inflammation, parenchymal necrosis, and
ductal proliferation including bridging of the portal tracts in group II (H and E, 320). Portal inflammation, parenchymal
necrosis, and ductal proliferation attenuated markedly in group III compared with group II (H and E, 310). (Color version of
figure is available online.)
Table 4 e Evaluation of portal inflammation and focalnecrosis in the liver tissues of rats.
Percentile of grades Mean grades
0 1 2
Liver portal inflammation
Group I 37.5% 50% 12.5% 0.75 � 0.71 (0e2)
Group II 0% 25% 75% 1.75 � 0.38 (1e2)a
Group III 0% 100% 0% 1.00 � 0.00 (1e1)b
Liver focal necrosis
Group I 100% 0% 0% 0.00 � 0.00 (0e0)
Group II 0% 62.5% 37.5% 1.38 � 0.53 (1e2)a
Group III 25% 50% 25% 1.00 � 0.76 (0e2)b
The results are given as mean � SD (min-max).aP < 0.05, group I compared with group II and group III.bP < 0.05, group II compared with group III.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 2 ( 2 0 1 3 ) 2 8 5e2 9 5 291
intermediates. These free radicals can cause damage to
various biological targets, such as proteins, DNA, and lipids
[42,43]. Free radicals can induce tissue injury by causing lipid
peroxidation, and lipid peroxidation product accumulation in
human tissues is one of themain causes of tissue dysfunction
[44]. Tissues have a variety of defense mechanisms, including
the non-enzymatic GSH and the enzymatic SOD scavenger
systems against the oxidative injuries [45]. The damage
caused by free radicals can be protected by scavenger mole-
cules such as natural or synthetic antioxidant [46].
Lycopene, the most effective singlet oxygen quencher [47],
is an antioxidant carotenoid without provitamin A activity. It
has been shown to be a more potent antioxidant than alpha-
or beta-carotene in both human and animal studies [22].
Lycopene may protect in vivo against the oxidation of lipids,
proteins, and DNA [48]. In animal studies, it has been shown
that lycopene has a large margin of safety. No observed effect
level of lycopene was found to be 586mg/kg body weight/d for
males and 616 mg/kg body weight/d for females in the diet in
the 90-d oral toxicity study in Wistar albino rats [49].
OJ is greatly assumed to increase hepatic oxidative stress, as
indicated by elevations in hepatic plasma enzymes and bili-
rubin fractions. Bilirubin, an end-product of heme catabolism,
is formed in reticulo-endothelial cells. The potentially cytotoxic
lipid soluble bilirubin is transported in plasma tightly bound to
albumin. Conjugated bilirubins are formed by liver enzymes,
and these conjugatesmay increase in cholestasis [46]. The rises
in bilirubin and enzyme activities are indicators of liver injury,
cholestasis, and hepatic dysfunction [50,51]. In our study, the
levels of D-Bil, AST, ALT, GGT, and AP as indicatives of hepatic
functions were found to increase significantly in OJ, indicating
that OJ has affected liver functions. Bignotto et al. [52] found
that daily administration of lycopene (25 mg/kg/d) during the
14 d that preceded the experiments significantly reduced the
liver injury markers (AST, ALT, lactate dehydrogenase, and
GGT) induced by ischemia-reperfusion injury in rats. In our
study, we also found that lycopene ameliorated plasma hepa-
tocellular damage parameters (D-Bil, AST, ALT, GGT, and AP) in
rats with OJ.
MDA accumulation in tissues is indicative of the extent of
lipid peroxidation and oxidative stress [26,53,54]. GSH, an
antioxidant, prevents damage to important cellular compo-
nents caused by reactive oxygen species such as free radicals
and peroxides [55]. OJ has been associated with an increase in
systemic, hepatic, and renal MDA formation. Orellana et al.
[56] reported that the GSH and MDA levels of kidney and liver
tissues increased in the cholestasis-induced rats, indicating
that cholestasis produced oxidative stress in both organs.
Regarding the effect of BDL on the kidney, there are contra-
dictory reports. The content of total renal GSH was increased
as a tissue response to the increased oxidative stress in rats
[57]. Tajiri et al. [58] reported high levels of MDA and GSH in
kidney of 5-d BDL rats. However Gonzalez-Correa et al. [51]
reported normal MDA level and a decrease in GSH level in
kidney of 7-d BDL rats. Panozzo et al. [59] reported that
extrahepatic cholestasis reduced bioavailability of blood GSH
in rats. Sheen et al. [60] also reported that BDL-induced liver,
kidney, and brain tissue damages were associated with
increased oxidative stress, represented by decreased total
GSH levels in BDL rats. In our study, consistent with some of
the previous studies, we found a decrease in GSH levels and an
increase in MDA levels of the liver and renal tissues in BDL
rats.
Table 5e Evaluation of bile ductal proliferation in the livertissues of rats.
Grades Percentile (%) Mean grades
Group I 0 100% 0.00 � 0.00 (0e0)
1e6 0%
Group II 0e3 0% 5.13 � 0.69 (4e6)a
4 12.5%
5 62.5%
6 25.5%
Group III 0e3 0% 4.50 � 0.53 (4e5)a,b
4 50%
5 50%
6 0%
The results are given as mean � SD (min-max).aP < 0.05, group I compared with group II and group III.bP < 0.05, group II compared with group III.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 2 ( 2 0 1 3 ) 2 8 5e2 9 5292
Plasma antioxidant levels such as lycopene, retinol, alpha-
and beta-carotene, total carotenoid, lutein in cholestatic liver
disease (86 and 19 patients with primary sclerosing chol-
angitis and primary biliary cirrhosis, respectively) were found
to decrease significantly comparedwith the control group [61].
Daily administration of lycopene (25 mg/kg/d) during 14 d was
found to decrease liver MDA levels in rats with ischemia-
reperfusion injury [52]. Lycopene (1 mg/kg) treatments for 4
wk was also found to increase protection as detected by
a significant reduction in MDA levels and an elevation in
antioxidant GSH levels of the kidney in ratswith induced renal
toxicity by carbon tetrachloride administration [62]. Lycopene
treatment was also reported to improve the biochemical
parameters such as MDA and GSH in both plasma and kidney
tissues against cisplatin-induced nephrotoxicity and oxida-
tive stress in rats [63]. In our study, lycopene treatment
decreasedMDA and increased GSH levels of the liver and renal
tissues in 14 d in BDL rats, indicating that lycopene reduced
the oxidative stress induced by OJ.
High levels of bile salts during cholestasis disrupt the
intestinalmucosal barrier, causing the translocation of enteric
bacteria to the mesenteric lymph nodes and the liver [3],
which caused the endotoxemia is responsible for increased
NO synthesis by inducible NO synthase [64,65]. This excessive
generation of NO has been observed both in experimental
cholestasis [3,65] and in primary biliary cirrhosis patients
Fig. 4 e Histopathology of renal tissue in group I (A), group II (B),
in group I (H and E, 320). There were glomerular and tubular d
attenuated markedly in group III compared with group II (H an
[65,66]. The increase in hepatic and plasma levels of NO and
cytokines leads to the hepatocellular injury and the rapid
progression of hepatic dysfunction in cholestatic settings. In
our study, NO levels in the liver and renal tissues were found
to increase in OJ. Our data also demonstrated that lycopene
reduced NO levels induced by BDL.
The antioxidant enzymes (CAT, SOD, and GST) play an
essential role in cellular defense against free radicals. The
liver and renal antioxidant systems are also injured by
oxidative stress in OJ [14]. Montilla et al. [67] found that the
activity of CAT, SOD, glutathione peroxidase, glutathione
reductase, and GST decreased in the liver of BDL rats. Anti-
oxidant enzyme levels such as SOD, GGT glutathione peroxi-
dase, and CAT were reported to increase in some types of
chemical-induced damage [62,63]. In our study, CAT, SOT,
and GST levels in the liver and renal tissues were found to
decrease in OJ, which might indicate the oxidative imbalance
of these tissues. Our study also showed that lycopene might
play an important role since the levels of CAT, SOD, and GST
were found to increase in both liver and kidney tissues in
lycopene-treated BDL-rats, which is consistent with the
recent studies showing the beneficial effects of lycopene.
Proinflammatory cytokine TNF-a exerts a considerably
amplifying effect in hepatic inflammatory response and cau-
ses severe hepatic tissue damage. The severity of liver injury
induced by OJ is correlated with TNF-a level, and there is
a relation between inflammation and oxidative stress in OJ
[68,69]. Wang et al. [70] studied the effects of lycopene in
nonalcoholic steatohepatitis induced rats with high fatty diet.
Lycopene decreased proinflammatory cytokines and lipid
peroxidation in rats that are fed with high fatty diet for 6 wk
[70]. In the present study, lycopene significantly reduced TNF-
a level in the cholestatic liver, which suggests that the
protective effects of lycopene on liver injury might be medi-
ated by the suppression of the excessive hepatic inflammatory
response and its cascade induced by OJ.
There aremany studies on the intake of fruits or vegetables
showing significant reduction in oxidative damage to DNA. It
has been reported that lycopene has an effect on the
prevention of oxidative damage in many cells, including
lymphocyte DNA [71,72]. Matos et al. [73] demonstrated that
lycopene decreased lipid peroxidation and oxidative DNA
damage in monkey kidney fibroblast cell line. Pool-Zobel et al.
[24] found that supplementation of the diet with tomato,
and group III (C). Normal histologic structure was observed
amages in group II (H and E, 320). The degenerations
d E, 320). (Color version of figure is available online.)
Table 6 e Histopathologic analysis of the renal tissues inrats.
Percentile of grades Mean grades
0 1 2 3
Glomerular damage
Group I 100% 0% 0% 0% 0.00 � 0.00 (0e0)
Group II 12.5% 87.5% 0% 0% 0.88 � 0.35 (0e1)a
Group III 100% 0% 0% 0% 0.00 � 0. 00 (0e0)b
Interstitial inflammation
Group I 87.5% 12.5% 0% 0% 0.13 � 0.35 (0e1)
Group II 0% 100% 0% 0% 1.00 � 0.00 (1e1)a
Group III 100% 0% 0% 0% 0.00 � 0.00 (0e0)b
Hydropic degeneration
Group I 87.5% 12.5% 0% 0% 0.13 � 0.35 (0e1)
Group II 0% 62.5% 37.5% 0% 1.75 � 1.04 (1e3)a
Group III 87.5% 12.5% 0% 0% 0.13 � 0.35 (0e1)b
Tubular necrosis
Group I 100% 0% 0% 0% 0.00 � 0.00 (0e0)
Group II 25.0% 62.5% 12.5% 0% 0.88 � 0.64 (0e2)a
Group III 87.5% 12.5% 0% 0% 0.13 � 0.35 (0e1)a,b
Tubular desquamation
Group I 100% 0% 0% 0% 0.00 � 0.00 (0e0)
Group II 0% 0% 62.5% 37.5% 2.38 � 0.52 (2e3)a
Group III 0% 87.5% 12.5% 0% 1.13 � 0.35 (1e2)a,b
Tubular dilatation
Group I 100% 0% 0% 0% 0.00 � 0.00 (0e0)
Group II 0% 62.5% 37.5% 0% 1.38 � 0.52 (1e2)a
Group III 87.5% 12.5% 0% 0% 0.13 � 0.35 (0e1)a,b
The results are given as mean � SD (min-max).aP < 0.05, group I compared with group II and group III.bP < 0.05, group II compared with group III.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 2 ( 2 0 1 3 ) 2 8 5e2 9 5 293
carrot, or spinach products resulted in significantly decreased
levels of endogenous strand breaks in lymphocyte DNA. In
another study, Zhao et al. [74] found significant decreases in
endogenous DNA damage after taking supplements like
lutein, b-carotene, lycopene, and combination of all three, for
57 d. In our study, DNA damages in the lymphocytes, and the
liver and renal tissue cells increased in rats with OJ. However,
lycopene treatment prevented DNA damages induced by OJ.
Intrahepatic accumulation of toxic bile salts is thought to
be one of the important causes in the pathogenesis of liver
damage from OJ. Toxic bile acids may activate hepatocyte
death receptors and start oxidative damage, whichmay cause
mitochondrial dysfunction and induce endoplasmic retic-
ulum stress. Also regarding extrahepatic organs, accumula-
tion of bile acids in the systemic circulation can end up with
endothelial injury in the kidney and lungs. Hepatic fibrosis
usually develops in 2 or 3 wk in rats after bile duct ligation [9].
Liver fibrosis during acute and chronic cholestasis involves
the stepwise process of ductal reaction, which refers to an
increasing number of ducts and an increase in matrix, leading
to periportal fibrosis and eventually biliary cirrhosis [75]. Dirlik
et al. [9] found that ductal proliferation increased progressively
after common bile duct ligation and reaches a peak level after
5 d in rats [9]. In the present study, liver portal inflammation,
focal necrosis, and bile ductal proliferation were elevated in
the rats with OJ, indicating that OJ increased liver tissue
damage.
The fact that the levels of TNF-a and NOwere decreased in
the liver of rats treated with lycopene indicates that lycopene
might reduce the liver portal inflammation. In our study, the
improvements of ductal proliferation and focal necrosis
observed in lycopene treatment of 100 mg/kg/d for 14 d is
likely related to the reduction of MDA and NO levels, and the
induction of GSH level in the liver.
Panozzo et al. [59] reported that extrahepatic cholestasis
might play a role in the pathogenesis of acute renal injury in
rats due to the reduced bioavailability of blood GSH. Increased
renal MDA might play a role in renal damage induced by OJ
[57,76]. Holt et al. [8] reported that obstructive jaundice was
characterized by changes in the tubular handling of electro-
lytes. Raised natriuresis accompanied by decreased in tubu-
lointerstitial fibrosis and the vasodilation of inner medullary
capillaries takes place in the initial 2 wk following BDL, which
is due to mainly tubular effects. Tubular epithelial cells also
seem to be the targets of a systemic response to the liver
dysfunction [77]. In our study, glomerular damage, interstitial
inflammation, hydropic degeneration, and necrosis, desqua-
mation, and dilatation of renal tubule were found to increase
in 14-d BDL rats, indicating that OJ increased renal injury.
Renal damage induced by extrahepatic cholestasis was found
to be decreased by lycopene 100 mg/kg/d orally for 14 d. The
imbalance levels of MDA, GSH, NO, CAT, SOD, and GST might
bring about renal damage induced by OJ. Lycopene may
protect renal damage by ameliorating the antioxidant
parameters and antioxidant enzymes.
In conclusion, the study demonstrates that lycopene, one
of the most powerful scavengers of free radicals, reduces lipid
peroxidation, protects DNA damage, and protects peripheral
lymphocytes and renal and liver tissues from oxidative
damages in rats subjected to BDL. Having no side effects,
lycopene should be further investigated on other organs as
well as kidney and liver to determine its efficacy during the
cholestasis.
Acknowledgments
The authors declare that they have no conflicts of interest.
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