Antioxidant and Antigenotoxic Effects of Lycopene in Obstructive Jaundice

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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.

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http://dx.doi.org/10.1016/j.jss.2012.10.031




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.


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.


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.


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.


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.


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.


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%



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



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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Antioxidant and Antigenotoxic Effects of Lycopene in Obstructive Jaundice