Food Chemistry 141 (2013) 1587–1596

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Food Chemistry

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Protective role of Punica granatum (pomegranate) peel and seed oilextracts on diethylnitrosamine and phenobarbital-induced hepaticinjury in male rats

0308-8146/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.04.134

⇑ Corresponding author. Address: Biochemistry Department, Faculty of Science,Alexandria University, Moharambek, Alexandria, Egypt. Tel.: +20 3 4265345; fax:+20 3 3911794.

E-mail address: nshaban2001@yahoo.co.uk (N.Z. Shaban).

Nadia Z. Shaban ⇑, Mohamed A.L. El-Kersh, Fatma H. El-Rashidy, Noha H. HabashyBiochemistry Department, Faculty of Science, Alexandria University, Alexandria, Egypt

a r t i c l e i n f o

Article history:Received 1 November 2012Received in revised form 19 February 2013Accepted 30 April 2013Available online 23 May 2013

Keywords:PomegranateDiethylnitrosoamineHepatic injuryLipid peroxidationAntioxidantApoptosisDNA fragmentationCaspase-3

a b s t r a c t

The present study is an attempt to reveal the protective role of Punica granatum peel and seed oil extractsagainst diethylnitrosamine (DEN) and phenobarbital (PB) induced hepatic injury in rats. DEN administra-tion increased the levels of malondialdehyde (MDA), DNA fragmentation, caspase-3 and glutathionereductase (GSR) activities, while the level of reduced glutathione (GSH) and the activities of superoxidedismutase (SOD), glutathione S-transferase (GST) and total glutathione peroxidase (t-GPx) weredecreased compared with the control. Treatment with peel and seed oil extracts pre, during and postDEN administration improved liver functions, decreased the levels of MDA, DNA fragmentation, cas-pase-3 and GSR activities with an elevation in levels of GSH, SOD, GST and t-GPx activities. This indicatesthat these extracts reduced the oxidative stress and apoptosis induced by DEN. Also the effect of admin-istration of PE and SOE separately for a long time (23 weeks) on healthy rats was studied.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

In the human body, Nitrosamines such as diethylnitrosamine(DEN), are produced from the metabolism of certain therapeuticdrugs or from the reaction of nitrite with amines and amides(Mittal, Bar, & Soni, 2006). Previous studies have shown thatnitrosamines can be formed in the gastric juice of the human stom-ach by a process commonly referred to as endogenous nitrosation.The bacteria in the mouth chemically reduce nitrate, found inmany vegetables, to nitrite, which in turn can form nitrosatingagents. Many foods that contain amines can react with these nitro-sating agents in the acidic environment of the stomach to formnitrosamines (Pradeep, Mohan, Gobianand, & Karthikeyan, 2007).In addition, DEN, is widely distributed in processed meats, tobaccosmoke, whiskey (Sivaramakrishnan & Devaraj, 2009), smoked,salted and dried fish, cheese, soy bean and ground water havinga high level of nitrates. DEN and its metabolites induce oxidativestress, possibly due to generation of reactive oxygen species(ROS), which are capable of initiating peroxidative damage to thecell (Mittal et al., 2006). Intermediate reactive compounds

originating from the bioactivation of DEN, such as alkylatingagents, are known to form covalent bonds with important cell con-stituents, thus inducing the onset of mutations, cancer, and necro-sis (Nakae, Kobayashi, & Akai, 1997). Alkylating agents bind toeither DNA, forming promutagenic adducts that may initiate livercarcinogenesis, or RNA, that inhibits protein synthesis (Nakaeet al., 1997). Apoptosis is a highly organised and genetically con-trolled type of cell death, essential during embryonic developmentto ensure proper organogenesis as well as for the health of adultorganisms. It is characterised by a number of distinct morphologi-cal alterations, such as chromatin condensation and marginalisa-tion, cell shrinkage and plasma membrane blebbing, which areaccompanied by biochemical features, such as DNA fragmentation,membrane alterations, and degradation of specific cellular pro-teins, as a result of the massive activation of a large number ofintracellular proteases and endonucleases. In the latest stages,the dying cell is fragmented into membrane-bound vesicles con-taining relatively intact organelles and chromatin residues named‘‘apoptotic bodies’’ which are readily engulfed by neighbouringcells and professional phagocytes, such as Kupffer cells in the liver(Faubel & Edelstein, 2005).

Several studies have suggested that phytochemical productscould be a useful strategy to prevent the deleterious effects of car-cinogens and mutagens through the modulation of genes that arerelated to the control of cell proliferation, cell cycle, apoptosis,

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signal transduction, oncogenesis and transcription regulation (Cil-la, González-Sarrías, Tomás-Barberán, Espín, & Barberá, 2009).Pomegranate (P. granatum) is used in the traditional medicine ofdifferent Asian cultures for the treatment of a variety of ailments.The wide range of therapeutic benefits of pomegranate has beenattributed to its antioxidant and anti-inflammatory properties(Bishayee et al., 2011). Pomegranate seeds are rich in polyunsatu-rated fatty acids, vitamins, polysaccharides, polyphenols and min-erals. The seed oil contains 80% punicic acid, iso-flavone genistein,phytoestrogen coumestrol, and sex steroid hormones. Pomegran-ate peels are rich in polyphenols and polysaccharides (Lansky &Newman, 2007). The studies showed that seed oil extract, SOE,caused a significant reduction in the multiplicity of colon carci-noma (D’Aquino et al., 2010) and skin tumor development. It hasa cytotoxic effect on human monocytic leukaemia. Peel extract,PE, has a protective role against liver damage induced with CCl4

(Murthy, Jayaprakasha, & Singh, 2002). It inhibits gastric ulcerationand facilitates both wound healing and skin repair. PE and SOEhave shown suppressive effects on human breast cancer (Lansky& Newman, 2007).

The present study was designed to discover: (1) whether thepomegranate extracts (PE and SOE) have a protective role againsthepatic injuries induced by DEN and phenobarbital, (2) whethera long period of administration of PE and SOE has a toxic effecton healthy rat liver. The study was focused on the assessment ofapoptotic markers, especially caspase-3 activity and DNA fragmen-tation, as well as lipid peroxidation, enzymatic and non-enzymaticantioxidants in the liver.

2. Materials and methods

2.1. Chemicals

Kit for caspases-3 assay was obtained from Bio SourceInternational, Inc, 542 Flynn road Camarillo, CA 93012 USA.Diethylnitrosamine (DEN), phenobarbital, cumene-H2O2, 5,50 dithi-obis (2-nitrobenzoic acid) (DTNB), reduced glutathione (GSH),oxidised glutathione (GSSG), NADPH, standard SOD, thiobarbituricacid (TBA) and tetramethoxypropane (TMP) were purchased fromSigma–Aldrich, USA. Diethylene triaminopentaacetic acid (DTPA)and sodium dodecyl sulfate (SDS) were purchased from Fluka,Switzerland.

2.2. Preparation of pomegranate extracts

Pomegranate, from the family Rutaceae, was purchased fromthe local market. The fruits were peeled mechanically and good-quality peels were air-dried under shadow and then ground intofine powder by pulverization. For the preparation of PE, the driedpowder was extracted in 6 volumes of methanol at 30 �C for 72 hand filtered (Singh & Kar, 2007). The filtrate was evaporated at40 �C and stored in a dark bottle until used. SOE was prepared asdescribed by Kim et al. (2002), with some modifications. Thepomegranate hard white seeds were separated manually fromthe pericarps following juice extraction. The seed powder was ex-tracted with n-hexane (1:5, w/v) for 72 h and filtered. The filtratewas evaporated at 60 �C to dryness and stored in a dark bottlefor future use.

2.3. Characterization of pomegranate extracts (PE and SOE)

2.3.1. Total phenolic contentTotal phenolic compounds in PE and SOE were determined as

mg gallic acid equivalents according to the method of Taga, Miller,and Pratt (1984), using gallic acid as standard.

Chromatographic separation of 20 ll of PE or SOE was carriedout on a 150 � 4.6 mm, 5 lm Eclipse XDB-C18 column (AgilentTechnologies 1200S, Germany) at 320 nm and at a flow rate of0.75 ml/min. The mobile phase was formic acid (1%, v/v in aqueoussolution):acetonitrile:2-propanol (70:22:8), pH 2.5.

2.3.2. Total flavonoid contentTotal flavonoid compounds in PE and SOE were determined as

(+)-catechin equivalents in mg/ml of the extract according to thecolorimetric method of Zhishen, Mengcheng, and Jianming(1999), using (+)-catechin as a standard.

2.3.3. Antioxidant activities of PE and SOE in vitroTotal antioxidant capacities of PE and SOE were measured,

using ascorbic acid as standard, and the results were expressedas ascorbic acid equivalents in mg/g of dry extract (Tyagi et al.,2010).

2.3.4. Anti-lipid peroxidation of PE and SOE in vitroThe degree of lipid peroxidation in liver homogenate of non-

treated rats was assayed in the presence and absence of differentconcentrations of PE or SOE by estimating malondialdehyde(MDA) level, the main product of lipid peroxidation, according tothe method of Ohkawa, Ohishi, and Yagi (1979).

2.3.5. Determination of nitric oxide (NO)-scavenging activity of PE andSOE

NO was measured by the Griess reaction (Marcocci, Maguire,Droy-Lefaix, & Packer, 1994).

2.4. Animals

Forty-two adult male Sprague–Dawely rats, weighing100–110 g, were obtained from the Experimental Animals Breed-ing Centre of the Holding Company for Biological Products andVaccines (Helwan, Cairo, Egypt). All rats were examined for healthstatus and their room was designed to maintain the temperature at25 �C, relative humidity at approximately 50% and 12 h light/darkphotoperiod for 2 weeks prior to handling. The animals were thenhoused in stainless-steel cages, given standard diet and water adlibitum throughout the study and observed daily for abnormalsigns. After acclimatisation, rats were divided into six groups of se-ven rats each. Fig. 1 summarizes the experimental design. Groupcontrol (C): Untreated rats. Group DEN: rats were left for 4 weekswithout treatment, then they were injected subcutaneously (S.C)with DEN with a dose of 200 mg of saline/kg body mass/week for3 successive weeks (Katayama et al., 2003). Then rats were leftfor one week with no treatment. At the beginning of the fifth weekthe animals were given drinking water containing 500 ppm of phe-nobarbital (PB) for 19 weeks (Katayama et al., 2003); i.e. the totalexperimental period was 23 weeks. Group (PE): rats were treatedorally (using oral gavage) in a daily dose 0.2 ml of PE/kg bodymass/day (b.m./d) for 23 weeks according to Ajaikumar, Asheef,Babu, and Padikkala (2005). Group (PE–DEN): rats were treatedwith PE for 4 weeks and, at the beginning of the fifth week, theywere treated with DEN (as mentioned previously) beside PE treat-ment. At the beginning of the ninth week, rats were treated withPB with continuous intake of PE until the 23rd week of the exper-iment. Group (SOE): rats were treated orally with SOE (2 ml/kgb.m./d) for 23 weeks according to Kohno et al. (2004). Group(SOE–DEN): rats were treated with SOE for 4 weeks then treatedwith DEN and PB, as mentioned above, with continuous treatmentwith SOE during the period of the experiment.

At the end of the experimental period, feeding was stopped 12 hprior to killing. Rats were anaesthetized by diethylether and sacri-ficed. Livers were removed immediately and small portions were

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Experimental period (23 weeks)

Fig. 1. An illustration of the experimental design.

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fixed in 10% formalin for histopathological examination. Theremaining liver was washed with cold saline solution (0.9% NaCl),weighed, divided into two parts and kept at �80 �C prior to analy-sis. The first part was used for determination of DNA fragmenta-tion, caspase-3, GPx and GSR activities. The second part washomogenised in 5 vol of cold 0.1 M sodium phosphate buffer con-taining 0.9% NaCl (pH 7.4), using a glass–Teflon homogenizer. Theliver homogenate was divided into four portions and kept at�80 �C for subsequent determination of lipid peroxidation, proteincontent, GSH, GST and SOD activities. All processes were carriedout at 4 �C.

Unheparinized blood samples were collected, kept for 15 min atroom temperature and then sera were separated by centrifugationat 3000 rpm for 20 min and stored at �20 �C until used for thedetermination of ALT, AST, total bilirubin (TB), total proteins (TP)and albumin.

2.5. Biochemical assays

2.5.1. Caspase-3 assay (EC 3.4.22.56)Liver tissues were homogenised in 4 volumes of cold cell lysis

buffer (50 mM Tris–HCl buffer containing 0.2 M NaCl and 1% TritonX-100, pH 6.8), using a Teflon-glass homogenizer. The homoge-nates were centrifuged at 20,000 rpm for 3 min at 4 �C and thesupernatants were kept at �80 �C. Caspase-3 activity was deter-mined using a colorimetric kit (Talanian et al., 1997), briefly, thesupernatant (150 lg protein), reaction buffer and substrate wereincubated in a micro plate reader at 37 �C in the dark for 2 h. Theproduced yellow colour was measured at 405 nm against a blank,using a microplate reader (Bio-Tex. Instrument-Germany). Fold-in-crease in caspase-3 activity was determined by direct comparisonto the control.

2.5.2. DNA Fragmentation (DNAF)Fragmentation was determined in the liver homogenate, using

agarose gel electrophoresis (Li et al., 2003). Liver tissues werehomogenised in (1:5 w/v) 50 mM Tris–HCl buffer containing 20%sucrose and 50 mM EDTA, pH 7.6. DNA was isolated, using a DNApurification kit. Then DNA (15 lg/lane) was separated by electro-phoresis on 1% agarose gel containing ethidium bromide, at 5 V/1 cm for 2–3 h and then visualised under UV light using the Con-sorte 86 s, BE/Gium Instrument.

2.5.3. Lipid peroxidation (LP) levelMalondialdehyde (MDA) level, the main product of lipid perox-

idation, was determined as described by Ohkawa et al. (1979).MDA is defined as nmol per mg of protein.

2.5.4. Reduced glutathione (GSH)GSH in the deproteinized supernatant was determined as de-

scribed by Ellman (1959). GSH is expressed as mg/g of tissue.

2.5.5. Total glutathione peroxidase (t-GPx, EC 1.11.1.19)Liver was homogenised in 0.05 M potassium phosphate buffer

(1:4, w/v) containing 1.15% KCl (pH 7.6), using a Teflon–glasshomogenizer. The homogenate was centrifuged at 3200g, 4 �C for20 min. The activity of t-GPx was determined in the supernatantat 37 �C, using cumene hydroperoxide as a substrate (Rotrucket al., 1973). The specific activity was expressed as lg consumedGSH/mg protein/min.

2.5.6. Glutathione reductase (GSR, EC 1.8.1.7)Liver was homogenised in 0.05 M potassium phosphate buffer,

pH 7.2 (1:10, w/v), and centrifuged at 3200g, 4 �C for 20 min.GSR activity was determined in the supernatant by following the

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oxidation of NADPH to NADP+ during the reduction of GSSG (Gold-berg & Spooner, 1983) and expressed as lmol of NADPH oxidised/mg of protein/min.

2.5.7. Glutathione-S-transferase (GST, EC 2.5.1.18)GST was measured spectrophotometrically, using p-nitrobenzyl

chloride in 95% ethanol as substrate (Habig, Pabst, & Jakoby, 1974).The specific activity was defined as lmol/mg of protein/min.

2.5.8. Superoxide dismutase (SOD, EC 1.15.1.1)Cu–Zn–SOD activity in the liver homogenate was determined as

described by Marklund and Marklund (1974). The unit of activitywas defined as the amount of enzyme that inhibits the rate ofautoxidation of pyrogallol by 50% under standard conditions andexpressed as IU/mg protein.

2.5.9. Liver function testsTotal protein (TP) levels in serum and liver were determined by

means of the of biuret reaction (Gornall, Bardawill, & David, 1949).Serum albumin and total bilirubin (TB) were determined by themethods of Doumas, Watson, and Biggs (1971) and Jendrassikand Grof (1938), respectively. Aspartate aminotransferase (AST;EC 2.6.1.1) and alanine aminotransaminase (ALT; EC 2.6.1.2) activ-ities were assayed by the method of Reitman and Frankel (1957).

2.5.10. Histopathological studyLiver tissues were fixed, processed and embedded in paraffin

wax. Sections of 5 lm in thickness were cut and stained withhematoxylin and eosin.

2.5.11. Statistical analysisAll data are presented as means (X) ± standard deviation (S.D.).

Comparisons between the means of various treatment groups wereanalysed using the least significant difference (LSD) test. Differ-ences were considered significant at p < 0.05. All statistical analy-ses were performed using the statistical software, SPSS, version16.0.

3. Results

3.1. Characterization of PE and SOE

3.1.1. Total phenolic contents in PE and SOE and their analysisThe results showed that PE and SOE contain 2.9 and 0.21 mg/ml

of extract, respectively in terms of gallic acid equivalents. Fig. 2 (aand b) shows HPLC analysis of polyphenolic compounds in PE andSOE and their concentrations are listed in Table 1.

3.1.2. Total flavonoid contentPE and SOE contain 0.813 and 0.027 mg/ml extract, respectively

in terms of catechin equivalents.

3.1.3. Antioxidant, anti-lipid peroxidation and NO-scavengingactivities of PE and SOE in vitro

The results showed that the total antioxidant of PE and SOEwere 16.0 and 0.249 mg/g of extract, respectively in terms of ascor-bic acid equivalents. Fig. 2c and d shows the anti-lipid peroxidationand the nitric oxide-scavenging activities of PE and SOE,respectively.

3.2. TP level in liver

Administration of DEN increased TP level non-significantly byapproximately 8% compared to C (Table 2). Treatment with (PE–DEN) and (SOE–DEN) groups decreased TP levels significantly by

approximately 36% and 15%, respectively, compared with DEN.Administration of PE and SOE, separately, decreased TP levels byabout 44% (significantly) and 5% nonsignificantly, respectively, ascompared with the C group.

3.3. DNAF level

Gel electrophoresis of the hepatocytes of the control, C, showedvery low or undetectable DNA laddering (Fig. 3a) since the DNA in-tact band appears to be condensed near the application point withno DNA smearing, suggesting no DNAF. DEN administration in-creased DNAF as compared to the C group, while, treatment with(PE–DEN) and (SOE–DEN) decreased it as compared with theDEN group. PE administration increased DNAF, while SOE adminis-tration decreased it compared to the C group.

3.4. MDA level

DEN administration increased MDA level significantly, by about112% compared to the C group (Fig. 3b), while its levels were de-creased significantly in rats treated with (PE–DEN) and (SOE–DEN) by about 50% and 49%, respectively, compared with theDEN group. PE and SOE administration increased MDA levelsnon-significantly by about 22% and 19.6%, respectively, comparedwith the C group.

3.5. GSH level

DEN administration decreased GSH level significantly, by about42% compared with C (Fig. 3c). Rats treated with (PE–DEN) and(SOE–DEN) showed an elevation in GSH levels by about 214%and 11%, respectively, compared to the DEN group. GSH level inrats treated with SOE was decreased significantly by about 33%,while its level after PE administration was significantly increasedby about 108% compared to C.

3.6. Caspase-3 activities

Caspase-3 activity after DEN administration increased by about130% compared to C (Fig. 3d). Treatment with (PE–DEN) and(SOE–DEN) decreased caspase-3 activity by approximately 45%and 64%, respectively, compared with the DEN group. Administra-tion of PE increased caspase-3 activity by about 31%, while, SOEadministration decreased it by about 16% compared to the C group.

3.7. T-GPx activity

DEN decreased t-GPx activity by about 44% as compared with theC group (Fig. 3e), while, treatment with (PE–DEN) and (SOE–DEN)increased t-GPx activity by about 84% and 89%, respectively,compared to the DEN group. PE and SOE administration caused anelevation in t-GPx activities by about 71% and 26%, respectively,compared to the C group.

3.8. SOD activity

DEN administration decreased SOD activity by about 36% com-pared to the C group (Fig. 3f). SOD activity was increased in ratstreated with (PE–DEN) and (SOE–DEN) by about 39% and 40%,respectively, compared to the DEN group. Administration of PE in-creased SOD activity non-significantly by about 14.1%, while SOEadministration decreased it by about 6.9% compared to the Cgroup.

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Fig. 2. HPLC analysis of polyphenolic compounds in PE and SOE (a and b), the anti-lipid peroxidation activity of PE and SOE (c), nitric oxide-scavenging activity of PE and SOE(d).

N.Z. Shaban et al. / Food Chemistry 141 (2013) 1587–1596 1591

3.9. GSR activity

Administration of DEN elevated GSR activity by about 110% of C(Fig. 3g). Treatment with (PE–DEN) and (SOE–DEN) caused a de-cline in GSR activities by about 39% and 54%, respectively, com-pared to the DEN group. Administration of PE decreased GSRactivity by about 11.5%, while SOE increased it by about 37% com-pared to the C group.

3.10. GST activity

DEN administration decreased GST activity by about 29% com-pared to the C group (Fig. 3h). Rats treated with (PE–DEN) and(SOE–DEN) showed an elevation in GST activities by about 32%

and 21%, respectively, compared to the DEN group. Administrationof PE and SOE decreased GST activities by about 24% and 35%,respectively, compared to the C group.

3.11. Liver function tests

ALT and AST activities were decreased by about 50% and 14%,respectively, after DEN treatment compared to the C group (Table2). Treatment with (PE–DEN) and (SOE–DEN) caused an elevationin ALT (by about 177% and 194%, respectively) and AST activities(by about 22% and 57%, respectively) compared to the DEN group.PE administration increased ALT and AST activities by about 62%and 29%, respectively, while SOE decreased ALT and AST activitiesby about 49% and 18%, respectively, compared with the C group.

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Table 1HPLC analysis of polyphenolic compounds in PE and SOE and their concentrations.

Compound Concentration(mg/ml SOE)

Concentration(mg/ml PE)

Gallic acid 3.02 43.2Chlorogenic acid 0.003 0.171Caffeic acid 0.015 0.4123,5-Dicaffeoyl quinic acid – 0.3414,5-Dicaffeoyl quinic acid 0.011 0.105Rutin 2.79 17.5Tannic acid 3.01 19.7

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DEN administration elevated bilirubin levels by about 1902%and decreased the levels of albumin and TP in serum by about25% and 54%, respectively, compared to the C group (Table 2).

Table 2Effect of DEN and pomegranate peel and seed oil extracts (PE and SOE) on total protein in

Particulars C DEN PE

ALT (U/l) 31.9 ± 3.42a 15.8 ± 2.26b 51.1 ±AST (U/l) 134 ± 4.26a 116 ± 9.52b 163 ±STP (g/dl) 8.16 ± 0.56a 3.75 ± 0.86b 7.21 ±LTP (mg/g tissue) 614 ± 76.7ab 705 ± 113b 344 ±Albumin (g/dl) 3.43 ± 0.18a 2.57 ± 0.69b 3.89 ±Bilirubin (mg/dl) 0.45 ± 0.03a 9.04 ± 0.38b 0.13 ±

Values are expressed as means ± S.D. for seven rats. Within each row, values with differGroup C – control rats; group DEN – rats treated with DEN; group (PE–DEN) – rats treatewith SOE pre, during and post DEN administration, group (SOE) – rats treated with SOEALT, Alanine aminotransaminase; AST, Aspartate aminotransferase; STP, Serum total pro

Treatment with (PE–DEN) and (SOE–DEN) decreased serum biliru-bin (by about 92% and 90%, respectively) and increased the levelsof albumin (by about 25% and 35%, respectively) and TP (by about70% and 92%, respectively) in serum compared to the DEN group.Administration of PE decreased bilirubin and TP levels by about71% and 11.6%, respectively, but increased albumin by about 13%compared to the C group. SOE administration decreased albuminand TP levels nonsignificantly by about 1.1% and 8.2%, respectively,while, it had no effect on bilirubin level.

3.12. Histopathologic results

Histopathological examination of the control group showednormal rat liver with no remarkable pathological changes(Fig. 4a). DEN administration showed liver cells with mild dysplas-tic nuclei, prominent nucleoli and binucleated cells (Fig. 4b).

liver and liver function tests in serum of the experimental animals.

PE-DEN SOE SOE-DEN

4.16c 43.8 ± 5.17d 16.3 ± 2.2b 46.5 ± 3.87d

6.48c 141 ± 4.74d 111 ± 8.6b 181 ± 3.06e

0.43c 6.38 ± 0.65d 7.50 ± 0.28c 7.19 ± 0.52c

107c 424 ± 68.4c 582 ± 3.1a 558 ± 121a

0.40c 3.22 ± 0.34a 3.39 ± 0.14a 3.46 ± 0.28a

0.02c 0.74 ± 0.04d 0.45 ± 0.074a 0.92 ± 0.14d

ent letters are significantly different at p < 0.05.d with PE pre, during and post DEN administration; group (SOE–DEN) – rats treatedonly and group (PE) – rats treated with PE only.tein; LTP, Liver total protein.

C DEN PE PE – DEN SOE SOE – DEN1 2 3 4 5 6 7 8 9 10 11 12

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bbc

de

Fig. 3. Treatment effects on the levels of: DNA fragmentation in all groups (a)[control rats (lane 1), DEN (lanes 2, 3, 4), PE (lanes 5, 6), PE-DEN (lanes 7, 8), SOE(lane 9) and SOE-DEN (lanes 10, 11, 12)]; MDA (b); GSH (c); caspases-3 (d); GPx (e);SOD (f); GSR (g) and GST (h) activities in rat liver tissues. Group C – control rats;group DEN – rats treated with DEN; group (PE–DEN) – rats treated with PE pre,during and post DEN administration; group (SOE–DEN) – rats treated with SOE pre,during and post DEN administration, group (SOE) – rats treated with SOE only andgroup (PE) – rats treated with PE only. Results are given as means ± S.D. for sevenrats. Values with different letter are significantly different at p < 0.05.

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Treatment with (PE–DEN) revealed liver tissue with a moderateperiportal collection of chronic inflammatory cells, mainly lym-phocytes (Fig. 4c), while treatment with (SOE–DEN) showed earlycirrhotic nodule changes with a mild inflammatory collection ofchronic lymphocytes (Fig. 4d1). It also, showed loss of architecture,nodules of variable sizes surrounded by fibrous tissue and chronicnodules, (Fig. 4d2). Examination of rat liver treated with PE showednormal preserved architecture with very mild portal inflammation(Fig. 4). SOE administration showed hepatic tissue with mild peri-portal inflammatory reactions (Fig. 4).

4. Discussion

The present study showed an elevation in the number of Kupp-fer cells, mild dysplastic nuclei, prominent nucleoli and binucle-ated cells after DEN administration, indicating that DEN releasedmediators which activated macrophage (Laskin & Pendino, 1995).The data showed a significant elevation in MDA level and GSRactivity with a significant decline in GSH level in rat liver homog-enate after DEN administration compared to the control. MDA is amajor oxidation product of peroxidised polyunsaturated fatty acidsand increased MDA content is an important indicator of lipid per-oxidation and this may be due to an enhanced generation of O��2and H2O2 radicals that accelerated peroxidation of native mem-brane lipids (Shaban, Helmy, El-Kersh, & Mahmoud, 2003). Perox-idation of mitochondrial membrane lipids led to loss of cellintegrity, increase in membrane permeability and alteration ofCa2+ homeostasis that contribute to cell death due to alterationin the inner membrane potential (Shaban et al., 2003). The declinein GSH level may possibly be due to increased demand of the tri-peptide for lipid hydroperoxide metabolism by GPx and conjuga-tion of GSH with DEN or its metabolites to terminate free radicalreactions. GSH depletion could also contribute to the activationof lipid peroxidation (Shaban et al., 2003). GSR plays a key role incellular defence against oxidative stress by preventing accumula-tion of GSSG and thus maintaining the redox state. The increasein GSR activity after DEN administration probably reflects an adap-tation to oxidative conditions (Shaban et al., 2003). The decrease inthe specific activities of antioxidant enzymes, SOD and t-GPx andGST, a drug metabolising enzyme, after DEN administration maybe related to their inhibition by DEN through direct interactionwith the enzyme molecules or modification of the post-transcrip-tional and post-translational steps in the enzyme synthesis (Pra-deep et al., 2007). Another explanation for the inhibition of SODmay be the action of O��2 or, after transformation to H2O2, oxidationof the cysteine of the enzyme (Shaban et al., 2003). Otherwise, theelevation in the activity of caspase-3 after DEN administration wasconfirmed by analysis of DNA fragmentation at the nucleosomal le-vel. Caspase-3 is a major mediator of both apoptotic and necroticcell death and increases in the activities of caspases-1 and -3 havebeen described in ischemic injury to various organs (Faubel & Edel-stein, 2005). A hallmark feature of apoptosis is the observation thatnuclear DNA, extracted from apoptotic cells, is often degraded inan internucleosomal pattern. The internucleosomal fragmentationof DNA is catalysed by endonucleases. The ladder pattern of thefragmentation in apoptosis might be a consequence of the stateof the chromatin at the time of fragmentation rather than of thenature of the endonucleases involved (Yakovlev et al., 2000). Innecrosis, however, DNA degradation is a later phenomenon aftercell rupture and the chromatin is digested by endonucleases andproteases into a smear pattern instead of a ladder pattern (Lansky& Newman, 2007). Our results showed that the endonuclease-dependent fragmentation shows mixed smearing and ladderingDNA fragments. This indicates that, exposure to DEN generates

a

b

c

d1

d2

e

f

Fig. 4. Microscopic examination of hepatic tissues. The control group (a) showing preserved architecture with no remarkable pathological changes (H & E stain � 100). DENgroup (b) showing mild dysplastic nuclei with prominent nucleoli, and binucleated cells (H & E stain � 250). PE–DEN group (c) showing moderate periportal collection ofchronic inflammatory cells (H & E stain � 250). SOE–DEN group (d1) shows early cirrhotic nodular changes with mild inflammatory collection of chronic lymphocytes (H & Estain � 100) and (d2) showing loss of architecture, nodules of variable sizes surrounded by fibrous tissues, chronic nodules (H & E stain � 100). PE group (e) showing normalpreserved architecture with very mild portal inflammation (H & E stain � 200). SOE group (f) showing preserved architecture with mild periportal inflammatory reaction (H &E stain � 100).

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ROS which trigger DNA damage. We have also shown that DENadministration caused a significant elevation in serum total biliru-bin level. Also, the data showed an elevation in liver total proteinlevel, probably due to increase of protein expression towards apop-tosis and liver injury induced by DEN (Kaufman, James, Sheedy,Harper, & Matsumoto, 2006). The results show a significant declinein the levels of AST, ALT, total protein and albumin in serum com-pared to control. This indicates that liver cell death occurs by apop-tosis, as well as by necrosis. Hepatocytes dying by apoptosispresumably synthesize less AST and ALT as they wither away.Our results are similar to those of Pradeep et al. (2007) who re-ported that, DEN-induced hepatotoxicity elevated levels of serumbilirubin, AST and ALT and a fall in their levels after 30 days. Previ-ous studies have demonstrated that hepatocyte necrosis in acutehepatitis, toxic injury or ischemic injury results in the leakage ofenzymes into the blood circulation (Pradeep et al., 2007) However,in chronic liver diseases, such as hepatitis C and cirrhosis, the ser-um ALT level correlates only moderately well with liver inflamma-tion. In hepatitis C, liver cell death occurs by apoptosis andnecrosis. This probably explains why at least one third of patientsinfected with hepatitis C virus have persistently normal serum ALTlevels, despite the presence of inflammation on liver biopsy. Pa-tients with cirrhosis often have normal or only slightly elevatedserum AST and ALT levels. Thus, AST and ALT lack some sensitivityin detecting chronic liver injury.

The results showed an attenuation of hepatic injury induced byDEN in rats treated with PE and SOE before, during and after DENadministration. This was confirmed with a significant improve-ment in liver function tests since the data showed a decline in lev-els of serum total bilirubin and liver total protein, with an elevationin the levels of serum AST, ALT, albumin and total protein (com-pared to DEN) to reach the values of the control. In addition, thesetreatments decreased lipid peroxidation and apoptosis induced byDEN since the levels of MDA, DNAF, the activities of GSR and cas-pase-3 were decreased with an elevation in the levels of GSH,GST, t-GPx and SOD activities compared with the DEN group. Thisis because both PE and SOE possess antioxidant and radical-scav-enging properties which attenuate programmed cell death. Asshown from our results, the total antioxidant activity of PE is veryhigh since it contains high amounts of total flavonoids and poly-phenolic compounds. Therefore, the antioxidant and antiapoptoticeffects of PE are related to the effects of its polyphenols, such asflavonoids (epicatechin, epigallocatechin gallate, quercetin, luteo-lin and naringinin), phenolic acids (chlorogenic and caffeic acids)ellagitannin (tannic acid) and corilagin (Lansky & Newman,2007). Previous studies showed that the antioxidative propertiesof plant polyphenols may arise from their reactivity as hydrogenor electron donors, their ability to stabilize unpaired electronsand their ability to terminate Fenton reactions (Bishayee et al.,2011). The polyphenolic compounds are good inhibitors of the N-nitrosation reaction and can prevent oxidative damage as a resultof their ability to scavenge ROS. The polyphenolic compounds en-hance GSH level and the activities of t-GPx and SOD, but they inhi-bit GSR activity (Moskaug, Carlsen, Myhrstad, & Blomhoff, 2005).Otherwise, the polysaccharide fraction in PE has free radical-scav-enger properties and its exact mechanism of action is still un-known (Route & Banerjee, 2007).

Our results showed that SOE has low contents of total flavo-noids and phenolic compounds. This indicates that the antioxidantand antiapoptotic effects of SOE are related to the effect of flavo-noids and phenolic compounds beside other components havingmajor effects, such as triterpenoids, gamma-tocopherol, 17-a-estradiol, estrogens ‘‘estrone and estriol’’, testosterone, b-sitosterol,coumesterol, campesterol, stigmasterol, punicic acid and flavo-noids ‘‘genistein and diadzein’’ (Lansky & Newman, 2007). Triterp-enoids, e.g. ursolic acid, have hepatoprotective effects against

chronic ethanol-mediated hepatotoxicity in rats (Saravanan,Viswanathan, & Pugalendi, 2006). Genistein and diadzein werefound to enhance the activities of t-GPx and SOD (Sivaramakrish-nan & Devaraj, 2009). Estrogens may modulate intracellular Ca2+,prevent inflammation and inhibit cell death in spinal cord injury.Estradiol attenuates programmed cell death after stroke-injury(Sribnick, Matzelle, Ray, & Banik, 2006).

Administration of rats with PE and SOE, separately, for a longperiod (23 weeks) caused hepatic tissue to show mild inflamma-tory reaction as compared to the C group, as shown from the his-topathological results. The biochemical results showed that theanimals treated with PE for 23 weeks have a non-significant in-crease in the levels of MDA, DNAF and caspase-3 activity as com-pared to the C group. This indicates that certain polyphenols andalkaloids in PE accumulated in the liver and produced only smallamounts of ROS and damaged DNA. It has been observed that, die-tary polyphenolics with phenol rings were metabolised by peroxi-dase to form prooxidant phenoxyl radicals (Cilla et al., 2009).Flavonoids undergoing autooxidation catalysed by transition met-als produce O��2 radicals which are transformed to H2O2 and formOH�� radicals via Fenton reaction (Lansky & Newman, 2007). Theprevious studies reported that polyphenols, such as gallic acid, epi-gallocatechin gallate and quercetin, generate H2O2 when added tocell culture. Tannins (punicalagin and punicalin) and alkaloids actas proapoptotic agents (Lansky & Newman, 2007). Also, tannic acid,in the presence of Cu(II), causes DNA degradation through genera-tion of ROS. It exhibits antimutagenic and anticarcinogenic activi-ties, as well as inducing apoptosis in animal cells. Ellagic acidexhibited both antioxidant activity in V79-4 cells and apoptosis-inducing activity in human osteogenic sarcoma cells, through theup-regulation of Bax and activation of caspase-3 (Lansky & New-man, 2007).

The data showed an elevation in GSH level, t-GPx and SODactivities and reduction in GSR and GST activities in rats adminis-tered with PE alone, as compared with C. This may be due to theeffects of certain polyphenols in PE, such as flavonoids, luteolin,naringinin, quercetin, ellagic acid, corilagin, ferulic acid, tannic acidand chlorogenic acid (Lansky & Newman, 2007). Flavonoids and el-lagic acid can enhance the expression of c-glutamyl cysteine syn-thetase, a rate-limiting enzyme in biosynthesis of GSH (Moskauget al., 2005). The increase in GSH level may increase the activityof t-GPx which can also be up-regulated at the gene level by nar-inginin. Also, these polyphenols can enhance SOD activity (Lansky& Newman, 2007). Otherwise, GST activity can be inhibited by cor-ilagin, luteolin, quercetin, ellagic acid, ferulic acid and chlorogenicacid (Cilla et al., 2009; Dahlawi, Jordan-Mahy, Clench, & Le Maitre,2012). Also, GSR activity is inhibited by tannic acid and flavonoids(Bishayee et al., 2011; Lansky & Newman, 2007).

Administration of rats with SOE for a long period caused a non-significant elevation in MDA level and the activities of t-GPx andGSR, while the levels of GSH, DNAF, GST, SOD and caspase-3 activ-ities were decreased as compared with the C group. This may dueto the effects of estrogens, punicic acid, genistein and diadzein inSOE (Sivaramakrishnan & Devaraj, 2009). These compounds formquinone radicals during their metabolism which oxidise GSH andother antioxidants, leading to oxidative stress and depletion ofthe antioxidants within the cell (Blair, 2006).

Administration of PE and SOE, separately, for a long period,caused a decline in total protein level in liver as compared to theC group and this may be due to inhibition of matrix metallopro-teinases by different compounds of these extracts which accumu-lated in liver tissues. These administrations elevated serumalbumin level and depressed the levels of serum total proteinsand total bilirubin compared to C; all of these changes may bedue to the effects of different contents of PE or SOE that accumu-lated in rat liver. Our results agree with Vidal et al. (2003) who

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reported that, the repeated administration of the ethanolic extractof the whole pomegranate fruit for 5 weeks decrease the total pro-tein level in serum. Otherwise, PE administration elevated ALT andAST levels compared to C. This may be due to the side effect of thepolyphenolic compounds in PE extract that accumulated in livertissues. In contrast, SOE administration depressed ALT and AST ascompared to C.

5. Conclusion

The present study demonstrated that pomegranate extracts (PEand SOE) exert significant protective effects against DEN-inducedoxidative stress and apoptosis in hepatocytes by augmenting hostantioxidant defence mechanisms. These extracts are promisingagents for the prevention of chemical- and drug-induced toxicitythrough enhancing the antioxidative and drug-metabolising en-zymes, as well as lowering the extent of lipid peroxidation. Admin-istration of PE and SOE for long period should be avoided.

Acknowledgements

We would like to thank the late Dr. Mohammed Magdy, Lectureof Biochemistry, Faculty of Science, Alexandria University, for hisgreat help, providing the unavailable research chemicals, adviceand encouragement. Also, we thank Dr. Sozan Ahmad Kato, Profes-sor of Pathology Department, Faculty of Medicine, Alexandria Uni-versity. Who patiently devoted much of her precious time forcompleting the histopathological part of this work.

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