Experimental and Toxicologic Pathology 63 (2011) 433–441

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Experimental and Toxicologic Pathology

0940-29

doi:10.1

n Corr

E-m

journal homepage: www.elsevier.de/etp

Protective effect of date palm fruit extract (Phoenix dactylifera L.) ondimethoate induced-oxidative stress in rat liver

Emna Behija Saafi a, Mouna Louedi a, Abdelfattah Elfeki b, Abdelfattah Zakhama c, MohamedFadhel Najjar d, Mohamed Hammami a, Lotfi Achour e,n

a Laboratoire de Biochimie, UR Nutrition Humaine et Desordres Metaboliques, Faculte de Medecine, 5000 Monastir, Tunisiab Laboratoire d’Ecophysiologie Animale, Faculte de Sciences, 3018 Sfax, Tunisiac Laboratoire d’Anatomopathologie, Faculte de Medecine, 5000 Monastir, Tunisiad Laboratoire de Biochimie, Hopital Universitaire, CHU Fattouma Bourguiba, 5000 Monastir Tunisiae Institut Superieur de Biotechnologie, Avenue Tahar Hadded, BP 74, 5019 Monastir, Tunisia

a r t i c l e i n f o

Article history:

Received 5 March 2009

Accepted 7 March 2010

Keywords:

Oxidative stress

Liver

Dimethoate

Date fruit (Phoenix dactylifera L.) extract

Antioxidant

93/$ - see front matter & 2010 Elsevier Gmb

016/j.etp.2010.03.002

esponding author. Tel.: +216 73 465 405; fax

ail address: lotfiachour@yahoo.fr (L. Achour).

a b s t r a c t

Nowadays, people’s exposure to chemical compounds such as organophosphorus insecticides is

continuously on the rise more and more. Theses compounds have induced an excessive production of

free radicals which are responsible for several cell alterations in the organism. Recent investigations

have proved the crucial role of nutritional antioxidants to prevent the damage caused by toxic

compounds. In this study, we investigate the role of date palm fruit extract (Phoenix dactylifera L.) in

protection against oxidative damage and hepatotoxicity induced by subchronic exposure to dimethoate

(20 mg/kg/day). Oral administration of dimethoate caused hepatotoxicity as monitored by the increase

in the levels of hepatic markers enzymes (transaminases, alkaline phosphatase, gamma-glutamyl

transferase and lactate dehydrogenase), as well as in hepatic malondialdehyde thus causing drastic

alteration in antioxidant defence system. Particularly, the activities of superoxide dismutase (SOD) and

glutathione peroxidase (GPx) were found increased by dimethoate while catalase (CAT) activity was

reduced significantly. These biochemical alterations were accompanied by histological changes marked

by appearance of vacuolization, necrosis, congestion, inflammation, and enlargement of sinusoids in

liver section. Pretreatment with date palm fruit extract restored the liver damage induced by

dimethoate, as revealed by inhibition of hepatic lipid peroxidation, amelioration of SOD, GPx and CAT

activities and improvement of histopathology changes. The present findings indicate that in vivo date

palm fruit may be useful for the prevention of oxidative stress induced hepatotoxicity.

& 2010 Elsevier GmbH. All rights reserved.

1. Introduction

For several years, a special attention has been paid to oxidativestress; situation of an excessive production of reactive oxygenspecies (e.g. the famous ‘‘free radical’’) in the organism. A largenumber of experimental and epidemiological studies haveindicated that the reactive oxygen species (ROS) contribute toorgan injury in many systems (Halliwell et al., 1992; Cadet et al.,2002; Del Rio et al., 2005; Beaudeux et al., 2006; Goetz and Luch,2008). Reactive oxygen species are constantly formed as a by-product of normal metabolic reaction and their generation isaccelerated by accidental exposure to occupational chemicals likepesticides. Nowadays the hazards of using such chemicalcompounds have been accentuated by the sharp rise in their useby house-holders and governments, farmers and industrialists

H. All rights reserved.

: +216 73 465 404.

alike. Organophosphorus insecticides (OPI) represent one group ofpesticides that is widely used and has proved to have toxic effectsin humans and animals (De-Bleecker et al., 1993; Betrosian et al.,1995; Tsatsakis et al., 1998; Hagar and Fahmy, 2002).

Dimethoate (O,O-dimethyl S-methyl carbamoyl phosphoro-dithioate) is one of the most important OPI used extensively on alarge number of crops against several pests. Dimethoate poison-ing is usually associated with neuromuscular transmission blockin both animals and humans (De-Bleecker et al., 1993; Dongrenet al., 1999). Immunotoxicological effects due to dimethoate havealso been reported (Institoris et al., 1999). Moreover, dimethoateinduce hyperglycemia and cause various toxic effects on ratpancreas following acute, subchronic and chronic exposure(Hagar and Fahmy, 2002; Kamath and Rajini, 2007; Kamathet al., 2008). Earlier studies have shown that acute and subchronicexposure to dimethoate alters the antioxidant status and thehistology of liver and brain in rats (Sharma et al., 2005a, 2005b;Sayim, 2007). Involvement of oxidative stress following expo-sure to dimethoate and to OPI in general has been reported

E.B. Saafi et al. / Experimental and Toxicologic Pathology 63 (2011) 433–441434

(Banerjee et al., 2001; Sivapiriya et al., 2006) and it has beendemonstrated that lipid peroxidation mediated by free radicalsis one of the molecular mechanisms involved in OPI-inducedtoxicity (Akhgari et al., 2003). The cellular antioxidant statusdetermines the susceptibility to oxidative damage and is usuallyaltered in response to oxidative stress. The cellular antioxidantpool comprises antioxidant free radical scavenging enzymes likecatalase (CAT), superoxide dismutase (SOD) and glutathioneperoxidase (GPx) (Pincemail et al., 2002). The cellular antioxidantaction is reinforced by the presence of dietary antioxidants(Prior and Cao, 2000; Pincemail et al., 2002; Kiefer et al., 2004).Accordingly, interest has recently grown in the role and usage ofnatural antioxidants as a strategy to prevent oxidative damage invarious health disorders with oxidative stress as a factor in theirpathophysiology (Khan et al., 2005; Koechlin-Ramonatxo, 2006;Kasdallah-Grissa et al., 2007; Mehmetc- ik et al., 2008; Shireenet al., 2008).

Fruits of the date palm (Phoenix dactylifera L.) are verycommonly consumed in many parts of the world and a vitalcomponent of the diet and a staple food in most of the Arabiancountries. The Deglet Nour is an important date variety thatmakes up about 62.5% of Tunisia date production (GIF, 2008).Dates are rich in certain nutrients and provide a good source ofrapid energy due to their high carbohydrate content (�70–80%).Most of the carbohydrates in dates are in the form of fructose andglucose, which are easily absorbed by the human body (Al-Hootiet al., 1995; Myhara et al., 1999; Al-Farsi et al., 2005a; Saafi et al.,2008). The good nutritional value of dates is also based on theirdietary fiber and on their essential minerals such as calcium, iron,magnesium, phosphorus, potassium, zinc, selenium and manga-nese (Sawaya et al., 1982; Al-Showiman et al., 1994; Mohamed,2000; Al-Shahib and Marshall, 2002, 2003; Al-Farsi et al., 2005a;Elleuch et al., 2008). The date fruit is listed in folk remedies for thetreatment of various infectious diseases and cancer (Duke, 1992).Experimentally, it has been shown that, depending on the type ofextract used, date fruit and pit extracts significantly increase ordecrease gastrointestinal transit in mice (Al-Qarawi et al., 2003).Moreover, the aqueous and ethanolic extracts, were effective inameliorating the severity of gastric ulceration in rats (Al-Qarawiet al., 2005). Researchers also, found that the consumption ofdates might be of benefit in glycaemic and lipid control of diabeticpatients (Miller et al., 2003). Recent studies have indicate that theaqueous extracts of dates have potent antioxidant and antimuta-genic activity (Vayalil, 2002; Al-Farsi et al., 2005b, 2007;Mohamed and Al-Okbi, 2005; Allaith, 2007; Biglari et al., 2008;Saafi et al., 2009). The antioxidant activity is attributed to thewide range of phenolic compound in dates including p-coumaric,ferulic, and sinapic acids, flavonoids and procyanidins (Regnalut-Roger et al., 1987; Al-Farsi et al., 2005b; Mansouri et al., 2005;Hong et al., 2006) and also to the presence of vitamin C (Allaith,2007; Mrabet et al., 2008).

Muslims believe that ‘‘He who eats seven dates every morningwill not be affected by poison or magic on the day he eats them’’(cited by Miller et al., 2003). Accordingly, we hypothesized thatdate extract may prevent the oxidative stress and the hepatoxicityin rats induced by dimethoate.

2. Materials and methods

2.1. Date palm extract preparation

Fresh ripened Deglet Nour variety was collected from thestation of Douz (Kebili, Tunisia). Fruit flesh was extracted twotimes with distilled water (1/10, w/v) by grinding with a mortarand pestle. It was centrifuged at 4 1C for 20 min at 4000g and the

supernatant was collected. We selected an aqueous extractbecause most of the antioxidant components in dates areextracted in water (Vayalil, 2002; Al-Farsi et al., 2005b).

During the experience, the aqueous date fruit extract of DegletNour (DNE) was daily prepared and administrated to rats.

2.2. Chemicals

Formulation grade dimethoate (40%, Aragol L40, CHIMIC AGRI,Tunisia, Homologation I. 96060) was used. It was in the form of anemulsion and was diluted in saline in order to obtain an effectiveconcentration of 20 mg/kg body weight. The test concentration ofdimethoate was calculated from the percentage of the reactiveingredients. Solution was freshly made immediately before use.

2.3. Animals

This study was conducted on males, adult Wistar albino rats,180–200 g purchased from the Central Pharmacy (SIPHAT, Tunis,Tunisia). Before any experience, all animals were maintained 2weeks under the same laboratory conditions of temperature(2273 1C), relative humidity (5575%) and a 12/12 h light/darkcycle and received a nutritionally standard diet (SICO, Sfax,Tunisia) and tap water ad libitum.

The experimental procedures were carried out according to theNational Institute of Health Guidelines for Animal Care andapproved by the local Ethics Committee.

2.4. Experimental design

After an acclimation period, rats were randomly divided into sixgroups of ten each. The first group served as untreated control andreceived saline (0.9%, w/v) daily by oral gavage for 2 months. Thesecond group (D) received a daily oral dose of 20 mg/kg bodyweight of dimethoate in saline for 2 months. Rats in the third group(DNE) were given a daily oral dose of aqueous date extract of thevariety Deglet Nour (4 ml/kg) for 2 months. Rats of group four(DNE+D) were also administered daily DNE (4 ml/kg) 30 minbefore the administration of the daily oral dose of dimethoate(20 mg/kg) for 2 months. Animals of group five (Vit C+D) receiveda daily oral dose of vitamin C (100 mg/kg), 30 min before theadministration of the daily oral dose of dimethoate (20 mg/kg) for2 months. Rats of group six (D+DNE) were given the daily oral doseof dimethoate (20 mg/kg) for the first month. During the secondmonth the animals received DNE (4 ml/kg), 30 min after the dailyoral dose of dimethoate. The dose of dimethoate used in this studyrepresents 1/20 of the LD50 (380 mg/kg) which has been usedpreviously by other investigators since it is toxic but not lethal torats (Hagar and Fahmy, 2002; Sayim, 2007; Kamath et al., 2008).The DNE dose used in this study 4 ml/kg/day contains a sufficientamount of antioxidant compounds such as polyphenols which cangive a good protection against the toxicity caused by dimethoate(Saafi et al., 2009). The vitamin C dose used (100 mg/kg) gives aprotection against the toxicity (Ambali et al., 2007) and was usedlike a positive control for protection against dimethoate-inducedoxidative stress.

2.5. Preparation of serum and tissue extract

After completion of treatment period, blood samples fromrats were collected under anesthesia by cardiac puncture inheparinized tubes and centrifuged at 3000g for 15 min at 4 1C.Plasma samples were stored at �20 1C in aliquots until analysis.Livers were excised immediately, washed with ice-cold physio-logic saline solution (0.9%, w/v), blotted and weighed. Small

E.B. Saafi et al. / Experimental and Toxicologic Pathology 63 (2011) 433–441 435

representative slices were fixed in 10% buffered-neutral formalinfor routine histopathology. About 1 g of the remaining liver wascut into small pieces, homogenized with an Ultra Turraxhomogenizer in 2 ml ice-cold appropriate buffer (TBS, pH 7.4)and centrifuged at 9000g for 15 min at 4 1C. Supernatants (S1)were collected, aliquoted and stored at �80 1C until use forenzyme assays.

2.6. Biochemical assays

2.6.1. Biochemical indicators of liver function

Plasma aspartate aminotransferase (AST), alanine aminotran-saminase (ALT), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT) and total bilirubin and lactate dehydrogenase(LDH) activities were determined spectrophotometrically usingcommercial diagnostics kits (Biomaghreb, Tunisia; Randox,United Kingdom).

2.6.2. Measurement of TBARS levels

According to Buege and Aust (1978), lipid peroxidation wasestimated by measuring thiobarbituric acid reactive substances(TBARS) and expressed in terms of malondialdehyde (MDA)content. For the assay, 125 ml of supernatant (S1) were mixedwith 50 ml of saline buffer (TBS, pH 7.4), 125 ml of 20%trichloroacetic acid containing 1% butylhydroxytoluene andcentrifuged (1000g, 10 min, 4 1C). Then, 200 ml of supernatant(S2) was mixed with 40 ml of HCl (0.6 M) and 160 ml ofTris–thiobarbituric acid (120 mM) and the mixture was heatedat 80 1C for 10 min. The absorbance was measured at 530 nm. Theamount of TBARS was calculated using an extinction coefficient of1.56�10�5 M�1 cm�1 and expressed in nmol of MDA/mgprotein.

2.6.3. Catalase activity

Hepatic catalase activity was measured according to Aebi(1984). Hydrogen peroxide (H2O2) disappearance was monitoredkinetically at 240 nm for 1 min at 25 1C. The enzyme activity wascalculated using an extinction coefficient of 0.043 mM�1 cm�1.One unit of activity is equal to the mmol of H2O2 destroyed/min/mg protein.

2.6.4. Superoxide dismutase activity

Superoxide dismutase (SOD) activity in liver homogenate wasassayed spectrophotometrically as described by Beyer andFridovich (1987). This method is based on the capacity of SODto inhibit the oxidation of nitroblue tetrazolium (NBT). One unit ofSOD represents the amount of enzymes required to inhibit therate of NBT oxidation by 50% at 25 1C. The activity was expressedas units/mg protein.

2.6.5. Glutathione peroxidase activity (GPX)

GPX activity was assayed according to the method of Flohe andGunzler (1984). The activity was expressed as mmol of GSHoxidized/min/mg of protein, at 25 1C.

2.6.6. Protein content

Protein content in tissue extracts was determined according toLowry’s method (1951) using bovine serum albumin as standard.

2.7. Histopathological examination

Histological assessment was used to complete the study ofliver damage. For this purpose, each liver tissue was fixed in 10%buffered-neutral formalin, routinely processed, embedded inparaffin and sections of 5 mm thick were cut. Hematoxylin and

eosin (H&E) were used for staining. The sections were analyzed bya certified pathologist ignoring the sample assignments toexperimental groups. A minimum of three fields of each liverslide was morphologically evaluated.

2.8. Statistical analysis

The results were expressed as means 7standard deviation. Alldata were done with the Statistical Package for Social Sciences(SPSS 11.0 for windows). The results were analyzed using one wayanalysis of variance (ANOVA) followed by Duncan’s multiplerange test (DMRT) for comparison between different treatmentgroups. Statistical significance was set at po0.05.

3. Results

3.1. Growth performance and liver weight

During the experiment, rats in the control group (C) and in theDeglet Nour extract (DNE) treated group did not show any sign oftoxicity or death. However, dimethoate treated rats (D) showedvarying degrees of clinical signs few minutes after dosing. Thesigns included huddling, depression, conjunctivitis, mild tremor,piloerection diarrhea and dyspnea, and two rats died in thesecond and third weeks of dosing, respectively. The observedsigns were related to the cholinergic crisis; a consistent sign inacute organophosphate poisoning. Except for the huddling, noother significant clinical manifestation was observed in theDNE+D, vitamin C+D, and in the D+DNE-treated rats. However,death was observed in one of the rats in each of the groups by thethird week of dosing.

At the end of the experiment, control and dimethoate-treatedrats with or without Deglet Nour extract and vitamin C gainedweight (Table 1). The mean body weight gain of dimethoate-treated rats was 29.5871.89% against 40.2772.46% in controlrats (po0.05). Pretreatment of dimethoate-treated rats by DNEtends to ameliorate growth performance and body weight gain,which was 35.9972.59% (po0.05 when compared with control).Nevertheless, no significant changes were observed in parametercited above between (D), (vit C+D) and (D+DNE) groups.Throughout the 2 months, we found that food intake wasunchanged between all the groups of treated rats and theaverage was 7.6470.07 g/100 g/day.

On the other hand, results showed that oral administration ofdimethoate (D group), significantly decreased the absolute andthe relative liver weights compared with those of control group(po0.05). But no significant changes were observed between (D),(DNE+D), (vit C+D) and (D+DNE) groups at the end of theexperimental period (Table 1).

3.2. Biochemical indicators of liver function

Figs. 1–3 showed the plasma hepatic marker enzyme levelsand bilirubin of control and experimental rats. Oraladministration of dimethoate caused abnormal liver function intreated rats. The levels of plasma hepato-specific enzymes such asalanine transaminase (ALT), aspartate transaminase (AST),alkaline phosphatase (ALP), lactate dehydrogenase (LDH) andgamma glutamyl transferase (GGT) but not the level of totalbilirubin were significantly increased (po0.05) in dimethoate-intoxicated rats, when compared with control rats. Treatmentswith Deglet Nour extract (pretreatment and post-treatment) andwith vitamin C significantly (po0.05) restored these parameterswhen compared with dimethoate-alone-treated rats.

Table 1Effects of Deglet Nour extract and vitamin C on growth parameters of rats exposed to dimethoate.

Parameters and treatments Weight gain (%) Food intake (g/100 g b.w/d) Absolute liver weight (g) Relative liver weight (g/100 g b.w)

Control (C) 40.2772.46a 7.5170.15 9.9470.41a 3.5170.08a

Dimethoate (D) 29.5871.89b 7.8970.25 7.9770.33b 3.2570.06b

DNE 40.0072.75a 7.3970.20 9.0370.42ac 3.3870.08ab

DNE+D 35.9972.59ab 7.5870.10 7.9170.28b 3.2570.05b

Vit C+D 30.5872.09b 7.5870.14 8.1070.21bc 3.3870.08ab

D+DNE 29.0072.46b 7.8870.08 7.7370.33b 3.2870.07b

Values are mean 7 SD for ten rats in each group.a,b,c Values are not sharing a common superscript letter (a, b, c) differ significantly at po0.05 (DMRT).

a

b

a ac c

0

10

20

30

40

50

60

70

80

Control D DNE DNE+D Vit C+D D+DNE

ALT

(UI/L

)

a

b

a ac cd

0

20

40

60

80

100

120

140

160

180

Control D DNE DNE+D Vit C+D D+DNE

AS

T (U

I/L)

A

B

Fig 1. Effects of Deglet Nour extract and vitamin C on dimethoate-induced changes in hepatic functional markers: (A) plasma ALT and (B) plasma AST levels of rats. Data

are reported to mean 7 SD of ten animals in each group. a,b,c,dBars not sharing a common superscript letter (a, b, c, d) differ significantly at po0.05 (DMRT).

E.B. Saafi et al. / Experimental and Toxicologic Pathology 63 (2011) 433–441436

3.3. Lipid peroxidation of the liver

After a 2-month exposure to dimethoate, a significant increasein hepatic MDA levels occurred in the dimethoate-treated group,indicating an enhancement in the lipid peroxidation potential ofthe liver (po0.05) (Fig. 4). Although this increase did more thandouble, Deglet Nour extract and vitamin C administration todimethoate-treated rats showed an efficiency to attenuate MDAformation in the liver.

3.4. Activities of liver antioxidant enzymes

Results of liver antioxidant enzymes have been depicted inTable 2. The exposure of rats to dimethoate for 2 months caused asignificant increase in hepatic SOD and GPx activities comparedwith those of control group (po0.05). Conversely, dimethoate-rats pretreated orally with Deglet Nour extract or with vitamin Cshowed a spectacular restoration of these hepatic activities citedabove which attain control values (po0.05 when compared withdimethoate group). However, the post-treatment with Deglet

Nour extract after 1 month exposure to dimethoate (D+DNE)showed a little amelioration in the GPx and SOD activities.

For catalase activity, in contrast to SOD and GPx, the oraladministration of dimethoate for 2 months induced a markeddecrease of this activity (po0.05) (Table 2). So, this alterationslightly improved (po0.05) by pretreatment with DNE but notwith vitamin C or via post-treatment with DNE.

3.5. Histological assessment of the liver

In light microscopic examinations, histopathological changeswere observed in the livers of all experiment groups comparedwith those of controls. In the control and Deglet Nour extracttreated rats, normal liver histologic aspect with central vein andradiating hepatic cords was seen (Fig. 5A–C). Dimethoateintoxication exhibited severe histopathological changes such asmononuclear cells infiltration in the parenchymatous tissue andportal area (Fig. 6A), congestion, enlargement of the hepaticsinusoids (Fig. 6B) and enlargement of the central and the portalveins and hepatocellular damage. The parenchymatous cells

a

b

ac c c

0

50

100

150

200

250

Control D DNE DNE+D Vit C+D D+DNE

ALP

(UI/L

)

a

b

a

c

d d

0

2

4

6

8

10

12

14

Control D DNE DNE+D VitC+D D+DNE

GG

T (U

I/L)

a

b

a ac c

0

100

200

300

400

500

600

700

800

900

Control D DNE DNE+D Vit C+D D+DNE

LDH

(UI/L

)

B

C

A

Fig. 2. Effects of Deglet Nour extract and vitamin C on dimethoate-induced changes in hepatic functional markers: (A) plasma ALP, (B) plasma GGT and (C) plasma LDH

levels of rats. Data are reported to mean 7 SD of ten animals in each group. a,b,c,dBars not sharing a common superscript letter (a, b, c, d) differ significantly at po0.05

(DMRT).

E.B. Saafi et al. / Experimental and Toxicologic Pathology 63 (2011) 433–441 437

showed cytoplasmic vacuolization and degeneration of nuclei(Fig. 6C). An increase in the number of Kupffer cells in the liverparenchyma was also observed. In contrast, the histologicalexamination of tissue sections from rats exposed to dimethoateand pretreated with DNE (Fig. 7A) or with vitamin C (Fig. 7B)showed an improvement of liver morphology except for mildinflammation. Necrotic cells and vacuolization are nearly absent.Rats post-treated with DNE (D+DNE group) present a similarhistopathological change compared with dimethoate treated ratswith attenuate severity (Fig. 7C).

4. Discussion

This study was undertaken to determine whether a dietaryregimen reinforced with date palm fruit extract (Phoenix

dactylifera L.) could attenuate some of the toxic effects of dimeth-oate (20 mg/kg/day) in Wistar rats treated for 2 months. Duringthe experiment, the clinical signs observed in the dimethoategroup were consistent with cholinergic symptoms associatedwith cholinesterase inhibition; the principal mode of action oforganophosphorus compounds (Pope et al., 1991; Sarkar et al.,2003; Sharma et al., 2005b). The signs observed in this group werethe most severe in comparison to those observed in DNE pre andpost-treated rat and in those pretreated with vitamin C. Thereduction in the severity of clinical signs reveals that oxidativestress played a primary role in the toxicity induced by pesticides(Khan et al., 2005). Observation in the present study demonstratesthat the subchronic dimethoate administration produced toxicityin rats as monitored by weight loss and decrease in absolute andrelative liver weight. These observations were in accordance withthose obtained by previous studies (Sayim, 2007; Fetoui et al.,

0

1

2

3

4

5

6

7

Control D DNE DNE+D Vit C+D D+DNE

Bili

rubi

n (u

mol

/L)

Fig. 3. Effects of Deglet Nour extract and vitamin C on total plasma bilirubin of

rats exposed to dimethoate. Data are reported to mean 7 SD of ten animals in

each group. No significant differences were observed in this parameter between

the groups.

ab

c

a

dbd d

0.00

0.05

0.10

0.15

0.20

0.25

0.30

C D DNE DNE+D Vit C+D D+DNE

nmol

MD

A/m

g pr

otei

n

Fig. 4. Effects of Deglet Nour extract and vitamin C on the MDA levels in rat liver

after dimethoate administration for 2 months. Data are reported to mean 7 SD of

ten animals in each group. a,b,c,dBars not sharing a common superscript letter (a, b,

c, d) differ significantly at po0.05 (DMRT).

Table 2Effects of Deglet Nour extract and vitamin C on antioxidant enzyme activities

(SOD, GPx and CAT) on rat liver exposed to dimethoate.

Parameters SODx GPxy CATz

Control (C) 3.6470.17a 6.8070.37a 521.17718.00a

Dimethoate (D) 4.6170.26b 8.0370.50b 431.06717.00b

DNE 3.6070.11a 6.5670.23a 527.90716.36a

DNE+D 3.9570.20ac 7.0170.37ab 490.1079.54ac

Vit C+D 3.8570.24ac 6.7570.28a 458.2677.03bc

D+DNE 4.4470.20bc 7.2970.32ab 442.25719.64b

Values are mean 7SD for ten rats in each group.a,b,c Values are not sharing a common superscript letter (a, b, c) differ significantly

at po0.05 (DMRT).

x Units/mg protein.y mmol of GSH oxidized/min/mg protein.z mmol H2O2 degraded/min/mg protein.

C

A

CV

B

Fig. 5. Normal liver histologic aspect from a control ((A) 20� and (B) 50� ) and

DNE extract treated rats ((C) 50� ). It is composed of hexagonal or pentagonal

lobules with central veins (CV) and peripheral hepatic triads embedded in

connective tissue. Hepatocytes are arranged in trabecules running radiantly from

the central vein and are separated by sinusoids containing Kuppfer cells.

E.B. Saafi et al. / Experimental and Toxicologic Pathology 63 (2011) 433–441438

2009). However, pretreatment with DNE or with vitamin C causeda significant increase in weight and on absolute and relative liverweight. Therefore, the weight loss observed in the dimethoate-treated group may be a result of the combination of cholinergicand oxidative stress.

Our results have also shown that oral administration ofdimethoate-induced hepatotoxicity. This is clearly evident bysubstantial augmentation in plasma levels of transaminases, ALP,LDH and GGT. Besides a significant increase of MDA levels wasregistered revealing an increase in the lipid peroxidation potentialof the liver accompanied by histological alteration includingmono- and poly-nuclear cell infiltration in the portal area addedto congestion and necrosis in the liver. In addition, the results

have shown an intense cytoplasm vacuolization, enlargement ofthe sinusoids and veins, increase in the number of Kuppfer cellsand hepatocellular damage in the parenchymatous tissue.

Our results are in agreement with similar data reported indifferent experimental models of rats exposed to dimethoate andother pesticides (Selmanoglu and Akay, 2000; Sivapiriya et al., 2006;Sayim, 2007; Fetoui et al., 2009) and confirm the pathogenic role ofoxidative stress in the liver. Several studies illustrate the mechanismby which dimethoate and OPI in general, could promote oxidativestress. Sharma et al. (2005b) proved that dimethoate acts as aninducer of P450 isoenzyme. This induction of P450 enzyme systemmay be responsible for dimethoate’s increased biotransformation toP¼O analogue (Kaloyanova et al., 1984). The dimethoate-inducedenhancement in liver microsomal Cytochrome P450 content andoxygen radical production together with an augmented lipidperoxidation index as made evident by the significant increase inTBARS detected in the liver. Lipid peroxidation explain a number ofdeleterious effects such as increased membrane rigidity, osmoticfragility, decreased cellular deformation, reduced erythrocyte survivaland membrane fluidity (Thampi et al., 1991). The increase in thelevels of TBARS indicates an enhanced lipid peroxidation leading totissue injury and failure of the antioxidant defence mechanisms toprevent the formation of excess free radicals (Comporti, 1985). Theprotective action of antioxidant may be due to an inhibition ofreactive oxygen species (ROS) inducing a chain reaction mediated by

A

B

C

Fig. 6. Liver from dimethoate-treated rats. Panel A (H&E 50� ): photomicrograph

of mononuclear cells infiltration in portal triad region. Panel B (H&E 50� ):

photomicrograph of degenerated hepatocytes, focal necrosis, and - conges-

tion and enlargement at sinusoids in liver. Panel C (H&E 100� ): photomicrograph

of abundance cytoplasm vacuolization in parenchymatous cells of the liver.

A

B

C

Fig. 7. Effects of Deglet Nour extract and vitamin C on dimethoate-induced

hepatic injury in rats (H&E stain 50� ): (A) DNE+dimethoate group shows hepatic

aspect similar to the control group, steatosis and necrosis were absent. (B) Vitamin

C + dimethoate group shows hepatic aspect similar to the control group except for

a slight infiltration of inflammatory cells. (C) Dimethoate + DNE-treated rat liver

shows a similar histopathological change compared with dimethoate treated rats

with attenuate severity.

E.B. Saafi et al. / Experimental and Toxicologic Pathology 63 (2011) 433–441 439

several antioxidant enzymes including SOD, GPx and catalase. In thecurrent study, the significant increase in SOD and Gpx hepaticactivities after dimethoate treatment showed an activation of thecompensatory mechanism through the effect of the insecticide onprogenitor cells, and its extent depends on the magnitude of theoxidative stress and hence on the dose of stressor (Prakasam et al.,2001). However, the decrease of catalase activity may cause theaccumulation of the O�2 , H2O2 or their product of decomposition. Lossof catalase activity results in oxygen intolerance and triggers anumber of deleterious reactions such as protein and DNA oxidation,and cell death (Halliwell and Gutteridge, 1999). Several reportssuggest that natural antioxidants constitute efficient treatment oftoxicity induced by xenobiotics. Nonenzymatic antioxidants such asvitamins E and C, and polyphenolic compounds represent some ofthese natural antioxidants that could act to overcome the oxidativestress. In addition, the results of our study show that pretreatmentwith date palm fruit extract (DNE+D group) fixes the liver damagecaused by dimethoate exposure as revealed by remarkable decreasein plasma ALT, AST, and LDH levels. Moreover, the results have shownthat DNE has a high potent protective effect against oxidative stress;as demonstrated by the significant decrease of lipid peroxidation, aswell as the amelioration of enzymes’ antioxidant status and by

normal liver morphology except for mild inflammation. Similarly,pretreatment with vitamin C, has shown the same protection againstdimethoate exposure but at a lower degree. Furthermore, post-treatment with DNE after 1 month of exposure to dimethoate did notshow total protection of the liver. Similarly to rats exposed todimethoate alone, some parameters of toxicity such as the increase inSOD activity and histological (mild vacuolization and inflammation)were present. This result was attributed to the excessive toxicityrevealed by dimethoate treatment in the first month. Perhaps, if post-treatment with DNE were prolonged, the liver damage could beutterly restored.

The mechanism by which the aqueous date palm fruitextract induces its hepato-protective activity against oxidativedamage caused by dimethoate is not certain. However, it ispossible that polyphenolic compounds (flavonoids, anthocyaninsand phenolic acids), and trace elements (selenium, copper,zinc and manganese), in addition to vitamin C present in thedate palm fruit (Al-Farsi et al., 2005a, 2005b; Mansouri et al.,2005; Hong et al., 2006; Allaith, 2007; Saafi et al., 2009) are theresponsible compounds for this protection. In fact, Vayalil (2002)proved the antioxidant and the antimutagenic activity of theaqueous date palm fruit extract, as monitored by the inhibitionof lipid peroxidation and protein oxidation and also by theaptitude to scavenge superoxide and hydroxyl radicals in vitro.

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The antioxidant mechanism of DNE may be related to the abilityof its active compounds to detoxify free radicals and to inhibitlipid peroxidation in the liver. Anti-inflammatory effect ofpolyphenols is also demonstrated by its ability to inhibit theproduction of nitric oxide and tumor necrosis factor a (TNF-a)(Kawada et al., 1998).

In conclusion, the present study demonstrates the capacity of theaqueous date palm fruit extract to heal the hepatotoxicity and cellulardamage in rat liver after subchronic exposure to dimethoate.However, the accurate mechanism is not yet clear. To be able topropose the potential therapeutic use of date palm fruit in preventingthe liver from xenobiotic-induced oxidative damage further studiesare needed. Muslims believe that ‘‘seven dates every morning keeppoison or magic away’’ (cited by Miller et al., 2003).

Acknowledgments

This work was supported by a grant from the Tunisian MinistryHigher Education, Scientific Research and Technology trough theResearch Unit of Human Nutrition and Metabolic DisordersUR03ES08. We would like to thank Pr. Zohra Haouas andDr. Fadwa Hssin for their technical assistance and Mr. FethiChahata for his constructive criticism of manuscript.

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