Experimental and Toxicologic Pathology 63 (2011) 433–441

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Experimental and Toxicologic Pathology

journal homepage: www.elsevier.de/etp

Protective effect of date palm fruit extract (Phoenix dactylifera L.) on

dimethoate induced-oxidative stress in rat liver
Emna Behija Saafi a, Mouna Louedi a, Abdelfattah Elfeki b, Abdelfattah Zakhama c, Mohamed
Fadhel Najjar d, Mohamed Hammami a, Lotfi Achour e,n
a
Laboratoire de Biochimie, UR Nutrition Humaine et Désordres Métaboliques, Faculté de Médecine, 5000 Monastir, Tunisia
b
Laboratoire d’Ecophysiologie Animale, Faculté de Sciences, 3018 Sfax, Tunisia
c
Laboratoire d’Anatomopathologie, Faculté de Médecine, 5000 Monastir, Tunisia
d
Laboratoire de Biochimie, Hôpital Universitaire, CHU Fattouma Bourguiba, 5000 Monastir Tunisia
e
Institut Supérieur de Biotechnologie, Avenue Tahar Hadded, BP 74, 5019 Monastir, Tunisia

a r t i c l e in fo abstract

Article history: Nowadays, people’s exposure to chemical compounds such as organophosphorus insecticides is
Received 5 March 2009 continuously on the rise more and more. Theses compounds have induced an excessive production of
Accepted 7 March 2010 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
Keywords: compounds. In this study, we investigate the role of date palm fruit extract (Phoenix dactylifera L.) in
Oxidative stress protection against oxidative damage and hepatotoxicity induced by subchronic exposure to dimethoate
Liver (20 mg/kg/day). Oral administration of dimethoate caused hepatotoxicity as monitored by the increase
Dimethoate in the levels of hepatic markers enzymes (transaminases, alkaline phosphatase, gamma-glutamyl
Date fruit (Phoenix dactylifera L.) extract
transferase and lactate dehydrogenase), as well as in hepatic malondialdehyde thus causing drastic
Antioxidant
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 alike. Organophosphorus insecticides (OPI) represent one group of

For several years, a special attention has been paid to oxidative
stress; situation of an excessive production of reactive oxygen
species (e.g. the famous ‘‘free radical’’) in the organism. A large
number of experimental and epidemiological studies have
indicated that the reactive oxygen species (ROS) contribute to
organ 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 is
accelerated by accidental exposure to occupational chemicals like
pesticides. Nowadays the hazards of using such chemical
compounds have been accentuated by the sharp rise in their use
by house-holders and governments, farmers and industrialists
n
Corresponding author. Tel.: + 216 73 465 405; fax: + 216 73 465 404.
E-mail address: lotfiachour@yahoo.fr (L. Achour).
pesticides that is widely used and has proved to have toxic effects
in 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 a
large number of crops against several pests. Dimethoate poison-
ing is usually associated with neuromuscular transmission block
in both animals and humans (De-Bleecker et al., 1993; Dongren
et al., 1999). Immunotoxicological effects due to dimethoate have
also been reported (Institoris et al., 1999). Moreover, dimethoate
induce hyperglycemia and cause various toxic effects on rat
pancreas following acute, subchronic and chronic exposure
(Hagar and Fahmy, 2002; Kamath and Rajini, 2007; Kamath
et al., 2008). Earlier studies have shown that acute and subchronic
exposure to dimethoate alters the antioxidant status and the
histology 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

0940-2993/$ - see front matter & 2010 Elsevier GmbH. All rights reserved.
doi:10.1016/j.etp.2010.03.002
434 E.B. Saafi et al. / Experimental and Toxicologic Pathology 63 (2011) 433–441
(Banerjee et al., 2001; Sivapiriya et al., 2006) and it has been
demonstrated that lipid peroxidation mediated by free radicals
is one of the molecular mechanisms involved in OPI-induced
toxicity (Akhgari et al., 2003). The cellular antioxidant status
determines the susceptibility to oxidative damage and is usually
altered in response to oxidative stress. The cellular antioxidant
pool comprises antioxidant free radical scavenging enzymes like
catalase (CAT), superoxide dismutase (SOD) and glutathione
peroxidase (GPx) (Pincemail et al., 2002). The cellular antioxidant
action 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 of
natural antioxidants as a strategy to prevent oxidative damage in
various health disorders with oxidative stress as a factor in their
pathophysiology (Khan et al., 2005; Koechlin-Ramonatxo, 2006;
Kasdallah-Grissa et al., 2007; Mehmetc- ik et al., 2008; Shireen
et al., 2008).
Fruits of the date palm (Phoenix dactylifera L.) are very
commonly consumed in many parts of the world and a vital
component of the diet and a staple food in most of the Arabian
countries. The Deglet Nour is an important date variety that
makes up about 62.5% of Tunisia date production (GIF, 2008).
Dates are rich in certain nutrients and provide a good source of
rapid energy due to their high carbohydrate content (  70–80%).
Most of the carbohydrates in dates are in the form of fructose and
glucose, which are easily absorbed by the human body (Al-Hooti
et 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 their
dietary 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 the
treatment of various infectious diseases and cancer (Duke, 1992).
Experimentally, it has been shown that, depending on the type of
extract used, date fruit and pit extracts significantly increase or
decrease gastrointestinal transit in mice (Al-Qarawi et al., 2003).
Moreover, the aqueous and ethanolic extracts, were effective in
ameliorating the severity of gastric ulceration in rats (Al-Qarawi
et al., 2005). Researchers also, found that the consumption of
dates might be of benefit in glycaemic and lipid control of diabetic
patients (Miller et al., 2003). Recent studies have indicate that the
aqueous 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 the
wide 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 morning
will not be affected by poison or magic on the day he eats them’’
(cited by Miller et al., 2003). Accordingly, we hypothesized that
date extract may prevent the oxidative stress and the hepatoxicity
in rats induced by dimethoate.
2. Materials and methods
2.1. Date palm extract preparation
Fresh ripened Deglet Nour variety was collected from the
station of Douz (Kébili, Tunisia). Fruit flesh was extracted two
times with distilled water (1/10, w/v) by grinding with a mortar
and pestle. It was centrifuged at 4 1C for 20 min at 4000g and the
supernatant was collected. We selected an aqueous extract
because most of the antioxidant components in dates are
extracted in water (Vayalil, 2002; Al-Farsi et al., 2005b).
During the experience, the aqueous date fruit extract of Deglet
Nour (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 an
emulsion and was diluted in saline in order to obtain an effective
concentration of 20 mg/kg body weight. The test concentration of
dimethoate was calculated from the percentage of the reactive
ingredients. 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 2
weeks under the same laboratory conditions of temperature
(2273 1C), relative humidity (5575%) and a 12/12 h light/dark
cycle and received a nutritionally standard diet (SICO, Sfax,
Tunisia) and tap water ad libitum.
The experimental procedures were carried out according to the
National Institute of Health Guidelines for Animal Care and
approved by the local Ethics Committee.
2.4. Experimental design
After an acclimation period, rats were randomly divided into six
groups of ten each. The first group served as untreated control and
received saline (0.9%, w/v) daily by oral gavage for 2 months. The
second group (D) received a daily oral dose of 20 mg/kg body
weight of dimethoate in saline for 2 months. Rats in the third group
(DNE) were given a daily oral dose of aqueous date extract of the
variety Deglet Nour (4 ml/kg) for 2 months. Rats of group four
(DNE+D) were also administered daily DNE (4 ml/kg) 30 min
before the administration of the daily oral dose of dimethoate
(20 mg/kg) for 2 months. Animals of group five (Vit C+D) received
a daily oral dose of vitamin C (100 mg/kg), 30 min before the
administration of the daily oral dose of dimethoate (20 mg/kg) for
2 months. Rats of group six (D+DNE) were given the daily oral dose
of dimethoate (20 mg/kg) for the first month. During the second
month the animals received DNE (4 ml/kg), 30 min after the daily
oral dose of dimethoate. The dose of dimethoate used in this study
represents 1/20 of the LD50 (380 mg/kg) which has been used
previously by other investigators since it is toxic but not lethal to
rats (Hagar and Fahmy, 2002; Sayim, 2007; Kamath et al., 2008).
The DNE dose used in this study 4 ml/kg/day contains a sufficient
amount of antioxidant compounds such as polyphenols which can
give a good protection against the toxicity caused by dimethoate
(Saafi et al., 2009). The vitamin C dose used (100 mg/kg) gives a
protection against the toxicity (Ambali et al., 2007) and was used
like a positive control for protection against dimethoate-induced
oxidative stress.
2.5. Preparation of serum and tissue extract
After completion of treatment period, blood samples from
rats were collected under anesthesia by cardiac puncture in
heparinized 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
representative slices were fixed in 10% buffered-neutral formalin
for routine histopathology. About 1 g of the remaining liver was
cut into small pieces, homogenized with an Ultra Turrax
homogenizer 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 for
enzyme 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-glutamyl
transferase (GGT) and total bilirubin and lactate dehydrogenase
(LDH) activities were determined spectrophotometrically using
commercial diagnostics kits (Biomaghreb, Tunisia; Randox,
United Kingdom).
2.6.2. Measurement of TBARS levels
According to Buege and Aust (1978), lipid peroxidation was
estimated by measuring thiobarbituric acid reactive substances
(TBARS) and expressed in terms of malondialdehyde (MDA)
content. For the assay, 125 ml of supernatant (S1) were mixed
with 50 ml of saline buffer (TBS, pH 7.4), 125 ml of 20%
trichloroacetic acid containing 1% butylhydroxytoluene and
centrifuged (1000g, 10 min, 4 1C). Then, 200 ml of supernatant
(S2) was mixed with 40 ml of HCl (0.6 M) and 160 ml of
Tris–thiobarbituric acid (120 mM) and the mixture was heated
at 80 1C for 10 min. The absorbance was measured at 530 nm. The
amount of TBARS was calculated using an extinction coefficient of
1.56  10  5 M  1 cm  1 and expressed in nmol of MDA/mg
protein.
2.6.3. Catalase activity
Hepatic catalase activity was measured according to Aebi
(1984). Hydrogen peroxide (H2O2) disappearance was monitored
kinetically at 240 nm for 1 min at 25 1C. The enzyme activity was
calculated 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 was
assayed spectrophotometrically as described by Beyer and
Fridovich (1987). This method is based on the capacity of SOD
to inhibit the oxidation of nitroblue tetrazolium (NBT). One unit of
SOD represents the amount of enzymes required to inhibit the
rate of NBT oxidation by 50% at 25 1C. The activity was expressed
as units/mg protein.
2.6.5. Glutathione peroxidase activity (GPX)
GPX activity was assayed according to the method of Flohe and
Gunzler (1984). The activity was expressed as mmol of GSH
oxidized/min/mg of protein, at 25 1C.
2.6.6. Protein content
Protein content in tissue extracts was determined according to
Lowry’s method (1951) using bovine serum albumin as standard.
2.7. Histopathological examination
Histological assessment was used to complete the study of
liver damage. For this purpose, each liver tissue was fixed in 10%
buffered-neutral formalin, routinely processed, embedded in
paraffin and sections of 5 mm thick were cut. Hematoxylin and
435
eosin (H&E) were used for staining. The sections were analyzed by
a certified pathologist ignoring the sample assignments to
experimental groups. A minimum of three fields of each liver
slide was morphologically evaluated.
2.8. Statistical analysis
The results were expressed as means 7standard deviation. All
data were done with the Statistical Package for Social Sciences
(SPSS 11.0 for windows). The results were analyzed using one way
analysis of variance (ANOVA) followed by Duncan’s multiple
range test (DMRT) for comparison between different treatment
groups. Statistical significance was set at p o0.05.
3. Results
3.1. Growth performance and liver weight
During the experiment, rats in the control group (C) and in the
Deglet Nour extract (DNE) treated group did not show any sign of
toxicity or death. However, dimethoate treated rats (D) showed
varying degrees of clinical signs few minutes after dosing. The
signs included huddling, depression, conjunctivitis, mild tremor,
piloerection diarrhea and dyspnea, and two rats died in the
second and third weeks of dosing, respectively. The observed
signs were related to the cholinergic crisis; a consistent sign in
acute organophosphate poisoning. Except for the huddling, no
other significant clinical manifestation was observed in the
DNE+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 the
third week of dosing.
At the end of the experiment, control and dimethoate-treated
rats with or without Deglet Nour extract and vitamin C gained
weight (Table 1). The mean body weight gain of dimethoate-
treated rats was 29.58 71.89% against 40.2772.46% in control
rats (p o0.05). Pretreatment of dimethoate-treated rats by DNE
tends to ameliorate growth performance and body weight gain,
which was 35.99 72.59% (po0.05 when compared with control).
Nevertheless, no significant changes were observed in parameter
cited above between (D), (vit C +D) and (D + DNE) groups.
Throughout the 2 months, we found that food intake was
unchanged between all the groups of treated rats and the
average was 7.647 0.07 g/100 g/day.
On the other hand, results showed that oral administration of
dimethoate (D group), significantly decreased the absolute and
the relative liver weights compared with those of control group
(p o0.05). But no significant changes were observed between (D),
(DNE+D), (vit C+ D) and (D + DNE) groups at the end of the
experimental period (Table 1).
3.2. Biochemical indicators of liver function
Figs. 1–3 showed the plasma hepatic marker enzyme levels
and bilirubin of control and experimental rats. Oral
administration of dimethoate caused abnormal liver function in
treated rats. The levels of plasma hepato-specific enzymes such as
alanine transaminase (ALT), aspartate transaminase (AST),
alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and
gamma glutamyl transferase (GGT) but not the level of total
bilirubin were significantly increased (p o0.05) in dimethoate-
intoxicated rats, when compared with control rats. Treatments
with Deglet Nour extract (pretreatment and post-treatment) and
with vitamin C significantly (p o0.05) restored these parameters
when compared with dimethoate-alone-treated rats.
436 E.B. Saafi et al. / Experimental and Toxicologic Pathology 63 (2011) 433–441

Table 1
Effects 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) 40.27 7 2.46a 7.51 70.15 9.94 7 0.41a 3.51 7 0.08a
Dimethoate (D) 29.58 7 1.89b 7.89 70.25 7.97 7 0.33b 3.25 7 0.06b
DNE 40.00 72.75a 7.39 70.20 9.03 7 0.42ac 3.38 7 0.08ab
DNE + D 35.99 7 2.59ab 7.58 70.10 7.91 7 0.28b 3.25 7 0.05b
Vit C + D 30.58 7 2.09b 7.58 70.14 8.10 7 0.21bc 3.38 7 0.08ab
D +DNE 29.00 7 2.46b 7.88 70.08 7.73 7 0.33b 3.28 7 0.07b

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 p o 0.05 (DMRT).

80
b
A
70
c
60 c
a a
50 a
ALT (UI/L)

40

30

20

10

0
Control D DNE DNE+D Vit C+D D+DNE
180
B b
160
140
d
120
AST (UI/L)

ac c
100 a a
80
60
40
20
0
Control D DNE DNE+D Vit C+D D+DNE

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 p o 0.05 (DMRT).

3.3. Lipid peroxidation of the liver
After a 2-month exposure to dimethoate, a significant increase
in hepatic MDA levels occurred in the dimethoate-treated group,
indicating an enhancement in the lipid peroxidation potential of
the liver (p o0.05) (Fig. 4). Although this increase did more than
double, Deglet Nour extract and vitamin C administration to
dimethoate-treated rats showed an efficiency to attenuate MDA
formation in the liver.
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 oral
administration of dimethoate for 2 months induced a marked
decrease of this activity (p o0.05) (Table 2). So, this alteration
slightly improved (p o0.05) by pretreatment with DNE but not
with vitamin C or via post-treatment with DNE.
3.5. Histological assessment of the liver

3.4. Activities of liver antioxidant enzymes
Results of liver antioxidant enzymes have been depicted in
Table 2. The exposure of rats to dimethoate for 2 months caused a
significant increase in hepatic SOD and GPx activities compared
with those of control group (p o0.05). Conversely, dimethoate-
rats pretreated orally with Deglet Nour extract or with vitamin C
showed a spectacular restoration of these hepatic activities cited
above which attain control values (p o0.05 when compared with
dimethoate group). However, the post-treatment with Deglet
In light microscopic examinations, histopathological changes
were observed in the livers of all experiment groups compared
with those of controls. In the control and Deglet Nour extract
treated rats, normal liver histologic aspect with central vein and
radiating hepatic cords was seen (Fig. 5A–C). Dimethoate
intoxication exhibited severe histopathological changes such as
mononuclear cells infiltration in the parenchymatous tissue and
portal area (Fig. 6A), congestion, enlargement of the hepatic
sinusoids (Fig. 6B) and enlargement of the central and the portal
veins and hepatocellular damage. The parenchymatous cells
 E.B. Saafi et al. / Experimental and Toxicologic Pathology 63 (2011) 433–441 437

250
A
b
200
c c c
a

ALP (UI/L)
150 a

100

50

0
Control D DNE DNE+D Vit C+D D+DNE
14
B b
12
d d
10
c
GGT (UI/L)

8
a a
6

0
Control D DNE DNE+D VitC+D D+DNE
900 b
800
C c
c
700 a a a
600
LDH (UI/L)

500
400
300
200
100
0
Control D DNE DNE+D Vit C+D D+DNE

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 p o 0.05
(DMRT).

showed cytoplasmic vacuolization and degeneration of nuclei
(Fig. 6C). An increase in the number of Kupffer cells in the liver
parenchyma was also observed. In contrast, the histological
examination of tissue sections from rats exposed to dimethoate
and pretreated with DNE (Fig. 7A) or with vitamin C (Fig. 7B)
showed an improvement of liver morphology except for mild
inflammation. Necrotic cells and vacuolization are nearly absent.
Rats post-treated with DNE (D +DNE group) present a similar
histopathological change compared with dimethoate treated rats
with attenuate severity (Fig. 7C).
4. Discussion
This study was undertaken to determine whether a dietary
regimen 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. During
the experiment, the clinical signs observed in the dimethoate
group were consistent with cholinergic symptoms associated
with cholinesterase inhibition; the principal mode of action of
organophosphorus compounds (Pope et al., 1991; Sarkar et al.,
2003; Sharma et al., 2005b). The signs observed in this group were
the most severe in comparison to those observed in DNE pre and
post-treated rat and in those pretreated with vitamin C. The
reduction in the severity of clinical signs reveals that oxidative
stress played a primary role in the toxicity induced by pesticides
(Khan et al., 2005). Observation in the present study demonstrates
that the subchronic dimethoate administration produced toxicity
in rats as monitored by weight loss and decrease in absolute and
relative liver weight. These observations were in accordance with
those obtained by previous studies (Sayim, 2007; Fetoui et al.,
438 E.B. Saafi et al. / Experimental and Toxicologic Pathology 63 (2011) 433–441

7
A
6

5
Bilirubin (umol/L)

2
CV
1

0
Control D DNE DNE+D Vit C+D D+DNE
B
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.

0.30

0.25 c
nmol MDA/mg protein

0.20 d d
bd
0.15
ab C
0.10 a

0.05

0.00
C D DNE DNE+D Vit C+D D+DNE

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 po 0.05 (DMRT).
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
Table 2 lobules with central veins (CV) and peripheral hepatic triads embedded in
Effects of Deglet Nour extract and vitamin C on antioxidant enzyme activities connective tissue. Hepatocytes are arranged in trabecules running radiantly from
(SOD, GPx and CAT) on rat liver exposed to dimethoate. the central vein and are separated by sinusoids containing Kuppfer cells.

Parameters SODx GPxy CATz

Control (C) 3.64 70.17a 6.80 70.37a 521.177 18.00a have shown an intense cytoplasm vacuolization, enlargement of
Dimethoate (D) 4.61 70.26b 8.03 70.50b 431.06 7 17.00b the sinusoids and veins, increase in the number of Kuppfer cells
DNE 3.607 0.11a 6.56 70.23a 527.90 7 16.36a and hepatocellular damage in the parenchymatous tissue.
DNE + D 3.95 70.20ac 7.01 70.37ab 490.10 79.54ac Our results are in agreement with similar data reported in
Vit C + D 3.85 70.24ac 6.75 70.28a 458.267 7.03bc
D + DNE 4.44 70.20bc 7.29 70.32ab 442.257 19.64b
different experimental models of rats exposed to dimethoate and
other pesticides (Selmanoglu and Akay, 2000; Sivapiriya et al., 2006;
Values are mean 7 SD for ten rats in each group. Sayim, 2007; Fetoui et al., 2009) and confirm the pathogenic role of
a,b,c
Values are not sharing a common superscript letter (a, b, c) differ significantly oxidative stress in the liver. Several studies illustrate the mechanism
at po 0.05 (DMRT).
by which dimethoate and OPI in general, could promote oxidative
x
Units/mg protein. stress. Sharma et al. (2005b) proved that dimethoate acts as an
y
mmol of GSH oxidized/min/mg protein.
z inducer of P450 isoenzyme. This induction of P450 enzyme system
mmol H2O2 degraded/min/mg protein.
may be responsible for dimethoate’s increased biotransformation to
2009). However, pretreatment with DNE or with vitamin C caused P¼ O analogue (Kaloyanova et al., 1984). The dimethoate-induced
a significant increase in weight and on absolute and relative liver enhancement in liver microsomal Cytochrome P450 content and
weight. Therefore, the weight loss observed in the dimethoate- oxygen radical production together with an augmented lipid
treated group may be a result of the combination of cholinergic peroxidation index as made evident by the significant increase in
and oxidative stress. TBARS detected in the liver. Lipid peroxidation explain a number of
Our results have also shown that oral administration of deleterious effects such as increased membrane rigidity, osmotic
dimethoate-induced hepatotoxicity. This is clearly evident by fragility, decreased cellular deformation, reduced erythrocyte survival
substantial augmentation in plasma levels of transaminases, ALP, and membrane fluidity (Thampi et al., 1991). The increase in the
LDH and GGT. Besides a significant increase of MDA levels was levels of TBARS indicates an enhanced lipid peroxidation leading to
registered revealing an increase in the lipid peroxidation potential tissue injury and failure of the antioxidant defence mechanisms to
of the liver accompanied by histological alteration including prevent the formation of excess free radicals (Comporti, 1985). The
mono- and poly-nuclear cell infiltration in the portal area added protective action of antioxidant may be due to an inhibition of
to congestion and necrosis in the liver. In addition, the results reactive oxygen species (ROS) inducing a chain reaction mediated by
 E.B. Saafi et al. / Experimental and Toxicologic Pathology 63 (2011) 433–441
A A
B
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.
several antioxidant enzymes including SOD, GPx and catalase. In the
current study, the significant increase in SOD and Gpx hepatic
activities after dimethoate treatment showed an activation of the
compensatory mechanism through the effect of the insecticide on
progenitor cells, and its extent depends on the magnitude of the
oxidative stress and hence on the dose of stressor (Prakasam et al.,
2001). However, the decrease of catalase activity may cause the
accumulation of the O2 , H2O2 or their product of decomposition. Loss
of catalase activity results in oxygen intolerance and triggers a
number of deleterious reactions such as protein and DNA oxidation,
and cell death (Halliwell and Gutteridge, 1999). Several reports
suggest that natural antioxidants constitute efficient treatment of
toxicity induced by xenobiotics. Nonenzymatic antioxidants such as
vitamins E and C, and polyphenolic compounds represent some of
these natural antioxidants that could act to overcome the oxidative
stress. In addition, the results of our study show that pretreatment
with date palm fruit extract (DNE+D group) fixes the liver damage
caused by dimethoate exposure as revealed by remarkable decrease
in plasma ALT, AST, and LDH levels. Moreover, the results have shown
that DNE has a high potent protective effect against oxidative stress;
as demonstrated by the significant decrease of lipid peroxidation, as
well as the amelioration of enzymes’ antioxidant status and by
439
B
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.
normal liver morphology except for mild inflammation. Similarly,
pretreatment with vitamin C, has shown the same protection against
dimethoate exposure but at a lower degree. Furthermore, post-
treatment with DNE after 1 month of exposure to dimethoate did not
show total protection of the liver. Similarly to rats exposed to
dimethoate alone, some parameters of toxicity such as the increase in
SOD activity and histological (mild vacuolization and inflammation)
were present. This result was attributed to the excessive toxicity
revealed by dimethoate treatment in the first month. Perhaps, if post-
treatment with DNE were prolonged, the liver damage could be
utterly restored.
The mechanism by which the aqueous date palm fruit
extract induces its hepato-protective activity against oxidative
damage caused by dimethoate is not certain. However, it is
possible that polyphenolic compounds (flavonoids, anthocyanins
and phenolic acids), and trace elements (selenium, copper,
zinc and manganese), in addition to vitamin C present in the
date palm fruit (Al-Farsi et al., 2005a, 2005b; Mansouri et al.,
2005; Hong et al., 2006; Allaith, 2007; Saafi et al., 2009) are the
responsible compounds for this protection. In fact, Vayalil (2002)
proved the antioxidant and the antimutagenic activity of the
aqueous date palm fruit extract, as monitored by the inhibition
of lipid peroxidation and protein oxidation and also by the
aptitude to scavenge superoxide and hydroxyl radicals in vitro.
440 E.B. Saafi et al. / Experimental and Toxicologic Pathology 63 (2011) 433–441
The antioxidant mechanism of DNE may be related to the ability
of its active compounds to detoxify free radicals and to inhibit
lipid peroxidation in the liver. Anti-inflammatory effect of
polyphenols is also demonstrated by its ability to inhibit the
production of nitric oxide and tumor necrosis factor a (TNF-a)
(Kawada et al., 1998).
In conclusion, the present study demonstrates the capacity of the
aqueous date palm fruit extract to heal the hepatotoxicity and cellular
damage in rat liver after subchronic exposure to dimethoate.
However, the accurate mechanism is not yet clear. To be able to
propose the potential therapeutic use of date palm fruit in preventing
the liver from xenobiotic-induced oxidative damage further studies
are needed. Muslims believe that ‘‘seven dates every morning keep
poison or magic away’’ (cited by Miller et al., 2003).
Acknowledgments
This work was supported by a grant from the Tunisian Ministry
Higher Education, Scientific Research and Technology trough the
Research Unit of Human Nutrition and Metabolic Disorders
UR03ES08. We would like to thank Pr. Zohra Haouas and
Dr. Fadwa Hssin for their technical assistance and Mr. Fethi
Chahata for his constructive criticism of manuscript.
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