e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 218–227

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/etap

Sub-chronic exposure to chlorpyrifos induces

hematological, metabolic disorders and oxidative stress in
rat: Attenuation by glutathione

Eman E. Elsharkawy a , Doha Yahia a , Neveen A. El-Nisr b

a Department of Forensic Medicine and Toxicology Faculty of Veterinary Medicine, Assuit University, Egypt
b Animal Health Institute of Research, Egypt

a r t i c l e i n f o a b s t r a c t

Article history: The current work aimed to investigate the different toxic effects of chlorpyrifos (CPF) in
Received 15 June 2012 subchronic exposure. Two groups of Sprague-Dawley male rats were exposed to CPF alone
Received in revised form in a dose of 30 mg/kg body weight, or CPF dose as previous plus glutathione (GSH) in a
8 December 2012 dose of 100 mg/kg body weight, for 90 days, twice weekly, orally. Another two groups of rat
Accepted 19 December 2012 were given corn oil (control) or GSH. There is a significant decrease in hemoglobin concen-
Available online 27 December 2012 tration, haematocrit percentage, thrombocytic indices, total protein and albumin levels in
CPF-exposed group. CPF induced hyperglycemia and significant increase in total cholesterol,
Keywords: but a significant decrease in triglyceride levels was obtained. A significant increase in the
Chlorpyrifos levels of lipid peroxidation was obtained while a significant decrease of the total antioxidant
Glutathione was recorded. The decrease in glycogen content and some histopathological changes were
Hematological observed in liver after CPF exposure. Furthermore, co-administration of GSH can restore
Oxidative stress some of these alterations.
Metabolic © 2012 Elsevier B.V. All rights reserved.
Histochemical

pesticides used extensively throughout the world, including

1. Introduction
Occupational exposure to pesticides is becoming a com-
mon and increasingly alarming world-wide phenomenon. The
World Health Organization estimates that the incidence of
pesticide poisoning in developing countries doubled between
1987 and 1997 (WHO, 1997). Organophosphate (OP) pesti-
cides are among the most widely used synthetic chemicals
for controlling a wide variety of pests. Residual amounts of
(OP) pesticides have been detected in the soil, water bod-
ies, vegetables, grains, and other food products (IARC, 1983;
Poet et al., 2004). Chlorpyrifos (O,O -dithyl-O-3,5,6-trichloro-2-
pyrydyl phosphorothionate, or CPF) is among the leading OP
Egypt. CPF is classified as a moderately hazardous, Class II
insecticide by the WHO (1997). Its acute toxicity varies accord-
ing to the species and route of exposure. For example, acute
oral LD50 for male rats is estimated to be 80 mg/kg bodyweight
(Karanth et al., 2004).
The chief action mechanism of OP pesticides occurs by
inhibiting neuronal cholinesterase activity, a key enzyme
involved in neurotransmission (Richardson et al., 1993).
CPF elicits a number of other effects, including: hepatic
dysfunction, genotoxicity, embryotoxicity, teratogenicity, neu-
robehavioral, and neurochemical changes (Yasmashita et al.,
1997; Goel et al., 2000). Acute and chronic exposure to CPF
has resulted in considerable liver damage, as evidenced by

E-mail address: medicine1971@yahoo.com (E.E. Elsharkawy).

1382-6689/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.etap.2012.12.009
 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 218–227 219

changes in several liver enzymes (Goel et al., 2000). Several

2. Materials and methods
studies reported that hyperglycaemia is one of the side effects
of poisoning by OP in acute treatment and in sub-chronic
2.1. Chemicals
exposure via glycogenolysis and gluconeogenesis. There are
reports of incidents of transient glycosuria and hyperglycemia
Chlorpyriphos (CPF) of purity 96.5% (Technical Grade) and
in humans following acute exposure to OPs (Zadik et al., 1983;
glutathione 100% (pure powdered form) were purchased
Shobha and Prakash, 2000).
from Sigma Chemicals Co. (St. Louis, USA). Total antioxidant
The liver plays a major role in blood-glucose homeosta-
capacity and lipid peroxide malondialdehyde (MDA) and 4-
sis by maintaining a balance between the uptake and storage
hydroxyalkenals (HAE) were measured using commercial test
of glucose via glycogenesis and the release of glucose via
kits supplied Bio-diagnostics (Bio-diagnostics, Cairo, Egypt).
glycogenolysis and gluconeogenesis (Hers, 1990; Nordlie et al.,
Glucose, total cholesterol, triglycerides, total proteins were
1999; Abdollahi et al., 2003a,b). These toxic effects proba-
determined using colorimetric kits purchased from Stanbio
bly occur through the generation of reactive oxygen species
laboratories, USA. Histochemical determination of glycogen
(ROS) causing damage to the various membranous compo-
content was done using a Periodic Acid-Schiff Stain kit (PAS)
nents of the cells. ROS had been implicated in important
supplied from Dia-Technology, Egypt. All other chemicals were
pathologies such as cardiovascular, pulmonary and autoim-
of the highest grade available commercially.
mune diseases, inherited metabolic disorders, cancer, and
aging (Halliwell and Gutteridge, 1999). Pesticides are known
to produce oxidative stress, and extensive data suggest that
2.2. Animals and treatments
oxygen free-radical formation can be a major contributor to
the toxicity of pesticides (Lodovici et al., 1994; Bagchi et al.,
Sprague-Dawley rats, weighing between 100 and 150 g,
2002). Several studies point to the production of ROS as a sec-
obtained from the Animal Laboratory House of Assiut Uni-
ondary means of toxicity (Bebe and Panemangalore, 2003).
versity, Assiut, Egypt were used for the study. The animals
These include hydroxyl, peroxyl radicals and hydrogen per-
were housed in plastic cages and allowed to adjust to the new
oxide that target and inactivate biological macromolecules
environment for a week before starting the experiment. Rats
eventually damaging membranes and other tissues (Meister,
were fed on standard food pellets and tap water ad libitum. The
1998). Cells succumb to oxidative damage when the indige-
rats were housed at 24–25 ◦ C and in a daily dark/light cycle.
nous store of antioxidants is used up by the oxidant exposure.
The design of the study was in accordance with the ethical
The antioxidant machinery is composed of enzymes and non-
guidelines prescribed by the Institution. Rats were free from
enzymatic components.
infection.
The enzymatic component is made of free radical scaven-
Animals were randomly divided into four groups of seven
gers like catalase (CAT) and superoxide dismutase (SOD) as
animals each. Animals in CPF-treated group: were given
well as the glutathione-dependent enzymes like glutathione
twice/weekly a dose of CPF alone (30 mg/kg body weight,
peroxidase (GPx), glutathione reductase (GR) and glutathione
oral) (1/45 LD50 ) for 90 days. The acute oral LD50 of CPF was
S-transferase (GST) (Gutteridge and Halliwell, 2000). The non-
136 mg/kg body weight for rats (Suleiman et al., 2010). Animals
enzymatic component is primarily composed of thiols and
in CPF plus GSH-treated group: were simultaneously given glu-
glutathione (GSH). In vitro, exposure to CPF has been shown
tathione as an aqueous solution in a dose of (100 mg/kg body
to affect activities of all these enzymes (Gultekin et al.,
weight, oral); twice/weekly, during the 90 days and exposed
2000).
to the CPF at the previous dose. The GSH dose was used
Glutathione is a major water-soluble antioxidant, low
according to Sharma and Mishra (2006). Animals in GSH-
molecular-weight thiol compound of living cells and plays
treated group: were given aqueous solution of glutathione
an important role in the detoxification of potentially toxic
alone; twice/weekly for 90 days. Control group: were given only
compounds by its conjugation with xenobiotic or their
standard pellet diet and corn oil.
metabolites (Selvam, 2002). Its net negative charge and over-
all hydrophilic nature greatly increases the aqueous solubility
of the lipophilic moieties with which it becomes conjugated
(Rana et al., 2002). GSH may bind to free-radicals with their 2.3. Necropsy
sulfhydryl groups resulting in an activation of the brain antiox-
idant enzymes and thus limiting the free-radical action more At the end of the study period, rats were fasted overnight;
extensively. Therapy with this agent was found to normalize anesthetized by inhalation of diethyl ether and blood sam-
the cellular antioxidant system, enzyme scavengers, inter- ple was drawn by puncturing the orbital sinus of the animals
rupted membrane LPO reaction and ATPase inactivation in rats under light anesthesia. Blood samples were put immediately
(Rana et al., 2002). The aim of this study was to determine the into silicon disposable glass tubes with ethylenediamine tetra
effect of a subchronic 90-days exposure to CPF on the hema- acetic acid (EDTA) as the anticoagulant to determine the dif-
tological, metabolic parameters and oxidative status of rats. ferent hematological parameters and in a clean glass test tube
And also was to evaluate the role of water soluble antioxidant and allowed to stand for 30 min at room temperature to clot
glutathione, to attenuate these changes following subchronic and centrifuged at 4000 × g for 10 min for serum separation.
exposure to CPF. This is the first time to our knowledge that the Rats were sacrificed after anesthetizing with chloroform, liv-
antioxidant glutathione has been employed to protect against ers were immediately excised and fixed in 10% neutral buffer
CPF’s toxic effects. formalin and were processed for light microscopy.
220 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 218–227
2.4. Hematological parameters
Hematological parameters [red blood cell count (RBC),
hemoglobin (HB), haematocrite (HCT), red Blood cell dis-
tribution width (RDW), mean corpuscular volume (MCV),
mean corpuscular hemoglobin (MCH), mean corpuscular
hemoglobin concentration (MCHC), white blood cell count
(WBC) and differential leucocytic count]. Thrombocytic
indices [total platelet count (PLT), mean platelet volume (MPV),
total platelet crit (PCT) and platelet distribution width (PDW)]
were analyzed by the automated parameter called Electronic
Blood Cell Counter (Medonic vet. CA620, Sweden).
2.5. Metabolic parameters
2.5.1. Glucose assay
Glucose was measured by the glucose oxidase and peroxi-
dase using quinoneimine as a chromogen. The amount of
plasma glucose is related to the amount of quinoneimine,
which is measured spectrophotometrically at 505 nm accord-
ing to Bergmeyer et al. (1974).
2.5.2. Protein assay
Total protein (TP) and albumin (Alb) were determined in serum
according to Doumas, 1971.
2.5.3. Total cholesterol and triglycerides assay
Total cholesterol and triglycerides were determined in serum
according to Tietz, 1994 and Carlson, 1963, respectively.
2.6. Determination of lipid peroxidation
Measurements of MDA and HAE have been used as an indi-
cator of lipid peroxidation. The colorimetric kit was used to
determine the levels of oxidized lipid according to Buege and
Aust, 1978. This assay is based on the reaction of chromogenic
reagent with MDA and HAE at 45 ◦ C to yield a stable chro-
mophore with maximum absorbance at 586 nm. The rate of
lipid peroxidation was expressed as nmol of reactive sub-
stance formed/min/mg protein.
2.7. Determination of total antioxidant
Total antioxidant status was assessed by using commercial
test kit and using spectrophotometer, according to the meth-
ods described by Koracevic et al. (2001). The basis of latter
method is a change of 2,2 azino-di (3-ethylbenzthiazoline) sul-
fonate (ABTS), incubated with peroxidase (metmyoglobin) and
H2 O2 into its cation form. This leads to a change in color of
the sample examined proportional to the concentration. After
addition of 1 mL of ABTS incubated with metmyoglobin to
20 mL of the supernatant, the sample was centrifuged and the
absorbance was read at a wavelength of 600 nm. Then, 200 mL
of H2 O2 was added, and another reading was obtained after
3 min. The data are presented as mmol/L.
2.8. Histopathological study
Liver samples were dissected and fixed in 10% neutral forma-
lin, dehydrated in ascending grades of alcohol and embedded
in paraffin wax. Paraffin sections (5 ␮m thick) were stained for
routine histological study using hematoxylin and eosin (H&E).
2.9. Histochemical determination of glycogen content
Paraffin sections were stained by Periodic acid Schiff’s reagent
technique (PAS) for demonstrating glycogen content (Pears,
1985).
2.10. Statistical analysis
Data were analyzed using one-way analysis of variance
(ANOVA) followed by Duncan’s multiple range test. All data
were expressed as mean ± SD for all experimental and con-
trol animals. P < 0.05 was considered significant compared to
control.
3. Results
3.1. Changes in hematological parameters
When the GSH-treated animals were compared with the con-
trol animals at the end of the 90 days, they did not differ
significantly in any terms of the hematological parameters.
When the CPF-treated group and the CPF plus GSH-treated
group were compared with the control group, they did not
differ significantly in RBC counts but significantly lower in
terms of HB, HCT, MCV, MCH, MCHC and RDW values (Table 1).
CPF-treated group had significantly higher WBC, lymphocyte
and monocyte counts but significantly lower in granulocyte
counts than the control animals. CPF plus GSH-treated group
was significantly more lymphocytes and significantly fewer
granulocytes than the control animals (Table 2). CPF-treated
group and the CPF plus GSH-treated group were significantly
decreased in terms of PLT, MPV, PCT and PDW values than
the control. However, the CPF plus GSH-treated group was
significantly differed in most previous parameters than the
CPF-treated group (Tables 1–3).
3.2. Changes in glucose assay
The CPF-treated group and the CPF plus GSH-treated group
had significantly higher glucose levels than the control group.
There is a significant difference between the CPF and CPF plus
GSH-treated group (Table 4).
3.3. Changes in protein assay
In comparison with the control group, the CPF-treated group
and the CPF plus GSH-treated group had significantly lower
total protein and albumin levels, when the CPF plus GSH-
treated group was compared with the CPF-treated group;
they had significantly higher total protein and albumin levels
(Table 4).
3.4. Changes in total cholesterol and triglycerides
The CPF-treated group and the CPF plus GSH-treated group
both had significantly higher total cholesterol levels and
 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 218–227 221

Table 1 – Effect of sub-chronic exposure to chlorpyrifos on the hematological parameters and the protective effect of
glutathione.
RBCs (×106 /mm3 ) HB (g/dl) HCT (%) MCV (␮m) MCH (pg) MCHC (g/dl) RDW (%)
CPF 8.40 ± 0.35 14.22 ± 0.57*bc 40.55 ± 1.80*c 48.27 ± 1.21*c 16.97 ± 0.33*c 35.20 ± 0.21*c 35.60 ± 1.11*c
CPF + GSH 8.77 ± 0.38 15.20 ± 0.47a 42.30 ± 1.41*c 48.23 ± 0.82*c 17.36 ± 0.23*c 36.00 ± 0.20*c 35.80 ± 1.26*c
GSH 8.34 ± 0.15 15.78 ± 0.31a 48.65 ± 1.35ab 49.41 ± 0.56ab 18.25 ± 0.01ab 37.65 ± 0.22ab 36.83 ± 1.24ab
Control 8.64 ± 0.19 15.58 ± 0.31 47.65 ± 1.35 50.50 ± 0.46 18.15 ± 0.02 37.05 ± 0.25 36.63 ± 1.54
Data are expressed as means ± S.D. of seven animals per group.*denotes P < 0.05 as compared to control group, a denotes P < 0.05 as compared
to CPF-group. b denotes P < 0.05 as compared to CPF + GSH-group. C denotes P < 0.05 as compared to GSH-group (One-way ANOVA/Duncan).

Table 2 – Effect of sub-chronic exposure to chlorpyrifos on the total and differential leucocytes counts (103 mm−3 ) and the
protective effect of glutathione.
WBCs Lymphocytes Granulocytes Moncytes
CPF 11.05 ± 2.18*bc 6.92 ± 1.01*bc 3.10 ± 0.98*bc 1.02 ± 0.19*bc
CPF + GSH 9. 93 ± 0.90ac 5.10 ± 0.60*ac 2.35 ± 0.27*ac 0.60 ± 0.34a
GSH 10.55 ± 0.24ab 4.23 ± 0.80ab 5.98 ± 0.80ab 0.88 ± 0.19a
Control 10.53 ± 0.34 4.13 ± 0.12 5.50 ± 0.25 0.80 ± 0.05
Data are expressed as means ± S.D. of seven animals per group.*denotes P < 0.05 as compared to control group, a denotes P < 0.05 as compared
to CPF-group. b denotes P < 0.05 as compared to CPF + GSH-group. C denotes P < 0.05 as compared to GSH-group (One-way ANOVA/Duncan).

Table 3 – Effect of sub-chronic exposure to chlorpyrifos on the thrombocytic indices and the protective effect of
glutathione.
PLT (103 /ml) MPV (␮m) PDW (␮m) PCT (%)
CPF 468.6 ± 62.4*c 5.45 ± 0.25 c 8.35 ± 0.41*c 0.24 ± 0.02*bc
CPF + GSH 497.4 ± 41.7*c 5.50 ± 0.11*c 8.45 ± 0.17*c 0.27 ± 0.04*ac
GSH 698.6 ± 15.8 ab 6.48 ± 0.38ab 9.29 ± 0.15ab 0.33 ± 0.01ab
Control 696.3 ± 15.8 6.06 ± 0.08 9.16 ± 0.17 0.30 ± 0.01
Data are expressed as means ± S.D. of seven animals per group.*denotes P < 0.05 as compared to control group, a denotes P < 0.05 as compared
to CPF-group. b denotes P < 0.05 as compared to CPF + GSH-group. C denotes P < 0.05 as compared to GSH-group (One-way ANOVA/Duncan).

Table 4 – Effect of sub-chronic exposure to chlorpyrifos on the biochemical parameters and the protective effect of
glutathione.
TP (g\dl) Alb (g\dl) Total cholesterol (mg/dl) Triglycerides (mg/dl) Glucose (mg/dl)
CPF 5.98 ± 0.1*bc 4.33 ± 0.45*bc 115.00 ± 4.25*bc 80.65 ± 5.6*bc 81.5 ± 4.8*bc
CPF + GSH 7.40 ± 0.9*ac 4.82 ± 0.91*ac 93.72 ± 3.11*ac 105.08 ± 5.2*ac 72.6 ± 5.5*ac
GSH 7.86 ± 0.27ab 5.47 ± 0.56ab 79.42 ± 3.51ab 125.20 ± 6.8 ab 60.0 ± 4.2ab
Control 7.88 ± 0.17 5.67 ± 0.56 81.07 ± 3.23 123.93 ± 7.15 63.8 ± 5.2
Data are expressed as means ± S.D. of seven animals per group.*denotes P < 0.05 as compared to control group, a denotes P < 0.05 as compared
to CPF-group. b denotes P < 0.05 as compared to CPF + GSH-group. C denotes P < 0.05 as compared to GSH-group (One-way ANOVA/Duncan).

significantly lower triglyceride than the control group. When

Table 5 – Effect of sub-chronic exposure to chlorpyrifos
the CPF plus GSH-treated groups were compared to the CPF- on the oxidative status and the protective effect of
treated group, they had significantly lower total cholesterol glutathione.
levels and significantly higher triglyceride’s levels (Table 4).
Total antioxidant MDA&HAE
(mM/L) (nmol/mg protein)
3.5. Lipid peroxidation and total antioxidant CPF 0.36 ± 0.02*bc 20.58 ± 0.51*bc
CPF + GSH 0.45 ± 0.01*ac 14.53 ± 0.44*ac
Table 5 shows that the CPF-treated group and the CPF GSH 0.1 ± 0.12ab 13.80 ± 0.21ab
plus GSH-treated group in rats caused significantly marked Control 0.98 ± 0.14 13.50 ± 0.31
increase in the levels of MDA and 4HNE in blood when com- Data are expressed as means ± S.D. of seven animals per
pared with control. While the total antioxidant status was group.*denotes. P < 0.05 as compared to control group, a denotes
statistically significantly lower than the control group. In P < 0.05 as compared to CPF-group. b denotes P < 0.05 as compared
to CPF + GSH-group. C denotes P < 0.05 as compared to GSH-group
contrast, treatment with GSH resulted in a significant ame-
(One-way ANOVA/Duncan).
lioration of the activities of both the levels of MDA and 4HNE
and the total antioxidant, when compared with CPF-treated
group and the CPF plus GSH-treated group.
222 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 218–227

Fig. 1 – Livers from the control (a) exhibited normal arrangement and structure of the hepatocytes’ H&E X40. Liver sections
of CPF-treated rat showed (b) sever vacuolar degeneration H&E X40, (c) disarrangement of the hepatic cord H&E X25, (d)
congestion of the portal blood vessels which, surrounded with thick connective tissue and monocytic cellular infiltration
H&E X10, (e) the portal area blockaded with fibrous strands associated with kupffer cell infiltration H&E X40. Liver of rat of
CPF plus GSH-group showed (f) more or less normal hepatocytes associated with regular architecture’s H&E X25.

3.6. Histopathological study CPF plus GSH-treated animals exhibited histopathological

Light microscopic examination of the livers revealed that
those of the control exhibited normal arrangement of the
hepatocytes and there were visible sinusoids in most places.
The nuclei of the hepatocytes had a normal vesicular struc-
ture, and the cytoplasm appeared uniform and regular (Fig. 1a).
In contrast, the livers of the CPF-treated animals and the
changes: The livers of the CPF-treated animals displayed hem-
orrhage, vacuolar degeneration, dilation of sinusoids (Fig. 1b).
Hepatocytes lost their radial arrangement (Fig. 1c) with mono-
nuclear cell infiltration, vascular congestion, and necrosis
(Fig. 1d–e), while the livers of the CPF plus GSH-treated animals
exhibited regular arrangement of the hepatocytes with little
mononuclear cell infiltration and slight vascular congestion
 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 218–227 223

Fig. 2 – Glycogen in paraffin sections of rat liver stained with PAS. Original magnification is ×400. (a) Control liver showed
the homogenous feature of glycogen that was arranged in regular lines in all hepatocytes. (b) Liver from CPF-treated groups
showed marked disturbance of glycogen content that was polling in one side of most the hepatocytes. (c) The livers of the
CPF plus GSH-treated animals showed less polling, decrease and disturbance of glycogen content than CPF-treated animals.

(Fig. 1f). However, the latter livers did not display the vacuolar
degeneration, calcification and necrosis that were detected in
the livers of CPF-treated animals.
3.7. Histochemical study
Glycogen in paraffin sections of rat liver stained with PAS is
illustrated in (Fig. 2) containing three figures (a, b and c liver).
Control liver showed the homogenous feature of glycogen
that was arranged in regular lines in all hepatocytes (Fig. 2a).
While sections from animals exposed to CPF and CPF plus GSH
showed disturbance of glycogen content that was polling in
one side of most the hepatocytes (Fig. 2b). However, the livers
of the CPF-treated animals showed more obvious disturbance
and pooling of glycogen content than CPF plus GSH-treated
animals (Fig. 2c).
4. Discussion
Several studies have shown that OP insecticides can alter
hematological parameters in experimental animals (Kalender
et al., 2006; Yehia et al., 2007; Celik and Suzek, 2008) and in
clinical cases (Patil and Govindwar, 2003). The present results
revealed that exposure to CPF was associated with significant
decreases in HB, HCT, MCV, MCHC and RDW values but no sig-
nificant change was observed in RBC counts. A similar trend
has been observed in mice treated with Nuvacron and Furadan
(Gupta et al., 1982), and in rats treated with CPF (Shalby, 1998).
In this study, no significant change was observed in RBC counts
but HB content was decreased in CPF-treated rats. Because
of this a significant decease was observed in both MCH and
MCHC contents. As MCH shows hemoglobin content in eryth-
rocytes, and MCHC means hemoglobin rate to RBC (Nussey
et al., 1995), the reduction in HB content was suggested to be
due to an adverse effect of OP on the bone marrow (Seiverd,
1972). MCV has been reported to provide information about
the size and status of erythrocytes (Nussey et al., 1995). Thus,
the observed decrease in the mean of MCV and RDW in rats
exposed to CPF was a manifestation of atrophied erythrocytes
and microcytic anemia. Patil and Govindwar, 2003) stated that
many steps in the heme biosynthesis are inhibited by pesticide
residues, and this might be a possible physiological reason for
the inverse proportion to the result obtained. They also sug-
gested that the poisoning by pesticide residue induce anemia
due to interference of hemoglobin biosynthesis and short-
ening of the life span of circulating erythrocytes. Therefore,
the obtained decrease in the hemoglobin concentration might
be due to the effect of pesticide on erythropoietic tissue and
haem biosynthesis in rats. Our findings suggested that the
exposure of rats to CPF result in microcytic hypochromic ane-
mia, which may be due to the inhibition of erythropoiesis and
haemosynthesis.
Another study has shown that some OP insecticides can
increase WBC counts (Celik et al., 2009). These findings are
consistent with our results, which revealed that CPF in sub-
chronic exposure elevated WBC, lymphocyte and monocyte
counts. It has been shown that leukocytosis may be due to
increased leukocyte mobilization and can be directly pro-
portional to the severity of the causative stress condition
(Celik et al., 2009). Thus, the leukocytosis seen in the CPF-
treated rats may reflect the response of the immune system
to the stress imposed by CPF. It is possible that the toxic
effects of CPF weaken the constitution of the rats, which
then acquire infections that provoke an immune response
(Celik and Suzek, 2008). In addition, OP insecticides have been
shown to induce hemorrhage, inflammatory cell infiltration
(Morowati, 1998; Elhalwagy et al., 2008), tissue damage, and
necrosis (Kalender et al., 2006), which could provoke increased
WBC and lymphocyte counts. This possibility is supported
by our histopathological analysis of the CPF-treated rats in
this study, which revealed the occurrence of hepatic hem-
orrhage, inflammatory cell infiltration, edema and necrosis.
The results obtained from this study revealed that the CPF-
treated group exhibited decreases in PLT, MPV, PCT and PDW
values compared to the control. Similarly, Kalender et al.
(2006) reported that a significant decrease was observed in
thrombocyte counts at the end of 1st and 4th weeks of diazi-
non exposure. And also, a significant decrease was observed
in platelets counts indices in diazinon sub-acute exposure
(Hariria et al., 2010 and Hariria et al., 2011). In contrast,
Kalender et al. (2009) reported that malathion acute exposure
induced increase in thrombocyte’s count. Our results indi-
cated that CPF induced thrombocytopenia in exposed rats. It
224 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 218–227
should be noted that it is the first time we know a study discuss
the effect of CPF on thrombocytic indices. The thrombocy-
topenia may develop due to antibody production against viral
antigens attached to platelet surfaces or to nonspecific binding
of antigen-antibody complexes to platelet surfaces (Cheung
et al., 2005). Furthermore, drug and xenobiotic-induced throm-
bocytopenia has been reported in dogs, cats, and horses. One
mechanism is marrow suppression of megakaryocytes or pos-
sibly even generalized marrow stem cell suppression after
administration of xenobiotics (Gibbins and Mahaut-Smith,
2004). Another mechanism is increased platelet destruction
and consumption resulting in impair of platelet function.
Numerous xenobiotics were reported to block platelet receptor
binding or to change platelet membrane charge or permeabil-
ity (Page et al., 2002). Quantitative platelet disorders have been
reported in liver disease with or without coagulation protein
deficiencies (Tkaczyk and Baj, 2002).
In this study, the CPF-treated animals exhibited signifi-
cantly lower total protein and albumin levels than the control
animals. Albumin, which is the most abundant blood plasma
protein, is produced by the liver and several studies have
shown that its production can be decreased by OP such as CPF
(Kalender et al., 2005; Yousef et al., 2006; Ogutcu et al., 2008).
Since reductions in albumin levels are generally suggestive
of liver disease, it is possible that OP alters protein and free
amino acid metabolism and their synthesis within the liver
(Ncibi et al., 2008).
OP insecticides generally increase the total cholesterol and
total lipid levels (Kalender et al., 2005; Ogutcu et al., 2008;
Lasram et al., 2009) and indeed, in the present study, CPF ele-
vated the total cholesterol levels of the rats. This increase
in serum cholesterol can be attributed to the effect of pes-
ticides on the permeability of the liver cell membrane (Yousef
et al., 2006). In addition, the increase in serum total choles-
terol levels may be due to the blockage of the liver bile ducts,
which reduces or stops cholesterol secretion into the duode-
num (Zaahkouk et al., 2000; Ogutcu et al., 2008). Increased
serum cholesterol levels may be a sign of liver damage. Some
pesticides also decrease the triglyceride levels (Kalender et al.,
2005; Ogutcu et al., 2008). It has been shown that parenchymal
liver diseases are associated with decreases in triglyceride lev-
els (Kalender et al., 2005). In the present study, CPF exposure
reduced the triglyceride levels. These observations are consis-
tent in the presence of liver cell damage that was seen in the
CPF-exposed rats by light microscopy.
Additionally, several studies indicate that OP exposure
causes hyperglycemia, as an immediate consequence of
malathion administration (Gupta, 1974; Rodrigues et al., 1987),
which was explained by stimulation of glycogenolysis in sev-
eral organs (Pournourmohammadi et al., 2004; Abdollahi et al.,
2004) and gluconeogenesis in liver (Abdollahi et al., 2004),
provoking the release of glucose into the blood. Treatment
of rats with acute diazinon also resulted in hyperglycemia,
increased activity of the hepatic gluconeogenic enzyme and
phosphoenolpyruvate carboxykinase (PEPCK) (Matin et al.,
1990). In the present study, CPF induced hyperglycemia
and a significant disturbance in hepatic glycogen distri-
bution rate after 90 days of sub-chronic exposure. It has
been shown that acute exposure to malathion leads to an
increase of glycogen deposition in the liver during 6–24 h
after malathion treatment (Gupta, 1974). It is well estab-
lished that the increase in the hepatic glycogen rate is due to
the stimulation of the key enzyme of glycogenesis (glycogen
synthetase) (Rezg et al., 2006). One possible explanation for
these results could be the turnover of glucose by its release
through a succession of glycogenolysis and gluconeogenesis
(Pournourmohammadi et al., 2004), which involves abnormal
hyperglycemia and its storage via glycogenesis (Rezg et al.,
2006). This is proven by marked changes to the overall histo-
architecture of the liver (Gupta, 1974). Hepatic atrophy has
also been observed when repeated doses of CPF are given. In
addition, Radiger et al. (2003) stated that the decline of glyco-
gen content may reflect a disruption in enzyme pathways of
glycolysis.
Pesticides are known to produce oxidative stress, and
extensive data suggest that oxygen free-radical formation can
be a major contributor to the toxicity of pesticides (Lodovici
et al., 1994; Bagchi et al., 2002). It has been previously reported
that acute exposure to OP pesticides may induce oxidative
stress in rats (Koner et al., 1997). Our result strengthens the
hypothesis and suggests that induction of oxidative stress
is perhaps the central mechanism by which OP pesticides
exert their cellular action. Oxidative damage primarily occurs
through production of reactive oxygen species, including
hydroxyl radicals and hydrogen peroxide that subsequently
react with biological molecules as well as causing damage
to membranes and other tissues (Banerjee et al., 1999). A
significant decrease in the total antioxidant level and a con-
comitant increase in LPO level following administration of the
pesticide dose were observed in the present study. Suppres-
sion in the total antioxidant capacity is due to the decrease
in biologic antioxidant enzymes. The results are in agree-
ment with previous studies by Vidyasagar et al. (2004), where
alterations in glutathione, total thiols, lipid peroxidation and
changes in the activity of SOD and catalase in erythrocytes
have been noted following exposure to OP pesticides. Deple-
tion in biologic antioxidant enzymes is responsible for the
most cytotoxic effects of OP (John et al., 2001). CPF induced
oxidative stress (Verma et al., 2007), and effected on thio-
barbituric acid reactive substances, scavenging enzymes and
glutathione in rat tissues (Verma and Srivastava, 2003). The
basis of OP toxicity in the production of oxidative stress may
be due either to: (a) their “redox-cycling” activity, where they
readily accept an electron to form free radicals and then trans-
fer them to oxygen to generate superoxide anions and hence
hydrogen peroxide through disputation reactions, or (b) to ROS
generation via changes in normal antioxidant homeostasis
resulting in antioxidant depletion, if the requirement of con-
tinuous antioxidants is not maintained (Banerjee et al., 1999;
Kovacic, 2003; Vidyasagar et al., 2004).
OP insecticides are known to induce various histopatho-
logical changes in the liver tissues (Goel et al., 2005; Gokcimen
et al., 2007; Sayım, 2007; Yehia et al., 2007) and indeed,
we found that CPF-induced inflammatory cell infiltration,
hemorrhage, calcification, vacuolar degeneration, dilation of
sinusoids, vascular congestion and necrosis in the rat liver.
These changes are entirely consistent with the changes in var-
ious biochemical parameters that were also observed. Such
liver damage may arise from the toxic effects of CPF, which dis-
turbs the detoxification mechanisms of the liver. In addition,
 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 218–227
it is possible that CPF, like several other insecticides, adversely
affects the cytochrome P450 system or the mitochondrial
membrane transport system of hepatocytes. Exposure to CPF
may lead to membrane damage in liver cells and eventual
loss of membrane integrity (Gokcimen et al., 2007). It has
been reported previously that during liver damage, there is an
observed decrease in antioxidant defenses in the liver (Seven
et al., 2004). Glycogen is the major storage form of carbo-
hydrates in the animal liver (Murray et al., 2000). A marked
decrease of hepatocyte’s glycogen was noticed after CPF expo-
sure, focally to the central vein region indicating the increase
of vacuolated cells that may affect hepatocytes function. How-
ever, marked increases in glycogen deposits in one side of
some hepatocytes may be due to the detoxification by hepato-
cytes. These findings are in good agreement with the results of
a previous study by Radiger et al. (2003). They stated that, the
decline of glycogen content may reflect a disruption in enzyme
pathways of glycolysis.
Antioxidants have a number of biological activities, includ-
ing immune stimulation and alteration of the metabolic
activities of carcinogens. These vitamins can also prevent
genetic changes by inhibiting the DNA damage induced by
reactive oxygen metabolites (Verma et al., 2007). Glutathione,
the major water-soluble antioxidant, is known to protect cel-
lular membranes and lipoproteins from peroxidation (Rana
et al., 2002), may minimize lipid peroxidation in biological
systems (Selvam, 2002) and acts to protect both cytosol and
membranes against free-radical attack Hogg, 1998. Its high
electron donating capacity combined with its intensive intra-
cellular concentration endows GSH with great reducing power,
which is used to regulate a complex thiol exchange system
(Meister, 1994).
The hematological, metabolic disorders and the changes
of the oxidative status that were induced by CPF exposure
were at least partially normalized when the GSH antioxidant
was given with CPF. Moreover, our light microscopic anal-
yses revealed that the antioxidant-treated animals did not
exhibit the hepatic calcification, vacuolar degeneration and
necrosis seen in the livers of the CPF-treated group. Further-
more, while both groups exhibited hepatic inflammatory cell
infiltration, hemorrhage, dilation of sinusoids, and vascular
congestion, these changes were less severe in the CPF plus
GSH-treated group. Thus, GSH may be could ameliorate the
liver damage induced by CPF exposure. In conclusion, our
results demonstrate that sub-chronic exposure to CPF leads
to significant hematological, metabolic disorders and oxida-
tive damage. Administration of non-enzymatic water soluble
antioxidant GSH exerts a significant protective role against the
CPF toxic effect in rat.
Conflicts of interest
None declared.
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