Pesticide Biochemistry and Physiology 98 (2010) 128–134

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Pesticide Biochemistry and Physiology

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

Protective effects of Nigella sativa oil on propoxur-induced toxicityand oxidative stress in rat brain regions

Ahmed M. Mohamadin a,*, Bassem Sheikh b, Amany A. Abd El-Aal c, Ahmed A. Elberry d, Fahad A. Al-Abbasi e

a Department of Clinical Biochemistry, Faculty of Medicine, Taibah University, Al-Madinah Al-Munawwarah, Saudi Arabiab Department of Surgery, Faculty of Medicine, Taibah University, Al-Madinah Al-Munawwarah, Saudi Arabiac Department of Parasitology, Faculty of Medicine, Taibah University, Al-Madinah Al-Munawwarah, Saudi Arabiad Department of Clinical Pharmacy, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabiae Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia

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

Article history:Received 25 February 2010Accepted 21 May 2010Available online 31 May 2010

Keywords:PropoxurBrain regionsOxidative stressNigella sativa oil

0048-3575/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.pestbp.2010.05.011

* Corresponding author. Address: Department of CliMedicine, Taibah University, P.O. Box 30001, Al-MadArabia. Fax: +966 48461407.

E-mail address: amohamadin@yahoo.com (A.M. M

Propoxur (PPr) is a widely used broad spectrum carbamate insecticide mainly used to control householdpests. Because of the widespread use of pesticides for domestic and industrial applications, evaluation oftheir neurotoxic effects is of major concern to public health. The aim of the present study was to evaluatethe possible protective effects of Nigella sativa oil (NSO), an antioxidant agent, against PPr-induced tox-icity and oxidative stress in different brain regions of rats including cerebellum, cortex and hippocampus.In the present study, 32 male Sprague–Dawley rats were used and divided into four equal groups. Group1 was allocated as the control group. Groups 2–4 were orally administered 1 ml/kg/bw/day NSO, 8.51 mg/kg/bw/day PPr or NSO plus PPr, respectively, for 30 days. Lipid peroxidation (LPO), protein carbonyl con-tent (PCC) and acetylcholine esterase activity (AChE) were determined. Enzymatic antioxidant activities[superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione-S-transferase(GST)] and non-enzymatic antioxidants [reduced glutathione (GSH)] were determined. PPr treatment sig-nificantly increased the levels of LPO, PCC and oxidized glutathione (GSSG) in brain regions. On the con-trary, levels of GSH and the activities of SOD, CAT, GSH-Px, GST and AChE were significantly decreased.NSO treatment to PPr intoxicated rats restored such biochemical parameters to within control levelsexcept GST activity, emphasizing its antioxidant role. We conclude that NSO significantly reduces PPr-induced toxicity and oxidative stress in rat brain regions via a free radicals scavenging mechanism.

� 2010 Elsevier Inc. All rights reserved.

1. Introduction

Propoxur (2-(1-methylethoxy) phenol methyl carbamate;Fig. 1) is a carbamate insecticide developed by Bayer AG, Germany,and registered for use against ants, cockroaches, crickets, fleas,flies, mosquitoes, wasps, and ticks in many countries [1]. Propoxur(PPr) was found to be neurotoxic, with massive reversible inhibi-tion of acetylcholine esterase (AChE) as the main mechanism ofaction [2]. As a consequence, the risk of human exposure by re-peated low doses of these substances is relatively high [1,3]. Theexact mechanism(s) of PPr-induced toxicity have yet to be fully de-fined. Involvement of reactive oxygen species (ROS) has been pos-ited as a likely mechanism of PPr-induced toxicity [4,5]. ROS areclosely implicated in several diseases of nervous system includingParkinson’s disease, schizophrenia and Alzheimer’s disease [6].

ll rights reserved.

nical Biochemistry, Faculty ofinah Al-Munawwarah, Saudi

ohamadin).

Carbamate insecticides may induce oxidative stress leading to gen-eration of free radicals with alteration in enzymatic and non-enzy-matic antioxidant systems [7,8]. Lipid peroxidation (LPO) has beensuggested as one of the molecular mechanisms involved in carba-mate-induced toxicity [4]. Enzymatic antioxidants and glutathionemetabolism-regulating enzymes may protect the cellular systemagainst various deleterious effects of free radicals induced by pes-ticides [9,4].

The brain exhibits distinct variations in cellular as well as regio-nal distribution of antioxidant biochemical defenses [10]. Thus,neural cells and/or brain regions are likely to differentially respondto changes in metabolic rates associated with the generation ofROS [11]. Indeed, there is abundant evidence invoking regionalsensitivity to oxidative stress that is dependent on cellular and re-gional redox status [12].

More attentions have been paid to the protective effects of nat-ural antioxidants against chemicals-induced toxicities especiallywhenever free radical generations are involved. Nigella sativa L.(N. sativa) is a plant of Ranunculaceae family that grows spontane-ously and widely in several southern Mediterranean and Middle

Fig. 1. Propoxur; chemical name: 2-(1-methylethoxy) phenol methyl carbamate;trade name: BAYGON�; CAS No. 114-26-1; empirical formula: C11H15NO3.

A.M. Mohamadin et al. / Pesticide Biochemistry and Physiology 98 (2010) 128–134 129

Eastern countries [13]. N. sativa seed has over 100 different chem-ical constituents, including abundant sources of all the essentialfatty acids. Although it is the oil that most often used medicinally,the seeds are a bit spicy and are often used whole in cooking cur-ries, pastries and Mediterranean cheeses [13]. The seeds of N.sativa, also known as black seed or black cumin, are often usedas a spice but are also used extensively in the traditional medicineof many countries [14]. N. sativa has been used traditionally for thetreatment of many diseases owing to the reported antiviral [15],anti-schistosomiasis [16], anti-inflammatory [17], and immuno-modulatory [18] activities. Furthermore, it was found that N. sativaextract has anti-tumor properties [19], attenuates toxic side effectscaused by several chemotherapeutic agents [20] and protectsagainst gentamicin-induced nephrotoxicity [21]. In addition, it pre-vents hippocampal neurodegeneration after chronic toluene expo-sure in rats [22].

The high lipid content of the brain makes it particularly suscep-tible to free radicals mediated insult [23]. Antioxidant enzymes areinvolved in the defense system against free radical mediated tissueor cellular damage [24]. Therefore, the present study was plannedto evaluate the role of NSO as a protective agent against propoxur-induced toxicity and oxidative stress in rat brain regions.

2. Materials and methods

2.1. Chemicals

Propoxur (isopropoxiphenylmethylcarbamate) of 99.4% puritywas purchased from ChemService (West Chester, PA, USA). Thio-barbituric acid (TBA), reduced glutathione (GSH), 5,5-dithio bis(2-nitrobenzoic acid), 1-chloro 2,4 dinitrobenzene (CDNB), gluta-thione reductase (GR), were purchased from Sigma–Aldrich Chem-ical Company (St. Louis, MO, USA). N. sativa seeds were purchasedlocally, and identity was confirmed by professional botanists(Department of Natural Products and Alternative Medicine, Facultyof Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia). Allthe other chemicals used were of analytical grade.

2.2. Preparation of the decoction

The dried seeds of N. sativa were purchased from a local marketand authenticated in the Department of Natural Products andAlternative Medicine, Faculty of Pharmacy, King Abdulaziz Univer-sity, Jeddah, Saudi Arabia and voucher specimens are deposited atthe Pharmacognosy Department. The seeds were crushed and coldmacerated in petroleum ether (40–60 �C) for three days. The ex-tract was taken out, petroleum ether evaporated and the oil wasfiltered. Yield was 17.50% v/w with reference to dried seeds. Theextracted oils were kept in screw-capped tubes in the dark at�20 �C until use. This dose corresponds to normal therapeutic dose

administered to adult humans as calculated based on relative sur-face areas of human and rat [25].

2.3. Experimental groups

Thirty-two adult male Sprague–Dawley rats (250–300 g) wererandomly assigned into four groups. The first group was adminis-tered groundnut oil as a vehicle (1 ml/kg/day), the second groupwas administered N. sativa oil (NSO), the third group was adminis-tered PPr and the fourth group was administered a combination ofNSO and PPr. The selected dose of the insecticide was based on pre-vious studies in which 1/10 LD50 of propoxuor induced biochem-ical alterations in rats without morbidity [26]. Propoxur wasdissolved in groundnut oil of pharmaceutical quality and wasadministered orally in a dose of 8.51 mg/kg body weight/day for30 days with intragastric tube. NSO was administered (1 ml/kg/day) orally at least 1 h before the administration of PPr.

2.4. Tissue preparation

Twenty-four hours after last treatment, the animals wereeuthanized using carbon dioxide asphyxiation and brains wereimmediately removed and washed in ice-cold physiological salinerepeatedly and dissected over ice-cold glass slides to the followingregions: cerebellum, cerebral cortex and hippocampus [27]. Pooledregions from each of the brain tissue were blotted, weighed accu-rately, and placed in chilled 0.01 mol/l Tris–HCl buffer (pH 7.4). Thesamples were homogenized using a Potter–Elvehjem homogenizer(Wheaten Science Products, Milliville, NJ, USA) to produce 10%homogenates and used for determining the biochemical parame-ters described below. Protein concentrations of the tissue homog-enates were determined by the standard method of Lowry et al.[28], using bovine serum albumin as the standard.

2.5. Biochemical assays

2.5.1. Acetylcholine esterase (AChE) assayAChE activity was determined by the method of Ellman et al.

[29]. Total assay volume of 355 ll, consisted of 5 ll of tissue,300 lL of chromogen/buffer (0.3 mM 5,50-dithio-bis (2-nitroben-zoic acid); final DTNB concentration in assay 0.25 mM) and 50 llof substrate (8.45 mM acetylthiocholine iodide; final concentrationin assay 1.2 mM). There was a 5-min pre-incubation period and thereaction was conducted at 37 �C. Specific activity of AChE was ex-pressed as nmoles of substrates hydrolyzed/min/mg protein.

2.5.2. Reduced and oxidized glutathione (GSH and GSSG) assayGSH level in the selected brain regions was determined by the

method of Ellman [30]. Briefly, an aliquot of 1 ml of suspensionprecipitated with 10% trichloroacetic acid (TCA). The precipitatewas removed by centrifugation. The supernatant was then dividedinto two aliquots. One was directly used for total GSH assayaccording to the modified method of Ellman, 1959, and the otherfor GSSG. One hundred microliters of supernatant fractions with2 ll vinyl pyridine were incubated at room temperature for 1 hto scavenge GSH for the GSSG determination. The GSSG was thensubtracted from the total glutathione to evaluate the GSH levels.GSH was determined by the DTNB-glutathione reductase recyclingmechanism.

2.5.3. Lipid peroxidation (LPO) assayLPO was determined in brain tissues using the method of Deva-

sagayam and Tarachand [31]. Briefly, the reaction mixture con-sisted of 1 ml 0.15 mol/l Tris–HCl buffer (pH 7.4), 0.3 ml 10 mmol

130 A.M. Mohamadin et al. / Pesticide Biochemistry and Physiology 98 (2010) 128–134

KH2PO4 and 0.2 ml of tissue extract in a total volume of 2 ml. Thetubes were incubated at 37 �C for 20 min with constant shaking.The reaction was stopped by the addition of 1 ml 10% TCA. Thetubes were shaken well and 1.5 ml TBA was added, and heated ina boiling water bath for 20 min. The color developed was measuredat 532 nm and the malondialdehyde (MDA) content of the samplewas expressed as nmol of MDA formed/mg protein.

2.5.4. Protein carbonyl content (PCC) assayThe PCC in each of the regional brain samples was determined

spectrophotometrically by a method based on reaction of the car-bonyl group with 2,4-dinitrophenylhydrazine to form dini-trophenylhydrazone [32]. The PCC was expressed as nmol/mgprotein.

2.5.5. Superoxide dismutase (SOD) assayBrain SOD activity was determined by the method of Marklund

and Marklund [33]. Briefly, the assay mixture contained 2 ml of0.1 mol/l Tris–HCl buffer (pH 8.2), 0.5 ml of 2 mmol/l pyrogallol,0.75 ml of 1:10 diluted aliquot of the enzyme extract and waterto give a final volume of 4 ml. The rate of inhibition of pyrogallolauto-oxidation after the addition of enzyme extract was noted.The amount of enzyme required to give 50% inhibition of pyrogallolauto-oxidation was considered as one unit of enzyme activity. Theenzyme activity was expressed as U/mg protein.

Table 1Effect of Nigella sativa oil (NSO) on body weight and brain regions weight in propoxur-

2.5.6. Catalase (CAT) assayBrain CAT activity was determined according to Aebi method

[34]. The principle of the assay is based on the determination ofthe H2O2 decomposition rate at 240 nm. Results were expressedas U/mg protein. One unit is the amount of enzyme that utilizes1 mmol of hydrogen peroxide/min.

treated rats.

Control N. sativa Propoxur N. sativa + PPr

Body weight (g) 187 ± 4.5 185 ± 5.0 142 ± 5.3* 168 ± 4.0*,#

Brain regions weight (mg)Cerebellum 218.3 ± 3.6 215.7 ± 4.5 158 ± 7.1* 227 ± 8.6

#

Cortex 341.5 ± 5.8 344.2 ± 4.8 260 ± 9.3* 349 ± 10.2#

Hippocampus 186.2 ± 7.1 187.6 ± 5.3 105 ± 3.7* 191.3 ± 7.5#

Data are presented as means ± SE of eight animals.* Significantly different from control group at P < 0.05.# Significantly different from propoxur group at P < 0.05.

2.5.7. Glutathione peroxidase (GSH-Px) assayThe activity of GSH-Px was determined in brain tissues by the

method of Rotruck et al. [35]. The enzymatic reaction in the tube,which contains NADPH, GSH, sodium azide, and glutathione reduc-tase, was initiated by addition of H2O2 and the change in absor-bance at 340 nm was monitored by a spectrophotometer. Resultswere expressed as U/mg protein (one unit is the amount of enzymethat converts 1 mg GSH to GSSG in the presence of H2O2/min/mgprotein).

Ach

E ac

tivity

oles

of s

ubst

rate

yzed

/min

/mg

prot

ein)

10

15

20Control

N.sativa

PPr

N.sativa+PPr

* *

# #

#

2.5.8. Glutathione-S-transferase (GST) assayBrain GST activity was assayed by the method of Habig et al.

[36]. To 0.4 ml potassium phosphate buffer (0.5 mol/l; pH 6.5),0.1 ml 1:10 diluted homogenate, 1.2 ml water and 0.1 ml CDNB(1-chloro-2,4 dinitrobenzene, 30 mmol/l) conjugate were addedand incubated in a water bath at 37 �C for 10 min. After incubation,0.1 ml of reduced glutathione (30 mmol/l) was added. The changein optical density was measured against a reagent blank at 340 nmat 30 s interval. Activity of GST was expressed as U/mg protein (oneunit is the amount of enzyme that conjugate 1 mmol of CDNB withGSH/min).

(nm

hydr

ol

0

5

Cerebellum Cortex Hippocampus

*

Fig. 2. Effect of propoxur (PPr) and Nigella sativa oil (NSO) on acetylcholine esterase(AChE) activity in cerebellum, cortex and hippocampus in rats. Results are shown asmeans ± SE (n = 8). *Significantly different from control group at P < 0.05. #Signif-icantly different from propoxur group at P < 0.05.

2.6. Statistical analysis

Data are expressed as means ± S.E. The differences betweengroups were analyzed by one-way analysis of variance (ANOVA)followed by the Tukey–Kramer test for multiple comparisons. A0.05 level of probability was used as the criterion for significance.All univariate analysis of variance (ANOVA) was completed usingSPSS 10.0 software (SPSS Inc., Chicago, IL, USA).

3. Results

3.1. Body and organ weights

The effects of PPr and NSO on body weight and various brain re-gions such as cerebellum, cortex and hippocampus weights weresummarized in Table 1. The body weight and weights of brain re-gions were significantly (p < 0.05) decreased in PPr-exposed rats.However, the pretreatment with NSO restored significantly thebody weight and the weights of various brain regions. Excessivesalivation and soiled appearances were also noted only in thePPr-treated rats. Though there were some apparent dose-depen-dent behavioral changes, no quantitative measurements weremade for assessment of neurobehavioral characterizations inducedby PPr, as this was deemed to be beyond the scope of the presentresearch.

3.2. AChE activity

Fig. 2 shows the effect of PPr and pre-administration of NSOwith PPr on the AChE activity in the selected brain regions of rats.The activity of AChE was significantly decreased in cerebellum,cerebral cortex and hippocampus of PPr-treated rats by 50%, 39%and 54%, respectively. NSO administration restored significantlyAChE activity in all the three brain regions, by 82%, 87% and 90%,respectively.

3.3. Reduced and oxidized glutathione

Fig. 3 shows the effect of PPr and pretreatment with NSO onGSH, GSSG levels and GSSG/GSH ratio in the three brain regions.

GSH

(nm

oles

/ mg

prot

ein)

0

25

50

75

100 Control

N.sativa

PPr

N.sativa+PPr

Cerebellum Cortex Hippocampus

A

#

# #*

* *

GSS

G (n

mol

es/m

g pr

otei

n)

0

5

10

15

20

25 Control

N.sativa

PPr

N.sativa+PPr

Cerebellum Cortex Hippocampus

B*

#*

#*

#

GSS

G/G

SH ra

tio (%

)

0

25

50

75

100 ControlN.sativa

PPrN.sativa+PPr

Cerebellum Cortex Hippocampus

C

*

#

*

#

*

#

Fig. 3. Effect of propoxur (PPr) and NSO on (A) reduced GSH, (B) oxidizedglutathione (GSSG) and (C) GSSG/GSH ratio in cerebellum, cortex and hippocampusin rats. Results are shown as means ± SE (n = 8). *Significantly different from controlgroup at P < 0.05. #Significantly different from propoxur group at P < 0.05.

MD

A (n

mol

es/ m

g pr

otei

n)

0

1

2

3ControlN.sativa

PPrN.sativa+PPr

Cerebellum Cortex Hippocampus

A

*

#

*

#

*

#

Prot

ein

carb

onyl

con

tent

(nm

oles

/mg

prot

ein)

0.00

0.25

0.50

0.75

1.00 ControlN.sativa

PPrN.sativa+PPr

Cerebellum Cortex Hippocampus

B

*

#

*

#

* #

Fig. 4. Effect of propoxur (PPr) and NSO on (A) malondialdehyde (MDA) and (B)protein carbonyl content (PCC) in cerebellum, cortex and hippocampus in rats.Results are shown as means ± SE (n = 8). *Significantly different from control groupat P < 0.05. #Significantly different from propoxur group at P < 0.05.

A.M. Mohamadin et al. / Pesticide Biochemistry and Physiology 98 (2010) 128–134 131

GSH levels were significantly decreased in PPr-treated rats. GSHlevels were decreased in PPr-treated group in the studied brain re-gion by about 50%. However, co-treatment with NSO significantlyincreased the levels of GSH in all the three brain regions(Fig. 3A). The levels GSSG (Fig. 3B) and the ratio of GSSG/GSH(Fig. 3C) were significantly increased in all the three brain regionsin PPr-treated group compared with control group. These levelswere restored to control values.

3.4. Lipid peroxidation and protein oxidation

MDA is a marker of lipid peroxidation. MDA level significantly(P < 0.05) increased in PPr-treated group in all the three brain re-gions compared to control group (Fig. 4A). Co-treatment withNSO to the rats intoxicated with PPr significantly decreased MDAlevel, but still was higher than that of the control. Protein carbonyl

levels were significantly (P < 0.05) elevated in PPr-treated rats inall the three brain regions compared with control group (Fig. 4B).Nevertheless, co-treatment with NSO significantly (P < 0.05) de-creased the brain PCC content as compared to PPr-treated group.

3.5. Antioxidant enzymes

Fig. 5A–D represents the effects of PPr and NSO on the activitiesof different antioxidant enzymes in various brain regions of rats.The activities of SOD, CAT, GSH-Px and GST decreased significantly(P < 0.05) in all the brain regions of PPr-exposed rats. Whereas, thetreatment with NSO restored the activities of antioxidant enzymessuch as SOD, CAT and GSH-Px. Activity of GST was not significantlyaltered in NSO-treated rats.

4. Discussion

In the present study, increases in LPO, protein oxidation andGSSG levels due to toxic effects of PPr were accompanied by signif-icant reductions in GSH levels of the three brain regions, implicat-ing oxidative tissue damage. Furthermore, oxidative stress statusevidenced by the decreased enzymatic antioxidants; SOD, CAT,GSH-Px and GST demonstrated the severity of PPr-induced brainoxidative stress. NSO, as an antioxidant agent, ameliorated oxida-tive injury in the tissues and functional deteriorations. These find-ings are also consistent with recent reports which demonstratethat insecticides-induced oxidative stress is attenuated by antiox-idant vitamins such as vitamins A, C and E [37–39], adding furthersupport to the role of ROS in mediating PPr toxicity.

The brain has a high rate of oxidative metabolism, consuming�20% of the cardiac output. Brain tissue is rich in polyunsaturated

Cat

alas

e (U

/mg

prot

ein)

0

5

10

15Control

N.sativa

PPr

N.sativa+PPr

Cerebellum Cortex Hippocampus

A

*

#

*

#

*

#

GSH

-Px

(U/m

g pr

otei

n)

0.0

0.5

1.0

1.5

2.0 Control

N.sativa

PPr

N.sativa+PPr

Cerebellum Cortex Hippocampus

C

*

#*

# *

#

SOD

(U/m

g pr

otei

n)

0

2

4

6

8Control

N.sativa

PPr

N.sativa+PPr

Cerebellum Cortex Hippocampus

B

*

#

*

#

*

# GST

(U/m

g pr

otei

n)

0.0

0.5

1.0

1.5

2.0Control

N.sativa

PPr

N.sativa+PPr

Cerebellum Cortex Hippocampus

D

* * **

* *

Fig. 5. Effect of propoxur (PPr) and NSO on the (A) CAT, (B) SOD, (C) GSH-Px and (D) GST activities in cerebellum, cortex and hippocampus in rats. Results are shown asmeans ± SE (n = 8). *Significantly different from control group at P < 0.05. #Significantly different from propoxur group at P < 0.05.

132 A.M. Mohamadin et al. / Pesticide Biochemistry and Physiology 98 (2010) 128–134

fatty acids (PUFAs). These PUFAs are especially vulnerable to oxi-dation [40]. Certain brain regions are highly enriched in non hemeiron, which is catalytically involved in the production of oxygenfree radicals [41] thus increasing the risk of neurodegenerative dis-eases. Mandavilli and Rao [42] showed that cortex and hippocam-pus are more susceptible to oxidative damage when compared tocerebellum which is in corroboration with the present study.Meanwhile, the brain, compared to liver, lung and other organs,contains relatively low levels of enzymatic and non-enzymaticantioxidants and high amounts of peroxidizable unsaturated lipids,rendering it more vulnerable to oxidative stress compared to othertissues [43].

Antioxidant enzymes are essential part of the cellular defenseagainst ROS. Decreased activities of antioxidant enzymes and in-creased level of LPO and PCC generation in brain regions of PPr-treated animals indicate increased oxidative stress in those re-gions. Our results are parallel with those of Eraslan et al. [44]who reported that bee pollen exhibitd a positive influence on oxi-dative stress markers in rats that displayed oxidative stress due toadministration of PPr for 14 days.

ROS may propagate the initial attack on lipid membranes of thebrain to cause LPO [45]. LPO is one of the main manifestations ofoxidative damage and has been found to play an important rolein the toxicity of many xenobiotics [46]. The increased LPO mayalso be due to decreased activities of SOD and CAT which are thefree radical scavenging enzymes. SOD protects against oxygen free

radicals by catalyzing the removal of superoxide radical ðO��2 Þ,which damage the membrane and biological structures. The de-creased activity of SOD in the selected brain regions of PPr-exposedanimals documented in our study is in corroboration with previousinvestigations [47]. CAT has been shown to be the main scavengerof H2O2 at high concentration [48]. Decreased CAT activity hasbeen shown to be associated with oxidative stress in brain regions[49]. Along with CAT, GSH-Px is also involved in the scavenging ofH2O2. GSH-Px is the most important antioxidant enzyme in thebrain that metabolizes peroxides such as H2O2 and protects cellmembranes from LPO [49]. In the present study, the observed de-cline in the activity of GSH-Px in PPr-exposed animals may be as-cribed to the reduction in the level of substrate, i.e., GSH and anincrease in the level of peroxides.

GSTs are a family of enzymes that catalyze the addition of thetripeptide glutathione to endogenous and xenobiotic substrateswhich have electrophilic functional groups. They play an importantrole in the detoxification and metabolism of many xenobiotic com-pounds [50]. Along with GSH-Px, GST activity was found to be low-ered in brain regions of PPr-exposed animals. Free radicals inducetissue damage by initiating and propagating LPO [50]. The increasein LPO in brain regions observed in present experiment could bedue to the significant increase in the generation of ROS in thoseregions of PPr-exposed rats. It may be one of the reasons fordecreased weight of cerebellum, cortex and hippocampus in PPr-exposed animals. Therefore it can be said that increase in ROS

A.M. Mohamadin et al. / Pesticide Biochemistry and Physiology 98 (2010) 128–134 133

observed in the present experiment might have inhibited the AChEactivity in those regions of PPr-treated animals. Tsakiri et al. [51]showed that the activity of AChE was inhibited by free radical for-mation. This may upset the pro-oxidant/antioxidant balance with-in the brain, which could be one of the reasons for decreased AChEactivity. Vincent et al. [52] reveals that the polychlorinated biphe-nyl exposure affects the cholinergic system in experimental ani-mals. In the present study also, the activity of AChE wasdecreased in the selected brain regions of PPr-treated rats.

It is well known that endogenous antioxidant enzymes areresponsible for preventing and neutralizing the free radical-in-duced oxidative damage. The antioxidant enzymes, such as CAT,SOD, GSH-Px, GR and GST, constitute a major supportive team ofdefense against free radicals [53]. Administration of NSO restoredthe activities of SOD, CAT, GSH-Px, AChE and decrease the genera-tion of LPO and PCC in the selected brain regions to normal levels. Arelationship between oxidative stress and neurotoxicity has beenspeculated in many experimental animal models. The implicationof ROS in PPr neurotoxicity was strengthened by the fact that manyfree radical scavengers provide marked functional and histopathol-ogical protection against PPr neurotoxicity [44]. Pretreatment withNSO in the present study significantly increased the brain tissue le-vel of enzymatic antioxidants compared to PPr-treated rats. Thismight indicate the usefulness of NSO, as an excellent source ofantioxidants, in modulating PPr-induced neurotoxicity. The protec-tive effect of antioxidant-rich diets in diseases involving oxidativedamage has been reported. Burits and Bucar [54] showed that N.sativa has appreciable antioxidant and free radical scavenger prop-erties but no prooxidant effect. Yaman and Balikci [55] showedthat N. sativa have a marked protective effect against gentami-cin-induced nephrotoxicity in rats. It has been also shown thatNSO and thymoquinone may have protective effects on lipid perox-idation process during ischemia–reperfusion injury in rat hippo-campus [56].

According to the results of the present study, it can be con-cluded that PPr induced lipid and protein peroxidation and de-creased acetylcholine esterase activity in the selected three brainregions. Also, the current study demonstrates that the enzymaticantioxidant (SOD, CAT, GSH-Px and GST) activities and non-enzy-matic antioxidant (GSH) levels were significantly decreased byPPr. Additionally, treatment with NSO can ameliorate the toxicityand oxidative stress in all the three brain regions. Thus, sufficientintake of NSO by individuals, who regularly are exposed to theseinsecticides, is beneficial in combating the adverse effects ofpropoxur.

Acknowledgments

The authors thank the Dr. Ashraf Abdel Naim, Professor of Phar-macology and Toxicology at the University of King Abdulaziz, forproviding the animals’ house facility for the study and Dr. EssamAbdel-Sattar Department of Natural Products, Faculty of Pharmacy,King Abdulaziz University, Jeddah, Saudi Arabia for his assistanceand identifying N. sativa.

References

[1] L. Institoris, O. Siroki, I. Desi, U. Undeger, Immunotoxicological examination ofrepeated dose combined exposure by dimethoate and two heavy metals inrats, Human Exp. Toxicol. 18 (1999) 88–94.

[2] A.P. Alvares, Pharmacology and toxicology of carbamates, in: B. Ballantyne,T.C. Marrs (Eds.), Clinical and Experimental Toxicology of Organophosphatesand Carbamates, Butterworth and Heinemann, Oxford, 1992, pp. 40–46.

[3] J.P. Groten, E.D. Schoen, P.J. Bladeren, C.F. Kuper, J.A. Zorge, V.J. Feron, Subacutetoxicity of a mixture of nine chemicals in rats: detecting interactive effectswith a fractionated two-level factorial design, Fundam. Appl. Toxicol. 36(1997) 15–29.

[4] B.D. Banerjee, V. Seth, A. Bhattacharya, S.T. Pasha, A.K. Chakraboty, Biochemicaleffects of some pesticides on lipid peroxidation and free-radical scavengers,Toxicol. Lett. 107 (1999) 33–47.

[5] J.C. Tharappel, E.Y. Lee, L.W. Robertson, B.T. Spear, H.P. Glanet, Regulation ofcell proliferation, apoptosis and transcriptional activities during the promotionof liver carcinogenesis by PCB, Toxicol. Appl. Pharmacol. 179 (2002) 172–184.

[6] J. Smythies, The neurotoxicity of glutamate, dopamine, iron and reactiveoxygen species: functional interrelationship in health and diseases,Neurotoxicol. Res. 1 (1999) 27–39.

[7] V. Seth, B.D. Banerjee, A.K. Chakroborty, L. Institoris, L. Desi, Effect of propoxuron humoral and cell-mediated immune responses in albino rats, Bull. Environ.Contam. Toxicol. 68 (2002) 369–376.

[8] B.D. Banerjee, V. Seth, R.S. Ahmed, Pesticides induced oxidative stress:perspectiveness and trends, Rev. Environ. Health 16 (2001) 1–40.

[9] A.M. El-Sharkawy, S.Z. Abdul Rahman, A. A Hassan, S.M. el-Zoghby, S.M. el-Sewedy, Biochemical effects of some insecticides on the metabolic enzymesregulating glutathione metabolism, Bull. Environ. Contam. Toxicol. 52 (1994)505–510.

[10] R.S. Verma, N. Srivastava, Chlorpyrifos induced alterations in levels ofthiobarbituric acid reactive substances and glutathione in rat brain, Indian J.Exp. Biol. 39 (2001) 174–177.

[11] S. Hussain, W. Slikker Jr., S.F. Ali, Age-related changes in antioxidant enzymes,superoxide dismutase, catalase, glutathione peroxidase and glutathione indifferent regions of mouse brain, Int. J. Dev. Neurosci. 13 (1995) 811–817.

[12] B.S. Baek, H.J. Kwon, K.H. Lee, M.A. Yoo, Y. Ikeno, B.P. Yu, H.Y. Chung, Regionaldifference of ROS generation, lipid peroxidation, and antioxidant enzymeactivity in rat brain and their dietary modulation, Arch. Pharm. Res. 22 (1999)361–366.

[13] M. Tariq, Nigella sativa seeds: folklore treatment in modern day medicine,Saudi J. Gastroenterol. 14 (2008) 105–106.

[14] B. Meddah, R. Ducroc, M. El Abbes Faouzi, B. Eto, L. Mahraoui, A. Benhaddou-Andaloussi, L.C. Martineau, Y. Cherrah, P.S. Haddad, Nigella sativa inhibitsintestinal glucose absorption and improves glucose tolerance in rats, J.Ethnopharmacol. 121 (2009) 419–424.

[15] M.L. Salem, M.S. Hossain, Protective effect of black seed oil from Nigella sativaagainst murine cytomegalovirus, Int. J. Immunopharmacol. 22 (2000) 729–740.

[16] N.S. El Shenawy, M.F. Soliman, S.I. Reyad, The effect of antioxidant propertiesof aqueous garlic extract and Nigella sativa as anti-schistosomiasis agents inmice, Rev. Inst. Med. Trop. Sao Paulo 50 (2008) 29–36.

[17] V. Hajhashemi, A. Ghannadi, H. Jafarabadi, Black cumin seed essential oil, as apotent analgesic and antiinflammatory drug, Phytother. Res. 18 (2004) 195–199.

[18] I. Tekeoglu, A. Dogan, L. Ediz, M. Budancamanak, A. Demirel, Effects ofthymoquinone (volatile oil of black cumin) on rheumatoid arthritis in ratmodels, Phytother. Res. 21 (2007) 895–897.

[19] L. Ait MbarekL, H. Ait Mouse, N. Elabbadi, M. Bensalah, A. Gamouh, R.Aboufatima, A. Benharref, A. Zyad, Anti-tumor properties of blackseed (Nigellasativa L.) extracts, Braz. J. Med. Biol. Res. 40 (2007) 839–847.

[20] E. Uz, O. Bayrak, E. Uz, A. Kaya, R. Bayrak, B. Uz, F.H. Turgut, Nigella sativa oil forprevention of chronic cyclosporine nephrotoxicity: an experimental model,Am. J. Nephrol. 28 (2008) 517–522.

[21] I. Yaman, E. Balikci, Protective effects of Nigella sativa against gentamicin-induced nephrotoxicity in rats, Exp. Toxicol. Pathol. 62 (2010) 183–190.

[22] M. Kanter, Nigella sativa and derived thymoquinone prevents hippocampalneurodegeneration after chronic toluene exposure in rats, Neurochem. Res. 33(2008) 579–588.

[23] S.B. Pajovic, Z.S. Saicic, M.B. Spasic, M.B. Petrovic, The effect of ovarianhormones on antioxidant enzyme activities in the brain of male rats, Physiol.Rev. 52 (2003) 189–194.

[24] J. Chaudiere, R. Ferrari-iliou, Intracellular antioxidants: from chemical tobiochemical mechanisms, Food Chem. Toxicol. 37 (1999) 949–962.

[25] G.E. Paget, J.M. Barnes, Interspecies dosage conversion scheme in evaluation ofresults and quantitative application in different species, in: D.R. Laurence, A.L.Bacharach (Eds.), Evaluation of Drug Activities: Pharmacometrics, vol. 1,Academic Press, London and New York, 1964, pp. 160–167.

[26] L. Instito’ris, A. Papp, O. Siroki, B.D. Banerjee, I. Desi, Immuno- andneurotoxicological investigation of combined subacute exposure with thecarbamate pesticide propoxur and cadmium in rats, Toxicology 178 (2002)161–173.

[27] L.J. Pelligrino, A.S. Pelligrino, A.J. Cushman, A stereotaxic atlas of the rat brain.Plenum, New York. Pouyatos, B., Gearhart, C., Nelson-Miller, A., Fulton, S.,Fechter, L., 2007. Oxidative stress pathways in the potentiation of noise-induced hearing loss by acrylonitrile, Hear. Res. 224 (1979) 61–74.

[28] O.H. Lowry, N.J. Risebrough, A.L. Farr, Randal, protein measurement with Folinphenol reagent, J. Biol. Chem. 193 (1951) 265–275.

[29] G.L. Ellman, K.D. Courtney, V. Andres, V. Feather Stone, A new and rapidcolorimetric determination of AchE activity, Biochem. Pharmacol. 7 (1961) 88–95.

[30] G.L. Ellman, Tissue sulphydryl groups, Arch. Biochem. Biophys. 82 (1959) 70–77.

[31] T.P. Devasagayam, U. Tarachand, Decreased lipid peroxidation in rat kidneysduring gestation, Biochem. Biophys. Res. Commun. 145 (1987) 134–138.

[32] R.L. Levine, D. Garland, C.N. Oliver, A. Amici, I. Climent, A.G. Lenz, B.W. Ahn, S.Shalteil, E.R. Stantman, Determination of carbonyl content in oxidativelymodified proteins, in: L. Packer, A.N. Glazer (Eds.), Methods in Enzymology, V186, Oxygen Radicals in Biological Systems, Academic Press, New York, 1990,pp. 464–478.

134 A.M. Mohamadin et al. / Pesticide Biochemistry and Physiology 98 (2010) 128–134

[33] S. Marklund, G. Marklund, Involvement of superoxide anion in theautooxidation of pyrogallol and a convenient assay for superoxidedismutase, Eur. J. Biochem. 47 (1974) 469–474.

[34] H. Aebi, Catalase in vitro, in: L. Packer, F.L. Orlando (Eds.), Methods inEnzymology, vol. 105, Academic Press, New York, 1984, pp. 121–126.

[35] J.T. Rotruck, A.L. Pope, H.E. Ganther, W.G. Hoekstra, Selenium: biochemicalroles as a component of glutathione peroxidase, Science 179 (1973) 588–590.

[36] W.H. Habig, M.J. Pabst, W.B. Jacoby, Glutathione-S-transferase; the first step inmercapturic fermentation, J. Biochem. 249 (1973) 7130–7139.

[37] T.A. Slotkin, F.J. Seidler, Comparative developmental neurotoxicity oforganophosphates in vivo: transcriptional responses of pathways for braincell development, cell signaling, cytotoxicity and neurotransmitter systems,Brain Res. Bull. 72 (2007) 232–274.

[38] R.S. Verma, A. Mheta, N. Srivastava, In vivo chlorpyrifos induced oxidativestress: attenuation by antioxidant vitamins, Pesticide Biochem. Physiol. 88(2007) 191–196.

[39] F. Yu, Z. Wang, B. Ju, Y. Wang, J. Wang, D. Bai, Apoptotic effect oforganophosphorus insecticide chlorpyrifos on mouse retina in vivo viaoxidative stress and protection of combination of vitamins C and E, Exp.Toxicol. Pathol. 59 (2008) 415–423.

[40] B.A. Wagner, G.R. Buettner, G.P. Burna, Free radical mediated peroxidation incells: oxidability is a function of cell lipid bis-allylic hydrogen content,Biochemistry 33 (1994) 4449–4453.

[41] J.M. Hill, R.C. Switzer, The regional distribution and cellular localization of ironin the rat brain, Neuroscience 11 (1984) 595–603.

[42] B.S. Mandavilli, K.S. Rao, Neurons in the cerebral cortex are more susceptible toDNA-damage in ageing rat brain, Biochem. Mol. Biol. Int. 40 (1996) 507–514.

[43] S.C. Bondy, Free-radical-mediated toxic injury to the nervous system, in: K.B.Wallace (Ed.), Free Radical Toxicology, Taylor & Francis, Oxford, 1997, pp. 221–248.

[44] G. Eraslan, M. Kanbur, S. Silici, B.C. Liman, S. Altınordulu, Z. Soyer Sarıca,Evaluation of protective effect of bee pollen against propoxur toxicity in rat,Ecotoxicol. Environ. Saf. 72 (2009) 931–937.

[45] M. Das, S.K. Kanna, Clinico epidemiological and toxicological and safetyevaluation studies on argensone oil, Crit. Rev. Toxicol. 27 (1997) 273–297.

[46] R. Anane, E.E. Creppy, Lipid peroxidation as pathway of aluminiumcytotoxicity in human skin fibroblast cultures: prevention by superoxidedismutase + catalase and vitamins E and C, Hum. Exp. Toxicol. 20 (2001) 477–481.

[47] P. Matos, A. Fontaınhas-Fernandes, F. Peixoto, J. Carrola, E. Rocha, Biochemicaland histological hepatic changes of Nile tilapia Oreochromis niloticus exposed tocarbaryl, Pestic. Biochem. Physiol. 89 (2007) 73–80.

[48] Y. Kono, I. Fridorich, Superoxide radical inhibits catalase, J. Biol. Chem. 257(1982) 5751–5754.

[49] S. Shila, V. Kokilavani, M. Subathra, C. Panneerselvam, Brain regional responsesin antioxidant system to a-lipoic acid in arsenic intoxicated rat, Toxicology210 (2005) 25–36.

[50] C. Mylonas, D. Kouretas, Lipid peroxidation and tissue damage, In Vivo 13(1999) 295–309.

[51] S. Tsakiri, P. Angelogianni, K.H. Schulpis, C. Stavridis, Protective effect of L

phenylalanine on rat brain acetylcholine esterase inhibition induced by freeradicals, Clin. Biochem. 33 (2000) 103–106.

[52] D.R. Vincent, W.S. Bradshaw, G.H. Booth, R.E. Seegmiller, S.D. Allen, Effect ofPCB and DES on rat monoamine oxidase, acetylcholine esterase, testosterone,and estradiol ontogeny, Bull. Environ. Contam. Toxicol. 48 (1992) 884–893.

[53] H. Sung, J. Nah, S. Chun, H. Park, S.E. Yang, W.K. Min, In vivo antioxidant effectof green tea, Eur. J. Clin. Nutr. 54 (2000) 527–529.

[54] M. Burits, F. Bucar, Antioxidant activity of Nigella sativa essential oil, Phytother.Res. 14 (2000) 323–328.

[55] _I. Yaman, E. Balikci, Protective effects of Nigella sativa against gentamicin-induced nephrotoxicity in rats, Exp. Toxicol. Pathol. 62 (2010) 183–190.

[56] H. Hosseinzadeh, S. Parvardeh, M.N. Asl, H.R. Sadeghnia, T. Ziaee, Effect ofthymoquinone and Nigella sativa seeds oil on lipid peroxidation level duringglobal cerebral ischemia-reperfusion injury in rat hippocampus,Phytomedicine 14 (2007) 621–627.