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Novel neuroprotective and hepatoprorective effects of citric acid in acute malathion
intoxication
Omar M.E. Abdel-Salam, Eman R. Youness, Nadia A. Mohammed, Noha N. Yassen,
Yasser A. Khadrawy, Safinaz Ebrahim El-Toukhy, Amany A. Sleem
PII: S1995-7645(16)30462-X
DOI: 10.1016/j.apjtm.2016.11.005
Reference: APJTM 372
To appear in: Asian Pacific Journal of Tropical Medicine
Received Date: 18 July 2016
Revised Date: 19 August 2016
Accepted Date: 18 September 2016
Please cite this article as: Abdel-Salam OME, Youness ER, Mohammed NA, Yassen NN, Khadrawy
YA, El-Toukhy SE, Sleem AA, Novel neuroprotective and hepatoprorective effects of citric acid
in acute malathion intoxication, Asian Pacific Journal of Tropical Medicine (2016), doi: 10.1016/
j.apjtm.2016.11.005.
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Title:
Novel neuroprotective and hepatoprorective effects of citric acid in acute malathion intoxication
Authors:
Omar M.E. Abdel-Salam
1*
, Eman R. Youness
2
, Nadia A. Mohammed
2
, Noha N. Yassen
3
, Yasser
A. Khadrawy
4
, Safinaz Ebrahim El-Toukhy
2
, Amany A. Sleem
5
Affiliations:
1
Department of Toxicology and Narcotics, National Research Centre, Tahrir St., Dokki, Cairo,
Egypt
2
Medical Biochemistry, National Research Centre, Tahrir St., Dokki, Cairo, Egypt
3
Pathology, National Research Centre, Tahrir St., Dokki, Cairo, Egypt
4
Physiology, National Research Centre, Tahrir St., Dokki, Cairo, Egypt
5
Pharmacology, National Research Centre, Tahrir St., Dokki, Cairo, Egypt
*First and corresponding author: Omar M.E. Abdel-Salam, Department of Toxicology and
Narcotics, National Research Centre, Cairo, Egypt.
E-mail: omasalam@hotmail.com
This paper has 13 Figures.
Article history:
Received 18 July 2016
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Received in revised form 19 August 2016
Accepted 18 September 2016
Available online 16 November 2016
Key words
Citric acid
Malathion
Oxidative stress
Paraoxonase 1
Cholinesterase
Comet assay
Abstract
Objective: To study the effect of citric acid given alone or combined with atropine on brain
oxidative stress, neuronal injury, liver damage, and DNA damage of peripheral blood
lymphocytes induced in the rat by acute malathion exposure. Methods: Rats were received
intraperitoneal (i.p.) injection of malathion 150 mg/kg along with citric acid (200 or 400 mg/kg,
orally), atropine (1 mg/kg, i.p.) or citric acid 200 mg/kg+atropine 1 mg/kg and euthanized 4 h
later. Results: Malathion resulted in increased lipid peroxidation (malondialdehyde) and nitric
oxide concentrations accompanied with a decrease in brain reduced glutathione, glutathione
peroxidase (GPx) activity, total antioxidant capacity (TAC) and glucose concentrations.
Paraoxonase-1, acetylcholinesterase (AChE) and butyrylcholinesterase activities decreased in
brain as well. Liver aspartate aminotransferase and alanine aminotransferase activities were
raised. The Comet assay showed increased DNA damage of peripheral blood lymphocytes.
Histological damage and increased expression of inducible nitric oxide synthase (iNOS) were
observed in brain and liver. Citric acid resulted in decreased brain lipid peroxidation and nitric
oxide. Meanwhile, glutathione, GPx activity, TAC capacity and brain glucose level increased.
Brain AChE increased but PON1 and butyrylcholinesterase activities decreased by citric acid.
Liver enzymes, the percentage of damaged blood lymphocytes, histopathological alterations and
iNOS expression in brain and liver was decreased by citric acid. Meanwhile, rats treated with
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atropine showed decreased brain MDA, nitrite but increased GPx activity, TAC, AChE and
glucose. The drug also decreased DNA damage of peripheral blood lymphocytes,
histopathological alterations and iNOS expression in brain and liver. Conclusions: The study
demonstrates a beneficial effect for citric acid upon brain oxidative stress, neuronal injury, liver
and DNA damage due to acute malathion exposure.
1. Introduction
Oxygen derived free radicals are produced in the cell from many sources. One important
source is the mitochondrial electron transport chain where electrons that leaked from O
2
result in
generation of superoxide anion radical. (O
2
•-). The redox state of the cell is kept in balance due
to a number of antioxidant mechanisms. These include both enzymatic (eg., catalases, speroxide
dismutases, and glutathione peroxidase) and non-enzymatic free radical scavengers (eg.,
glutathione, α-tocopherol, ascorbic acid)
[1,2]
. Oxidative stress develops when there is an increase
in oxidants and/or in adequate antioxidants
[3]
. Oxidative stress contributes to the development of
several disease processes eg., diabetes mellitus, cardiovascular disease, cancer,
neurodegenerative and psychiatric disorders
[4-7]
. Owing to its high metabolic demand, the brain
utilizes much O
2
with the consequent increased generation of reactive oxygen metabolites.
Moreover, auto-oxidation of brain neurotransmitters generating O
2
and quinones and the
presence of redox-active metals capable of catalyzing free radical reactions increase the brain’
oxidant burden. The brain is also rich in polyunsaturated fatty acids, which is the preferred
substrate for free radical attack. These factors coupled with modest antioxidant mechanisms
make the brain tissue particularly susceptible to oxidative stress
[1,2,8]
.
Reactive oxygen metabolites are likely to contribute to the neurotoxic effects of
organophosphate insecticides. In this context, exposure to malathion caused increased lipid
peroxidation
[9,10]
increased nitric oxide, and decreased reduced glutathione (GSH)
[10]
in the rat
brain. Lipid peroxidation increased in blood, liver
[11,12]
and in the rat erythrocytes as well
[13]
.
Studies also indicated decreased activities of the antioxidant enzymes glutathione reductase and
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glutathione peroxidase in rat cerebral cortex
[14]
and activities of superoxide dismutase, catalase
and glutathione peroxidase in human erythrocytes
[15]
also decreased after exposure to malathion.
Moreover, the chain breaking antioxidants α-tocopherol and ascorbate were able to reduce lipid
peroxidation and ameliorate the changes in antioxidant enzymes caused by malathion in rat and
human erythrocytes
[13,15]
.
Citric acid (2-hydroxy-1,2,3-propane-tricarboxylic acid) is a weak organic acid found in all
animal tissues
[16]
. Cellular citrate is synthesized inside the mitochondria
[17]
while rich dietary
sources include lemon, orange, tangerine and grapefruit
[18]
. Intracellular citric acid is important
in the intermediary energy metabolism of the cell. Citrate is produced in the mitochondria from
acetyl-CoA and oxalacetate and enters the citric acid cycle (tricarboxylic acidcycle or Krebs
cycle). The resulting high-energy intermediates; the reduced coenzymes nicotinamide adenine
dinucleotide and flavin adenine dinucleotide are then utilized in the respiratory chain in the inner
mitochondrial membrane to make ATP (adenosine 5’-triphosphate) for the cell’s energy needs.
Citric acid released into the cytoplasm via specific mitochondrial carriers is converted to acetyl
CoA for the biosynthesis of fatty acids, lipids, and cholesterol
[19]
.
Besides its role in the generation of energy, citrate have other important actions including
down regulation of inflammation and reduction of lipid peroxidation
[20-23]
. Citrate reduces
polymorphonuclear cell degranulation and attenuate the release of inflammatory mediators eg.,
myeloperoxidase, platelet factor 4, interleukin 1β
[18-20]
and tumour necrosis factor-alpha
[23]
.
Citric acid displayed hepatoprotective effects where it reduced hepatocellular damage evoked by
carbon tetrachloride in rats
[24,25]
. It also decreased brain lipid peroxidation and inflammation and
liver damage in mice treated with bacterial lipopolysaccharide endotoxin
[23]
.
A defect in mitochondrial bioenergetics might be involved in the neurotoxic effects of
malathion. This is because organophosphates can cause mitochondrial impairment
[9,26-29]
.
Moreover, the administration of methylene blue, an antioxidant
[30]
and an enhancer of the
electron transport chain
[31]
protected against the malathion-induced neurotoxicity
[10]
. Thus, in
view of the bioenergetic, antioxidant and anti-inflammatory effects reported above for citrate, it
looked pertinent to investigate the effect of citric acid administration on oxidative stress and
brain damage in rats intoxicated with the organophosphate malathion. We also examined the
possible modulation by citric acid of the effect of atropine, the muscarinic receptor antagonist
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and the antidote employed in the management of acute organophosphate poisoning
[32,33]
. Since,
malathion has been shown to cause hepatocellular damage
[34,35]
, the study was extended to
include the liver tissue.
2. Materials and methods
2.1. Animals
Male rats of the Sprague-Dawley strain with body weight of (130-140) g were used. Rats were
obtained from Animal House Colony of the National Research Centre. Rats allowed free access
to standard laboratory food and water. Animal procedures were done in accordance to the
recommendations of the institutional Ethics Committee and the National Institutes of Health
Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985).
2.2. Drugs and chemicals
Malathion (Commercial grade, 57%) was purchased from El-Naser Chemical Co., Cairo.
Citric acid and atropine were obtained from Sigma-Aldrich (St Louis, MO, USA). Other
chemicals and reagents were of analytical grade and purchased from Sigma-Aldrich.
2.3. Study design
Rats were randomly divided into different groups (6 rat/group). Group 1 was treated with i.p.
saline (0.2 mL/rat) and served as negative control. Group 2-6 were i.p. treated with malathion at
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a dose of 150 mg/kg, along with saline (group 2), citric acid at 200 or 400 mg/kg (groups 3&4),
atropine at 1 mg/kg (group 5) or citric acid at 200 mg/kg + atropine at 1 mg/kg (group 6). Rats
were euthanized by decapitation 4h after drug administration. Their brains were quickly removed
on ice-plate and washed with ice-cold phosphate-buffered saline at pH 7.4. Brains were weighed
and stored at -80 ℃ for later biochemical analyses. Homogenization of brain tissues were carried
out using 0.1M phosphate buffer saline (pH 7.4) to give a final concentration of 20% w/v for the
biochemical assays.
2.4. Biochemical analyses
2.4.1. Lipid peroxidation
Malondialdehyde (MDA), a product of lipid peroxidation was determined in tissue
homogenates by the method of Nair and Turne
[36]
. In this assay thiobarbituric acid reactive
substances (TBA) react with thiobarbituric acid to form TBA-MDA adduct which can be
measured colorimetrically at 532 nm.
2.4.2. Reduced glutathione
Reduced glutathione (GSH) was determined in tissue homogenates using the method of
Ellman et al.
[37]
. The procedure is based on the reduction of Ellman’s reagent [DTNB; 5, 5’-
dithiobis (2-nitrobenzoic acid)] by the free sulfhydryl group on GSH to form yellow colored 5-
thio-2-nitrobenzoic acid which can be determined using spectrophotometer at 412 nm.
2.4.3. Nitric oxide
Nitric oxide was determined using colorimetric assay where nitrate is converted to nitrite via
nitrate reductase. Griess reagent then act to convert nitrite to a deep purple azo compound that
can be determined using spectrophotometer
[38]
.
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2.4.4. Glutathione peroxidase activity
Glutathione peroxidase (GPx) activity was determined in supernatants using colorimetric
glutathione peroxidase kit (Biodiagnostic, Egypt). The activity of GPx is expressed as mU/mL.
2.4.5. Paraoxonase-1 activity
Paraoxonase-1 arylesterase activity was measured using phenylacetate as a substrate and the
formation of phenol was measured spectrophotometrically by monitoring the increase in
absorbance at 270 nm at 25 ℃. One unit of arylesterase activity is defined as 1 µM of phenol
formed per minute. Enzyme activity was calculated based on the extinction coefficient of phenol
of 1 310 M
-1
cm
-1
at 270 nm, pH 8.0 and 25 ℃. and expressed kilo International Unit/Liter
(kU/L)
[39,40]
.
2.4.6. Acetylcholinesterase activity
Acetylcholinesterase activity was measured using the method of Ellman et al.
[41,42]
. The
method uses DTNB to measure the amount of thiocholine produced as acetylthiocholine is
hydrolyzed by AChE. The color of DTNB adduct can be measured using spectrophotometric at
412 nm. AChE activity was expressed as µmol sulfhydryl (SH) groups/g tissue/min.
2.4.7. Butyrylcholinesterase activity
Butyrylcholinesterase (EC 3.1.1.8; BChE) activity was measured in brain supernatants using
commercially available kit from Ben Biochemical Enterprise (Milan, Italy). The method is that
of Ellman et al.
[41]
. Butyrylcholinesterase catalyzes the hydrolysis of butyrylthiocholine as
substrate into butyrate and thiocholine. Thiocholine reacts with 5, 5’-dithiobis (2-nitrobenzoic
acid) (DTNB) forming a yellow chromophore which can be quantified using spectrophotometer.
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2.4.8. Total antioxidant capacity
Total antioxidant capacity (TAC) was measured in brain homogenates using a colorimetric kit
obtained from Biodiagnostic (Egypt). In this method antioxidants in the sample react with a
defined amount of exogenously provided hydrogen peroxide. The remaining hydrogen peroxide
will be determined calorimetrically by an enzymatic reaction which involves the conversion of 3,
5-dichloro-2-hydroxy benzensulfate to a colored product
[29]
.
2.4.9. Glucose
Glucose oxidase catalyzes the oxidation of glucose to gluconic acid with the formation of
hydrogen peroxide. The latter reacts with phenol and 4-amino-antipyrine in the presence of
peroxidase resulting in a colored quinonemine which can be measured using spectrophotometer.
2.4.10. Liver enzymes
Reitman-Frankel procedure (Crowley 1967) was used for the colorimetric determination of
alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in liver.
Alkaline phosphatase (ALP) activity was determined colorimetrically according to Belfield and
Goldberg (1971). Commercially available kits from BioMérieux (France) were used for this
purpose.
2.5. Comet assay
Isolation of peripheral blood leukocytes were done by centrifugation (30 min at 1 300 g) using
Ficoll-Paque density gradient (Pharmacia LKB Biotechnology, Piscataway, NJ, USA). After
centrifugation, leukocytes were represented as a buffy coat, aspirated and washed twice with
phosphate buffered saline (pH 7.4).
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The comet assay was done at low temperature to minimize spontaneous DNA damage
[44,45]
. In
brief, 100 µL of normal melting point agarose (at 0.7%) was applied onto a pre-cleaned
microscope charged slide and a coverslip was gently applied. The coverslip was removed after
the agarose solidified at 4 ℃ and 100 µL of low melting point agarose at 0.5% containing 1 500
peripheral blood lymphocytes were then added. The coverslip was replaced and the slide placed
at 4 ℃ for solidification. The coverslip was then removed and a final layer of low melting
agarose was added, coversliped, and left to solidify for 10 min.
The coverslip was removed and the slide was immersed in 100 mL of fresh lysis buffer (2.5
mol/L NaCl, 100 mmol/L EDTA, 1% sodium hydroxide, 10 mmol/L Tris, 1% Triton X-100,
10% DMSO (pH10) at 4 ℃ for 1 h. The microgels slides were then removed from the solution
and rinsed with DNA unwinding solution (300 mmol/L NaOH and 1 mmol/L EDTA, pH 13) for
30 min at 4°C. Thereafter, the slides were placed on a horizontal gel electrophoresis chamber
that is filled with DNA-unwinding solution. Gels were run for 30 min with constant current of
300 mA at 4 ℃. Following electrophoresis, the microgels were neutralized with 0.4 M Trisma
base at pH 7.5 for10 min and finally, the slides were stained with 20 µL ethidium bromide
(Sigma) at 10 µg/mL.
Examination of the slides was done at 400× magnification using a fluorescence microscope
(IX70; Olympus, Tokyo, Japan). The apparatus was equipped with an excitation filter of 549 nm
and a barrier filter of 590 nm and attached to an ‘Olympus’ video camera. The damaged cell had
the appearance of a comet, with a brightly fluorescent head and a tail to one side. The latter
formed by the DNA containing strand breaks that were drawn away during electrophoresis.
Samples were studied by counting the number of damaged cell per 100 cells as to calculate the
percent of damaged cells.
2.6. Histological studies
The rats were killed by decapitation, and their brains and livers were quickly removed out.
Slices were then were fixed in 10% formalin (pH 7.4) for a minimum of 72 h, washed in tap
water for 30 min, dehydrated using ascending grades of alcohol, cleared in xylene and embedded
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in paraffin. Serial sections (6 µm thick) were stained with haematoxylin and eosin (Hx&E),
examined and photographed under light microscope using a digital camera (DP70, Tokyo).
Adobe Photoshop version 8.0 was used for images processing.
2.7. Immunohistochemistry
Immunohistochemistry for iNOS was done on paraffin-embedded sections that were
deparaffinized and rehydrated. Sections were incubated in 0.3% H
2
O
2
solution in methanol at
room temperature for 30 min to block endogenous peroxidase activity. For antigen retrieval,
sections were heated in a microwave oven at 720 W for 25 min and incubated with mouse
monoclonal iNOS antibodies (dilution 1:50) at 4 overnight. Sections were then washed with
phosphate buffered saline, pH 7.4, followed by incubation with biotinylated goat-anti-rabbit-
immunoglobulin G secondary antibodies (dilution 1:200) and streptavidin/alkaline phosphatase
complex (dilution 1:200) (for 30 min at room temperature). The binding sites of antibody were
visualized with 3, 3’-diaminobenzidine. Sections were then rinsed with phosphate buffered
saline, counterstained with H&E for (2-3) min, and dehydrated in ascending grades of ethanol.
Slices were then soaked twice in xylen at room temperature for 5 min, mounted, and examined
by light microscope.
2.8. Immunomorphometric analysis
Quantitative assessment of iNOS immunoreactivity was done with an Image Analyzer system
(Leica Qwin 500IW, Cambridge, England).
2.9. Statistical analysis
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Data are expressed as mean±SE. Statistical significance was determined using one-way
analysis of variance (ANOVA), followed by Duncan’s multiple range test (SPSS software; SAS
Institute Inc., Cary, NC). A probability value of less than 0.05 was considered statistically
significant.
3. Results
3.1. Oxidative stress
3.1.1. Lipid peroxidation
In brain tissue, lipid peroxidation measured as malondialdehyde (MDA) showed significant
increase by 45% after exposure to malathion compared with the saline control group,
(29.00±1.20) vs. (20.00±0.84) nmoL/g tissue. In rats treated with malathion, citric acid at 200
and 400 mg/kg resulted in significant decrease in the level of MDA by 14.8% and 18.6%,
respectively (24.70±0.92) and (23.60±1.10) vs. (29.00±1.20) nmoL/g tissue. No significant effect
was observed for atropine but the combined treatment with atropine and citric acid reduced
MDA by 41% compared with the malathion only group (17.10±1.30) vs. (29.00±1.20) nmoL/g
tissue ) (Figure 1A).
In liver tissue, malathion induced a marked increase in membrane lipid peroxidation, as shown
by the level of MDA (55.5% increase: saline control, (51.9±2.1); malathion (80.7±4.9) nmoL/g
tissue). A significant decrease in MDA by 16.4% and 26.9% was observed after treatment with
200 mg/kg citric acid and after both atropine and citric acid (59.0±3.7) and (67.4±3.6) vs.(80.7 ±
4.9) nmoL/g tissue) (Figure 2A).
3.1.2. Nitrite
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Malathion induced a significant rise in the level of brain nitrite by 42% compared with the
saline group (27.4±2.2) vs. (19.3±1.6) µmol/g tissue). Treatment with citric acid significantly
reduced brain nitrite by 28.8% and 33.9%, respectively, compared with the malathion only
treatment group (19.5±0.9) and (18.1±1.3) vs. (27.4±2.2) µmol/g tissue. A significant decrease in
brain nitrite by 37.2% and 35% was also observed in rats treated with either atropine or atropine
and citric acid (17.2±1.0) and (17.80 ± 0.68) vs. (27.4±2.2) µmoL/g tissue) (Figure 1B).
In the liver tissue of malathion only treated rats, a significant increase in nitrite was also
observed as 39.3% increase: saline control, (44.0±2.6); malathion (61.3±4.0) µmoL/g tissue. The
administration of citric acid, atropine or their combination, however, had no significant effect on
liver MDA (Figure 2B).
3.1.3. Reduced glutathione
Compared with the saline control group, the level of brain GSH decreased by 29.8% in the
malathion only treated rats (3.40±0.11) vs. (4.83±0.24) µmol/g tissue. Brain GSH showed
significant rise by 19.1% and 23.5% after treatment with citric acid at 200 and 400 mg/kg,
respectively (4.05±0.12) and (4.20±0.18) vs. (3.40±0.11) µmol/g tissue. The administration of
atropine alone or combined with citric acid, however, had no significant effect on brain GSH
(Figure 1C).
A significant decrease in GSH by 30.4% was also observed in the liver of malathion only
treated rats (8.00±0.25) vs. (11.50±0.61) µmol/g tissue. Rats treated with 200 m/kg citric acid or
both atropine and citric acid had 17% and 17.5% increments in liver GSH compared with the
malathion only treated group (9.36±0.21) and (9.42±0.3) vs. (8.00±0.25) µmol/g tissue) (Figure
2C).
3.2. Glutathione peroxidase activity
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Malathion resulted in a significant decrease in brain GPx activity by 44.9% saline control,
(83.9±3.5); malathion (46.2±2.9) mU/mL. Rats treated with citric acid showed significant
increments in GPx activity by 67.3% and 79%, respectively, compared with the respective
malathion only control group (77.3±4.0) and (82.7±4.3) vs. (46.2±2.9) mU/mL. A significant
increase in brain GPx activity by 47.2% and 47.4% was also observed in rats treated with
malathion along with atropine or both atropine and citric acid compared with the malathion only
control group (68 ± 3.0) and (68.1±3.6) vs.(46.2±2.9) mU/mL) (Figure 1D).
3.3. Total antioxidant capacity
Rats treated with only malathion had significantly decreased TAC in their brains by 62.5%
compared with their saline control counterparts (0.268±0.010) vs.(0.714±0.060) µmol/g tissue. A
significant increase in brain TAC by 27.2%, 58.9% and 37.3% was observed in rats treated with
malathion along with 400 mg/kg citric acid, atropine or both atropine and citric acid compared
with the malathion only control group (0.341±0.010), (0.426±0.030) and (0.368±0.020)
vs.(0.268±0.010) µmol/g tissue) (Figure 1E).
3.4. Acetylcholinesterase activity
Rats treated with only malathion showed 30.7% inhibition in brain AChE activity compared
with the saline control group (4.71±0.23) vs. (6.80±0.41) µmoL SH/g/min. A significant increase
in brain AChE activity by 47.1%, 32.9%, and 25.3% was observed in rats treated with malathion
along with 400 mg/kg citric acid, atropine or both atropine and citric acid compared with the
malathion only control group (6.93±0.47), (6.26±0.22) and (5.90±0.31) vs.(4.71±0.23) µmol
SH/g/min) (Figure 3).
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3.5. Butyrylcholinesterase activity
In malathion only-treated rats, brain BChE activity was inhibited by 38.2% compared with the
saline control group (120.51±7.60) vs. (195.14±13.70) U/L. Further significant decrease in brain
BChE activity by 24.4%, and 24.5% was observed in rats treated with malathion along with 400
mg/kg citric acid or both atropine and citric acid compared with the malathion only control group
(91.1±6.0) and (91.0±4.8) vs.(120.51±7.60) U/L) (Figure 4).
3.6. Paraoxonase-1 activity
Compared with the saline control group, malathion only-treated rats showed a significant
inhibition of brain PON1 activity by 36.9% (7.95±0.54) vs. (12.6±0.42) kU/L. No significant
effect for atropine was observed on PON1 activity in malathion-treated rats. The enzyme activity
in the brain, however, showed further inhibition by 19.1%, 23.3% and 24.9% following
treatment with citric acid at 200 or 400 mg/kg and both atropine and citric acid compared with
the malathion only control group (Figure 5A). In the liver tissue, malathion caused 34.8%
inhibition in PON1 activity (21.7±1.2) vs. (33.27±1.60) kU/L. There was no significant effect for
citric acid alone on PON1 activity in malathion-treated rats. Treatment with only atropine
inhibited the activity of the enzyme by 20.6% (17.23±0.83) vs. (21.7±1.2 kU/L), whereas citric
acid in combination with atropine increased PON1 activity by 26.7% compared with the
malathion only control group (27.5±1.5) vs. (21.7±1.2) kU/L) (Figure 5B).
3.7. Brain glucose
Compared with the saline-treated rats, a significant decrease in brain glucose concentrations
by 30.6% was observed in the malathion only treated group (218.0±13.0) vs. (314.0±9.6) µg/g
tissue). A significant increase in glucose concentrations by 69.3%, 49.7%, 33.9%, and 97.2%,
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respectively, was observed in brain of rats treated with malathion along with (200-400) mg/kg
citric acid, atropine or both atropine and citric acid compared with the malathion only control
group (Figure 6).
3.8. Liver enzymes
Results are presented in Figure 7. Malathion caused significant elevation in liver ALT and
AST by 83.9% and 130.8%, respectively compared with saline-treated rats (84.6±4.1) vs.
(46.0±3.2) and (83.1±3.7) vs. (36.0±1.9) U/g tissue. Citric acid given at 200 and 400 mg/kg
significantly decreased liver AST by 22.7% and 47.4%, respectively, compared with malathion
only group (65.4±5.2) and (44.5±2.1) vs. (84.6±4.1) U/g tissue. Meanwhile, there was 26%
decrease in liver ALT by citric acid at 400 mg/kg (61.5±4.2) vs. (83.1±3.7) U/g tissue. In
contrast, significant increase in AST and ALT by 30% and 67.3% was observed in malathion +
atropine compared with malathion only group. On the other hand, rats treated with malathion +
citric acid+atropine showed 14.3% and 29% increments in AST and ALT compared with
malathion only group.
3.9. Comet assay
Malathion injection induced DNA fragmentation in blood lymphocytes. The comet percentage
of lymphocytes in malathion only treated rats was 83.5%±1.34% compared with saline control
value of 6.2%±0.47% (P<0.05). Citric acid administered at 200 or 400 mg/kg to malathion-
treated rats resulted in a dose-dependent decrease in the % of damaged cells by 68.9% and 87.2%
(26.00%±0.81%) and (10.70%±0.98%) vs. (83.50%±1.34%). The comet percentage of
peripheral blood lymphocytes after treatment with atropine and citric acid+atropine was
significantly reduced by 85% and 86.8% compared with the malathion only group
(12.50%±0.56%) and (11.00%±0.71%) vs. (83.5%±1.34%) (Figure 8 and 9).
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3.10. Histopathological results
3.10.1. Brain
Rats treated with saline (control group) showed normal histological picture of brain tissue
(Figure 10A). Rats treated with only malathion showed marked changes in the form of
meningitis and lymphocytic infiltration associated with destruction of most of the Purkinje cells
layer (Figure 10B). On the other hand there was marked improvement after the administration of
citric acid although congested blood vessels and reactive gliosis were seen (Figure 10C and
10D). Rats treated with malathion and only atropine exhibited near normal brain tissue except for
generalized astrogliosis as demonstrated by cellular hypertrophy (Figure 10E). Sections from rats
treated with malathion+citric acid+atropine showed only congested blood vessels (Figure 10F).
3.10.2. Liver
Saline-treated rats showed normal histology of hepatic tissue (Figure 11A). Rats treated with
malathion showed liver structural damage along with disarrangement of hepatic lobules with
formation of fibrotic strands. Necrosis and vacuole formation in hepatocytes, sinusoidal
enlargement, and leucocytic infiltration were seen. Moreover, dilation and congested blood
vessels with hemorrhage were noted (Figure 11B). Rats treated with both malathion and citric
acid (200 mg/kg) exhibited improved hepatic tissue structure, with minimal vacuolated hepatic
cells (Figure 11C). Treatment with the higher dose of citric acid (400 mg/kg) improved hepatic
cells except for minimal lymphocytic infiltration around the hepatic vessels (Figure 11D).
Meanwhile, rats given both malathion and atropine exhibited near normal liver tissue with
normal architecture and distinct hepatic cells except for blood vessel dilatation (Figure 11E).
Likewise, rats treated with malathion, atropine and citric acids showed only minimal congested
hepatic vessels (Figure 11F).
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3.11. Immunomorphometric analysis of iNOS immunoreactivity
3.11.1. Brain
Figure 12 shows the mean optical density measurements of iNOS immunoreactivity±SE (%) in
cerebral cortex of rats treated with saline, malathion and malathion along with citric acid and/or
atropine. There was negligible iNOS expression in the control group. Rats treated with only
malathion exhibited increased iNOS expression compared with the saline treated group (213.6%
increase: (4.92±0.35) vs. (0.22±0.10). In malathion-treated rats, citric acid caused a significant
and dose-dependent decrease in iNOS immunoreactivity (63% and 95.3% decrements:
(1.82±0.16), (0.23±0.05) vs. (4.92±0.35). A significant and marked decrease in iNOS expression
by 89.2% and 76.4% was also observed following atropine alone or combined with citric acid
(0.53±0.02), (1.162±0.17) vs. (4.92±0.35).
3.11.2. Liver
Quantitative measurements of iNOS immunoreactivity in the liver from rats exposed to
malathion revealed marked and significant increase in iNOS immunoreactivity compared to the
saline-treated group 741.7% increase: (7.55±0.17) vs. (0.897±0.120). Rats treated with malathion
and citric acid exhibited 92.4% and 89.8% decrements in iNOS immunoreactivity, respectively,
as compared with the malathion only group (0.57±0.10) and (0.773±0.130) vs. (7.55±0.17).
Treatment with atropine resulted in 98.4% decrease in iNOS immunoreactivity. Meanwhile,
atropine combined with citric acid resulted in 79.9% decrease in iNOS immunoreactivity as
compared with the malathion only group (1.516 ± 0.270) vs. (7.55±0.17) (Figure 13).
4. Discussion
Several recent studies indicated a role for reactive oxygen metabolites and increased oxidative
stress in the neurotoxic effects of organophosphate insecticides
[9,10,15]
. In this study, the decrease
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in reduced glutathione, GPx activity, TAC and the increase in the lipid peroxidation product
malondialdehyde is evidence of increased generation of reactive oxygen metabolites and other
free radicals capable of inducing lipid peroxidation of the cell membrane polyunsaturated fatty
acids
[4]
. Organophosphates result in increased free radicals either as a part of their
biotransformation or in the detoxification pathway
[46]
. These agents can also cause mitochondrial
impairment
[9,29]
, which could lead to increased reactive oxygen species. The latter in turn could
result in further damage to the mitochondria, thereby, generating a vicious circle
[47,48]
. In this
context, malathion caused the increased formation of superoxide anion in sub-mitochondrial
particles in hippocampus and inhibition of mitochondrial complex
[9]
. There was also a
decrease in complex activity in the hippocampus of malathion-exposed animals
[28]
. Other
organophosphates induced alterations in mitochondrial dynamics such as increased
mitochondrial length and reduced number of mitochondria as well as impaired axonal transport.
These changes occurred at insecticide concentrations that failed to affect acetylcholinesterase
activity
[27]
. In this study we hypothesized that citric acid by virtue of its antioxidant and
bioenergetic actions would be able to decrease the malathion-induced neurotoxicity. Our findings
validate this assumption. We have shown that the concurrent administration of malathion and
citric acid resulted in markedly decreased neuronal damage due to the insecticide. Brain MDA
decreased after citric acid administration and the decline in GSH, GPx activity and TAC were all
reduced by citric acid. DNA damage of peripheral blood lymphocytes was also markedly
decreased. Moreover, the histopathological alterations in the brain caused by malathion were
ameliorated by treatment with citric acid. The present study thus suggests a novel action for citric
acid in the protection against acute malathion intoxication.
Citrate occupies a central role in cellular intermediary metabolism. It occurs as an intermediate
in the tricarboxylic acid or Krebs cycle, which generates energy through the oxidation of acetate.
Energy in the form of reduced pyridine nucleotides are then used in the mitochondrial respiratory
chain for synthesis of adenosine 5’-triphosphate
[17]
. Energy derived through glycolysis and
tricarboxylic acid cycle is indispensable for maintaining neuronal and astrocytic functions and
integrity
[49,50]
. In their study, Ying et al.
[51]
have shown that, excessive activation of PRAP1.
Excessive activation of PRAP1 induces neuronal and astrocytes death by decreasing the levels of
cytosolic NAD+ and impairing glycolysis. Provision of the tricarboxylic acid cycle substrates
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eg., α-ketoglutarate or pyruvate (though not glucose) reduced this cell death
[51]
. In the same way,
it is possible that a reduction in brain metabolism is related to the neurotoxic actions of
malathion and this was amenable to treatment with citrate.
Besides its bioenergetic role, intracellular citrate is also important for the synthesis of fatty
acids, isoprenoids and cholesterol
[52]
. Citrate exerted anti-inflammatory actions eg., decreased
release of myeloperoxidase, interleukin 1β, platelet factor 4 and elastase
[20-22,53]
. Citrate also
displayed antioxidative effects reducing lipid peroxidation
[21,23]
. These actions of citrate are
likely to be involved in protecting brain neurons from deleterious effects of malathion. It has also
been suggested that citrate released by astrocytes may modulate neuronal excitability by
chelating Ca
2+
and Mg
2+
and thus regulating their extracellular concentrations
[54]
. This might
provide another mechanism for the ability of citrate to protect neurons from malathion. There is
also an evidence for the importance of cytoplasmic citrate availability in maintaining genome
stability. In their study, defects in the mitochondrial citrate carrier SLC25A1 that releases citrate
into the cytoplasm resulted in chromosomal breaks
[55]
.
In this study, malathion caused increased brain nitric oxide and iNOS expression which
decreased following treatment with citric acid. These observations suggest a pivotal role for
nitric oxide in the development of malathion neurotoxicity. The gaseous molecule nitric oxide
has an important role in the brain as intracellular messenger and in maintaining vascular tone
owing to its vasodilator action
[56]
. In brain tissue, the excessive formation of nitric oxide in
inflammatory and toxic states from astrocytes and microglia by the inducible nitric oxide
synthase can lead to neuronal death. Increased activation of neuronal nitric oxide synthase during
certain pathological conditions can also result in the release of excessive amounts of nitric oxide
and ensuing neurotoxicity. Nitric oxide itself is relatively non-toxic, but can react with
superoxide resulting in the formation of the peroxynitrite radical (ONOO-), and also with oxygen
to yield NO
2
and N
2
O
3
capable of inducing lipid peroxidation, oxidation or nitrosylation of thiols
e.g. glutathione or protein thiols and DNA damage
[57,58]
. Energy depletion occurs because of an
inhibitory action on cytochrome oxidase, mitochondrial respiration, glycolysis, and the induction
of mitochondrial permeability transition
[59]
.
In this study, we observed decreased arylesterase activity of PON1 in the brain and liver of
malathion exposed rats. The enzyme PON1 hydrolyzes several organophosphorus compounds,
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carbamates, nerve agents and many other xenobiotics
[60,61]
. The catalytic efficiency of PON1 in
hydrolyzing specific organophosphate insecticides determines the individual’s susceptibility to
these agents
[62-65]
. The activity of the enzyme in plasma is also affected by exposure to
organophosphate insecticides and is associated with marked inhibition of plasma BChE
[66-68]
.
The PON1 enzyme also has an antioxidant action
[40]
and a peroxidase-like activity
[69]
.
Paraoxonase-1 is inactivated by oxidants
[70,71]
. The decrease in enzyme activity by malathion
might thus involve oxidative modification and/or direct inhibition by the organophosphate. In
brain tissue of malathion-treated rats, the enzyme activity, however, showed further inhibition by
citric acid and by both atropine and citric acid. Similar changes were not observed in liver tissue
where citric acid alone had on PON1 activity in malathion-treated rats. These findings might
suggest that changes in enzyme activity are not related to the protective action of citric acid in
this study.
Organophosphate insecticides irreversibly bind to and inactivate the enzyme cholinesterase
[72]
.
In mammals, both AChE and BChE hydrolyze the neurotransmitter acetylcholine but with
differing specificity. This neurotransmitter is present in the post-synaptic neuronal membrane, at
the myoneural junction, autonomic ganglia, and at the terminal endings of the post-ganglionic
parasympathetic nerves
[73]
. Organophosphates thus results in acetylcholine accumulation at the
neuronal synapse and excessive central and peripheral cholinergic activity
[72,73]
. Whereas the role
for AChE in hydrolyzing acetylcholine is clear, the physiological role of BChE is less
obvious
[74]
. Butyrylcholinesterase also detoxifies or catabolize ester-containing drugs. Humans
with BChE deficiency are asymptomatic but exhibit increased sensitivity to the muscle relaxants
suxamethonium and mivacurium, two BChE substrates, used in anesthesia
[75]
. In managing
poisoning due to organophosphate, the antidote atropine, a cholinergic receptor antagonist, is
frequently used to prevent the effects of excess acetylcholine at the muscarinic cholinergic
synapses
[32,33]
. In this study, we demonstrate that atropine administered at time of exposure to
malathion was capable of increasing brain cholinesterase activity and ameliorating the neuronal
damage due to the insecticide. The agent also effectively inhibited DNA damage of peripheral
blood lymphocytes. In the brain of malathion-exposed rats, atropine inhibited iNOS expression
and the release of nitric oxide. Glutathione peroxidase activity and total antioxidant capacity also
increased following treatment with atropine. These findings suggest a link between excessive
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cholinergic stimulation and the development of oxidative stress due to the organophosphate and
that oxidative stress is a consequence to increased cholinergic stimulation.
Glucose is the main energetic fuel for energy-dependent brain functions and brain glucose
consumption accounts for 45%-60% of glucose used by the body
[76]
. In this study, a decrease in
brain glucose in malathion-exposed rats was observed. It is not clear whether this represents an
increase in energy production by neurons and astrocytes in face of the toxic challenge or is the
result of impaired glucose brain transport. Nevertheless, recovery of brain glucose in malathion-
exposed rat occurred following citrate, atropine or their combined administration, suggesting a
link between the restoration of brain glucose level and neuroprotection by these agents.
In this study, the ability of malathion to cause DNA damage of peripheral blood lymphocytes
was determined using the ‘Comet assay’. This assay is useful for the detection of DNA strand
breaks in mammalian cells
[44]
. Our results confirm previous studies indicating increased the
DNA damage in peripheral blood of malathion-treated rats
[77,78]
. The number of comets produced
by malathion in peripheral blood lymphocytes showed marked decrease in citric acid and/or
atropine-treated rats.
Studies have shown that exposure to malathion is able to cause liver tissue damage which is
mediated by free radicals
[34,35]
. We thus extended our observations in order to delineate an effect
for citric acid on liver injury in malathion intoxicated rats. Our results showed that malathion
increased liver lipid peroxidation and nitrite along with decreased GSH indicative of increased
oxidative stress. There were also increased liver transaminases, a marker of liver cell damage.
Only with citric acid at 200 mg/kg or citric acid-atropine combined treatment, there was
decreased lipid peroxidation and an increase in hepatic GSH while transaminases decreased after
treatment with citric acid at 400 mg/kg. These changes were not observed after atropine.
Nevertheless, histopathological studies indicated a clear protective effect for citric acid, atropine,
or their combination along with inhibition of iNOS expression in hepatocytes. The liver lobule
and hepatocytes thus regained their morphological integrity after treatment with citric and/or
atropine. It is likely; however, that functional recovery is not yet complete in view of the still
increased lipid peroxidation and tissue transaminaeses.
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Our results thus indicate that cholinergic receptor blockade with atropine was able to protect
against the histopathological alterations in the brain and liver and markedly inhibited DNA
damage in blood lymphocytes following acute malathion exposure in rats. The mechanism
probably involves decreased oxidative stress that accompanied cholinesterase inhibition and the
excessive central and peripheral cholinergic stimulation. The study also demonstrates for the first
time that the administration of citrate was able to ameliorate the neurotoxicity and hepatotoxicity
as well as DNA damage caused by malathion. These effects are likely to involve an antioxidant
as well as a bioenergetic action of citric acid. Citrate might thus find a role in treatment of
nervous system consequences following exposure to malathion and possibly other
organophosphates.
Conflicts of interest statement
We declare that we have no conflict of interest.
References
[1] Halliwell B. Biochemistry of oxidative stress. Biochem Soc Trans 2007; 35(Pt 5): 1147-1150.
[2] Weidinger A, Kozlov AV. Biological activities of reactive oxygen and nitrogen species:
oxidative stress versus signal transduction. Biomolecules 2015; 5(2): 472-484.
[3] Sies H. Oxidative stress: oxidants and antioxidants. Exp Physiol 1997; 82(2): 291-295.
[4] Solsona C, Kahn TB, Badilla CL, Álvarez-Zaldiernas C, Blasi J, Fernandez JM, et al. Altered
thiol chemistry in human amyotrophic lateral sclerosis-linked mutants of superoxide dismutase 1.
J Biol Chem 2014; 289(39): 26722-26732.
MANUS CRIP T
ACCEP TED
ACCEPTED MANUSCRIPT
23
[5] Markkanen E, Meyer U, Dianov GL. DNA Damage and repair in schizophrenia and autism:
implications for cancer comorbidity and beyond. Int J Mol Sci 2016; 17(6). doi:
10.3390/ijms17060856.
[6] Peiró C, Romacho T, Azcutia V, Villalobos L, Fernández E, Bolaños JP, et al. Inflammation,
glucose, and vascular cell damage: the role of the pentose phosphate pathway. Cardiovasc
Diabetol 2016; 15(1): 82.
[7] Huang WJ, Zhang X, Chen WW. Role of oxidative stress in Alzheimer’s disease. Biomed
Rep 2016; 4(5): 519-522.
[8] Schieber M, Chandel NS. ROS function in redox signaling and oxidative stress. Curr Biol
2014; 24(10): R453-R462.
[9] Delgado EH, Streck EL, Quevedo JL, Dal-Pizzol F. Mitochondrial respiratory dysfunction
and oxidative stress after chronic malathion exposure. Neurochem Res 2006; 31(8): 1021-1025.
[10]Abdel-Salam OM, Youness ER, Esmail RS El-N, Mohammed NA, Khadrawy YA, Sleem
AA, et al. Methylene blue as a novel neuroprotectant in acute malathion intoxication. Reactive
Oxygen Species 2016; 1: 165-177.
[11]El-Bini Dhouib I, Lasram MM, Annabi A, Gharbi N, El-Fazaa S. A comparative study on
toxicity induced by carbosulfan and malathion in Wistar rat liver and spleen. Pestic Biochem
Physiol 2015; 124: 21-28.
[12]Karabag-Cobana F, Buldukb I, Limana R, Incec S, Cigercid I, Hazmane O. Oleuropein
alleviates malathion-induced oxidative stress and DNA damage in rats. Toxicol & Environ Chem
2016; 98(1): 101-108.
[13]John S, Kale M, Rathore N, Bhatnagar D. Protective effect of vitamin E in dimethoate and
malathion induced oxidative stress in rat erythrocytes. J Nutr Biochem 2001; 12(9): 500-504.
[14]Trevisan R, Uliano-Silva M, Pandolfo P, Franco JL, Brocardo PS, Santos AR, et al.
Antioxidant and acetylcholinesterase response to repeated malathion exposure in rat cerebral
cortex and hippocampus. Basic Clin Pharmacol Toxicol 2008; 102(4): 365-369.
MANUS CRIP T
ACCEP TED
ACCEPTED MANUSCRIPT
24
[15]Durak D, Uzun FG, Kalender S, Ogutcu A, Uzunhisarcikli M, Kalender Y. Malathion-
induced oxidative stress in human erythrocytes and the protective effect of vitamins C and E in
vitro. Environ Toxicol 2009; 24(3): 235-242.
[16]German JB. Antioxidants. In: Food additives. Branen AL, Davidson PM, Salminen S,
Thorngate III JH, editors. 2nd edition. New York, Basel: Marcel Dekker, Inc.; 2002. p. 538.
[17]Fromm HJ, Hargrove MS. The tricarboxylic acid cycle. In: Essentials of Biochemistry.
Berlin Heidelberg: Springer-Verlag; 2012. p. 205-221.
[18]Penniston KL, Nakada SY, Holmes RP, Assimos DG. Quantitative assessment of citric acid
in lemon juice, lime juice, and commercially-available fruit juice products. J Endourol 2008;
22(3): 567-570.
[19]Franklin RB, Costello LC. Citrate metabolism in prostateand other cancers. In: Singh KK,
Costello LC, editors. Mitochondria and Cancer. Springer; 2009. p. 61-78.
[20]Gabutti L, Ferrari N, Mombelli G, Keller F, Marone C. The favorable effect of regional
citrate anticoagulation on interleukin-1beta release is dissociated from both coagulation and
complement activation. J Nephrol 2004; 17(6): 819-825.
[21]Gritters M, Grooteman MP, Schoorl M, Schoorl M, Bartels PC, Scheffer PG, et al. Citrate
anticoagulation abolishes degranulation of polymorphonuclear cells and platelets and reduces
oxidative stress during haemodialysis. Nephrol Dial Transplant 2006;21(1):153-159.
[22]Tiranathanagul K, Jearnsujitwimol O, Susantitaphong P, Kijkriengkraikul N,
Leelahavanichkul A, Srisawat N, et al. Regional citrate anticoagulation reduces
polymorphonuclear cell degranulation in critically ill patients treated with continuous
venovenous hemofiltration. Ther Apher Dial 2011; 15(6): 556-564.
[23]Abdel-Salam OME, Youness ER, Mohammed NA, Morsy SMY, Omara EA, Sleem AA.
Citric acid effects on brain and liver oxidative stress in lipopolysaccharide-treated mice. J Med
Food 2014; 17(5): 588-598.
[24]Abdel Salam OME, Sleem AA, Shaffie NM. Hepatoprotective effects of citric acid and
aspartame on carbon tetrachloride-induced hepatic damage in rats. EXCLI J 2009; 8: 41-49.
MANUS CRIP T
ACCEP TED
ACCEPTED MANUSCRIPT
25
[25]Abdel Salam OME, Sleem AA, Shaffie NM. Protection against carbon tetrachloride-induced
liver damage by citric acid. Cell Biol: Res Ther 2015; 4: 1.
[26]Kaur P, Radotra B, Minz RW, Gill KD. Impaired mitochondrial energy metabolism and
neuronal apoptotic cell death after chronic dichlorvos (OP) exposure in rat brain.
Neurotoxicology 2007; 28(6): 1208-1219.
[27]Middlemore-Risher ML, Adam BL, Lambert NA, Terry AV Jr. Effects of chlorpyrifos
andchlorpyrifos-oxon on the dynamics and movement of mitochondria in rat cortical neurons. J
Pharmacol Exp Ther 2011; 339(2): 341-349.
[28]Karami-Mohajeri S, Hadian MR, Fouladdel S, Azizi E, Ghahramani MH, Hosseini R, et al.
Mechanisms of muscular electrophysiological and mitochondrial dysfunction following exposure
to malathion, an organophosphorus pesticide. Hum Exp Toxicol 2014; 33(3): 251-263.
[29]dos Santos AA, Naime AA, de Oliveira J, Colle D, dos Santos DB, Hort MA, et al. Long-
term and low-dose malathion exposure causes cognitive impairment in adult mice: evidence of
hippocampal mitochondrial dysfunction, astrogliosis and apoptotic events. Arch Toxicol 2016;
90(3): 647-660.
[30]Bozkurt B, Dumlu EG, Tokac M, Ozkardes AB, Ergin M, Orhun S, et al. Methylene blue as
an antioxidant agent in experimentally-induced injury in rat liver. Bratisl Lek Listy 2015; 116(3):
157-161.
[31]Roy Choudhury G, Winters A, Rich RM, Ryou MG, Gryczynski Z, Yuan F, et al. Methylene
blue protects astrocytes against glucose oxygen deprivation by improving cellular respiration.
PLoS One 2015; 10(4): e0123096.
[32]Jokanović M. Medical treatment of acute poisoning with organophosphorus and carbamate
pesticides. Toxicol Lett 2009; 190(2): 107-115.
[33]Eddleston M, Chowdhury FR. Pharmacological treatment of organophosphorus insecticide
poisoning: the old and the (possible) new. Br J Clin Pharmacol 2016; 81(3): 462-470.
MANUS CRIP T
ACCEP TED
ACCEPTED MANUSCRIPT
26
[34]Possamai FP, Fortunato JJ, Feier G, Agostinho FR, Quevedo J, Wilhelm Filho D, et al.
Oxidative stress after acute and sub-chronic malathion intoxication in Wistar rats. Environ
Toxicol Pharmacol 2007; 23(2): 198-204.
[35]Zidan Nel-H. Hepato- and nephrotoxicity in male albino rats exposed to malathion and
spinosad in stored wheat grains. Acta Biol Hung 2015; 66(2):133-148.
[36]Nair V, Turner GA. The thiobarbituric acid test for lipid peroxidation: structure of the adduct
with malondialdehyde. Lipids 1984; 19: 804-805.
[37] Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959; 82(1): 70-77.
[38]Archer S. Measurement of nitric oxide in biological models. FASEB J 1993; 7(2): 340-360.
[39]Eckerson HW, Wyte CM, La Du BN. The human serum paraoxonase/arylesterase
polymorphism. Am J Hum Genet 1983; 35(6): 1126-1138.
[40]Haagen L, Brock A. A new automated method for phenotyping arylesterase (EC 3.1.1.2)
based upon inhibition of enzymatic hydrolysis of 4-nitrophenyl acetate by phenyl acetate. Eur J
Clin Chem Clin Biochem 1992; 30(7): 391-395.
[41]Ellman GL, Courtney KD, Andres V Jr, Feather-Stone RM. A new and rapid colorimetric
determination of acetylcholinesterase activity. Biochem Pharmacol 1961; 7: 88-95.
[42]Gorun V, Proinov I, Baltescu V, Balaban G, Barzu O. Modified Ellman procedure for assay
of cholinesterases in crude enzymatic preparations. Anal Biochem 1978; 86(1): 324-326.
[43]Trinder P. Determination of glucose in blood using glucose oxidase with an alternative
oxygen acceptor. Ann Clin Biochem 1969; 6: 24-25.
[44]Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low
levels of DNA damage in individual cells. Exp Cell Res 1988; 175(1): 184-191.
[45]Blasiak J, Gloc E, Drzewoski J, Wozniak K, Zadrozny M, Skórski T, et al. Free radical
scavengers can differentially modulate the genotoxicity of amsacrine in normal and cancer cells.
Mutat Res 2003; 535(1): 25-34.
MANUS CRIP T
ACCEP TED
ACCEPTED MANUSCRIPT
27
[46]Elersek T, Filipic M. (2011). Organophosphorous pesticides-mechanisms of their toxicity. In
pesticides-the impacts of pesticides exposure. Prof. Margarita Stoytcheva editor. doi:
10.5772/14020. Available from: http://www.intechopen.com/books/pesticides-the-impacts-of-
pesticidesexposure/organophosphorous-pesticides-mechanisms-of-their-toxicity.
[47]Sullivan LB, Chandel NS. Mitochondrial reactive oxygen species and cancer. Cancer Metab
2014; 2: 17.
[48]Holmström KM, Finkel T. Cellular mechanisms and physiological consequences of redox-
dependent signalling. Nat Rev Mol Cell Bio 2014; 15(6): 411-421.
[49]Attwell D, Laughlin SB. An energy budget for signaling in the grey matter of the brain. J
Cereb Blood Flow Metab 2001; 21(10): 1133-1145.
[50]Hertz L, Peng L, Dienel GA. Energy metabolism in astrocytes: high rate of oxidative
metabolism and spatiotemporal dependence on glycolysis/glycogenolysis. J Cereb Blood Flow
Metab 2007; 27(2): 219-249.
[51]Ying W, Chen Y, Alano CC, Swanson RA. Tricarboxylic acid cycle substrates prevent
PARP-mediated death of neurons and astrocytes. J Cereb Blood Flow Metab 2002; 22(7): 774-
779.
[52]Inoue K, Zhuang L, Ganapathy V. Human Na+ -coupled citrate transporter: primary
structure, genomic organization, and transport function. Biochem Biophys Res Commun 2002;
299(3): 465-471.
[53]Bryland A, Wieslander A, Carlsson O, Hellmark T, Godaly G. Jones LM, et al. Gustatory
processing: a dynamic systems approach. Curr Opin Neurobiol 2006; 16(4): 420-428.
[54]Brekke E, Morken TS, Sonnewald U. Glucose metabolism and astrocyte-neuron interactions
in the neonatal brain. Neurochem Int 2015; 82: 33-41.
[55]Morciano P, Carrisi C, Capobianco L, Mannini L, Burgio G, Cestra G, et al. A conserved
role for the mitochondrial citrate transporter Sea/SLC25A1 in the maintenance of chromosome
integrity. Hum Mol Genet 2009; 18(21): 4180-4188.
MANUS CRIP T
ACCEP TED
ACCEPTED MANUSCRIPT
28
[56]Sobrevia L, Ooi L, Ryan S, Steinert JR. Nitric oxide: A regulator of cellular function in
health and disease. Oxid Med Cell Longev 2016; 2016: 9782346.
[57]Wink DA, Feelisch M, Vodovotz Y, Fukuto J, Grisham MB. The chemical biology of nitric
oxide. In: reactive oxygen species in biological systems. Gilbert and Colton, editors. New York:
Kluwer Academic/PlenumPublishers; 1999. p. 245-291.
[58]Yuste JE, Tarragon E, Campuzano CM, Ros-Bernal F. Implications of glial nitric oxide in
neurodegenerative diseases. Front Cell Neurosci 2015; 9: 322.
[59]Brown GC. Nitric oxide and neuronal death. Nitric Oxide 2010; 23(3): 153-165.
[60]La Du BN. Human serum paraoxonase/arylesterase. In: Pharmacogenetics of Drug
Metabolism. Kalow W, editor. New York: Pergamon Press, Inc.; 1992. p. 51-91.
[61]Kulka M. A review of paraoxonase 1 properties and diagnostic applications. Pol J Vet Sci
2016; 19(1): 225-232.
[62]Jørgensen A, Nellemann C, Wohlfahrt-Veje C, Jensen TK, Main KM, Andersen HR.
Interaction between paraoxonase 1 polymorphism and prenatal pesticide exposure on metabolic
markers in children using a multiplex approach. Reprod Toxicol 2015; 51: 22-30.
[63]Carr RL, Dail MB, Chambers HW, Chambers JE. Species differences in paraoxonase
mediated hydrolysis of several organophosphorus insecticide metabolites. J Toxicol 2015; 2015:
470189.
[64]Holland N, Lizarraga D, Huen K. Recent progress in the genetics and epigenetics of
paraoxonase: why it is relevant to children’s environmental health. Curr Opin Pediatr 2015;
27(2): 240-247.
[65]Marsillach J, Costa LG, Furlong CE. Paraoxonase-1 and early-life environmental exposures.
Ann Glob Health 2016; 82(1): 100-110.
[66]McDaniel CY, Dail MB, Wills RW, Chambers HW, Chambers JE. Paraoxonase 1
polymorphisms within a Mississippi USA population as possible biomarkers of enzyme activities
associated with disease susceptibility. Biochem Genet 2014; 52(11-12): 509-523.
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[67]Bernal-Hernández YY, Medina-Díaz IM, Barrón-Vivanco BS, Robledo-Marenco Mde L,
Girón-Pérez MI, Pérez-Herrera NE, et al. Paraoxonase 1 and its relationship with pesticide
biomarkers in indigenous Mexican farmworkers. J Occup Environ Med 2014; 56(3): 281-290.
[68]Medina-Díaz IM, Ponce-Ruiz N, Ramírez-Chávez B, Rojas-García AE, Barrón-Vivanco BS,
Elizondo G, et al. Downregulation of human paraoxonase 1 (PON1) by organophosphate
pesticides in HepG2 cells. Environ Toxicol 2016. doi: 10.1002/tox.22253.
[69]Aviram M, Hardak E, Vaya J, Mahmood S, Milo S, Hoffman A, et al. Human serum
paraoxonases (PON1) Q and R selectively decrease lipid peroxides in human coronary and
carotid atherosclerotic lesions: PON1 esterase and peroxidase-like activities. Circulation 2000;
101(21): 2510-2517.
[70]Aviram M, Rosenblat M, Billecke S, Erogul J, Sorenson R, Bisgaier CL, et al. Human serum
paraoxonase (PON 1) is inactivated by oxidized low density lipoprotein and preserved by
antioxidants. Free Radic Biol Med 1999; 26(7-8): 892-904.
[71]Nguyen SD, Sok DE. Oxidative inactivation of paraoxonase1, an antioxidant protein and its
effect on antioxidant action. Free Radic Res 2003; 37(12): 1319-1330.
[72]Pohanka M. Inhibitors of acetylcholinesterase and butyrylcholinesterase meet immunity. Int
J Mol Sci 2014; 15: 9809-9825.
[73]Silman I, Sussman JL. Acetylcholinesterase: ‘classical’ and ‘non-classical’ functions and
pharmacology. Curr Opin Pharmacol 2005; 5(3): 293-302.
[74]Çokuğraş AN. Butyrylcholinesterase: structure and physiological importance. Turk J
Biochem 2003; 28(2): 54-61.
[75]Delacour H, Dedome E, Courcelle S, Hary B, Ceppa F. Butyrylcholinesterase deficiency.
Ann Biol Clin (Paris) 2016; 74(3): 279-285.
[76]Shrayyef MZ, Gerich JE. Normal glucose homeostasis. In: L. Poretsky, editor. Principles of
diabetes mellitus. Springer; 2010. p.19-34.
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[77]Réus GZ, Valvassori SS, Nuernberg H, Comim CM, Stringari RB, Padilha PT, et al. DNA
damage after acute and chronic treatment with malathion in rats. J Agric Food Chem 2008; 56:
7560-7565.
[78]Ojha A, Srivastava N. In vitro studies on organophosphate pesticides induced oxidative
DNA damage in rat lymphocytes. Mutat Res Genet Toxicol Environ Mutagen 2014; 761: 10-17.
Figure legends
Figure 1. Oxidative stress markers in the brain of malathion-treated rats and the effect of citric
acid, atropine or citric acid combined with atropine.
*P < 0.05 compared with saline group. +P < 0.05 compared with malathion only group. #P <
0.05 compared with malathion + atropine group.
Figure 2. Oxidative stress markers in the liver tissue of malathion-treated rats and the effect of
citric acid, atropine or citric acid combined with atropine.
*P<0.05 compared with saline group. +P<0.05 compared with malathion only group. #P<0.05
compared with malathion+ atropine group.
Figure 3. Acetylcholinesterase (AChE) activity in the brain of malathion-treated rats and the
effect of citric acid, atropine or citric acid combined with atropine.
*P < 0.05 compared with saline group. +P < 0.05 compared with malathion only group.
Figure 4. Butyrylcholinesterase (BChE) activity in the brain of malathion-treated rats and the
effect of citric acid, atropine or citric acid combined with atropine.
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*P < 0.05 compared with saline group. +P < 0.05 compared with malathion only group.
Figure 5. Paraoxonase-1 (PON-1) activity in the brain and liver of malathion-treated rats and the
effect of citric acid, atropine or citric acid combined with atropine.
+P < 0.05 compared with malathion only group. #P < 0.05 compared with malathion + atropine
group.
Figure 6. Brain glucose level in rats treated with malathion and the effect of citric acid, atropine
or citric acid combined with atropine.
*P<0.05 compared with saline group. +P<0.05 compared with malathion only group. #P<0.05
compared with malathion+atropine group.
Figure 7. AST and alanine ALT activities in the liver tissue of malathion-treated rats and the
effect of citric acid, atropine or citric acid combined with atropine.
*P<0.05 compared with saline group and between different groups as indicated in the figure.
+P<0.05 compared with malathion only group. #P<0.05 compared with malathion + citric acid.
Figure 8. Representative fluorescence images of comets from blood lymphocytes of malathion
and citrate/atropine-treated rats.
A and B: malathion (control); C: malathion+citric acid 200 mg/kg; D: malathion+citric acid 400
mg/kg; E and F: malathion+atropine 1 mg/kg; G and H: malathion+citric acid 200
mg/kg+atropine 1 mg/kg.
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Figure 9. The percentage of comets from blood lymphocytes of rats treated with malathion alone
or combined with citric acid and or atropine. means± SE.
*P < 0.05 compared with saline group. +P < 0.05 compared with malathion only group. #P <
0.05 compared with malathion + citric acid 200 mg/kg group.
Figure 10. Hematoxylin and eosin (H&E) stained sections of cerebral cortex of rats.
Treated with (A) Saline: normal structure; the granular layer (curved yellow arrow), Purkinje cell
layer (yellow arrow) and the molecular layer (yellow star). (B) Malathion 150 mg/kg:
lymphocytic infiltration (wavy arrows), destruction of Purkinje cells layer (short arrows). (C)
Malathion+citric acid 200 mg/kg: congested blood vessels (red arrow heads), and reactive gliosis
(green stars). (D) Malathion+citric 400 mg/kg: reactive gliosis (green stars). (E) Malathion +
atropine: generalized astrogliosis (thick red arrows). (F) Malathion+citric acid+atropine: normal
glial tissue with minimal congested blood vessels (red arrow heads) (H&E×200).
Figure 11. Hematoxylin and eosin (H&E) stained liver sections from rats.
Treated with (A) Saline: normal hepatic lobules; the central vein (long thin arrow), portal triad.
(B) Malathion (150 mg/kg): congested blood vessels (thick arrows), fibrotic strands (arrow
heads), leucocytic infiltration (wavy arrows), and vacuolar hepatocytes (thin arrows). (C)
Malathion+citric acid 200 mg/kg: minimal congestion of the central vein (thick arrow),
vacuolated hepatocytes (thin arrows). (D) Malathion+citric acid 400 mg/kg: leucocytic
infiltration around hepatic vessels (wavy arrows), and minimally congested vessels (thick arrow).
(E) Malathion+atropine: well formed hepatic tissue and dilated congested hepatic vessels (thick
arrow). (F) Malathion+citric acid+atropine: foci of inflammatory cells (wavy arrow), foci of
necrotic tissue (star), congested blood vessels (thick arrow) (H&E×200).
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Figure 12. Optical density measurements of iNOS immunoreactivity ± SE (%) in the cerebral
cortex of rats treated with malathion and the effect of citric acid, atropine or citric acid combined
with atropine.
*P < 0.05 compared with saline group and between different groups as shown on the graph. +P <
0.05 compared with malathion only group. #P < 0.05 compared with malathion + citric acid 200
mg/kg group.
Figure 13. Optical density measurements of iNOS immunoreactivity ± SE (%) in the liver of rats
exposed to malathion and the effect of citric acid, atropine or citric acid combined with atropine.
*P < 0.05 compared with saline group and between different groups as shown on the graph. +P <
0.05 compared with malathion only group. #P < 0.05 compared with malathion + citric acid 200
mg/kg group.
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