Anal. Methods Environ. Chem. J. 4 (2) (2021) 60-71
Research Article, Issue 2
Analytical Methods in Environmental Chemistry Journal
Journal home page: www.amecj.com/ir
AMECJ
Effects of malathion exposure on glucose tolerance test in
diabetic rats; emphasis on oxidative stress
and blood concentration of malathion by gas chromatography
mass spectrometry
Seyedeh-Azam Hosseini
a
, Ali Faghihi zarandi
b
and Somayyeh Karami-Mohajeri
a,c,
*
a
Pharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran.
b
Department of Occupational Health Engineering, School of Public Health, , Kerman University of Medical Sciences, Kerman, Iran.
c
Department of Toxicology and Pharmacology, School of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran
ABSTRACT
Malathion is one of the widely used broad-spectrum organophosphate
insecticides (OPI) in Iran. Malathion affects carbohydrate metabolism,
causes hyperglycemia and increases the risk of diabetes. The present
study was undertaken to investigate the potential of malathion to
exacerbate diabetes-induced oxidative stress and impairment in blood
glucose level and glucose tolerance in a sub-acute study. Malathion
concentration in blood was analyzed with gas chromatography mass
spectrometry (GC-MS) after sample preparation of blood samples
based on magnetic Fe
3
O
4
-supported graphene oxide (Fe
3
O
4
@ GO)
nanoparticles. Type 1 diabetes was experimentally induced by
intraperitoneal administration of streptozocin (65 mg kg
-1
). Diabetic
and non-diabetic rats were treated with malathion at the dose of 150
mg kg
-1
day
-1
or 0.5-4.0 mg L
-1
in blood for 4 weeks. Fasting blood
glucose was measured every week. At the end of the study, blood
samples were investigated for markers of oxidative stress. Exposure
to multiple doses of malathion decreased the total antioxidant capacity
of plasma and the activity of catalase and superoxide dismutase
enzymes in diabetic rats. Blood glucose and glucose tolerance test
(GTT) and oxidative damages did not change signicantly in diabetic
rats exposed to malathion. However, malathion concentration in
blood caused to increase GTT in malathion-treated non-diabetic rats.
Taking together, our ndings provide evidence that daily exposure
to malathion for 4 weeks tends to exacerbate the decrease in blood
antioxidant status and protein carbonylation in diabetic rats.
Keywords:
Malathion,
Blood samples,
Diabetes,
Oxidative stress,
Erythrocyte,
Magnetic graphene oxide,
Gas chromatography mass spectrometry
ARTICLE INFO:
Received 25 Feb 2021
Revised form 29 Apr 2021
Accepted 21 May 2021
Available online 29 Jun 2021
*Corresponding Author: Somayyeh Karami-Mohajeri
Email: s_karami@kmu.ac.ir, somayyehkarami@gmail.com
https://doi.org/10.24200/amecj.v4.i02.141
------------------------
1. Introduction
The agricultural application of pesticides in the
world has been linked to a wide range of human
health hazards through occupational, accidental,
and intentional exposures [1]. It seems that among
all pesticides, organophosphate insecticides (OPI)
are more toxic to vertebrates with low mammalian
toxicity [2, 3]. OPI inhibit acetylcholinesterase
(AChE), which leads to the accumulation of
acetylcholine in the cholinergic synapses and
interfere with the normal function of the nervous
system [4]. However, it has been shown that these
pesticides have different toxicities in vivo and in
vitro through AChE-independent mechanisms [5, 6].
61
OPI inuences normal glucose homeostasis and
carbohydrate metabolism and induces oxidative and
nitrosative stress [7, 8]. Many techniques such as,
UV-VIS, gas chromatography mass spectrometry,
High Performance Liquid Chromatography
(HPLC) and Liquid Chromatography [9, 10]
were used for OPI and pesticides determination.
The blood had difculty matrixes and so must be
treatment. Many sample preparation were used
for treatment of blood samples for determination
seven pesticides (malathion, methyl isofenphos,
dichlorvos, chlorpyrifos, phenthoste, p,p′-DDD,
p,p′-DDE) in blood samples based on a quick,
easy, cheap, effective, rugged and safe (QuECh-
ERS) sample preparation method. Occasionally,
the Fe
3
O
4
magnetic nanoparticles (MNPs) as the
new adsorbing material was used for treatment
of blood samples [11-14]. Although the foremost
mechanism for hyperglycemia induced by OPI has
not been recognized yet, some explanations are
mentioned such as physiological stress, oxidative
stress, paraoxonase enzyme inhibition, nitrosative
stress, pancreatitis, cholinesterase inhibition,
adrenal gland stimulation, and disturbance in
liver tryptophan metabolism [15]. The human
body is constantly exposed to various factors that
contribute to the production of reactive oxygen
species called free radicals. Imbalance between
free radicals production and antioxidant systems
lead to oxidative stress, which contributed
to occurrence of the pathological conditions
such as diabetes and development of diabetic
complications [16-19]. Many toxic chemicals can
generate reactive oxygen and trigger diabetes and
hyperglycemia by induction of apoptosis in beta
cells [20, 21]. Some evidence points to the long-
term effects of OPI on glucose metabolism and
increased risk of diabetes [15]. Malathion, one
the of most popular OPI, has been used widely
in agriculture, industry, and also for therapeutic
purposes in humans (anti-louse) and animals (anti-
ectoparasites) [22]. Malathion alters the pathways
of carbohydrate metabolism mainly though
increase in the activity of glycogen phosphorylase,
phosphofructokinase, phosphoenolpyruvate
carboxykinase, and hexokinase which affects
glycolysis, gluconeogenesis, and glycogenolysis
[15, 23]. Induction of oxidative and nitrosative
stress in hepatocytes and pancreas beta cells are
other contributing factors in hyperglycemia caused
by Malathion [7, 23]. Activation of redox sensitive
kinases and induction of oxidative stress in muscle
cells after exposure to sub-toxic dose of malathion
impairs insulin signaling and muscle glucose
uptake and consequently causes insulin resistance
state [24]. Hence, the present work has been
designed to determine whether sub-acute exposure
to repeated non-lethal dose of malathion can impair
blood glucose control and exacerbate oxidative
stress in diabetic rats. To do so, fasting blood
glucose (FBG), glucose tolerance test (GTT), and
biomarkers of oxidative damage were measured in
non-diabetic and diabetic rats treated orally by sub-
lethal dose of malathion for 4 weeks.
2. Materials and methods
2.1. Chemicals and methods
Technical-grade malathion, which contains
>96% malathion, was obtained from the Shimi-
Keshavarz Pesticides Production Company (Tehran,
Iran). The name of malathion based on IUPAC
(International Union of Pure and Applied Chemistry)
is diethyl(dimethoxythiophosphorylthio) succinate;
S-1,2-bis(ethoxycarbonyl) ethyl-O,O-dimethyl
phosphorodithioate (CAS N.: 121-75-5, Sigma,
Germany) and UV spectrum of malathion in
acetonitrile (CAS N.: 75-05-08 , Merck, ACN)
was shown in Schema 1. All other materials were
purchased from the Merck and Sigma-Aldrich
Chemical Company (St. Louis, MO). The HNO
3
,
HCl, polyoxyethylene octyl phenyl ether (MTX-
100, CAS N: 9002-93-1, Sigma, Germany),
acetone and toluene (CAS N: 108-88-3, Merck)
were purchased from Merck, Germany. Anhydrous
magnesium sulfate (CAS N: 10034-99-8), sodium
chloride was purchased from Sigma (Germany).
Acetonitrile (ACN) and methanol were purchased
from Sigma Company (Germany). GC–MS (Agilent
7890A/5975C, USA) with HP-5MS column (30 m
× 0.25 mm i.d.,) with ow of 1 mL per minute of
Malathion exposure on glucose and determination by GC-MS Seyedeh-Azam Hosseini et al
62
He was used for qualitatively and quantitatively
detecting pesticides in blood. Because blood is
a complex matrix, and pesticides in blood are
usually at low concentrations, the separation of
malathion and elimination of interference in blood
have needed a special sample treatment. Working
standard solutions were prepared in DW. All these
solutions were stored at 4 ºC without any light. The
range of this study of malathion in blood is 0.3–4.4
μg mL
−1
by GC-MS after dilution 1 mL of blood
with DW.
2.2. Synthesis of magnetic Fe
3
O
4
-supported
graphene oxide
The magnetic Fe
3
O
4
-supported graphene oxide
(MNGO, Fe
3
O
4
@NGO) were prepared by co-
precipitation of FeCl
2
·4H
2
O and FeCl
3
·6H
2
O, in
the presence of NGO [19]. rstly, a liquid solution
of FeCl
2
·4H
2
O / FeCl
3
·6H
2
O was prepared (molar
ratio= 1:2). The weight ratio of FeCl
3
/ NGO in
the product was mFeCl
3
: mGO = 20:1. To prepare
the magnetic graphene oxide (Fe
3
O
4
@NGO),
10 mg of graphene oxide mixed with 10 mL of
DW and ultrasonicated for 30 min [19]. Then,
12.5 mL solution of FeCl
2
·4H
2
O (125 mg) and
FeCl
3
·6H
2
O (200 mg) in DW was added to the
mixture. Finally, the pH of 11 was achieved by
30% ammonia solution and the temperature was
adjusted to 70
°
C (Fig.1).
Shema 1. The structure and UV spectrum of malathion
Fig.1. Synthesis of magnetic Fe
3
O
4
-supported graphene oxide [19]
Anal. Methods Environ. Chem. J. 4 (2) (2021) 60-71
63
2.3. Sample Extraction Procedure for malathion
in blood
The sample preparation of blood samples in
rat were prepared based on quick, easy, cheap,
effective, rugged and safe (QuECh- ERS) method
based on Fe
3
O
4
magnetic nanoparticles (MNPs)
functionalized with NGO. The free of DDC, DDT
and malathion pesticide in blood samples were
used as blank solution. 1 mL of rat blood sample
was added into 10 mL of vial. Standard volumes
of DDC, DDT and malathion pesticide were added
to the vial, and then shaken for 1 min. The samples
were extracted with 2 mL acetonitrile for 30 s.
Anhydrous NaCl (0.1g) / MgSO
4
(0.3 g) were added
to the mixture centrifuging at 4000 rpm for 5 min
and then, the supernatant moved to 10 mL of vial
include Fe
3
O
4
@NGO (0.04 g). The vial shake for 1
min, and the supernatant separated with an external
magnet. Finally, the sample was dissolved in 50μL
of acetonitrile and 1μL of solution was determined
by GC–MS. The detection limits (LOD)and linear
range(LR) of the QuECh- ERS method based on
Fe
3
O
4
@NGO obtained 0.1 μg mL
−1
and 0.3–4.4 μg
mL
−1
with recoveries more than 95% [9-12].
2.4. Animals
Male Wistar rats weighing 211.5 ± 10.6 grams were
fed with standard diet and kept under 12:12 hour
light:dark cycle, at the temperature of 20 °C and
relative humidity of 25 to 30%. This study received
ethical approval (Code: IR.79.KMU.REC.1395-
79) from the local ethical committee of the Kerman
University of Medical Sciences.
2.5. Pilot experiment
A pilot test is designed to determine an oral
dose of malathion which inhibits 30% of plasma
ChE activity without signicant physiological
consequences and mortality within 4 weeks [25].
The treated groups (Ten rats in each group) received
the oral doses of 75, 100, 150, and 300 mg/kg/day of
malathion dissolved in corn oil for 4 weeks, while
the controls received only corn oil. Blood samples
were taken at the end of each week for measurement
of plasma ChE activity according to the Ellman’s
colorimetric method with slight modication [26].
Briey, 300 µl of 5,5′-Dithiobis(2-nitrobenzoic
acid) (0.25 mM in 0.1M phosphate buffer, pH 7.4)
was added to 10 µl of plasma and after 5 minutes
10 µl of acetylthiocholine iodide (3 mM) was
added and the absorbance was measured at 412 nm
for 5 minutes. The activity of ChE was calculated
according to the molar extinction coefcient of
5-thio-2-nitrobenzoate (13.6 × 10
3
M
− 1
cm
− 1
) and
expressed as nMol min
-1
mL
-1
. As depicted in Table 1,
malathion at the dose of 150 mg kg
-1
day
-1
during
4 weeks inhibited 30% of the plasma ChE activity
(663.40±72.09) compared with the control group
(1029.67±84.52) with no mortality or acute toxic
effects in rats.
Table 1. Plasma cholinesterase activity as percent of inhibition (%) after 4 weeks of daily administration of oral
multiple doses of malathion.
Week
Malathion (mg kg
-1
day
-1
)
0 75 100 150 200
1st 99.4 ± 4.3 93.2 ± 1.8 86.6 ± 2.1*** 76.4 ± 5.7*** 63.8 ± 3.3***
2nd 101.4 ± 9.1 93.3 ± 1.4* 86.4 ± 2.2*** 77.3 ± 5.5*** 63.0 ± 2.6***
3rd 99.5 ± 5.8 91.4 ± 1.8* 85.2 ± 3.2*** 71.0 ± 4.2*** 59.1 ± 6.6***
4th 100.6 ± 7.5 84.5 ± 1.6*** 80.8 ± 1.9*** 66.7 ± 2.9*** 54.9 ± 4.8***
Data was expressed as mean ± SD; n = 10; * P < 0.05 and *** P < 0.001, signicantly different from the control values
(One-way ANOVA followed by multiple comparison test).
Malathion exposure on glucose and determination by GC-MS Seyedeh-Azam Hosseini et al
64
2.6. Measurement of malathion
GC–MS (Agilent 7890A/5975C, USA) with HP-
5MS column (30 m × 0.25 mm i.d.,) with ow of
1 mL per minute of He was used for DDC, DDT
and malathion pesticide in blood rats. The splitless
injector was used. By GC–MS, the main parameters
such as, the inlet and interface temperature set at
250 and 280 °C, respectively. The source of MS
tuned 220 °C and ionization energy was less than
65-eV. The oven temperature was rst at 100 °C
(1.5 min), and increased up to 200 - 280 °C (20 - 6
°C/min). The Chromatographic of DDC, DDT and
malathion pesticides was shown in Figure 2 [9-13].
2.7. Induction of diabetes in rats
Type 1 diabetes was induced by intraperitoneal
injection of a single dose streptozotocin (STZ)
solubilized in 0.1 M trisodium citrate buffer (pH,
4.5) at the dose of 65 mg kg
-1
, according to the
method described by Furman [27]. STZ-treated
rats received 10% of sucrose instead of water
for 48 h. Induction of diabetes was veried by
measurement of FBG four times (1, 3 and 28 days
after the beginning of treatment) to ensure that the
hyperglycemia (FBG>250 mg dL
-1
) was established.
Polyuria and polydipsia were also monitored by
observation of the amount of consumed water and
the frequency of bedding exchange.
2.8. Experimental design, animal treatment, and
sample collection
Forty rats were randomly allocated to four groups
of ten as follows:
Control: Healthy rats that only received corn oil
orally.
DM: Diabetic rats received corn oil orally.
MT: Healthy rats received malathion (150 mg kg
-1
day
-1
, oral) for 4 weeks.
DM+MT: Diabetic rats received malathion (150
mg kg
-1
day
-1
, oral) for 4 weeks.
At the end of the experiment, all rats were
anesthetized by ketamine (60 mg/kg) and xylazin (6
mg kg
-1
) and after collection of blood sample through
cardiac puncture sacriced by cervical dislocation.
Blood samples immediately centrifuged at 3000 g
for 15 minutes for separation of plasma. To prepare
hemolysate, 250 µl of distilled water was added
to 50 µl of packed RBCs and mixed thoroughly.
Plasma samples and hemolysate were kept at -80
°C for further experiments.
Fig.2. The Chromatographic of DDC, DDT and malathion pesticides by GC-MS
Anal. Methods Environ. Chem. J. 4 (2) (2021) 60-71
65
2.9. Measurement of FBG and GTT
Blood glucose was measured in the blood sample
obtained by a small cut on the tip of rat’s tail
immediately after overnight fasting using the
commercial glucose diagnostic kit of Pars Azmoon
Company (Tehran, Iran). For measurement of GTT,
blood glucose was recorded every 30 min after oral
administration of glucose (2% w/v). Area under the
curve (AUC 0-120 min) of glucose concentration
from 0 to 120 minutes after administration of
glucose was calculated by the trapezoidal method
[28].
2.10. Measurement of oxidative stress
biomarkers by spectrophotometry
2.10.1.Measurement of total antioxidant
capacity of plasma
Antioxidant capacity was measured by ferric
reducing antioxidant power (FRAP) method.
During the reaction in acidic pH, the colorless
ferric-tripyridyl triazine (Fe
3+
-TPTZ) is reduced to
blue ferrous-tripyridyl triazine (Fe
2+
-TPTZ) [29].
To perform this experiment, 10 µl of plasma was
added to 300 µl FRAP reagent (1:1:10 mixture
of FeCl3, 10mM TPTZ, and 0.3M acetate buffer at
pH 3.6). After incubation at 37 °C for 10 minutes,
the absorbance was read at 593 nm. Finally, FRAP
was expressed as mmol Fe
2+
/mg protein according
to the standard curve of FeSO
4
.
2.10.2.Measurement of glutathione (GSH)
content
According to the Elman method, thiol groups react
by 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB)
and produce yellow complex [30]. Briey, 250 μL
of TCA 10% were added to 500 μL of hemolysate
and centrifuged at 3500 g for 35 minutes. Then,
200 μL of Tris buffer and 500 μL DTNB (10 mM
in 0.1 M phosphate buffer, pH 8) were added to
the supernatant and incubated in the dark at room
temperature for 15 minutes. The absorbance was
read at 412 nm and total thiol was expressed as
nmol/mg protein according to the standard curve
of GSH.
2.10.3.Measurement of superoxide dismutase
(SOD) activity
SOD activity was measured based on autoxidation
rate of pyrogallol at 420 nm by the Worthington
method with minor modication [31]. Briey, the
absorbance of pyrogallol (2 mM pyrogallol in Tris-
HCl buffer, pH 8.2) was determined kinetically
alone and after the addition of 50 μl of hemolysate.
The amount of SOD needed for 50% inhibition
of the pyrogallol autoxidation was considered as
one unit of SOD activity and expressed as U/mg
protein.
2.10.4.Measurement of catalase (CAT) activity
According to Cohen method [32], 1 ml of 30 mM
H
2
O
2
and 50 µl of the hemolysate was added to 2
ml of phosphate buffer (50 mM, pH 7.0) and then
the absorbance was measured kinetically at 240
nm. One unit of catalase activity is equal to 1 μM
H
2
O
2
decomposed per minute. The concentration of
H
2
O
2
was calculated using the following equation:
H
2
O
2
(μM) = (Absorbance at 240 nm × 1000)/43.6
M
-1
cm
-1
). Catalase activity was expressed as U/mg
protein.
2.10.5.Measurement of lipid peroxidation
Malondialdehyde (MDA) as the end-product
of lipid peroxidation was measured based on
the absorbance of MDA-thiobarbituric acid
(TBA) complex in acidic and high-temperature
condition [33]. Briey, 100 μl of hemolysate was
deproteinized by TCA 10% and centrifuged at 3500
g for 35 minutes. One ml of sulfuric acid 0.05%
and 800 μl of TBA (0.2%) were then added to the
precipitant and boiled at 95 °C for 30 minutes.
Then, MDA-TBA complex was extracted by 800 μl
n-butanol and the absorbance was read at 532 nm.
The level of MDA was expressed as nmol MDA/
mg protein according to the MDA standard curve.
2.10.6.Measurement of protein carbonylation
Protein carbonylation was measured according to
Levin et al. method [34]. Throughout the method,
100 μl of hemolysate was added to 500 μl of TCA
20%, kept at room temperature for 10 minutes, and
Malathion exposure on glucose and determination by GC-MS Seyedeh-Azam Hosseini et al
66
centrifuged. The supernatant was discarded and
1ml 4-dinitrophenylhydrazine (DNPH, 10 mM)
was added to the pellet and incubated at 37 ºC for
50 minutes. Then, 1ml of TCA 20% was added and
centrifuged. The remaining pellet was washed with
1mL of ethanol and ethyl acetate solution (1:1 ratio).
Then, 1mL of guanidine hydrochloride 6 M was
added and incubated at 37 °C for 30 minutes. After
centrifugation, the supernatant was transferred to a
96-well plate and the absorbance was measured at
380 nm. The carbonyl content was calculated using
the molar extinction coefcient of 22,000 M
−1
cm
−1
and expressed as nmol/mg protein
2.11. Measurement of protein concentration
Protein concentration in the samples was measured
according to the Bradford’s method [35]. Briey,
200 µl of Bradford reagent (100 mg coomassie
brilliant blue G-250 was dissolved in 50 ml 95%
ethanol and then 100 ml 85% phosphoric acid and
850 ml of distilled water was added) were mixed
with 50 µl of samples and bovina serum albumin
(BSA) as standard in 96-well plate. After ve
minutes’ incubation at 37 °C, the absorbance was
measured at 595 nm and the protein concentration
was expressed as mg mL
-1
of samples according to
the standard curve of BSA.
2.12. Statistical analysis
Data were analyzed by using commercially
available SPSS software. Data was analyzed by
one-way ANOVA followed by Tukey’s multiple
comparison test. Results were presented as
mean ± SD (Standard Deviation) and p values
less than 0.05 were regarded as statistically
significant.
3. Results and discussion
3.1. Malathion blood concentration
The blood malathion rstly determined by GC-MS
analysis after oral intake in rats. The blood analysis
of malathion indicates that oral administration of
malathion at the dose of 50-150 mg kg
-1
day
-1
caused
a blood concentration of malathion in the range of
0.5–4.0 μg mL
-1
.
3.2. Induction of diabetes in rats
As shown in Table 2, administration of STZ at
the single dose of 65 mg kg
-1
caused signicant
hyperglycemia (FBG = 424.8 ± 28.9 mg dL
-1
, p
< 0.001) and loss of body weight was compared
with control group (FBG = 74.3 ± 25.5 mg dL
-1
).
Polyuria and polydipsia were other ndings were
observed in the diabetic rats within 4 weeks.
Table 2. Fasting blood glucose (FBG), Glucose tolerance test (GGT) and weight of rats on day 1, 3, and 28
in non-diabetic rats received corn oil (Control) and malathion (MT) and in diabetic rats received corn oil
(DM) and malathion (DM + MT).
Time Day 1 Day 3 Day 28
FBG (mg/dl)
Control 62.0 ± 9.7 74.3 ± 25.5 74.0 ± 14.7
DM 108.4 ± 18.6 424.8 ± 28.9 *** 395.4 ± 18.1***
MT 108.6 ± 19.3 89.1 ± 15.0 90.4 ± 21.0
DM + MT 133.25 ± 9.1 383.2 ± 19.1*** 413.7 ± 12.1***
GTT (AUC
0-120 min)
Control 9325 ± 409 NC 11990 ± 658.2
DM 11975 ± 521 NC 47574 ± 3758***
MT 10010.5 ± 688 NC 17886 ± 1438 *
DM + MT 10834 ± 674 NC 50577 ± 1256***
Weight (gr)
Control 211.5 ± 10.6 213.3 ± 9.2 263.7 ± 12.33
DM 189.4 ± 8.5 182.4 ± 12.3 158.4 ± 8.2**
MT 198.4 ± 8.3 198.8 ± 7.7 214.6 ± 7.1
DM + MT 212.6 ± 2.6 214.9 ± 3.1 162.7 ± 7.9***
Data was expressed as mean ± SD; n = 10; * p< 0.05, ** p< 0.01, and *** p < 0.001, signicantly different from the control
values (One-way ANOVA followed by multiple comparison test). NC: not calculated.
Anal. Methods Environ. Chem. J. 4 (2) (2021) 60-71
67
3.3. Effects on blood glucose
As shown in Table 2, malathion at the dose of 150
mg kg
-1
did not result in a signicant increase in FBG
in diabetic rats compared with non-treated diabetic
rats (p = 0.77) and in non-diabetic rats compared
with control group (p = 0.72). On the other hand,
malathion caused signicant (p = 0.04) increase
in AUC 0-120 of glucose concentration curve in
non-diabetic rats. most previous studies showed
that hyperglycemia in both short-term and long-
term exposure to OPI happened due to disruption
in glycolysis, glycogenolysis, and gluconeogenesis
pathways [7] and impairment in insulin signaling
and insulin-stimulated glucose uptake in muscle cells
[24]. Also, a meta-analysis conducted by Ramirez-
Vargas et al. (2018), revealed that blood glucose
concentrations were 3.3-fold higher in malathion-
exposed rats than in the control group [23]. In contrast,
it should be noted that some studies show gradual
increase in blood glucose and even hypoglycemia
after malathion exposure [36-38]. It has been also
reported that blood glucose in malathion-treated rats
increased (2.2-fold) after 2 h but gradually decreased
within 4 h [39]. It can be concluded that duration
of exposure, dose, experimental protocols, time of
blood sampling, and the mode of administration are
variables which affects the toxicity of malathion. As
the toxicity of malathion on carbohydrates, fats, and
protein metabolism pathways is approved previously,
signicant effects on FBG and GTT might be
obtained with increase in the number of examined
animal and duration of exposure to malathion.
3.4. Effects on antioxidants level
The total antioxidant capacity of plasma in diabetic
and non-diabetic rats exposed to malathion
decreased signicantly (p < 0.001) comparing to
control group. Moreover, a considerable difference
(p = 0.009) was detected in malathion-treated
diabetic rats compared to diabetic rats received
corn oil (Fig. 3a). GSH level in RBCs decreased
signicantly (p < 0.001) in all groups compared to
control group. However, there was no considerable
difference (p = 0.13) between diabetic and non-
diabetic rats received malathion (Fig. 3b). The
activity of SOD in RBCs decreased signicantly
in diabetic groups (p < 0.05), and in diabetic
and non-diabetic group received malathion (p
< 0.001) compared to control group. Malathion
decreased signicantly (p = 0.0008) the activity
of SOD in diabetic rats compared to diabetic
group received corn oil (Fig. 3c). The activity of
CAT in erythrocyte decreased signicantly (p <
0.001) in all groups in comparison with control
group. A signicant (p = 0.007) decrease was
also observed in diabetic rats received malathion
compared to diabetic groups (Fig. 3d). The results
of this study indicated that both diabetes and sub-
acute exposure to the sub-lethal dose of malathion
reduced the activity of CAT and SOD enzymes and
total antioxidant capacity of plasma and GSH level.
Interestingly, malathion in diabetic rats intensied
the reduction of total antioxidant capacity and the
activity of antioxidant enzymes. These results are
in agreement with previous studies, which have
indicated that diabetic condition and exposure to
OPI reduce the total antioxidant capacity of plasma
[7, 40, 41]. Reduction in total thiol content which
induces oxidative and nitrosative damages were
also reported in OPI exposure [42-45].
3.5. Effects on lipid peroxidation and protein
carbonylation
As shown in Table 3, lipid peroxidation and protein
carbonylation in diabetic rats as well as in diabetic and
non-diabetic rats exposed to malathion signicantly
(p < 0.001) increased compared to control group.
However, despite increase in the lipid peroxidation,
no signicant differences were observed between
lipid peroxidation level in diabetic rats and diabetic
rats exposed to malathion. Protein carbonylation
was signicantly (p = 0.042) increased in diabetic
rats exposed to malathion compared to diabetic rats
received corn oil. Generation of free radicals disables
antioxidant systems and consequently exerts further
destructive effects on cellular macromolecules [7,
15, 40]. Increase in protein carbonylation and lipid
peroxidation revealed in the current study was in
agreement with the ndings of other studies [7,
46]. OPI increases lipid peroxidation and protein
carbonylation in acute and sub-acute exposure
Malathion exposure on glucose and determination by GC-MS Seyedeh-Azam Hosseini et al
68
Table 3. Protein carbonylation and lipid peroxidation in non-diabetic rats received corn oil
(Control) and malathion (MT) and in diabetic rats received corn oil (DM)
and malathion (DM + MT) after 4 weeks.
Parameters Control DM MT DM + MT
Lipid peroxidation (nmol/mg protein)
0.64 ±
0.13
0. 86 ±
0.09***
0.92 ±
0.10***
0.99 ± 0.15***
Carbonyl protein formation (nmol/mg protein)
0.92 ±
0.15
1.12 ±
0.13**
1.20 ± 0.2** 1 45 ± 0.23***
Data was expressed as mean ± SD; n = 10; ** p< 0.01 and *** p < 0.001, signicantly different from the control
values (One-way ANOVA followed by multiple comparison test).
Anal. Methods Environ. Chem. J. 4 (2) (2021) 60-71
Fig. 3. Effects of malathion on antioxidants in the studied groups. a) Total antioxidant capacity of plasma, b)
RBCs glutathione content, c) RBCs superoxide dismutase activity, d) RBCs catalase activity in healthy rats
received corn oil (Control) and malathion (MT) and in diabetic rats received corn oil (DM) and malathion (DM
+ MT) after 4 weeks. Data was expressed as mean ± SD; n = 10; * p < 0.05, ** p < 0.01, and *** p < 0.001,
signicantly different between groups (One-way ANOVA followed by Tukey’s multiple comparison test).
[47-49]. Elevation in lipid peroxidation in diabetic
rats exposed to monocrotophos, an OPI, was also
reported by Vismaya and Rajini [44]. Increase in
the MDA level indicates the susceptibility of cell
membrane lipids against oxidative stress induced by
malathion in diabetic rats.
4. Conclusions
Taking together, the results of this study imply that
malathion aggravated decline in the enzymatic
antioxidant defense system. Impairment in
antioxidants capacity in long-term exposure to OPI
causes further oxidative damages as a proposed
69
mechanism for OPI-induced hyperglycemia.
According to high prevalence of diabetes, it is
recommended to conduct further in vivo, in vitro,
and clinical studies to investigate effects of OPI
on insulin release and blood glucose tolerance
in diabetic subjects exposed to OPI. It is also
recommended to evaluate effects of malathion on
tissues contributing to or affected by hyperglycemia.
Further consideration is also required to restrain
the utilization of OPI, which are widely used in
developing countries.
5. Acknowledgement
This manuscript was supported by Research Deputy
of Kerman University of Medical Sciences.
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