AHMED K. SALAMA AND OMRAN A. OMRAN
Medical Laboratories Dept., Faculty of Science, Majmaah University,
Kingdom of Saudi Arabia, 1434 H
This report is based upon work supported by the Essential and Health
Sciences Research Center , Scientific Research Deanship, Majmaah
University 1433.
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Pesticides are commonly used for achieving better quality
products, increased production rate and controlling pest
population.
On the other hand, pesticides are known to increase the
production of reactive oxygen species (ROS), including
hydrogen peroxide (H2O2 ), superoxide(O2-•), and hydroxyl
(•OH) radicals, which in turn prompt oxidative stress in
different tissues.
A major form of cellular oxidation damage is lipid
peroxidation, which is initiated by -OH free radical
through the extraction of hydrogen atom from
unsaturated fatty acids of membrane phospholipids
causing disturbance of the biochemical and physiological
functions of RBCs , damaging membranes and other
tissues . Free radical generation is expected to induce
hepatotoxicity.
The aim of the present study was planned to establish the antioxidant role of
selenium and a combination of vitamin E and vitamin C on oxidative stress
induced in rat RBCs and hepatocytes by some pesticides such as atrazine,
dimethoate, or endosulfan.
Vitamin E is a lipid soluble, chain-breaking antioxidant playing a major
protective role against oxidative stress and prevents the production of lipid
peroxides by scavenging free radicals in biological membranes.
Vitamin C has antiradical activity indicating that it could provide an important
dietary source of antioxidants. Vitamin C is a well-known low molecular weight
antioxidant that protects the cellular compartment from water-soluble oxygen
nitrogen radicals.
Selenium is an essential element for biological systems, It is present in the
active center of glutathione peroxidase (GPx), an antioxidant enzyme, which
protects lipid membranes and macromolecules from oxidative damage
produced by peroxides.
Selenium also has the ability to counteract free radicals and protect the
structure and function of proteins, DNA and chromosomes against oxidation
injury.
Selenium also controlling the deficiency of vitamin E and facilitate its
absorption.
Animals and treatments:
 Blood was obtained from rat by heart puncture using EDTA-Na salt
and centrifuged at 3000 rpm for 5 min at 4°C.
 RBC’s were taken and washed with phosphate buffered saline
(PBS), pH 7.2. The final red cell suspension was taken in test tubes
for treatment.
 Liver was also dissected out and homogenized in saline solution
(1:10 w/v). The homogenates were taken in a test tube for
treatment. Pesticides, vitamins and selenium (AT, DM, ES, VE, VC,
Se, AT + VE, DM + VE , ES + VE, AT + VC, DM + VC , ES + VC , AT +
VE + Se, DM + VE + Se , ES + VE + Se, AT + VC + Se, DM + VC + Se ,
ES + VC + Se) were dissolved in 5% DMSO.
 Also, 5% DMSO was dissolved/mixed in control group. RBC's or
liver homogenate were incubated for 3 hours at 37 °C in a shaking
water bath. At the end of incubation, the tubes will removed and
subjected to biochemical analysis.
Lipid peroxidation and GSH contents in erythrocytes and
hepatocytes:
The levels of lipid peroxides (LPOs) and GSH in erythrocyte
hemolysate and liver homogenate were determined. Lipid
peroxidation was calculated as nanomoles of malondialdehyde
(MDA)/mg protein. GSH was determined and calculated as
µmole/mg protein.
Antioxidant enzymes:
The erythrocyte and hepatocyte homogenate was used for analysis
of antioxidant enzymes. Activity of superoxide dismutase (SOD) was
measured as units/mg protein. Catalase (CAT) activity was
estimated as units/mg protein. Glutathione-S-transferase (GST-Px)
activity was estimated and expressed as units/mg protein.
Table (1): Levels of GSH, MDA and antioxidant enzymes of control and treated
erythrocytes with AT alone or its combination with VE or VC and Se.
GSH-Px
units/mg
protein
CAT
units/mg
protein
SOD
units/mg
protein
31.70 ± 2.54
25.2 ± 3.50
1.81 ± 0.08
40.35 ± 4.34
18.2 ± 1.57
1.50 ± 0.01
39.60 ± 2.56
26.3 ± 2.70
33.73 ± 3.22
MDA
nmoles/
mg protein
GSH content
µmole/mg
protein
Treatment
2.96 ± 0.11
Control
9.22 ± 0.39
1.92 ± 0.09
AT
1.80 ± 0.02
7.99 ± 0.90
2.50 ± 0.08
AT + VE
23.1 ± 3.22
1.83 ± 0.21
8.12 ± 1.05
2.61 ± 0.11
AT + VC
40.50 ± 1.19
26.5 ± 4.18
1.90 ± 0.03
7.43 ± 0.23
2.70 ± 0.10
AT + VE + Se
32.17 ± 3.44
24.2 ± 3.45
1.79 ± 0.05
7.85 ± 0.45
2.82 ± 0.31
AT + VC + Se
7.52 ± 0.09
Table (2): Levels of GSH, MDA and antioxidant enzymes of control and treated
erythrocytes with DM alone or its combination with VE or VC and Se.
GSH-Px
units/mg
protein
CAT
units/mg
protein
SOD
units/mg
protein
MDA
nmoles/
mg protein
GSH content
µmole/mg
protein
31.70 ± 2.54
25.2 ± 3.50
1.81 ± 0.08
7.52 ± 0.09
2.96 ± 0.11
Control
37.52 ± 3.28
19.9 ± 0.58
1.32 ± 0.04
12.12 ± 0.33
1.89 ± 0.09
DM
33.55 ± 1.50
24.8 ± 2.12
1.63 ± 0.06
9.00 ± 0.20
2.13 ± 0.08
DM + VE
32.77 ± 1.45
24.8 ± 1.90
1.79 ± 0.33
8.92 ± 0.05
2.10 ± 0.11
DM + VC
34.57 ± 3.98
26.9 ± 2.13
1.80 ± 0.12
7.73 ± 0.87
2.14 ± 0.10
DM + VE + Se
33.12 ± 4.33
26.1 ± 2.23
1.82 ± 0.09
7.85 ± 0.89
2.72 ± 0.31
DM + VC + Se
Treatment
Table (3): Levels of GSH, MDA and antioxidant enzymes of control and treated
erythrocytes with ES alone or its combination with VE or VC and Se.
GSH-Px
units/mg
protein
CAT
units/mg
protein
SOD
units/mg
protein
31.70 ± 2.54
25.2 ± 3.50
1.81 ± 0.08
38.22 ± 1.30
20.1 ± 1.22
34.23 ± 3.34
MDA
nmoles/
mg protein
GSH content
µmole/mg
protein
Treatment
7.52 ± 0.09
2.96 ± 0.11
Control
1.32 ± 0.04
10.26 ± 0.39
1.82 ± 0.01
ES
28.1 ± 2.00
1.60 ± 0.09
8.36 ± 0.90
2.70 ± 0.05
ES + VE
32.67 ± 3.13
26.4 ± 1.34
1.68 ± 0.11
8.19 ± 1.05
2.97 ± 0.02
ES + VC
35.10 ± 1.91
28.5 ± 3.10
1.80 ± 0.08
8.50 ± 0.23
2.82 ± 0.13
ES + VE + Se
33.55 ± 2.12
27.1 ± 1.15
1.77 ± 0.03
8.55 ± 0.45
2.96 ± 0.82
ES + VC + Se
Table (4): Levels of GSH, MDA and antioxidant enzymes of control and treated
hepatocytes with AT alone or its combination with VE or VC and Se.
GSH-Px
units/mg
protein
CAT
units/mg
protein
2.40 ± 0.54
85.30 ± 1.35
2.90 ± 0.14
66.15 ± 3.22
2.55 ± 0.59
SOD
units/mg
protein
GSH content
µmole/mg
protein
Treatment
7.53 ± 0.95
22.33 ± 1.45
Control
3.96 ± 0.33
10.13 ± 0.25
16.55 ± 2.92
AT
79.11 ± 2.10
4.70 ± 0.25
8.55 ± 0.99
20.13 ± 1.19
AT + VE
2.60 ± 0.44
81.00 ± 2.06
4.39 ± 0.20
8.13 ± 0.32
21.02 ± 3.44
AT + VC
2.49 ± 0.24
83.99 ± 0.40
5.20 ± 0.31
7.11 ± 0.11
22.10 ± 2.34
AT + VE + Se
2.50 ± 0.56
81.88 ± 9.22
5.10 ± 0.36
7.89 ± 0.54
21.88 ± 3.55
AT + VC + Se
6.30 ± 0.11
MDA
nmoles/
mg protein
Table (5): Levels of GSH, MDA and antioxidant enzymes of control and treated
hepatocytes with DM alone or its combination with VE or VC and Se.
GSH-Px
units/mg
protein
CAT
units/mg
protein
SOD
units/mg
protein
MDA
nmoles/
mg protein
GSH content
µmole/mg
protein
2.40 ± 0.54
85.30 ± 1.35
6.30 ± 0.11
7.53 ± 0.95
22.33 ± 1.45
Control
2.96 ± 0.90
61.12 ± 2.12
3.96 ± 0.33
10.13 ± 0.25
19.12 ± 1.22
DM
2.63 ± 0.81
73.19 ± 2.05
4.73 ± 0.13
7.51 ± 0.23
21.10 ± 1.13
DM + VE
2.78 ± 0.11
83.10 ± 1.36
4.50 ± 0.66
8.93 ± 0.31
22.12 ± 1.23
DM + VC
2.99 ± 0.22
83.93 ± 3.50
5.33 ± 0.30
7.98 ± 0.15
22.90 ± 1.23
DM + VE + Se
2.70 ± 0.13
84.76 ± 7.24
5.26 ± 0.27
9.11 ± 0.24
22.89 ± 2.56
DM + VC + Se
Treatment
Table (6): Levels of GSH, MDA and antioxidant enzymes of control and treated
hepatocytes with ES alone or its combination with VE or VC and Se.
GSH-Px
units/mg
protein
CAT
units/mg
protein
SOD
units/mg
protein
MDA
nmoles/
mg protein
GSH content
µmole/mg
protein
2.40 ± 0.54
85.30 ± 1.35
6.30 ± 0.11
7.53 ± 0.95
22.33 ± 1.45 Control
3.11 ± 0.09
61.11 ± 8.15
3.96 ± 0.90
10.13 ± 0.33
15.22 ± 0.82 ES
2.67 ± 0.09
79.66 ± 2.19
4.63 ± 0.12
8.31 ± 0.12
21.11 ± 1.67 ES + VE
2.98 ± 0.32
82.34 ± 6.11
4.88 ± 0.03
8.22 ± 0.80
21.15 ± 1.41 ES + VC
2.97 ± 0.31
83.05 ± 9.10
5.49 ± 0.90
9.13 ± 0.19
21.15 ± 0.94 ES + VE + Se
3.10 ± 0.08
83.98 ± 3.91
6.11 ± 0.22
10.09 ± 0.50
21.99 ± 2.65 ES + VC + Se
Treatment
The results of the study indicated that the in vitro lipo-peroxidative
effect induced by pesticides such as atrazine, dimethoate, or
endosulfan in male albino rat could be reduced using selenium
and a combination of vitamin E and vitamin C.
The treatment with selenium and a combination of vitamin E and
vitamin C is potentially reduced the free radicals in erythrocytes
or hepatocytes and ameliorated the oxidative stress as evidenced
from lower concentrations of LPOs, higher contents of GSH and
higher activities of SOD and CAT or GSH-Px in erythrocytes or
hepatocytes.
The efficacy of Vitamin E + Se in ameliorating pesticide-induced
oxidative stress was higher for dimethoate or endosulfan than for
Atrazin either in case of erythrocytes or hepatocytes.