Protective Effects of Vitamin E and Selenium

Against Dimethoate-Induced Cardiotoxicity

In Vivo: Biochemical and Histological Studies
Ibtissem Ben Amara,1 Nejla Soudani,1* Ahmed Hakim,2* Afef Troudi,1
Khaled Mounir Zeghal,2 Tahia Boudawara,3 Najiba Zeghal1
1
Animal Physiology Laboratory, Sfax Faculty of Science, BP1171, 3000 Sfax,
University of Sfax, Tunisia
2
Pharmacology Laboratory, Faculty of Medicine, 3029 Sfax, University of Sfax, Tunisia
3
Anatomopathology Laboratory, CHU Habib Bourguiba 3029 Sfax, University of Sfax, Tunisia

Received 28 January 2011; revised 7 July 2011; accepted 10 July 2011

ABSTRACT: There is considerable interest in the study of free radical-mediated damage to biological sys-
tems due to pesticide exposure. However, there is a lack of consensus as to which determinations are
best used to quantify future risks arising from xenobiotic exposure and natural antioxidant interventions.
Our study investigated the potential ability of selenium and/or vitamin E, used as nutritional supplements,
to alleviate cardiotoxicity induced by dimethoate. Female Wistar rats were exposed for 30 days either to
dimethoate (0.2 g L21 of drinking water), dimethoate1selenium (0.5 mg kg21 of diet), dimethoate1vitamin
E (100 mg kg21 of diet), or dimethoate1selenium1vitamin E. The exposure of rats to dimethoate pro-
moted oxidative stress with a rise in malondialdehyde, advanced protein oxidation, and protein carbonyl
levels. An increase of glutathione peroxidase, superoxide dismutase, and catalase activities was also
noted. A fall in acetylcholinesterase and Na1K1-ATPase activities, glutathione, nonprotein thiols, vitamins
C and E levels was observed. Plasma levels of cholesterol, triglycerides, and low density lipoprotein-cho-
lesterol increased and those of high density lipoprotein-cholesterol decreased. Coadministration of sele-
nium or vitamin E to the diet of dimethoate-treated rats ameliorated the biochemical parameters cited
above. The histopathological findings confirmed the biochemical results and the potential protective
effects of selenium and vitamin E against cardiotoxicity induced by dimethoate. # 2011 Wiley Periodicals, Inc.
Environ Toxicol 00: 000–000, 2011.
Keywords: dimethoate; selenium; vitamin E; rats; cardiotoxicity; antioxidant and lipid profiles;
histopathological studies

INTRODUCTION

Environmental pollution plays a crucial role in the occur-

*These authors contributed equally to this work. rence of diseases affecting plants, animals and human
Correspondence to: N. Zeghal; e-mail: naj_zgh@yahoo.fr or beings. The main factor causing this pollution is the irra-
najiba.zeghal@tunet.tn
tional use of pesticides (Al-Haj et al., 2005). Through, these
Contract grant sponsor: DGRST grants (Appui à la Recherche Univer-
sitaire de Base), Tunisia
compounds play a highly positive role in modern farming
Contract grant number: ARUB 99/UR/08-73 and food production, their release arising from nonap-
Published online in Wiley Online Library (wileyonlinelibrary.com). proved use, poor practice, illegal operations or misuse leads
DOI 10.1002/tox.20759 to potential health hazards (Fogg et al., 2003; WHO, 2009).


C 2011 Wiley Periodicals, Inc.

1
2 BEN AMARA ET AL.
Organophosphate (OP) pesticides constitute one of the
classes most widely employed in both agricultural and land-
scape pest control. Residual amounts of OP have been
detected in the soil, water bodies, vegetables, grains, and
other food products (John et al., 2001; Poet et al., 2004)
causing various complications due to their intoxication
(Hsiao et al., 1996). Generally, toxicity results from either
accidental or intentional ingestion or from exposure to agri-
cultural pesticides (Watson et al., 2002). Other potential
causes of OP toxicity include the ingestion of contaminated
fruits, flour, or cooking oil and wearing contaminated cloth-
ing (Wu et al., 2001). Dimethoate (O,O-dimethyl S-methyl
carbamoylphosphoro-dithioate), one of the major groups of
OP, has an insecticidal efficiency for killing a wide range
of insects, including aphids, thrips, planthoppers and white-
flies systemically and on contact (Hayes and Laws, 1990).
Dimethoate (DM) is highly soluble in water and can leach
into nearby water sources, where it becomes toxic to both
vertebrates and invertebrates (Tuduri et al., 2006). Several
abnormalities appeared, due to DM contamination, such as
neuromuscular transmission block in both animals and
humans (De-Bleecker et al., 1993; Dongren et al., 1999),
immunotoxicological effects (Institoris et al., 1999), soft
tissues impairment like liver (Sayim, 2007), brain (Sharma
et al., 2005a,b), kidney (Mahjoubi-Samet et al., 2008), and
heart (Lundebyel et al., 1997).
The cardiac muscle is of particular interest regarding the
oxidative stress implications on its energy metabolism. The
heart is composed primarily of long-lived, postmitotic cells,
which prefers fatty acids as substrate for energy production,
so it becomes more susceptible to oxidative damage than
other tissues (Sohal and Weindruch, 1996).
Conversely, oxidant balance has a very important role in
the protection of the heart to allow normal cardiac contract-
ile performance. In general, the amount of antioxidants in
the heart is sufficient to protect it from any oxidant produc-
tion that may occur under normal circumstances (McDo-
nough, 1999). These antioxidants are uniquely different
from one another and will work most effectively when they
are used together. In the proper combination, they can per-
form a wide range of metabolic activities, free radical scav-
enging and preventive actions (Upaganlawar and Balara-
man, 2010). Several in vitro and in vivo studies have
reported that the combination of vitamins with other antiox-
idants produces synergistic effects (Yogeeta et al., 2006;
Pour et al., 2008; Punithavathi and Prince, 2009). The
application of Se or vitamin E in the prevention of cardio-
vascular diseases has been under consideration for several
decades. It has been proposed that Se, a cofactor of GPx,
may prevent lipid peroxidation in mammals (Crespo et al.,
1995). It is also an effective means to protect against athe-
rogenicity (Wojcicki et al., 1991). Vitamin E, an important
biological free radical scavenger in the cell membranes
(Punithavathi and Prince, 2009), has been shown to inhibit
the oxidative modification of low density lipoproteins (Ver-
Environmental Toxicology DOI 10.1002/tox
langieri and Bush, 1992) and to reduce smooth muscle cell
proliferation (Meydani, 1995), platelet adherence and
aggregation (Steiner, 1991) and protein kinase C activation
(Bursell and King, 1992).
To date, most studies are focused on identifying protec-
tive antioxidant agents against oxidative stress induced by
pesticides, particularly OP compounds. To our knowledge,
there is little information available on cardiac oxidative
stress and its effects on lipid profile induced by DM.
Besides, the potential ability of vitamin E and/or Se to
attenuate cardiotoxicity induced by DM has not yet been
investigated. Therefore, the present study aims to determine
the efficiency of these elements in antagonizing oxidative
damage in adult rats exposed to DM during 30 days.
MATERIALS AND METHODS
Chemicals
Sodium selenite (Na2SeO3), Vitamin E (a-tocopherol ace-
tate), glutathione (oxidized and reduced), nicotinamide ade-
nine dinucleotide phosphate reduced form (NADPH), 5-50 -
dithio-bis-2-nitrobenzoic acid (DTNB) and thiobarbituric
acid (TBA) were purchased from Sigma (St. Louis; MO).
DM [O,O-dimethyl-S(N-methyl-carbomethyl) phosphorodi-
thioate] was delivered by BASF (Ludwigshafen, Germany)
with 98% purity. All other chemicals were of analytical
grade and were purchased from standard commercial
suppliers.
Animals and Treatment
Adult female rats of Wistar strain, weighing 160 6 10 g,
were purchased from the Central Pharmacy (SIPHAT,
Tunisia). They were housed at ambient temperature 22 6
38C in a 12-h light/dark cycle and a minimum relative hu-
midity of 40%. The animals had free access to commercial
pellet diet (SICO, Sfax, Tunisia) and water ad libitum. The
general guidelines for the use and care of living animals in
scientific investigations were followed (Council of Euro-
pean Communities, 1986). The handling of the animals was
approved by the Tunisian Ethical Committee for the Care
and Use of laboratory animals.
One week after acclimatization to laboratory conditions,
rats which have at the onset of the experimentation similar
weights (160 6 10 g) were randomly divided into seven
groups of six each:
1. Group of controls;
2. Group of dimethoate (DM): received DM (0.2 g L21) in
their drinking water;
3. Group of dimethoate1selenium (DM1Se): received
both DM (0.2 g L21) and Se (0.5 mg kg21 of diet);
4. Group of dimethoate1vitamin E (DM1Vit E): was treated
with DM (0.2 g L21) and Vit E (100 mg kg21 of diet);

5. Group of dimethoate1selenium1vitamin E (DM1Se1
Vit E): received DM (0.2 g L21) 1 Se (0.5 Na2SeO3 mg
kg21 of diet)1 Vit E (100 mg a-tocopherol acetate/kg
of diet)
6. Group of selenium (Se): received Se (0.5 Na2SeO3 mg
kg21 of diet)
7. Group of vitamin E (Vit E): received Vit E (100 mg a-
tocopherol acetate/kg of diet).
Treatments were carried out over a period of 30 days.
The animals of the different groups were killed, at the
end of treatments, by cervical decapitation to avoid stress.
Blood was collected in heparined tubes and centrifuged at
2200 3 g for 10 min. Plasma samples were drawn and
stored at 2808C until analysis. Hearts were dissected out,
cleaned and weighed. Some hearts were homogenized
(10% w/v) in phosphate buffer (0.1 M, pH 7.4) and centri-
fuged at 10,000 3 g for 20 min. For the determination of
ATPases activities, hearts were homogenized in a Tris HCl
buffer, pH 7.4, instead of a phosphate buffer, according to
the method of Kawamoto et al. (2005). The resulting super-
natants were stored at 2808C until biochemical assays.
Other hearts were immediately removed, cleaned, and fixed
in 10% formalin solution and embedded in paraffin for his-
tological studies.
Biochemical Estimations
Protein Quantification
Heart protein contents were measured according to the
method of Lowry et al. (1951) using bovine serum albumin
as standard.
Malondialdehyde Measurement
The heart malondialdehyde concentrations, index of lipid
peroxidation, were determined spectrophotometrically
according to Draper and Hadley (1990).
Determination of Advanced Oxidation
Protein Products Levels
Advanced oxidation protein products (AOPP) levels were
determined according to the method of Kayali et al. (2006).
Determination of Protein Carbonyl Content
Protein carbonyls (PCO) were measured using the method
of Reznick and Packer (1994).
Determination of Antioxidant Enzyme Activities
Catalase (CAT) activity was assayed by the method of Aebi
(1984).
Superoxide dismutase (SOD) activity was estimated
according to Beauchamp and Fridovich (1971).
PROTECTIVE EFFECTS OF VITAMIN E AND SELELENIUM 3
Glutathione peroxidase (GPx) activity was measured
according to Flohe and Gunzler (1984).
Heart Glutathione Levels
Glutathione (GSH) in the heart was determined by the
method of Ellman (1959) modified by Jollow et al.
(1974).
Hydrophilic Antioxidants
Ascorbic acid (vitamin C) determination was performed as
described by Jacques-Silva et al. (2001).
Lipophilic Antioxidants
Vitamine E (a-tocopherol) level was assayed by the extrac-
tion method of Katsanidis and Addis (1999) using high-per-
formance liquid chromatography (HPLC).
Heart Nonprotein Thiols Levels
Heart nonprotein thiols (NPSH) levels were determined by
the method of Ellman (1959).
Determination of Acetylcholinesterase Activity
Acetylcholinesterase (AchE) activity was measured im-
mediately in homogenates according to the method of
Ellman et al. (1961), using acetylthiocholine iodide as a
substrate.
Na1,K1-ATPase Assay
Activity of Na1, K1-ATPase in heart was determined using
the method of Kawamoto et al. (2005).
Plasma Lipid Profile
Plasma lipid parameters such as total cholesterol (TC), tri-
acylglycerol (TG) and high-density lipoprotein-cholesterol
(HDL-C) levels were determined by enzymatic methods
according to Richmond (1973), Fossati and Prencipe (1982)
and Burstein et al. (1970), respectively. The low-density
lipoprotein-cholesterol (LDL-C) fraction and atherogenic
index (AI) were determined according to the Friedewald
equations (Friedewald, 1972):
LDL-C 5 Total cholesterol concentration—Triglyceride
concentration -HDL-C concentration
AI 5 TC-HDL-C
Histological Studies
Some heart samples, intended for histological examination
by light microscopy, were immediately fixed in 10% of for-
malin and processed in a series of graded ethanol solutions.
They were then embedded in paraffin, serially sectioned at
3 lm and stained with hematoxylin-eosin. Six slides were
Environmental Toxicology DOI 10.1002/tox
4 BEN AMARA ET AL.
prepared from each heart. All sections were evaluated for
the degree of heart injury. Each heart slide was examined
and assigned for severity of changes using scores on a scale
of none (-), mild (1), moderate (11), and severe (111)
damages.
Statistical Analysis
The data were analyzed using the statistical package pro-
gram Stat view 5 Software for Windows (SAS Institute,
Berkley, CA). Statistical analysis was performed using one-
way Analysis of Variance (ANOVA) followed by Fisher’s
Protected Least Significant Difference (PLSD) test as a post
hoc test for comparison between groups. All values were
expressed as means 6 S.D. Differences were considered
significant if P \ 0.05.
RESULTS
Doses Selection
In our experiments, we have tested different doses of DM:
no toxic effects and no oxidative stress were observed in
adult rats treated with DM at doses used between 0.1 and
0.15 g L21. From 0.2 3 g L21, oxidative stress was identi-
fied in adult rats without lethal effects. But with doses over
0.2 3 g L21, DMprovokes severe signs of toxicity and mor-
tality. So, this study was designed to investigate the toxicity
of 0.2 g L21 DM administered to adult rats via drinking
water. This dose which corresponds to 1/4 of LD 50 is con-
sistent with that reported previously by other investigators
(Sharma et al., 2005a,b; Kamath and Rajini, 2007; Mah-
joubi-Samet et al., 2008). The Se dose (0.5 mg kg21 of
diet) used in our experiment and in other findings gave high
protection against stress conditions in several tissues (Ogn-
janovic et al., 2008; Ben Amara et al., 2010; Soudani et al.,
2010). The dose of vitamin E (100 mg kg21 of diet) used
by Chow (1990) gave high protection against toxicity.
Effects of DM on General Health
Death was not observed during the experimental period. In
fact, rats in the control group and in the Se or Vit E-treated
group did not show any sign of toxicity. However, DM
treated rats showed varying degrees of clinical signs includ-
ing huddling, depression, conjunctivitis, mild tremor,
piloerection, diarrhea, and dyspnea. The observed signs
were related to the cholinergic crisis, a consistent sign in
acute organophosphate poisoning. No other significant clin-
ical manifestations were observed in the Se1DM, Vit
E1DM, and in the Se1 Vit E1DM-treated rats. In addi-
tion, our results indicated that the absolute and relative
heart weights of treated groups (DM, DM1Se, DM1 Vit E
Environmental Toxicology DOI 10.1002/tox
and DM1Se1 Vit E) were similar to those of controls
(data not shown).
Estimation of Lipid Peroxidation
Our results revealed an increase of lipid peroxidation in the
heart of the DM-treated group as evidenced by the
enhanced malondialdehyde levels in the heart homogenates
of adult rats (136%) when compared to controls (Table I).
The supplementation of Se alleviated lipid peroxidation
without reaching normal values, while vitamin E totally
restored malondialdehyde levels. The coadministration of
Se and vitamin E significantly modulated heart malondial-
dehyde levels to control values.
Protein Oxidative Damage Markers
Table I shows the levels of advanced oxidation protein
products (AOPP) and protein carbonyls (PCO) indices of
protein oxidative damage in the heart tissue of normal and
experimental animals. In DM group, a significant increase
of PCO and AOPP levels in the heart tissue of adult rats
(153; 150%) was observed when compared to controls.
Cotreatment of rats with Se and/or vitamin E resulted in a
marked decrease in heart PCO and AOPP levels, when
compared with DM group.
Heart AchE Activity
Exposure to pesticides was estimated using AchE. Adult
rats treated with DM showed a significant inhibition
(238%) of heart AchE activity (Fig. 1). The administration
of Se or vitamin E in the DM group ameliorated this param-
eter. However, both Se and vitamin E added in the diet of
the DM-treated group normalized heart AchE activity.
Effects on the Activity of ATPases in the Heart
Na1-K1-ATPase activity in the heart significantly
decreased by 42% in adult rats exposed to DM, compared
to the control values. Treatment with Se and/or vitamin E
ameliorated Na1-K1-ATPase activity (Fig. 1). After treat-
ment with Se or vitamin E alone, no significant changes in
the Na1-K1-ATPase activity were observed when com-
pared to controls.
Enzymatic Antioxidant Status in the Heart
In the heart homogenates of DM-treated rats, glutathione
peroxidase (GPx), catalase (CAT) and superoxide dismu-
tase (SOD) activities increased significantly by 35, 45, and
33% in adult rats, when compared to controls (Table II).
Supplementation of Se in the diet of (DM1Se)-group alle-
viated glutathione peroxidase activity which was similar to
 PROTECTIVE EFFECTS OF VITAMIN E AND SELELENIUM 5

67.328 6 3.214
0.072 6 0.010
10.720 6 0.630

Comparisons are made between two groups: DM, (DM1Se), (DM1Vit E), (DM1Se1Vit E), Se, Vit E group vs control group: **P \ 0.01; ***P \ 0.001, (DM1Se), (DM1Vit E), (DM1Se1Vit E)
that of controls, whereas vitamin E supplementation par-
TABLE I. Malonaldialdehyde, advanced oxidation protein products (AOPP) and protein carbonyls levels in the heart of adult rat controls or treated during tially ameliorated GPx activity when compared with DM-

Vit E
group. The increase of SOD induced by DM treatment was
corrected totally either by Se or vitamin E, while catalase
activity remained high. The administration of both Se and
vitamin E in the diet of DM group improved glutathione
66.149 6 3.372
0.075 6 0.010
10.428 6 1.509
peroxidase, catalase, and superoxide dismutase activities,
reaching normal values.
Se

Lipophilic Antioxidant (Vitamin E)

a-Tocopherol is the major lipophilic antioxidant in lipopro-
68.477 6 4.858111
0.079 6 0.018111
11.080 6 1.560111

teins. The levels of nonenzymatic antioxidants like vitamin

DM1Se1Vit E

E markedly decreased in DM group (276%) when com-

pared with controls. Se supplemented in the diet of DM-
treated group improved this parameter when compared to
DM group without reaching normal values. Vitamin E
alone or associated with DM significantly increased vitamin
E levels and even exceeded normal values (Table III).
0.073 6 0.020 111
77.773 6 5.915111

11.747 6 0.766111
DM1Vit E

0.090 6 0.014 111

90.316 6 7.871**11

14.732 6 1.12511
DM1Se
30 days with DM, DM1Se, DM1Vit E, DM1Se1Vit E, Se, Vit E

110.910 6 3.694***
0.198 6 0.028***
22.215 6 1.555***

Values are expressed as means 6 S.D for six animals in each group.
DM

group vs. DM group: 11 P \ 0. 01; 111 P \ 0.001.

71.063 6 4.476
0.092 6 0.032
11.162 6 3.909
Malonaldialdehyde: nmoles of MDA/g tissue.
Control

Protein carbonyls: lmoles mg21 protein.

Fig. 1. Na1-K1-ATPase (lmoles Pi/h/g heart) and acetyl-

AOPP: lmoles mg21 protein.

cholinesterase (AchE) (lmoles/min/mg protein) activities in

Parameters and treatments

the heart of adult rat controls (CT) or treated during 30 days

with DM, DM1Se, DM1Vit E, DM1Se1Vit E, Se, Vit
E.Values are expressed as means 6 S.D for six animals in
Malonaldialdehydea

Protein carbonylsc

each group. DM, (DM1Se), (DM1Vit E), (DM1Se1Vit E), Se,

Vit E group versus control group: *P \ 0. 05; **P \ 0. 01;
***P \ 0.001, (DM1Se), (DM1Vit E), (DM1Se1Vit E) group
versus DM group: 1P \ 0. 05; 11P \ 0. 01 ; 111P \
AOPPb

0.001. [Color figure can be viewed in the online issue, which

b
a

is available at wileyonlinelibrary.com.]

Environmental Toxicology DOI 10.1002/tox

 6

TABLE II. Enzymatic antioxidant activities (glutathione peroxidase, catalase and superoxide dismutase) in the heart of adult rat controls or treated during
30 days with DM, DM1Se, DM1Vit E, DM1Se1Vit E, Se, Vit E

Parameters and treatments Control DM DM1Se DM1Vit E DM1Se1Vit E Se Vit E

a
BEN AMARA ET AL.

Catalase 37.206 6 7.757 68.302 6 8.672*** 46.049 6 8.540*1 43.800 6 5.616 11 36.783 6 7.641111 35.910 6 7.137 35.012 6 7.856
Superoxide dismutaseb 30.172 6 4.384 44.842 6 6.874*** 31.787 6 2.105111 34.128 6 2.132111 30.706 6 6.322111 29.428 6 4.080 29.531 6 2.025
Glutathione peroxidasec 97.472 6 9.065 149.840 6 13.343** 98.625 6 10.41311 119.778 6 13.735*1 93.015 6 13.312111 91.756 6 7.221 90.388 6 7.280

Environmental Toxicology DOI 10.1002/tox

a
Catalase: lmoles H2O2 degraded/min/mg protein.
b
Superoxide dismutase: units/mg protein.
c
Glutathione peroxidase: nmoles of GSH/min/mg protein.
Values are expressed as means 6 S.D for six animals in each group.
Comparisons are made between two groups: DM, (DM1Se), (DM1Vit E), (DM1Se1Vit E), Se, Vit E group vs control group: *P \ 0. 05; **P \ 0. 01; *** P \ 0.001, (DM1Se), (DM1Vit E),
(DM1Se1Vit E) group vs. DM group: 1 P \ 0. 05; 11 P \ 0. 01; 111 P \ 0.001.

TABLE III. Nonenzymatic antioxidant levels (glutathione, non protein thiols, vitamin C and E) in the heart of adult rat controls or treated during 30 days with
DM, DM1Se, DM1Vit E, DM1Se1Vit E, Se, Vit E

Parameters and treatments Control DM DM1Se DM1Vit E DM1Se1Vit E Se Vit E

Glutathionea 48.510 6 6.890 27.990 6 4.310*** 44.150 6 4.601111 37.020 6 3.879*11 46.910 6 5.328111 51.720 6 3.550 48.720 6 3.386
Nonprotein thiolsb 3.424 6 0.205 2.031 6 0.346*** 3.036 6 0.287111 2.892 6 0.319*1 3.427 6 0.294111 3.616 6 0.200 3.521 6 0.200
Vitamin Cc 152.374 6 12.650 77.488 6 7.566*** 121.461 6 10.093*11 133.561 6 15.78011 159.817 613.496111 152.968 6 11.868 157.534 6 8.444
Vitamin Ed 2.466 6 0.220 0.602 6 0.05*** 1.630 6 0.247*11 5.152 6 1.280**111 4.716 6 1.880*111 2.061 6 1.015 5.928 6 0.587***
a
Glutathione : lg g21 tissue.
b
Nonprotein thiols: lmoles g21 tissue
c
Vitamin C: lmoles g21 tissue.
d
Vitamin E: lg g21 tissue.
Values are expressed as means 6 S.D for six animals in each group
Comparisons are made between two groups: DM, (DM1Se), (DM1Vit E), (DM1Se1Vit E), Se, Vit E group vs. control group: *P \ 0. 05; **P \ 0. 01; ***P \ 0.001, (DM1Se), (DM1Vit E),
(DM1Se1Vit E) group vs. DM group: 1 P \ 0. 05; 11 P \ 0. 01; 111 P \ 0.001.
 PROTECTIVE EFFECTS OF VITAMIN E AND SELELENIUM 7

Hydrophilic Antioxidant (Vitamin C)

LDL-cholesterol (mg dL21) 97.365 6 8.800 136.568 6 8.064*** 115.894 6 13.70511 94.910 6 2.822111 92.740 6 5.120111 94.388 6 8.084 85.825 6 10.153

HDL-C, high density lipoprotein-cholesterol; LDL-C, low density lipoprotein-cholesterol. Values are expressed as means 6 S.D for six animals in each group. Comparisons are made between two
groups: DM, (DM1Se), (DM1Vit E), (DM1Se1Vit E), Se, Vit E group vs control group: *P \ 0. 05; **P \ 0. 01; ***P \ 0.001, (DM1Se), (DM1Vit E), (DM1Se1Vit E) group vs. DM group: 1 P
139.664 6 2.763111 136.610 6 5.361111 137.809 6 7.315 133.493 6 8.899
67.5106 7.936111 62.766 67.717 68.774 6 3.673

30.272 6 1.294111 30.852 6 2.439 33.912 6 2.256

2.959 6 0.469
The data presented in Table III showed the levels of vita-

Vit E
min C in controls and tested groups. The exposure of rats to
DM caused a significant decrease in heart vitamin C levels
(-49%) when compared to controls. Treatment with Se or
vitamin E ameliorated vitamin C levels (P \ 0.01) when

3.498 6 0.520
compared to DM group. Supplementation of both Se and
vitamin E in the diet of the DM-treated group restored the

TABLE IV. Lipid profile in plasma of adult rat controls or treated during 30 days with DM, DM1Se, DM1Vit E, DM1Se1Vit E, Se, Vit E

Se
parameter cited above to normal values.

Glutathione (GSH) and Nonprotein Thiol

3.515 6 0.148111
(NPSH) Levels in the Heart

DM1Se1Vit E
A significant decrease of glutathione (GSH) and non pro-
tein thiol (NPSH) levels in the heart was evident in DM
group (242% and 241%, respectively) when compared
with controls (Table III). Supplementation of Se alone or
combined with vitamin E in the diet of DM group amelio-
rated GSH and NPSH levels when compared with DM-

4.029 6 0.605111
71.936 6 7.286 115.283 6 7.622*** 85.533 6 4.261*11 83.320 6 6.885 *11

27.708 6 3.32111
group.

DM1Vit E
Lipid Profile in Plasma
The exposure of adult rats to DM produced a significant
increase in total cholesterol (123%) and triglyceride
(138%) levels. Low-density lipoprotein-cholesterol (LDL-
C) levels and atherogenic index (AI) were enhanced by 29

HDL-cholesterol (mg dL21) 29.197 6 2.652 16.211 6 1.733*** 24.317 6 3.27711

5.293 6 1.14711
Total cholesterol (mg dL21) 140.607 6 7.976 181.310 6 7.943** 157.234 6 11.9341
and 61%, respectively, while high-density lipoprotein-cho-
DM1Se

lesterol (HDL-C) decreased by 45% in DM treated-rats

compared to controls (Table IV). Results revealed that there
were no significant changes in the lipid profile of rats
treated with Se or vitamin E alone, while the presence of Se
or vitamin E with DM could alleviate the adverse effects of
9.933 6 1.303***

this pesticide. Se associated to vitamin E was more power-

ful to totally restore plasma lipid profile in (DM1Se1Vit
E)-group.
DM

Histological Studies
Light microscopic examination of the heart in the controls
3.854 6 0.574

indicated a normal structure [Fig. 2(A)]. In the DM-treated

Control

rats, histological sections showed abnormalities [Fig. 2(B)]

when compared to controls. In fact, heart exhibited vacuoli-
\ 0. 05; 11 P \ 0. 01; 111 P \ 0.001.

zation, hemorrhages as well as infiltration of lymphocytes

cells. Furthermore, apoptotic cells were noted, showing an
atrophied nucleus and eosinophilic cytoplasm [Fig.
Parameters and treatments

2(B1,B2)]. Coadministration of Se or vitamin E ameliorated

Triglycerides (mg dL21)

heart histological pictures [Figs. 2(C) and 3(D)], where leu-

kocyte infiltrations, index of cellular defense, were noted.
Atherogenic index

After addition of both Se and vitamin E in the diet of DM

group, heart damages significantly decreased [Fig. 2(E)].
The histological pattern was normal in rats treated only
with Se or vitamin E [Fig. 2(F,G)]. The histopathological
changes are graded and summarized in Table V. Histologi-

Environmental Toxicology DOI 10.1002/tox

Fig. 2. Histological findings in heart tissue of adult rats from the seven experimental groups:
(A) Control group—showed normal cardiac muscle fibers; (B1) DM group—showed apoptotic
cells (indicated by arrows); (B2) DM group—showed cardiac muscle fibers with muscle separa-
tion, inflammatory cells, vacuolization and hemorrhage, (indicated by arrows); (C) DM1Se
group—Histological damage decreased with leukocyte infiltrations, index of cellular defense;
(D) DM1Vit E group—Histological picture showed a significantly decrease in heart damages;
(E) DM1Se1Vit E group—Histological picture showed a significant amelioration of cardiac his-
toarchitecture; (F) Se or (G) Vit E groups—showed normal muscle fibers without any pathologi-
cal changes. Hematoxylin-eosin, X 400. Arrows indicate: : normal cardiac muscle fibers
: apoptic cells : hemorrhage : leukocyte infiltrations : vacuolization. [Color
figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
 PROTECTIVE EFFECTS OF VITAMIN E AND SELELENIUM 9

TABLE V. Grading of the histopathological changes in the heart sections of adult rat controls or treated during
30 days with DM, DM1Se, DM1Vit E, DM1Se1Vit E, Se, Vit E

Groups Hemorrhage Leukocyte infiltrations Apoptosis Vacuolization

Controls 2 2 2 2
DM 11 111 111 111
DM1Se 1 11 1 1
DM1Vit E 1 11 1 1
DM1Se1Vit E 2 1 2 2
Se 2 2 2 2
Vit Es 2 2 2 2
Scoring was done as follows: none (2), mild (1), moderate (11) and severe (111) damage.

cal grading was made according to four severity grades:
2(none); 1 (mild); 11 (moderate), and 111 (severe).
DISCUSSION
Currently, DM poses a risk to humans, especially to those
professionally involved in its production or those who work
in farms and to those who consume contaminated water and
food (IPCS/WHO, 1996). The major goal of this work was
to evaluate the potential benefit of vitamin E and Se dietary
supplies, compared with Se or vitamin E used alone, against
cardiotoxicity induced by DM, since this has not yet been
investigated.
The major route of exposure to DM for the general pop-
ulation is the oral way. The present study was designed to
investigate the toxicity of DM given to rats via the oral
route. In our experimental study, when the 0.1 3 g L21,
0.15 3 g L21, and 0.2 g L21 doses were tested, a dose-
response relationship was observed in adult rats. Yet, the
0.2 g L21 dose used in our experiment induced oxidative
stress without lethal effects. This is in accordance with pre-
vious studies of Kamath and Rajini (2007) which used two
increasing doses of DM (20 and 40 mg kg21) demonstrat-
ing the dimethoate-dose-response relationship.
Acetylcholinesterase (AChE) activity changes are fre-
quently used as a biomarker for OP pesticide contamination
(Ferda and Ugur, 2010). During our experiment, a decrease
in AChE was noted in DM group. Our findings are in ac-
cordance with previous studies reporting that DM is an anti-
cholinesterase agent (Ecobichon, 1991; Sayim, 2007). It
has been reported that OP inhibits AChE by targeting the
serine hydroxyl group on the AChE active site, where they
bind to and inactivate the enzyme (Abbas and Hayton,
1997, Stenerson, 2004). The reactivation of AChE by de-
phosphorylation is very slow and controls the overall rate
of the reaction (Marrs, 1993). The inhibition of AChE ac-
tivity, as obtained by our results and by Lundebye et al.,
(1997), probably resulted in the accumulation of Ach,
which may increase cardiototoxicity. Our results showed
amelioration in AchE activity after the supply of vitamin E
and/or Se to the diet of DM group. These elements probably
protected AchE activities via their antioxidant properties.
Our hypothesis is in agreement with the study of Delwing-
de Lima et al. (2010) that has shown that antioxidants, like
vitamins C and E, are able to prevent acetylcholinesterase
alteration.
Cardiotoxicity may also be the result of oxidative stress.
Lipid peroxidation (LPO) is one of the main manifestations
of oxidative damage and has been found to play an impor-
tant role in the toxicity and carcinogenicity of many xeno-
biotics (Stohs and Bagchi, 1995). DM has been reported to
induce LPO, and to alter the physiological and biochemical
characteristics of biological systems as demonstrated by us
and by others (Sharma et al., 2005b; Mahjoubi-Samet et al.,
2008). So, the elevation of LPO in the heart suggested the
participation of free-radical-induced oxidative cell injury in
mediating the toxicity of DM. In our investigation, it is pos-
sible to say that vitamin E treatment is more effective than
Se in decreasing DM-induced LPO in the heart, but it does
not protect completely. Moreover, these results are in ac-
cordance with those obtained by Ogutcu et al. (2006) who
have postulated the beneficial role of vitamin E in diazi-
non-induced heart injury in rats. In fact, vitamin E is a fam-
ily of lipid-soluble vitamins and acts as an antioxidant in
cells, interrupting the propagation of LPO, and thus, pre-
serving membrane integrity (Warren et al., 2000). LPO
may bring bound enzymes either through direct attach-
ment by free radicals or through chemical modification by
its end products, malondialdehyde and 4-hydroxynonenal
(Brownlee, 2001).
Conversely, it is widely established that various reactive
oxygen species (ROS) react with membrane lipids, result-
ing in altered cell membrane fluidity and in the formation
of end products which attack proteins and DNA bases,
therefore causing mutagenic lesions (Andersen et al.,
2006). Moreover, proteins can be modified by direct attack
of ROS, giving rise to carbonyl group formation into side
chains and/or to sulfhydryl groups’ reduction in susceptible
amino acids (Halliwell and Gutteridge, 2001). In the pres-
ent study ROS, probably generated by DM treatment,
induced a rise of protein carbonyls (PCO) products,

Environmental Toxicology DOI 10.1002/tox

10 BEN AMARA ET AL.
markers of protein oxidative injury. The occurrence of pro-
tein oxidative stress in the heart tissue of experimental rats
was also confirmed by a novel marker, advanced oxidation
of protein products (AOPP), which reflected an excess of
free radical generation and of protein oxidative damages
(Gallan et al., 2003). Whereas, when DM was associated to
the nutritional supply of Se or vitamin E, a significant
decrease in the parameters cited above was obtained com-
pared with DM group. Treatment with Se and vitamin E
could prevent LPO and protein oxidation induced by DM.
This finding could be explained by the important role of Se
in preventing hydroxyl radicals’ formation and in protect-
ing the integrity and the functions of tissues (Ognjanovic
et al., 2008; Li et al., 2001). It could also be explained by
the ability of vitamin E to allow free radicals and to abstract
a hydrogen atom from the antioxidant molecule rather than
from polyunsaturated fatty acids (Pascoe et al., 1987).
In addition, the generation of free radicals and subse-
quent oxidative-stress, induced by DM, mediated structural,
and functional changes in the heart tissue. There are three
critical biochemical events that chemicals, inflicting cell
death, may initiate: (i) ATP depletion; (ii) sustained rise in
intracellular calcium; and (iii) overproduction of ROS and
reactive nitrogen species (RNS). These events interact,
amplify each other and can progressively aggravate the sit-
uation until it becomes a clear damage (Gregus and Klaas-
sen, 2001). Our study demonstrated the first biochemical
event cited above after DM treatment. In fact, there was a
decrease of Na1-K1-ATPase activity speaking in favor of
DM-induced-oxidative damage in heart tissue. This could
be explained by the oxidation of polyunsaturated fatty
acids, a main component of the membrane, thereby destroy-
ing the special arrangement of the membrane and removing
biological activities. As a result, membrane fluidity
decreases, impairing the crucial membrane functions of
transport and permeability and also hindering homeostasis
(Hong et al., 2004). The inhibition of Na1-K1 ATPase ac-
tivity can cause a depletion of cellular ATP and an increase
in intracellular calcium and ROS/RNS production, which,
in turn, can cause further inhibition of ATPase activity
(Gregus and Klaassen, 2001). The coadministration of Se
or vitamin E through the diet of DM-treated rats improved
ATPase activity. Besides, with Se1vitamin E supply,
ATPases activities reached normal values, demonstrating
the significant role of these elements in the protection of tis-
sues from oxidative damage owing to pesticide toxicity.
According to Schwenke and Behr (1998) and Aslam et al.
(2009), the synergistic effect of Se and vitamin E is most
powerful in reducing the storage and toxicity of ROS
induced by xenobiotics. Other recent studies of Sridevi
et al. (2007) and Naziroğlu et al. (2008) showed the impor-
tance of antioxidants such as vitamin E or Se in protecting
the living organism against the toxic effects of environmen-
tal chemicals via the inhibition of free radical production
and regulation of ATPases activity as well as the antioxi-
Environmental Toxicology DOI 10.1002/tox
dant redox system. These findings constitute further evi-
dence of vitamin E and Se powerful antioxidant potential.
Generally, antioxidants are the foremost defense system
that limits the toxicity associated with free radicals. SOD
and catalase provide the first line of defense against oxygen
derived free radicals. In the current study, the significant
increase in SOD and catalase activities after DM treatment
showed an activation of the compensatory mechanism
through the effect of the pesticide on progenitor cells. Its
extent depends on the magnitude of the oxidative stress and
hence on the dose of stressor (Prakasam et al., 2001).
According to some investigators (Sengupta et al., 1990),
the induction of SOD and catalase activities could be
understood as an adaptative response to oxidative stress as
these enzymes have a protective role against oxygen free
radical-induced damage. Moreover, an increase of LPO,
obtained by us and by Aggarwal et al. (2009), indicated
that the production of free radicals exceeded the capacity of
detoxification mechanisms. Our observations concerning
the increase in antioxidants activities may not be surprising
and are in accordance with some studies conducted on rats
exposed to DM (Sharma et al., 2005a; Sayim, 2007; Attia
and Nasr, 2009). The present findings showed that treat-
ment with DM, associated to nutritional supply of vitamin
E or Se, caused a significant decrease in SOD and catalase
activities as compared with the DM group. Yet, as demon-
strated by our results, the joint effect of Se and vitamin E is
more powerful in antagonizing DM-induced oxidative
stress. According to Chappuis and Poupon (1991), the pro-
tection of cells against oxidative stress takes place with
both vitamin E and Se involved in the capture of free
radicals.
Another antioxidant is glutathione peroxidase (GPx)
which plays an important role against oxidative stress. It
converts H2O2 or other lipid peroxides to water or hydroxyl
lipids. In the process, glutathione (GSH), a cofactor of
GPx, is converted to oxidized glutathione. Nonprotein thi-
ols (NPSH) and GSH are considered as the most important
non enzymatic antioxidants that counteract cardiotoxicity
(Priscilla and Prince, 2009). After DM treatment, an
increase of GPx activity and a depletion of non enzymatic
antioxidants including GSH and NPSH levels were
obtained. Enhanced GPx activity might be the natural
mechanism to counteract the pro-oxidant effect of DM tox-
icity. Moreover, the reduced levels of GSH and NPSH
could be the result of either an increased utilization for con-
jugation and/or their participation as antioxidants in termi-
nating free radicals products. Likewise, Sharma et al.
(2005a) reported an increase of glutathione peroxidase ac-
tivity and a decrease in GSH levels in the liver and brain of
rats after treatment with DM. GPx activity was ameliorated
partially or totally, respectively, after vitamin E or Se sup-
ply. These elements decrease free radical-mediated LPO
and regenerate GSH and NPSH, as demonstrated by our
results and by others (Othman and El Missiry, 1998; Gan

et al., 2002). In fact, Se supplementation increased the
activities of selenoproteins, perhaps by the high incorpora-
tion of selenocysteine in selenoproteins, which may
decrease free radical-mediated LPO and regenerate gluta-
thione (Saito and Takahashi, 2002).
On the other hand, vitamin E appears to be the most
effective lipid soluble antioxidant in the biological system.
It is involved in the inhibition of LPO and regenerates the
reduced vitamin C and GSH. By protecting myocardial
membranes and inhibiting the oxidation of lipoproteins,
vitamin E inhibits membrane peroxidative damage and
atherogenesis (Bendich et al., 1986). In our study, the
decreased concentration of vitamin C in the heart of DM-
treated rats could be explained by the depletion of GSH
since it is directly involved to recycle this vitamin (Chen
et al., 2003). Another possible explanation might be due to
the increase of LPO, since the reduction of a-tocopherol
level, an antioxidant present in cell membranes which plays
a major role in maintaining their integrity, increased DM
likelihood to cause oxidative damage.
In addition to oxidative stress caused by DM treatment,
plasma levels of total cholesterol, triglyceride (TG), and
LDL cholesterol significantly increased and HDL choles-
terol decreased. According to Faine et al. (2006) the
increased TG levels, observed in the serum of rats exposed
to Butyl hydroxytoluene, resulted in the high TG uptake by
the heart. In fact, myocardial TG provides 10-50% of the
energy requirements (Sabbah and Stanley, 2003). Its accu-
mulation in the cardiac tissue is associated with cardiomy-
opathy, suggesting that excess TG may be toxic (Lewin and
Coleman, 2003) and the elevated LPO could play a role in
this process. It was evident that Se and vitamin E were suc-
cessfully able to elevate the HDL levels, as demonstrated
by our results. Consequently, high level of HDL may com-
pete with LDL receptor sites on arterial smooth muscle
cells and thus partially inhibit its uptake. Supporting our
observation, Se and vitamin E are also able to maintain the
tissues and cells integrity and function by decreasing
plasma cholesterol and triglycerides. It has been proposed
that Se may exert an antiatherogenic influence by lowering
oxidative stress in the endothelium via the regulation of the
arachidonic acid cascade and detoxification of hydroperox-
ides (Alissa et al., 2003). Likewise, vitamin E inhibits
membrane peroxidative damage and atherogenesis by pro-
tecting myocardial membranes and inhibiting the oxidation
of lipoproteins (Bendich et al., 1986).
To substantiate the biochemical findings, a histological
examination of the heart was undertaken. Histopathological
examination of the cardiac tissue revealed that DM treat-
ment caused abnormal ultrastructural changes such as
vacuolization, hemorrhage and apoptosis. Cell death, as a
comprehensive consequence of myocardial abnormalities,
is an important cause of various cardiomyopathies (Kang,
2001). It can cause loss of contractile tissue, compensatory
hypertrophy of myocardial cells, and reparative fibrosis
PROTECTIVE EFFECTS OF VITAMIN E AND SELELENIUM 11
(Swynghedauw, 1999). Regarding the different possible
mechanisms by which apoptosis may be triggered, it has
been postulated that oxidative stress could be a key player
in such induction (Cai et al., 2006). In fact, ROS such as
hydrogen peroxide (H2O2) can induce cell death through
mechanisms such as LPO, alteration of cellular proteins
and initiation of diverse stress-signaling pathways (Li et al.,
2003). Therefore, it is possible that ROS accumulation
occurring in myocardium, in which apoptosis may take
place, would be a result of DM exposure. Since apoptotic
foci in the heart may cause severe failure, vitamin E and/or
Se could serve as the potential protective agents against
heart tissue damage. Such an effect of vitamin E or Se has
been previously reported in cardiac tissue (Shirpoor et al.,
2009; Soudani et al., 2010). Vitamin E is able to compen-
sate or repair cardiac disturbances by abrogating apoptotic
signals, suppressing LPO and protein oxidation and by
improving the antioxidant defense system (Shirpoor et al.,
2009). Se also inhibits the oxidative processes of lipids and
lipoproteins in cell membranes (Orun et al., 2008; Talas
et al., 2008).
CONCLUSION
To our knowledge, this study constitutes the first attempt to
evaluate the effects of Se combined or not with vitamin E
on DM-caused cardiotoxicity in adult rats. But the results
of animal experiments are limited with regards to extrapo-
lating the data to humans. In this finding, Se and vitamin E
may ameliorate cardiac disturbances. The mechanisms
which contribute to their effectiveness involve the quench-
ing of free radicals and antioxidant status improvement.
Thus, Se and vitamin E appear to be promising agents for
protection against DM induced cardiotoxicity. Dietary sup-
plementation with antioxidants could be an easy, inexpen-
sive and useful means to protect whoever is exposed to DM
from its toxic effects.
The authors are indebted to Miss Dalenda Kchaou for her assis-
tance in histolological techniques and to Mr Bejaoui Hafedh,
teacher of English at Sfax Faculty of Science, who has proofread
and edited this article.
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