Toxicological & Environmental Chemistry

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The role of thymoquinone as antioxidant

protection on oxidative stress induced by
imidacloprid in male and female Swiss albino mice

Sinan Ince , Ismail Kucukkurt , Hasan Huseyin Demirel , Ruhi Turkmen ,

Fahriye Zemheri & Erten Akbel

To cite this article: Sinan Ince , Ismail Kucukkurt , Hasan Huseyin Demirel , Ruhi Turkmen ,
Fahriye Zemheri & Erten Akbel (2013) The role of thymoquinone as antioxidant protection on
oxidative stress induced by imidacloprid in male and female Swiss albino mice, Toxicological &
Environmental Chemistry, 95:2, 318-329, DOI: 10.1080/02772248.2013.764672

To link to this article: http://dx.doi.org/10.1080/02772248.2013.764672

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Toxicological & Environmental Chemistry, 2013
Vol. 95, No. 2, 318–329, http://dx.doi.org/10.1080/02772248.2013.764672

The role of thymoquinone as antioxidant protection on oxidative stress

induced by imidacloprid in male and female Swiss albino mice
Sinan Incea*, Ismail Kucukkurtb, Hasan Huseyin Demirelc, Ruhi Turkmena,
Fahriye Zemherid and Erten Akbele
a
Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Afyon Kocatepe
University, Afyonkarahisar, Turkey; bDepartment of Biochemistry, Faculty of Veterinary Medicine,
Afyon Kocatepe University, Afyonkarahisar, Turkey; cDepartment of Pathology, Faculty of
Veterinary Medicine, Afyon Kocatepe University, Afyonkarahisar, Turkey; dDepartment of Medical
Biology and Genetics, Faculty of Veterinary Medicine, Afyon Kocatepe University, Afyonkarahisar,
Turkey; e Usak Health Training School, Usak University, Usak, Turkey
(Received 21 November 2012; final version received 28 December 2012)

The aim of the present study was to evaluate the possible protective effects of thymo-
quinone (TQ), an antioxidant agent, against imidacloprid (IMI)-induced oxidative
stress in male and female mice. In total, 48 Swiss Albino male and female mice were
fed a standard rodent diet and divided into 3 equal groups: the animals in the control
group (vehicle treated) were given corn oil, the second group were orally administered
15 mg/kg/day IMI alone, and the third group were orally administered 15 mg/kg/day
IMI and with TQ at 10 mg/kg/day for 21 days. During the experimental period, there
were no significant changes between initial body weights and final body weights of
IMI treated male and female mice. IMI produced significant increase in blood, liver,
kidney, and heart malondialdehyde (MDA) levels and decrease in blood and liver glu-
tathione (GSH) levels. In addition, IMI treatment decreased erythrocyte, liver, and
kidney superoxide dismutase (SOD) activity in male mice and decreased erythrocyte
and liver SOD activity in female mice. Erythrocyte catalase (CAT) activities were
found to be low in male and female mice. However, treatment with TQ reversed
IMI-induced oxidative stress, lipid peroxidation, and activities of antioxidant enzymes.
Moreover, TQ exhibited protective action against the IMI-induced histopathological
changes in tissues of male and female mice. In conclusion, TQ was found to be effec-
tive in protecting mice against IMI-induced oxidative stress by enhancing antioxidant
defense mechanisms.
Keywords: imidacloprid; thymoquinone; lipid peroxidation; oxidative stress; mice

Introduction
Imidacloprid [IMI, 1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine], a
neonicotinoid insecticide which acts selectively on nicotinic acetylcholine receptors
(nAChR), is used worldwide for insect pest management and flea control in cat and dogs
(Ihara et al. 2006). Nicotinic acetylcholine receptors that play important roles in synaptic
transmission in the central nervous system belong to a superfamily of ligand-gated ion
channels (Tasei, Lerin, and Ripault 2000) The selective toxicity to insects over verte-
brates of neonicotinoids was shown to be attributed, at least in part, to selective action on
insect nAChR. Relative toxicity of several nicotinoids correlates closely with their

*Corresponding author. Email: since@aku.edu.tr

Ó 2013 Taylor & Francis

 Toxicological & Environmental Chemistry 319

relative affinity for insects nAChR and suggested that nAChR is the predominant site of
action of these compounds (Matsuda et al. 2005; Toor, Sangha, and Khera 2013).
Few studies have been performed in mammals with neonicotinoid insecticides and
their relationship with oxidative and inflammatory events (Wu, Lin, and Cheng 2001;
Proenca et al. 2005). Tomizawa and Casida (2000) suggested that stimulation of the ner-
vous system by acute or sustained exposure to these chemicals may lead to synaptic plas-
ticity or attenuated neuronal functions. Duzguner and Erdogan (2010) demonstrated that
acute exposure to IMI leads to oxidative and inflammatory effects in rats. Bal et al.
(2012) reported that treatment with oral 0.5, 2 or 8-mg/kg IMI induced morphological
changes, DNA fragmentation, antioxidant imbalance and apoptosis in the reproductive
system of developing male rats. Duzguner and Erdogan (2012) also reported that chronic
IMI exposure produced oxidative stress and inflammation by altering antioxidant systems
and inducing pro-inflammatory cytokine production in liver and central nervous system
of non-target organisms.
Thymoquinone (TQ) is the main constituent of the volatile oil obtained from Nigella
sativa seeds and exerts various pharmacological effects (Ali and Blunden 2003) including
antioxidant properties (Houghton et al. 1995). Oral administration of TQ protected
several organs against oxidative damage induced by free radical-generating agents includ-
ing, doxorubicin-induced cardiotoxicity (Nagi and Mansour 2000), carbon tetrachloride
evoked hepatotoxicity (Nagi et al. 1999), and nephropathy produced by cisplatin (Badary
et al. 2003). Several investigators reported that TQ acts as scavenger of superoxide, hy-
droxyl radical and singlet molecular oxygen (Nagi et al. 1999; Kruk et al. 2000; Nagi and
Mansour 2000). Previously Ince et al. (2012a) demonstrated that subchronic exposure to
cypermethrin produced oxidative and pathological effects in mice and TQ exerted protec-
tive effects in a dose-dependent manner.
The aim of this study was to determine the influence of orally administered TQ against
oxidative damage induced by IMI as evidenced by lipid peroxidation (LPO), antioxidant
status and histopathological changes in male and female Swiss albino mice. Studies also
examined whether TQ had any inductive effect on both genders of Swiss albino mice.

Materials and methods

Materials
Imidacloprid and TQ were purchased from Biyoteknik A.S. (Istanbul, Turkey) and
Sigma-Aldrich (Interlab A.S., Turkey), respectively. The acute oral LD50 (lethal dose,
50%) values for IMI were in the range of approximately 130–170 mg/kg body weight in
male and female mice (El-Gendy et al. 2010), and thus IMI at 15 mg/kg (1/10 of LD50)
administered orally was used in this study. All the other chemicals and reagents were of
analytical reagent grade, purchased from commercial sources.

Experimental protocol
Male and female albino mice of the Swiss, 60 days of age and weighing 30–35 g, were
purchased from The Animal Breeding Laboratories of the Experimental Animal Research
and Application Center (Afyon, Turkey). Animals were kept at room temperature (25
C)
and relative humidity (50–55%) at a 12 hr light/dark cycle with ad libitum access to stan-
dard rodent diet and water. Mice were allowed to acclimatize to the animal facility for at
least 7 days before the start of the experiments.
320 S. Ince et al.

In the study, a total of 48 male and female mice were randomly allocated into 3 groups,
of 8 animals each. All animals were fasted overnight before the experiment. The first group
(control group) received orally (po) 0.5 ml corn oil for both males and females. The second
group (the IMI group) was treated with IMI, dissolved in distilled water of pharmaceutical
quality and administered po at a dose of 15 mg/kg body weight for 28 days for both male
and females. The third group (the TQþIMI group) received po 10 mg/kg body weight TQ
dissolved in corn oil together with 15 mg/kg body weight/day IMI for 28 days for both
males and females. The experimental protocols were also approved by the Animal Care
and Use Committee at Afyon Kocatepe University and are in accordance with the National
Institute of Health Guide for the Care and Use of Laboratory Animals.

Body weights
Body weight of mice was taken alternate weeks during morning hours at a fixed time.

Blood collection
Blood samples from each group, were collected by cardiac puncture into heparinized and
non-heparinized tubes under light ether anaesthesia at the end of 28 days.

Erythrocytes preparation
Within 30 min of blood collection, erythrocytes were precipitated by centrifugation at
600 g for 15 min at 4
C, and the plasma and serum were removed. The erythrocytes
were washed thrice with isotonic saline and the puffy coat was discarded. Then, same vol-
ume of isotonic saline and erythrocyte were added and stored at -20
C in the deep freeze.
Then used erythrocytes suspension was destroyed by osmotic pressure, using 5 vol cold
deionized water. The erythrocyte lysate was stored at 4
C until measurements within
3 days (Winterbourn et al. 1975).

Preparation of homogenates
Animals were killed by cervical dislocation; liver, kidneys, heart, and brain tissues were
excised and washed immediately with ice cold 0.9% NaCl. Each tissue was trimmed free
of extraneous tissue rinsed in chilled 0.15 M Tris–HCl buffer (pH 7.4). These tissues
were blotted dry, and homogenized in 0.15 M Tris–HCl buffer (pH 7.4) to yield a 10%
(w/v) homogenate. Tissues were centrifuged at 2500 g for 10 min at 4
C. The pellets rep-
resented the nuclear fraction and the supernatants were subjected to centrifugation at
20,000g for 20 min at 4
C. The resultant pellets and the supernatants represented the mi-
tochondrial fraction and the cytosolic (including microsomal fraction) fraction respective-
ly. Reactive oxygen species generation was observed in all the fractions as well as the
whole homogenate (Kucukkurt et al. 2008).

Preparation of tissues for histopathological analysis

A sample of each tissue was excised and fixed in a 10% formalin solution for histopatho-
logical analysis. After necropsy, a portion of the liver, kidneys, heart, and brain from
each animal was fixed in 10% buffered formalin, processed, embedded in paraffin, sec-
tioned at 5 mm, and stained with hematoxylin-eosin (H&E). Finally, sections were blindly
 Toxicological & Environmental Chemistry 321

analyzed by a pathologist under the light microscope (Olympus BX51 and DP20 attached
Microscopic Digital Image Analyze System, Tokyo, Japan).

Measurement of LPO in whole blood and tissue homogenates

Malondialdehyde (MDA), as a marker for LPO, was determined by the methods of Draper
and Hardley (1990) in whole blood and Ohkawa, Ohishi, and Yagi (1979) in tissue homo-
genates. The principle of the methods is based on spectrophotometric measurement of the
color produced during the reaction of thiobarbituric acid (TBA) with MDA and its absor-
bance was measured spectrophotometrically at 532 nm. The concentration of MDA was
calculated by the absorbance coefficient of MDA-TBA complex and expressed in
nmol/ml blood or nmol/g wet tissue.

Measurement of GSH in whole blood and tissue homogenates

Glutathione (GSH) concentration was measured using the method described by Beutler,
Duron, and Kelly (1993) in whole blood and tissue homogenates. The optical density was
measured at 412 nm using spectrophotometer. Results were reported as nmol/ml blood or
nmol/g wet tissue.

Measurement of SOD activity in erythrocyte lysate and tissue homogenates

The antioxidant enzyme activity of superoxide dismutase (SOD) in erythrocyte lysate and
tissue homogenate was measured according to the method of Sun, Oberley, and Li
(1988). The absorbance obtained from NBT reduction to blue formazon by superoxide
was determined at 560 nm spectrophotometrically. SOD activity was expressed in
U/mgHb erythrocyte or U/mg protein tissue.

Measurement of CAT activity in erythrocyte lysate and tissue homogenate

Catalase (CAT) activities in erythrocyte lysate and tissue homogenate were determined
according to the method of Luck (1963) and Aebi (1974), respectively. One unit CAT is
the amount that enzyme degrades 1 mmol of H2O2 per min at pH 4.5 at 25
C, and CAT
activity (k; nmol/min) was expressed in k/mgHb erythrocyte or k/mg protein tissue.

Measurement of hemoglobin (Hb) and protein concentrations

Hemoglobin (Hb) was determined colorimetric by cyanomethemoglobin method accord-
ing to Drabkin and Austin (1935), and tissue protein content was assayed according to the
colorimetric method of Lowry et al. (1951).

Spectrophotometric measurements
The spectrophotometric measurements were performed by using a Shimadzu 1601
UV-VIS spectrophotometer (Tokyo, Japan).

Statistical analyses
Data obtained from experimental animals were expressed as means and standard
deviation of means ( SD) and analyzed using one-way analysis of variance (ANOVA),
322 S. Ince et al.

followed by Duncan post-hoc tests on the SPSS (11.5) software computer program. A dif-
ference in the mean values of p < 0.05 was considered to be significant.

Results
Effect on body weight, food and water intake
Oral administration of 15 mg/kg/day IMI did not markedly affect body weight of male
and female mice. In addition, TQ at dose of 10 mg/kg/day and 15 mg/kg/day IMI exerted
no significant effect on body weight of male and female mice. Food and water consump-
tion was affected in all groups.

Effect on lipid peroxidation and reduced glutathione

A significant elevation in MDA levels was observed in the whole blood, liver, kidney and
heart of male and female mice, male kidney, male heart, and female brain (Table 1).
However, whole blood, liver, kidney, heart, and brain tissue MDA were found to be sig-
nificantly lower in IMI plus TQ groups with respect to the IMI alone. In the whole blood
and liver of both genders GSH levels were markedly lower in the IMI group (Table 2). In
contrast, administration of TQ showed increased blood and liver GSH levels in male and
female mice compared to IMI alone. Kidney, heart, and brain GSH levels were not
markedly affected.

Effect on antioxidant enzymes

Antioxidant enzymes, SOD and CAT activities were determined in erythrocyte, liver,
kidney, heart, and brain tissue of male and female mice as shown in Tables 3 and 4,
respectively. In the IMI groups, SOD activities were found to be significantly reduced in
erythrocyte, liver, and kidney in male mice and erythrocyte and liver in female mice,
whereas unchanged in female kidney, heart and brain tissues in male and female mice.
CAT activities were decreased in erythrocyte of male and female mice but not significant
in liver, kidney, heart, and brain. In contrast, administration of TQ prior to the IMI
challenge was observed to reverse IMI-induced alterations of SOD and CAT activities.

Histopathological alterations
Histopathological changes in organs of experimental groups were largely described and
shown in Figures 1 and 2. In IMI groups, vacuole degeneration, dilatation of sinusoids,
dissociated remark cordons, pyknotic nuclei, and leukocyte infiltration were observed in
male (Figure 1-A2) and female livers (Figure 2-A2). There were shrinkage of glomeruli
and degeneration of epithelial cells in kidneys of male (Figure 1-B2) and female
mice (Figure 2-B2). Degeneration was found in hearts of male (Figure 1-C2) and
female (Figure 2-C2). Focal gliosis was found in brains of male (Figure 1-D2) and
females (Figure 2-D2).
In TQ groups, slight histopathological changes were noted in liver, kidney, heart, and
brain tissues of male (Figure 1– A3, B3, C3, and D3, respectively) and females (Figure 2–
A3, B3, C3, and D3, respectively). In the control group, no significant histopathological
changes were observed in liver, kidney, heart, and brain of male (Figure 1– A1, B1, C1,
and D1, respectively) and females (Figure 2– A1, B1, C1, and D1, respectively).
Table 1. Effects of imidacloprid (IMI) and imidacloprid (IMI) þ thymoquinone (TQ) on malondialdehyde levels in blood, liver, kidney, heart, and brain of male
and female mice.

Blood (nmol/ml) Liver (nmol/g tissue) Kidney (nmol/g tissue) Heart (nmol/g tissue) Brain (nmol/g tissue)

Treatment design Male Female Male Female Male Female Male Female Male Female

Control 7.05 ± 1.54 c 7.40 ± 1.30 c 2.23 ± 0.79 c 1.58 ± 0.54 c 1.77 ± 0.54 c 2.19 ± 1.24b 2.29 ± 0.90c 2.27 ± 0.67 5.02 ± 1.32 b 3.54 ± 0.23 b
IMI 12.74 ± 1.18 a 12.14 ± 1.62 a 10.93 ± 2.47 a 10.15 ± 2.27 a 8.18 ± 1.06 a 5.65 ± 2.41a 8.02 ± 1.78a 2.83 ± 0.68 7.19 ± 0.88 a 6.86 ± 1.07 a
IMI þ TQ 9.55 ± 1.42 b 9.51 ± 1.40 b 8.71 ± 1.44 b 6.91 ± 1.75 b 5.06 ± 1.54 b 3.91 ± 0.97b 5.85 ± 1.42b 2.37 ± 0.89 6.24 ± 0.68 ab 4.02 ± 0.35 b
Values are mean  Standard deviations; n ¼ 8.
a,b,c
: in the same column values with different letters show statistically significant differences in blood and liver of male and female mice, male kidney, male heart, female brain
(p < 0.001), female kidney, and male brain (p < 0.01).

Table 2. Effects of imidacloprid (IMI) and imidacloprid (IMI) þ thymoquinone (TQ) on glutathione levels in blood, liver, kidney, heart, and brain of male and
female mice.

Blood (nmol/ml) Liver (nmol/g tissue) Kidney (nmol/g tissue) Heart (nmol/g tissue) Brain (nmol/g tissue)

Treatment design Male Female Male Female Male Female Male Female Male Female
a a a a
Control 47.05 ± 6.12 44.79 ± 2.64 3.30 ± 0.24 4.32 ± 0.62 2.78 ± 0.36 3.02 ± 0.32 2.96 ± 0.38 3.54 ± 0.43 2.82 ± 0.21 4.01 ± 0.86
IMI 36.50 ± 5.12 b 36.61 ± 3.24 b 2.29 ± 0.22 b 2.82 ± 0.51b 2.19 ± 0.84 2.82 ± 0.54 2.60 ± 0.37 2.98 ± 0.36 2.13 ± 0.75 3. 84 ± 0.84
IMI þ TQ 40.97 ± 4.30 ab 39.36 ± 1.59 b 2.76 ± 0.49 c 3.24 ± 0.52b 2.32 ± 0.47 2.85 ± 0.19 2.68 ± 0.79 3.46 ± 0.49 2.52 ± 0.24 3.73 ± 0.61
Values are mean  Standard deviations; n ¼ 8.
a,b,c
: in the same column values with different letters show statistically significant differences in male blood (p < 0.05), female blood (p < 0.001), and liver of male and female mice
Toxicological & Environmental Chemistry

(p ¼ 0.001).
323
 324

Table 3. Effects of imidacloprid (IMI) and imidacloprid (IMI) þ thymoquinone (TQ) on superoxide dismutase activities in erythrocyte, liver, kidney, heart, and
brain of male and female mice.

Erythrocyte (U/mgHb) Liver (U/mg protein) Kidney (U/mg protein) Heart (U/mg protein) Brain (U/mg protein)
S. Ince et al.

Treatment design Male Female Male Female Male Female Male Female Male Female

Control 40.78 ± 1.80a 46.43 ± 4.88a 19.11 ± 1.92 a 18.20 ± 2.00 a 21.03 ± 2.86 a 18.29 ± 1.44 14.72 ± 3.02 17.87 ± 1.20 25.39 ± 2.68 18.21 ± 2.21
IMI 22.76 ± 3.22c 29.87 ± 4.49b 11.90 ± 2.92 b 13.89 ± 1.95 b 12.23 ± 1.77 c 14.85 ± 3.02 12.12 ± 2.30 14.42 ± 2.91 22.91 ± 2.11 16.87 ± 1.62
IMI þ TQ 30.94 ± 3.02b 34.57 ± 3.40b 17.54 ± 2.10 a 15.87 ± 1.69 ab 15.44 ± 1.59 b 16.54 ± 2.05 12.87 ± 1.55 15.95 ± 2.45 23.90 ± 2.12 16.98 ± 2.26
Values are mean  Standard deviations; n ¼ 8.
a,b,c
: in the same column values with different letters show statistically significant differences in erythrocyte of male and female mice, male kidney (p < 0.001), and female liver
(p < 0.01).

Table 4. Effects of imidacloprid (IMI) and imidacloprid (IMI) þ thymoquinone (TQ) on catalase activities in erythrocyte, liver, kidney, heart, and brain of male
and female mice.

Erythrocyte (k/mgHb) Liver (k/mg protein) Kidney (k/mg protein) Heart (k/mg protein) Brain (k/mg protein)

Treatment design Male Female Male Female Male Female Male Female Male Female
a a
Control 480.74 ± 28.21 520.32 ± 60.84 1.56 ± 0.74 1.69 ± 0.50 0.75 ± 0.29 0.54 ± 0.12 0.66 ± 0.28 0.58 ± 0.26 0.18 ± 0.09 0.14 ± 0.10
IMI 124.64 ± 26.77 c 172.76 ± 20.67 c 1.37 ± 0.57 1.52 ± 0.41 0.60 ± 0.30 0.38 ± 0.12 0.23 ± 0.86 0.40 ± 0.18 0.11 ± 0.07 0.08 ± 0.05
IMI þ TQ 166.68 ± 14.45 b 283.02 ± 34.90 b 1.40 ± 0.73 1.58 ± 0.28 0.75 ± 0.23 0.44 ± 0.11 0.52 ± 0.16 0.68 ± 0.25 0.15 ± 0.05 0.10 ± 0.04
Values are mean  Standard deviations; n ¼ 8.
a,b,c
: in the same column values with different letters show statistically significant differences in male and female erythrocyte (p < 0.001).
 Toxicological & Environmental Chemistry 325

Figure 1. The effect of thymoquinone (TQ) on imidacloprid (IMI) induced damage in liver
(A), kidney (B), heart (C), and brain (D) of male mice. Representative figures were stained with
H&E. The original magnification was x 20 and the scale bars represent 100 mm. Arrows indicate
dilatation of sinusoids, dissociated remark cordons, pyknotic nuclei, and leukocyte infiltration in
the liver (Figure 1-A2), shrinkage of glomeruli and degeneration of epithelial cells in the kidney
(Figure 2-B2), degeneration in the heart (Figure 1-C2), focal gliosis in the brain (Figure 1-D2)
of male mice, respectively. (1) Control group, (2) animals treated with 15 mg/kg/day IMI, and
(3) animals treated with 10 mg/kg/day TQ in olive oil and 15 mg/kg/day IMI.

Figure 2. The effect of thymoquinone (TQ) on imidacloprid (IMI) induced damage in liver (A),
kidney (B), heart (C), and brain (D) of female mice. Representative figures were stained with H&E.
The original magnification was x 20 and the scale bars represent 100 mm. Arrows indicate
vacuole degeneration, dilatation of sinusoids, dissociated remark cordons, pyknotic nuclei in the
liver (Figure 2-A2), shrinkage of glomeruli and degeneration of epithelial cells in the kidney
(Figure 2-B2), degeneration in the heart (Figure 2-C2), focal gliosis in the brain (Figure 2-D2)
of female mice, respectively. (1) Control group, (2) animals treated with 15 mg/kg/day IMI, and
(3) animals treated with 10 mg/kg/day TQ in olive oil and 15 mg/kg/day IMI.
326 S. Ince et al.

Discussion
In this study, there were no significant changes in body weight gains of mice in IMI
treated groups. Similarly, Kapoor et al. (2010) reported that po administration to female
rats of different doses of IMI (5 or 10 mg/kg/day) did not produce signs of toxicity and
mortality during 90 days exposure. However, animals exposed to 20 mg/kg/day showed
significant reduction in body weight during 90 days exposure. Bal et al. (2012) also found
that body weight gains of rats were significantly lower in the IMI-treated (po 0.5, 2 or
8-mg/kg for 90 days) rats. In addition, Mohamadin et al. (2010) reported that body weight
was significantly decreased in propoxur (carbamate insecticide)-exposed rats and pre-
treatment with Nigella sativa oil restored significantly body weight of rats. In this study,
receiving only IMI did not markedly affect body weights of mice and receiving IMI with
TQ exerted no significant changes in body weight, indicating that IMI (15 mg/kg/bw for
30 days) did not affect body weight of mice.
Increased oxidative stress represents an imbalance between intracellular production of
free radicals and the cellular defense mechanisms; notably, MDA is one of the most im-
portant markers of oxidative stress. Several studies demonstrated that synthetic pyreth-
roids produce oxidative stress in a dose- and time-dependent manner (Kanbur et al.
2008), and increase levels of MDA, deplete GSH levels, and decrease activities of antiox-
idant enzymes such as SOD and CAT (Ince et al. 2010; Kucukkurt et al. 2010). El-Gendy
et al. (2010) reported that po administration of 15 mg/kg IMI significantly elevated LPO
levels and the activities of antioxidant enzymes including CAT, SOD, glutathione peroxi-
dase, and glutathione-S-transferase in male mice. The enhanced production of blood and
tissue lipid peroxides observed in our study is in agreement with other studies. TQ supple-
mentation was found to produce significantly less lipid peroxides than IMI-treated mice.
Consistent with our results, TQ acted as scavenger of superoxide, hydroxyl radical and
singlet molecular oxygen (Nagi et al. 1999; Kruk et al. 2000; Badary et al. 2003). Previ-
ous studies showed that GSH plays a key role in the detoxification of the reactive toxic
metabolites of IMI, and that cell death begins when GSH stores are markedly depleted
(Duzguner and Erdogan 2010; Kapoor et al. 2010; Bal et al. 2012). Nagi and Almakki
(2009) also demonstrated that TQ supplementation induced phase-2 enzymes activity as
quinone reductase and glutathione transferase in mouse liver. In this study, the observed
increase in LPO and concomitant depletion of GSH levels in IMI groups suggest that
the increased peroxidation may be a consequence of depleted GSH stores. Treatment of
IMI-induced mice with TQ yielded a return to normal levels of GSH, which may have
resulted from the TQ-mediated reduction of peroxidative activity among cells.
Antioxidants inhibit free radical formation. Both SOD and CAT dismute O2 and de-
compose H2O2, resulting in a decrease in oxidative stress, which is an effective way of
protecting cells from damage (Nagi and Almakki 2009). These enzymes work together to
eliminate reactive oxygen species (ROS), and deviations in physiological concentrations
may exert a dramatic effect on resistance of cellular lipids, proteins, and DNA to oxida-
tive damage (Ince et al. 2012b). Decreased activities of SOD and CAT in animals were
also reported with different pesticides such as chlorpyrifos, cypermethrin, carbofuran, di-
methoate, malathion, and IMI which reduced SOD and CAT activities in rats and mice
(Khan, Sobti, and Kataria 2005; Rai and Sharma 2007; Kanbur et al. 2008; Mansour and
Mossa 2009; Ince et al. 2012a). In the present study, administering IMI for 28 days de-
creased SOD and CAT activities, consistent with these studies. In the IMI groups, low
levels of SOD and CAT might be related to consumption of these enzymes due to in-
creased oxidative stress in erythrocytes, liver, and male kidneys. SOD and CAT activities
 Toxicological & Environmental Chemistry 327

of mice increased significantly in the TQ groups as compared to IMI, suggesting that TQ

in a dose dependent manner has the ability to restore and maintain the activity of these
enzymes.
IMI at oral doses of 15 mg/kg/day produced apparent histopathological changes in
liver, kidneys, heart, and brain in male and female mice on 28th day of insecticide treat-
ment. Vacuole degenerations, dilatation of sinusoids, dissociated remark cordons,
pyknotic nuclei, and leukocyte infiltration were observed in livers of male and female
mice. There were shrinkage of glomeruli and degeneration of epithelial cells in kidneys,
degeneration in hearts, and focal gliosis in brains of male and female mice. Similarly,
Mohany et al. (2011) reported that animals treated with 0.21 mg/ kg IMI for 4 weeks
showed heavily congested central vein and blood sinusoids, widely distributed pyknotic
nuclei, and leukocyte infiltration in rat liver. IMI at 1/10th of LD50 treatment resulted in
dilatations of central vein and sinusoids between hepatocytes (Toor, Sangha, and Khera
2012). High dose of IMI (20 mg/kg/day) resulted in mild focal necrosis with swollen
cellular nuclei and cytoplasmic lesions in rat liver and slight degeneration of tubules and
glomeruli of kidney of the female rats (Bhardwaj et al. 2010). IMI produced similar
histopathological lesions in liver, kidneys, and brain of Japanese quail exposed to
chemical for 6 weeks (Omiama 2004) and in layer chickens exposed to 139 mg/kg IMI
(Kammon et al. 2010). In contrast, TQ, in a dose dependent manner protected liver,
kidneys, heart, and brain tissues against IMI-induced cellular damage.
In conclusion, the results of this study demonstrate that TQ was effective for inhibi-
tion of IMI-induced oxidative stress in male and female mice. The results of this study
show that the protective effects of TQ may be due to both an increase in the activity of
the antioxidant defense system, as well as an inhibition of LPO.

Conflict of interest statement

This study was supported by a grant from the Afyon Kocatepe University Scientific Re-
search Council, Afyonkarahisar, Turkey (Project no: 12.HIZ.DES.32).

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