Accepted Manuscript Title: Fipronil induced oxidative stress in kidney and brain of mice: Protective effect of vitamin E and vitamin C Author: Prarabdh C. Badgujar, Nitin N. Pawar, Gauri A. Chandratre, A.G. Telang, A.K. Sharma PII: DOI: Reference:
S0048-3575(14)00195-3 http://dx.doi.org/doi: 10.1016/j.pestbp.2014.10.013 YPEST 3727
To appear in:
Pesticide Biochemistry and Physiology
Received date: Accepted date:
6-7-2014 21-10-2014
Please cite this article as: Prarabdh C. Badgujar, Nitin N. Pawar, Gauri A. Chandratre, A.G. Telang, A.K. Sharma, Fipronil induced oxidative stress in kidney and brain of mice: Protective effect of vitamin E and vitamin C, Pesticide Biochemistry and Physiology (2014), http://dx.doi.org/doi: 10.1016/j.pestbp.2014.10.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Fipronil induced oxidative stress in kidney and brain of mice: Protective effect of vitamin E and
2
vitamin C
3 Prarabdh C. Badgujar 1†, Nitin N. Pawar 1, Gauri A. Chandratre 2, A. G. Telang 3*, A. K. Sharma 2
4 5 6
1
7
2
8
3
9
Izatnagar 243 122, India
Division of Pharmacology and Toxicology, Indian Veterinary Research Institute, Izatnagar 243 122, India Division of Pathology, Indian Veterinary Research Institute, Izatnagar 243 122, India Toxicology Laboratory, Centre for Animal Disease Research and Diagnosis, Indian Veterinary Research Institute,
10 11
* Corresponding author: Toxicology Laboratory, Centre for Animal Disease Research and
12
Diagnosis, Indian Veterinary Research Institute, Izatnagar 243 122, India. Ph: +91 581 2302188;
13
Fax: +91 581 2300361. E-mail: [email protected] (A. G. Telang)
14 15
†
16
Food Technology Entrepreneurship and Management, Kundli, Sonipat, Haryana 131 028, India
Present address: Food Toxicology, Department of Food Science and Technology, National Institute of
17 18 19 20 21
Highlights
22
Oxidative stress inducing potential of fipronil in vivo demonstrated for the first time
23
Oxidative damage to mice kidney & brain after 28 days oral exposure to fipronil
24
Kidney: prominent histological lesions & increase in serum biochemical markers
25
Brain: Characteristic and remarkable histopathological alterations were observed
26
Vitamin E or vitamin C pre-treatment showed protective effect
27 28 29 30
Page 1 of 25
31 32
Graphical Abstracts
33 34 35 36 37 38 39
Abstract
40
Fipronil is a relatively new insecticide of the phenpyrazole group. Fipronil-induced effects on
41
antioxidant system and oxidative stress biomarkers are yet to be studied in vivo. The present study was
42
undertaken to evaluate fipronil-induced alterations in the blood biochemical markers and tissue
43
antioxidant enzymes after oral exposure in mice and to explore possible protective effect of pre-treatment
44
of antioxidant vitamins against these alterations. Mice were divided into eight groups containing control,
45
test and amelioration groups. Mice in the test groups were exposed to different doses of fipronil, i.e., 2.5,
46
5 and 10 mg/kg bw, respectively for 28 days. Mice in the amelioration groups were treated with vitamin E
47
or vitamin C (each at 100 mg/kg) 2 h prior to high dose (10 mg/kg) of fipronil. Fipronil exposure at three
48
doses caused significant increase in the blood biochemical markers, lipid peroxidation and prominent
49
histopathological alterations; while level of antioxidant enzymes was severely decreased both in kidney
50
and brain tissues. Prior administration of vitamin E or vitamin C in the fipronil exposed mice led to
51
decrease in lipid peroxidation and significant increase in activities of antioxidants, viz., glutathione, total
52
thiol, superoxide dismutase and catalase. Vitamin E and vitamin C administration in fipronil exposed
53
mice also improved histological architecture of the kidney and brain when compared with fipronil alone
54
treated groups. Thus, results of the present study demonstrated that in vivo fipronil exposure induces
55
oxidative stress and pre-treatment with vitamin E or C can protect mice against this oxidative insult. 2
Page 2 of 25
56 57
Keywords: Fipronil, Vitamin E, Vitamin C, Oxidative stress, Kidney and brain, Histopathology
58 59 60 61 62 63 64
1. Introduction
65
The strong need for the development of novel and selective pesticides having action against
66
resistant pest strains was partly fulfilled by the fipronil. Fipronil is a member of the phenylpyrazole class
67
of pesticides, with broad spectrum activity [1]. Fipronil is used to control ants, beetles, cockroaches, fleas,
68
ticks, termites, mole crickets, thrips, rootworms, weevils, and other insects. It is also being used to control
69
fleas and ticks on the domestic animals. Fipronil is effective at low field application rate against insects
70
that are resistant to pyrethroids, organophosphates, and carbamate insecticides [2]. Thus, fipronil is being
71
extensively used in the agriculture and veterinary medicine and inadvertent use of it could result in
72
exposure to animals and humans. Populations at highest risk of high dose exposure are producers, public
73
health and pesticide workers and farm owners. Low dose exposure originates mainly from the household
74
application of insecticides, contaminated food and water [3].
75
Many researchers have suggested that toxic effects of pesticides could be associated with
76
increased production of reactive oxygen species (ROS) which in turn induces tissue (liver, kidney and
77
brain) damage [4, 5 and 6]. When the production of ROS overwhelms antioxidant capacity in the target
78
cell oxidative stress occurs. The endogenous antioxidant defense system neutralizes toxic effects of these
79
free radicals by donating electrons to these toxic species, thereby reducing their damaging abilities. An
80
imbalance between prooxidant and antioxidants forces are crucial determinants of health and disease. 3
Page 3 of 25
81
The enzymatic and non-enzymatic antioxidants protect cells against oxidative damage. The
82
enzymatic antioxidant molecules, i.e., catalase, superoxide dismutase, glutathione reductase (GR),
83
glutathione peroxidase, and glutathione-s-transferase and non-enzymatic antioxidant molecules such as
84
reduced glutathione and total thiol have been shown to be significantly affected by exposure to pesticides
85
[7, 8]. Major contributors to non-enzymatic protection against lipid peroxidation are vitamin E and
86
vitamin C. Antioxidant properties of vitamin E and C have been very well documented. Vitamin E has
87
been reported to be the most effective lipid soluble anti-oxidant found in the biological system that
88
scavenges ROS, thereby preventing LPO and the initiation of oxidative tissue damage [9, 10]. The role of
89
vitamin C in neutralizing free radicals has been attributed to its property of being water soluble, allowing
90
it to work both inside and outside cells to combat free radical damage [11]. Thus, free radicals seek out an
91
electron to regain their stability and vitamin C donates electrons to free radicals such as hydroxyl and
92
superoxide radicals and ultimately quenches their reactivity [12].
93
Fipronil induced neuronal cell death has been shown to be mediated by generation of ROS
94
leading to apoptosis [13]. In undifferentiated neuronotypic PC12 cells, fipronil evoked an oxidative stress
95
and subsequently inhibited DNA and protein synthesis [14]. In vitro, fipronil induced oxidative stress has
96
been characterized in neuronal cell lines. However, except these studies there are no reports about effect
97
of fipronil on oxidative stress in an animal model (in vivo study). Therefore, present study was carried out
98
to investigate the effect of fipronil on biomarkers of oxidative stress in the kidney and brain of mice after
99
28 days exposure. Another goal of the study was to evaluate the protective role of pre-treatment of
100
vitamin E and vitamin C against fipronil induced oxidative stress.
101
2. Materials and methods
102
2.1 Chemicals
103
Technical grade fipronil (98% purity) was a kind gift from M/s Gharda Chemicals Company Pvt.
104
Ltd. (Mumbai, India). Vitamin E (>99% pure), Thiazolyl blue tetrazolium bromide (MTT), 5,5-dithiobis-
105
2-nitrobenzoic acid (DTNB, Ellman’s reagent), 1-chloro-2,4-dinitrobenzene (CDNB) were purchased 4
Page 4 of 25
106
from Sigma-Aldrich (St. Luis, MO, USA). Thiobarbituric acid (TBA) and Trichloro acetic acid (TCA),
107
Dimethyl sulphoxide (DMSO), ascorbic acid (>99.99% pure) were obtained from Merck (Germany). All
108
other reagents were of highest purity analytical grade.
109
2.2 Experimental Animals
110
Male Swiss albino mice (6-8 weeks old) were obtained from Laboratory Animal Resource section
111
of the Indian Veterinary Research Institute, Izatnagar, India. Mice were housed in the polypropylene
112
cages (five/cage) with ad libitum access to standard pellet feed and filtered tap water. The room was
113
maintained under a 12/12 hr light–dark cycle, an ambient temperature of 20-25°C and a relative humidity
114
of 45 (± 15)%. All mice were housed1 week for acclimatization before initiation of any experiment. All
115
animal experiments were approved by the Institutional Animal Ethics Committee (IAEC) of the institute.
116
2.3 Experimental Design
117
Animals were weighed and randomly divided into eight groups (n=5 mice/dose group). Corn oil
118
was used as a vehicle to prepare the desired doses of fipronil and vitamin E, while vitamin C was
119
dissolved in the distilled water. Mice were treated with fipronil and/or antioxidant vitamins by oral
120
gavage daily for the period of 28 days. The doses and exposure schedule is given below in the Table 1.
121
Mice in the vehicle control group (group 1) received corn oil orally (0.2 ml/mouse/day). The dose of
122
vitamin E and vitamin C was selected based on the dose that was seen to be most effective in lowering
123
toxicity induced by various xenobiotics [15 and 16 respectively]; while doses of fipronil were based on its
124
oral LD50 of 95 mg/kg bw (as determined in our laboratory in the Swiss albino mice; data not shown). The
125
LD50 of technical grade fipronil has been reported to be 91 mg/kg in mice [1]. Thus, three test doses of
126
fipronil were 2.5 mg/kg (1/40th LD50), 5 mg/kg (1/20th LD50) and 10 mg/kg (1/10th LD50). The protective
127
effect of both antioxidants was evaluated against high dose of fipronil (10 mg/kg) by administrating
128
vitamin E or vitamin C, 2 h prior to the daily dose of fipronil.
129
2.4 Sample collection
5
Page 5 of 25
130
At the end of the exposure period, i.e., on day 29, mice were sacrificed by cervical dislocation.
131
Prior to sacrifice, blood was collected from retro-orbital plexus and serum was separated by
132
centrifugation at 1200 g for 10 min at 4°C. After sacrifice kidney and brain were collected, lightly blotted
133
and weighed to record the absolute and relative organ weights. Later on, these organs were used for
134
biochemical and histopathological evaluation.
135
2.5. Body weight and organ weight
136
Body weight of mice were recorded at the time of dosing and represented as weekly body weight
137
gain. As mentioned above, kidney and brain weight was recorded after sacrifice and used for representing
138
absolute and relative organ weight.
139
2.6. Serum biochemical parameters
140
For the estimation of extent of nephrotoxicity, its biomarkers, i.e. blood urea nitrogen (BUN) and
141
serum creatinine level were measured with the help of commercially available diagnostic kits (Span
142
Diagnostics Pvt. Ltd., Surat, India).
143
2.6 Oxidative stress estimation
144
Kidney and brain homogenates were prepared in chilled phosphate buffer saline (PBS), pH 7.4
145
(10 % w/v) and 0.02M ethylenediamine tetraacetic acid (EDTA) (used for reduced GSH estimation only)
146
under ice cold conditions. The homogenates were centrifuged at 4000 g for 10 min to yield a supernatant
147
that was stored at -20°C for the assay of oxidative stress related parameters. Supernatant from
148
homogenates prepared in the PBS was used for the estimation of SOD, catalase, lipid peroxidation (LPO),
149
Glutathione-S-Transferase (GST), total thiol and total protein. Each sample was analyzed in triplicate and
150
GENESYS 10 UV spectrophotometer (Thermo Electron Corporation, Wisconsin, USA) was used to
151
record the optical density.
152
LPO was determined by thiobarbituric acid reactive substances (TBARS) which measures
153
formation of the malondialdehyde (MDA) according to the method of Shafiq-Ur-Rehman [17]. The
154
results were expressed as nmol of MDA formed/g of tissue. 6
Page 6 of 25
155
2.6.1 Evaluation of antioxidant enzymes
156
Following antioxidant enzymes were estimated in the kidney and brain homogenate supernatants.
157
Reduced GSH was evaluated by the method of Sedlak and Lindsay [18] in EDTA homogenates. Total
158
thiol was estimated by DTNB (5, 5’-dithiobis (2-nitrobenzoic acid; Ellman’s reagent) as described by the
159
Silverstein [19] with modifications. Briefly, 240 µl of 0.1 M phosphate buffer containing 2mM disodium
160
EDTA (pH 8.0) was added in a microtiter plate followed by addition of 30 µl of tissue homogenate
161
supernatants. Then, 30 µl of DTNB reagent (10mM in 0.1 M phosphate buffer) was mixed with the
162
supernatant with incubation for 4 min at room temperature. Readings were taken at 412 nm against blank.
163
Superoxide dismutase (SOD) activity was determined as described by Madesh and Balasubramanian [20].
164
It involved generation of superoxide by pyrogallol auto-oxidation with concomitant inhibition of
165
superoxide dependent reduction of the tetrazolium dye MTT [3-(4-5 dimethyl thiazol 2-xl) 2, 5-diphenyl
166
tetrazolium bromide] to its formazan, which was then measured at 570 nm and expressed as SOD units.
167
Catalase activity was measured in the homogenates using hydrogen peroxide as a substrate by the method
168
of Aebi [21]. The rate of H2O2 decomposition was measured spectrophotometrically at 240 nm during 180
169
s. Total protein concentration of the supernatant was assayed by the method of Lowry et al [22], with
170
serum bovine albumin as standard.
171
2.7 Histopathology
172
Representative pieces of kidney and brain were collected and fixed in the 10% buffered neutral
173
formalin. After fixation, tissues were cut into thinner pieces (2–3 mm thick). These samples were then
174
embedded in paraffin blocks. Sections of about 5 µm thickness were cut, stained with hematoxylin and
175
eosin by the standard method and examined under the light microscope.
176
2.8 Statistical analysis
177
Data are given as mean ± SEM. Statistical analysis of the data was performed with the help of
178
SPSS 20 and graphs were drawn with the help of GraphPad Prism 5 software (San Diego, California,
7
Page 7 of 25
179
USA). Data were analyzed statistically by one-way analysis of variance (ANOVA), followed by Tukey’s
180
post-hoc test. Results were considered statistically significant at p < 0.05.
181 182
3. Results
183
3.1 General observations
184
Mice exposed to different doses of fipronil over a period of 4 weeks did not produce any apparent
185
signs of toxicity and symptoms. Also, no mortality was observed in any of the treatment groups. At the
186
time of necropsy, no obvious gross lesions were noted in the kidney and brain. Biochemical parameters
187
did not show any alterations in mice treated with only vitamins (Vit C control and Vit E control) when
188
compared with control.
189
3.2 Body weight and organ weight
190
There was no significant change in the body weight of mice treated with fipronil as compared to
191
control and vitamin treated mice groups (data not shown). Medium and high dose of fipronil caused
192
decrease in absolute and relative weight of the kidney and brain as compared to control, albeit non-
193
significantly (p>0.05) (Table 2).
194
3.3 Serum biochemical findings
195
The result of serum creatinine and BUN is summarized in the Table 3. Significant and dose
196
dependent increase (p
S0048-3575(14)00195-3 http://dx.doi.org/doi: 10.1016/j.pestbp.2014.10.013 YPEST 3727
To appear in:
Pesticide Biochemistry and Physiology
Received date: Accepted date:
6-7-2014 21-10-2014
Please cite this article as: Prarabdh C. Badgujar, Nitin N. Pawar, Gauri A. Chandratre, A.G. Telang, A.K. Sharma, Fipronil induced oxidative stress in kidney and brain of mice: Protective effect of vitamin E and vitamin C, Pesticide Biochemistry and Physiology (2014), http://dx.doi.org/doi: 10.1016/j.pestbp.2014.10.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Fipronil induced oxidative stress in kidney and brain of mice: Protective effect of vitamin E and
2
vitamin C
3 Prarabdh C. Badgujar 1†, Nitin N. Pawar 1, Gauri A. Chandratre 2, A. G. Telang 3*, A. K. Sharma 2
4 5 6
1
7
2
8
3
9
Izatnagar 243 122, India
Division of Pharmacology and Toxicology, Indian Veterinary Research Institute, Izatnagar 243 122, India Division of Pathology, Indian Veterinary Research Institute, Izatnagar 243 122, India Toxicology Laboratory, Centre for Animal Disease Research and Diagnosis, Indian Veterinary Research Institute,
10 11
* Corresponding author: Toxicology Laboratory, Centre for Animal Disease Research and
12
Diagnosis, Indian Veterinary Research Institute, Izatnagar 243 122, India. Ph: +91 581 2302188;
13
Fax: +91 581 2300361. E-mail: [email protected] (A. G. Telang)
14 15
†
16
Food Technology Entrepreneurship and Management, Kundli, Sonipat, Haryana 131 028, India
Present address: Food Toxicology, Department of Food Science and Technology, National Institute of
17 18 19 20 21
Highlights
22
Oxidative stress inducing potential of fipronil in vivo demonstrated for the first time
23
Oxidative damage to mice kidney & brain after 28 days oral exposure to fipronil
24
Kidney: prominent histological lesions & increase in serum biochemical markers
25
Brain: Characteristic and remarkable histopathological alterations were observed
26
Vitamin E or vitamin C pre-treatment showed protective effect
27 28 29 30
Page 1 of 25
31 32
Graphical Abstracts
33 34 35 36 37 38 39
Abstract
40
Fipronil is a relatively new insecticide of the phenpyrazole group. Fipronil-induced effects on
41
antioxidant system and oxidative stress biomarkers are yet to be studied in vivo. The present study was
42
undertaken to evaluate fipronil-induced alterations in the blood biochemical markers and tissue
43
antioxidant enzymes after oral exposure in mice and to explore possible protective effect of pre-treatment
44
of antioxidant vitamins against these alterations. Mice were divided into eight groups containing control,
45
test and amelioration groups. Mice in the test groups were exposed to different doses of fipronil, i.e., 2.5,
46
5 and 10 mg/kg bw, respectively for 28 days. Mice in the amelioration groups were treated with vitamin E
47
or vitamin C (each at 100 mg/kg) 2 h prior to high dose (10 mg/kg) of fipronil. Fipronil exposure at three
48
doses caused significant increase in the blood biochemical markers, lipid peroxidation and prominent
49
histopathological alterations; while level of antioxidant enzymes was severely decreased both in kidney
50
and brain tissues. Prior administration of vitamin E or vitamin C in the fipronil exposed mice led to
51
decrease in lipid peroxidation and significant increase in activities of antioxidants, viz., glutathione, total
52
thiol, superoxide dismutase and catalase. Vitamin E and vitamin C administration in fipronil exposed
53
mice also improved histological architecture of the kidney and brain when compared with fipronil alone
54
treated groups. Thus, results of the present study demonstrated that in vivo fipronil exposure induces
55
oxidative stress and pre-treatment with vitamin E or C can protect mice against this oxidative insult. 2
Page 2 of 25
56 57
Keywords: Fipronil, Vitamin E, Vitamin C, Oxidative stress, Kidney and brain, Histopathology
58 59 60 61 62 63 64
1. Introduction
65
The strong need for the development of novel and selective pesticides having action against
66
resistant pest strains was partly fulfilled by the fipronil. Fipronil is a member of the phenylpyrazole class
67
of pesticides, with broad spectrum activity [1]. Fipronil is used to control ants, beetles, cockroaches, fleas,
68
ticks, termites, mole crickets, thrips, rootworms, weevils, and other insects. It is also being used to control
69
fleas and ticks on the domestic animals. Fipronil is effective at low field application rate against insects
70
that are resistant to pyrethroids, organophosphates, and carbamate insecticides [2]. Thus, fipronil is being
71
extensively used in the agriculture and veterinary medicine and inadvertent use of it could result in
72
exposure to animals and humans. Populations at highest risk of high dose exposure are producers, public
73
health and pesticide workers and farm owners. Low dose exposure originates mainly from the household
74
application of insecticides, contaminated food and water [3].
75
Many researchers have suggested that toxic effects of pesticides could be associated with
76
increased production of reactive oxygen species (ROS) which in turn induces tissue (liver, kidney and
77
brain) damage [4, 5 and 6]. When the production of ROS overwhelms antioxidant capacity in the target
78
cell oxidative stress occurs. The endogenous antioxidant defense system neutralizes toxic effects of these
79
free radicals by donating electrons to these toxic species, thereby reducing their damaging abilities. An
80
imbalance between prooxidant and antioxidants forces are crucial determinants of health and disease. 3
Page 3 of 25
81
The enzymatic and non-enzymatic antioxidants protect cells against oxidative damage. The
82
enzymatic antioxidant molecules, i.e., catalase, superoxide dismutase, glutathione reductase (GR),
83
glutathione peroxidase, and glutathione-s-transferase and non-enzymatic antioxidant molecules such as
84
reduced glutathione and total thiol have been shown to be significantly affected by exposure to pesticides
85
[7, 8]. Major contributors to non-enzymatic protection against lipid peroxidation are vitamin E and
86
vitamin C. Antioxidant properties of vitamin E and C have been very well documented. Vitamin E has
87
been reported to be the most effective lipid soluble anti-oxidant found in the biological system that
88
scavenges ROS, thereby preventing LPO and the initiation of oxidative tissue damage [9, 10]. The role of
89
vitamin C in neutralizing free radicals has been attributed to its property of being water soluble, allowing
90
it to work both inside and outside cells to combat free radical damage [11]. Thus, free radicals seek out an
91
electron to regain their stability and vitamin C donates electrons to free radicals such as hydroxyl and
92
superoxide radicals and ultimately quenches their reactivity [12].
93
Fipronil induced neuronal cell death has been shown to be mediated by generation of ROS
94
leading to apoptosis [13]. In undifferentiated neuronotypic PC12 cells, fipronil evoked an oxidative stress
95
and subsequently inhibited DNA and protein synthesis [14]. In vitro, fipronil induced oxidative stress has
96
been characterized in neuronal cell lines. However, except these studies there are no reports about effect
97
of fipronil on oxidative stress in an animal model (in vivo study). Therefore, present study was carried out
98
to investigate the effect of fipronil on biomarkers of oxidative stress in the kidney and brain of mice after
99
28 days exposure. Another goal of the study was to evaluate the protective role of pre-treatment of
100
vitamin E and vitamin C against fipronil induced oxidative stress.
101
2. Materials and methods
102
2.1 Chemicals
103
Technical grade fipronil (98% purity) was a kind gift from M/s Gharda Chemicals Company Pvt.
104
Ltd. (Mumbai, India). Vitamin E (>99% pure), Thiazolyl blue tetrazolium bromide (MTT), 5,5-dithiobis-
105
2-nitrobenzoic acid (DTNB, Ellman’s reagent), 1-chloro-2,4-dinitrobenzene (CDNB) were purchased 4
Page 4 of 25
106
from Sigma-Aldrich (St. Luis, MO, USA). Thiobarbituric acid (TBA) and Trichloro acetic acid (TCA),
107
Dimethyl sulphoxide (DMSO), ascorbic acid (>99.99% pure) were obtained from Merck (Germany). All
108
other reagents were of highest purity analytical grade.
109
2.2 Experimental Animals
110
Male Swiss albino mice (6-8 weeks old) were obtained from Laboratory Animal Resource section
111
of the Indian Veterinary Research Institute, Izatnagar, India. Mice were housed in the polypropylene
112
cages (five/cage) with ad libitum access to standard pellet feed and filtered tap water. The room was
113
maintained under a 12/12 hr light–dark cycle, an ambient temperature of 20-25°C and a relative humidity
114
of 45 (± 15)%. All mice were housed1 week for acclimatization before initiation of any experiment. All
115
animal experiments were approved by the Institutional Animal Ethics Committee (IAEC) of the institute.
116
2.3 Experimental Design
117
Animals were weighed and randomly divided into eight groups (n=5 mice/dose group). Corn oil
118
was used as a vehicle to prepare the desired doses of fipronil and vitamin E, while vitamin C was
119
dissolved in the distilled water. Mice were treated with fipronil and/or antioxidant vitamins by oral
120
gavage daily for the period of 28 days. The doses and exposure schedule is given below in the Table 1.
121
Mice in the vehicle control group (group 1) received corn oil orally (0.2 ml/mouse/day). The dose of
122
vitamin E and vitamin C was selected based on the dose that was seen to be most effective in lowering
123
toxicity induced by various xenobiotics [15 and 16 respectively]; while doses of fipronil were based on its
124
oral LD50 of 95 mg/kg bw (as determined in our laboratory in the Swiss albino mice; data not shown). The
125
LD50 of technical grade fipronil has been reported to be 91 mg/kg in mice [1]. Thus, three test doses of
126
fipronil were 2.5 mg/kg (1/40th LD50), 5 mg/kg (1/20th LD50) and 10 mg/kg (1/10th LD50). The protective
127
effect of both antioxidants was evaluated against high dose of fipronil (10 mg/kg) by administrating
128
vitamin E or vitamin C, 2 h prior to the daily dose of fipronil.
129
2.4 Sample collection
5
Page 5 of 25
130
At the end of the exposure period, i.e., on day 29, mice were sacrificed by cervical dislocation.
131
Prior to sacrifice, blood was collected from retro-orbital plexus and serum was separated by
132
centrifugation at 1200 g for 10 min at 4°C. After sacrifice kidney and brain were collected, lightly blotted
133
and weighed to record the absolute and relative organ weights. Later on, these organs were used for
134
biochemical and histopathological evaluation.
135
2.5. Body weight and organ weight
136
Body weight of mice were recorded at the time of dosing and represented as weekly body weight
137
gain. As mentioned above, kidney and brain weight was recorded after sacrifice and used for representing
138
absolute and relative organ weight.
139
2.6. Serum biochemical parameters
140
For the estimation of extent of nephrotoxicity, its biomarkers, i.e. blood urea nitrogen (BUN) and
141
serum creatinine level were measured with the help of commercially available diagnostic kits (Span
142
Diagnostics Pvt. Ltd., Surat, India).
143
2.6 Oxidative stress estimation
144
Kidney and brain homogenates were prepared in chilled phosphate buffer saline (PBS), pH 7.4
145
(10 % w/v) and 0.02M ethylenediamine tetraacetic acid (EDTA) (used for reduced GSH estimation only)
146
under ice cold conditions. The homogenates were centrifuged at 4000 g for 10 min to yield a supernatant
147
that was stored at -20°C for the assay of oxidative stress related parameters. Supernatant from
148
homogenates prepared in the PBS was used for the estimation of SOD, catalase, lipid peroxidation (LPO),
149
Glutathione-S-Transferase (GST), total thiol and total protein. Each sample was analyzed in triplicate and
150
GENESYS 10 UV spectrophotometer (Thermo Electron Corporation, Wisconsin, USA) was used to
151
record the optical density.
152
LPO was determined by thiobarbituric acid reactive substances (TBARS) which measures
153
formation of the malondialdehyde (MDA) according to the method of Shafiq-Ur-Rehman [17]. The
154
results were expressed as nmol of MDA formed/g of tissue. 6
Page 6 of 25
155
2.6.1 Evaluation of antioxidant enzymes
156
Following antioxidant enzymes were estimated in the kidney and brain homogenate supernatants.
157
Reduced GSH was evaluated by the method of Sedlak and Lindsay [18] in EDTA homogenates. Total
158
thiol was estimated by DTNB (5, 5’-dithiobis (2-nitrobenzoic acid; Ellman’s reagent) as described by the
159
Silverstein [19] with modifications. Briefly, 240 µl of 0.1 M phosphate buffer containing 2mM disodium
160
EDTA (pH 8.0) was added in a microtiter plate followed by addition of 30 µl of tissue homogenate
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supernatants. Then, 30 µl of DTNB reagent (10mM in 0.1 M phosphate buffer) was mixed with the
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supernatant with incubation for 4 min at room temperature. Readings were taken at 412 nm against blank.
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Superoxide dismutase (SOD) activity was determined as described by Madesh and Balasubramanian [20].
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It involved generation of superoxide by pyrogallol auto-oxidation with concomitant inhibition of
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superoxide dependent reduction of the tetrazolium dye MTT [3-(4-5 dimethyl thiazol 2-xl) 2, 5-diphenyl
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tetrazolium bromide] to its formazan, which was then measured at 570 nm and expressed as SOD units.
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Catalase activity was measured in the homogenates using hydrogen peroxide as a substrate by the method
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of Aebi [21]. The rate of H2O2 decomposition was measured spectrophotometrically at 240 nm during 180
169
s. Total protein concentration of the supernatant was assayed by the method of Lowry et al [22], with
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serum bovine albumin as standard.
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2.7 Histopathology
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Representative pieces of kidney and brain were collected and fixed in the 10% buffered neutral
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formalin. After fixation, tissues were cut into thinner pieces (2–3 mm thick). These samples were then
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embedded in paraffin blocks. Sections of about 5 µm thickness were cut, stained with hematoxylin and
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eosin by the standard method and examined under the light microscope.
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2.8 Statistical analysis
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Data are given as mean ± SEM. Statistical analysis of the data was performed with the help of
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SPSS 20 and graphs were drawn with the help of GraphPad Prism 5 software (San Diego, California,
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USA). Data were analyzed statistically by one-way analysis of variance (ANOVA), followed by Tukey’s
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post-hoc test. Results were considered statistically significant at p < 0.05.
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3. Results
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3.1 General observations
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Mice exposed to different doses of fipronil over a period of 4 weeks did not produce any apparent
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signs of toxicity and symptoms. Also, no mortality was observed in any of the treatment groups. At the
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time of necropsy, no obvious gross lesions were noted in the kidney and brain. Biochemical parameters
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did not show any alterations in mice treated with only vitamins (Vit C control and Vit E control) when
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compared with control.
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3.2 Body weight and organ weight
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There was no significant change in the body weight of mice treated with fipronil as compared to
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control and vitamin treated mice groups (data not shown). Medium and high dose of fipronil caused
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decrease in absolute and relative weight of the kidney and brain as compared to control, albeit non-
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significantly (p>0.05) (Table 2).
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3.3 Serum biochemical findings
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The result of serum creatinine and BUN is summarized in the Table 3. Significant and dose
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dependent increase (p
