NTT-06536; No of Pages 9 Neurotoxicology and Teratology xxx (2015) xxx–xxx

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Neurotoxicology and Teratology journal homepage: www.elsevier.com/locate/neutera

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Hanan A. Ogaly a, A.A. Khalaf b, Marwa A. Ibrahim a, Mona K. Galal a,⁎, Reham M. Abd-Elsalam c a

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Article history: Received 22 December 2014 Received in revised form 13 May 2015 Accepted 13 May 2015 Available online xxxx

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Keywords: Deltamethrin Green tea extract Brain Apoptosis Oxidative damage

Department of Biochemistry and Chemistry of Nutrition, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt Department of Pathology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt

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In the present study, we investigated the protective effect of an aqueous extract of green tea leaves (GTE) against neurotoxicity and oxidative damage induced by deltamethrin (DM) in male rats. Four different groups of rats were used: the 1st group was the vehicle treated control group, the 2nd group received DM (0.6 mg/kg BW), the 3rd group received DM plus GTE, and the 4th received GTE alone (25 mg/kg BW). The brain tissues were collected at the end of the experimental regimen for subsequent investigation. Rats that were given DM had a highly significant elevation in MDA content, nitric oxide concentration, DNA fragmentation and expression level of apoptotic genes, TP53 and COX2. Additionally, a significant reduction in the total antioxidant capacity in the second group was detected. The findings for the 3rd group highlight the efficacy of GTE as a neuro-protectant in DMinduced neurotoxicity through improving the oxidative status and DNA fragmentation as well as suppressing the expression of the TP53 and COX2 genes. In conclusion, GTE, at a concentration of 25 mg/kg/day, protected against DM-induced neurotoxicity through its antioxidant and antiapoptotic influence; therefore, it can be used as a protective natural product against DM-induced neurotoxicity. © 2015 Published by Elsevier Inc.

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1. Introduction

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The use of pesticide is one of the most researched topics in the agro fields of our economy. Although pesticides can significantly elevate crop productivity, they cause serious hazards in non-target organisms (Yonar and Sakin, 2011). Pesticides of the pyrethroid class, such as deltamethrin (DM), a type II synthetic pyrethroid, are widely used as insecticides due to their low environmental persistence and toxicity. DM is successfully substituted for organophosphates in pest-control programs (Shukla et al., 2002). Although DM was initially thought to be the safest available insecticide, a numbers of recent studies have been published on its toxicity (Kim et al., 2010; Hines, 2013; Abdel-Daim et al., 2013). The negative effects of DM on hematological, urinary and respiratory systems have previously been reported. DM exposure leads to the pathophysiology of a broad spectrum of cerebrovascular and neurodegenerative diseases (Mani et al., 2014). The mechanism of DM neurotoxicity arises through calcium overload which in turn is provoked by the delayed opening of the voltage gates of Na channel (VGSC) and inhibition of γ-aminobutyric acid receptors (Hossain and Richardson, 2011). Additionally, other studies demonstrated that a VGSC-independent

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Influence of green tea extract on oxidative damage and apoptosis induced by deltamethrin in rat brain

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⁎ Corresponding author. E-mail addresses: [email protected] (H.A. Ogaly), [email protected] (A.A. Khalaf), [email protected] (M.A. Ibrahim), [email protected] (R.M. Abd-Elsalam).

mechanism attributes to the DM-induced disruption of neuronal activity at higher levels of organization such as hippocampal neurons (Meyer et al., 2008). There is abundant literature reporting that DM increases the generation of reactive oxygen species (ROS) and free radicals, causing extensive oxidative stress and excessive lipid peroxidation (LPO) as well as reducing antioxidant enzyme activity (Abdel-Daim et al., 2013), which could be the main cause of DM toxicity. Under normal conditions, the body is endowed with effective antioxidant systems to combat the overproduction of ROS. However, under some circumstances, the balance between ROS production and its elimination is disturbed, leading to oxidative stress (Lushchak, 2011). The damage to macromolecules, including proteins, lipids, and nucleic acids, is considered to result from ROS overproduction, which is believed to be involved in the etiology of many neurodegenerative disorders (Mani et al., 2014). Herbs and medicinal plants are considered to be a potential therapeutic option for various diseases, including neurodegenerative disease (Khan et al., 2012). Green tea is a widely consumed beverage worldwide. Green tea leaves are reported to be antimutagenic through reducing cancer formation and chromosomal damage (Bushman, 1998). Additionally, the National Cancer Institute selected its extract as a cancer chemopreventive (Steele et al., 1999). The tealeaf contains polyphenols as the main component, comprising approximately 30% to 42% of the dry weight, and most of these are catechins (Graham, 1992). Epicatechin, epicatechin gallate, epigallocatechin, and epigallocatechin gallate (EGCG) are the major catechins present in green tea (Sang et al.,

http://dx.doi.org/10.1016/j.ntt.2015.05.005 0892-0362/© 2015 Published by Elsevier Inc.

Please cite this article as: Ogaly, H.A., et al., Influence of green tea extract on oxidative damage and apoptosis induced by deltamethrin in rat brain, Neurotoxicol Teratol (2015), http://dx.doi.org/10.1016/j.ntt.2015.05.005

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2. Materials and methods

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2.1. Animals

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Forty-eight male albino rats, weighing 120–140 g, were used in the present study. They were maintained under standard conditions in accordance with the Ethical Principles for the Care and Use of Laboratory Animals (Guide for the care and use of laboratory animals, 1995). The Animal Care and Use Committee of Beni-Suef University approved the study. All efforts were made to minimize animal suffering.

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2.2. Chemicals

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Deltamethrin (N 99% pure) was obtained from the KZ pesticide company (Egypt). DM was dissolved in corn oil. The selected dose of DM was based on previous studies in which 1/10 of the LD50 induced biochemical alterations in rats without having morbidity (Oda and ElMaddawy, 2011). The green tea leaves were obtained from Lipton green tea Unilever brand, packed in the United Arab Emirates Unilever Gulf FZE. The GTE was prepared according to Maity et al. (Maity et al., 1998) by soaking 15 g of instant green tea powder in 100 ml of boiling distilled water for 5 min. The GTE contained epigallocatechin gallate (337 mg/l), epigallocatechin (268 mg/l), epicatechin (90 mg/l), epicatechin gallate (60 mg/l), and coffeic acid (35 mg/l) as determined by the HPLC method (Maiani et al., 1977).

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2.3. Experimental regimen

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The rats were randomly divided into four different groups (n = 12). The route of administration selected for the study was oral gavage, which was performed once daily for 30 consecutive days.

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• The 1st group was treated with corn oil (0.2 ml\ rat) and acted as the control group. • The 2nd group received DM (0.6 mg/kg BW). • The 3rd group was given DM (0.6 mg/kg) plus GTE (25 mg/kg BW). • The 4th group was given GTE alone (25 mg/kg BW).

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Throughout the experimental time, there were no signs of toxicity in the animals treated with DM. The animals were fasted overnight, anesthetized and sacrificed by cervical dislocation at the end of the experiment, and the brain tissue was collected for further analysis.

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2.4. Oxidative stress parameter assay

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Brain samples were homogenized in cold phosphate buffered saline (pH 7.4) using Teflon homogenizer. The homogenates were centrifuged at 14,000 ×g for 15 min at 4 °C. The supernatant was used to measure the neuronal MDA according to the method described in Mihara and Uchiyama (1978), nitric oxide concentration (NO) (Miranda et al., 2001), total antioxidant capacity (TAC) using a commercial kit

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2.5. DNA damage evaluation

Apoptotic changes in the brain tissue were assessed using three dif- 138 ferent techniques that included estimation of DNA fragmentation per- 139 centage, DNA laddering assay and Comet assay. 140 2.5.1. DNA fragmentation percentage DNA fragmentation assay is a quantitative method used for grading the DNA damage (Perandones et al., 1993). The brain tissues were lysed in 0.5 ml of hypotonic lysis buffer containing 10 mM Tris–HCl (pH 8), 1 mM EDTA and 0.2% Triton X-100, and centrifuged at 14,000 ×g for 20 min at 4 °C. The pellets were resuspended in hypotonic lysis buffer. To the resuspended pellets and the supernatants, 0.5 ml of 10% trichloroacetic acid (TCA) was added. The samples were centrifuged for 20 min at 10,000 ×g at 4 °C, and the pellets were suspended in 500 μl of 5% TCA. Subsequently, each sample was treated with a double volume of diphenylamine (DPA) solution [200 mg DPA in 10 ml glacial acetic acid, 150 μl of sulfuric acid and 60 μl acetaldehyde] and incubated at 4 °C for 48 h. The proportion of fragmented DNA was calculated from the absorbance reading at 578 nm using the equation: DNA fragmentation = OD of fragmented DNA (supernatant)/[OD of fragmented DNA (supernatant) + OD of intact DNA (pellet)] × 100.

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2.5.2. DNA laddering assay The genomic DNA was extracted from brain tissue according to Wu et al. (Wu et al., 2005) to estimate DNA damages. DNA samples (10 μg) were separately loaded into 1.5% agarose gel electrophoresis for 45 min at 80 V. After electrophoresis gel was photographed through a digital camera. The migration of the fragmented DNA on agarose gel results in a characteristic laddering pattern which is considered a distinctive feature of the apoptotic DNA damage.

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2.5.3. Comet assay (alkaline single-cell microgel electrophoresis) Comet assay was performed according to Singh et al. (Singh et al., 1988). Briefly, 100 mg of crushed brain samples was suspended in 1 ml ice cold PBS, stirred for 5 min and filtered. 100 μl of cell suspension was thoroughly mixed with 600 μl of low-melting agarose, followed by spreading of 100 μl of the mixture on agarose pre-coated slides. The slides were left to solidify at 4 °C, and then they were immersed in chilled lysing solution for 1 h at 4 °C. The slides were removed and placed in a horizontal electrophoresis chamber, filled with freshly prepared electrophoretic alkaline buffer for 20 min. After electrophoresis, the slides were washed gently in 0.4 M Tris–HCl buffer and stained with ethidium bromide. The DNA migration patterns of 100 cells for each sample were observed using fluorescence microscope, and images were captured by a Nikon CCD camera. The qualitative and quantitative extent of DNA damage in the cells was estimated using the Comet 5 image analysis software developed by Kinetic Imaging Ltd. (Liverpool, UK).

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2.6. Histopathological examination

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The brain tissues from the different groups were fixed in 10% neutral buffered formalin and routinely processed for paraffin embedding to obtain 4 μm sections. The sections were stained with hematoxylin and eosin and examined under optical microscope (Bancroft and Stevens, 1996).

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2.7. Apoptotic gene expression analysis

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(purchased from Bio Diagnostic Company, Egypt) and the total protein 135 concentration according to Bradford (1976). 136

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2011). Those catechins act directly as radical scavengers for reactive oxygen and nitrogen species. Chemically, they all possess multiple hydroxyl substituents responsible for their antioxidant activity (Sang et al., 2005). They also exert indirect effects through activation of antioxidant enzymes (Mandel and Youdim, 2012). Recent findings indicate that DM may induce toxic manifestations by enhancing the production of ROS and disrupting the balance between pro-oxidants and antioxidants. In addition, there are scarce data about its neurodegenerative toxicity, mechanism and the possible protective role of natural antioxidants. Therefore, the present investigation was designed to evaluate the potency of DM in inducing neurotoxicity and the neuroprotective influence of GTE (25 mg/kg/day) through monitoring its effect on the oxidative status, DNA damage and apoptotic gene expression.

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2.7.1. Immunohistochemistry for P53 and COX2 proteins 189 Brain tissue sections were deparaffinized in xylene and rehydrated 190 in graded alcohol. Drops of Hydrogen Peroxide Block (Thermo Scientific, 191

Please cite this article as: Ogaly, H.A., et al., Influence of green tea extract on oxidative damage and apoptosis induced by deltamethrin in rat brain, Neurotoxicol Teratol (2015), http://dx.doi.org/10.1016/j.ntt.2015.05.005

H.A. Ogaly et al. / Neurotoxicology and Teratology xxx (2015) xxx–xxx

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Parameters

1st Group

2nd Group

3rd Group

4th Group

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DNA fragmentation %

17.28 ± 2.92a

59.7 ± 1.29b

26.16 ± 1.5c

15.7 ± 4.1a

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Values are expressed as the mean ± S.E. Different superscript letters in the same row in- t2:6 dicate significant differences (p b 0.05). t2:7

2.8. Statistical analysis

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The data were statistically analyzed using SPSS version 16.0 statistical package. Data are expressed as the mean ± SE. The differences between the groups were assessed using one-way analysis of variance (ANOVA). Differences were considered statistically significant for p b 0.05.

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3. Results

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Table 2 t2:1 Influence of GTE administration on the DNA fragmentation percentage in rats intoxicated t2:2 with DM. t2:3

USA) were added to block the endogenous peroxidase activity. The tissues were pretreated with 10 mM citrate buffer, pH 6.0 in microwave oven at 500 W for 10 min for antigenic retrieval. The slides were washed with PBS, and blocked with ultra V Blocking solution (Thermo scientific, USA) for 5 min. Sections were incubated overnight at 4 °C in a humidified chamber with one of the following primary antibodies: rabbit anti-p53 polyclonal antibody at 1:100 dilution (Leica Microsystems, Bannockburn, IL) and rabbit anti-COX2 polyclonal antiserum (Cayman Chemical, Ann Harbor, MI) at a 1:50 dilution. The sections were rinsed again with PBS then incubated with a biotinylated goat anti rabbit antibody (Thermo Scientific, USA) for 10 min. The sections were rinsed again with PBS. Finally, sections were incubated with Streptavidin peroxidase (Thermo scientific, USA). To visualize the reaction, slides were incubated for 10 min with 3,3′-diaminobenzidine tetrahydrochloride (DAB, Sigma). The slides were counterstained with hematoxylin then dehydrated and mounted. Primary antibodies were omitted and replaced by PBS for negative controls.

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Table 1 Influence of GTE administration on the oxidative stress parameters in rats intoxicated by DM.

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2.7.3. Quantitative real-time RT-PCR analysis The mRNA expression level of the TP53 and COX2 genes was assessed using real-time PCR standardized by co-amplification with the housekeeping gene GAPDH as an internal control. Total RNA was purified from 100 mg of rat brain tissue using the Qiagen Rneasy Mini Kit according to the manufacturer's protocol. The concentration of the total RNA was measured spectrophotometrically (Thermo Scientific, USA). The isolated RNA was reverse transcribed into cDNA and used for PCR with primers specific. The sequences of the primers used are as follows: TP53 forward 5′-GTG GTA CCG TAT GAG CCA CC-3′, reverse 5′-CAA CCT GGC ACA CAG CTT CC-3′; COX2 forward 5′-AAA GCC TCGTCCAGATGC TA-3′, reverse 5′-ATGGTGGCTGTCTTGGTAGG-3′; and GAPDH forward 5′-ACCACAGTCCATGCCATCAC-3′, reverse 5′-TCCACCACCCTGTTG CTGTA-3′. Real-time PCR was performed in color for research Laboratory (Qiagen, Egypt). cDNA was added to a SYBR Green qPCR Master Mix (Qiagen) containing 30 pg/ml of each primer. The cDNA was amplified by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s and extension at 72 °C for 45 s. During the first cycle, the 95 °C step was extended to 1 min. The GAPDH gene was amplified in the same reaction to serve as the reference gene. Every sample was analyzed in triplicate. The gene expression levels were calculated and determined according to the method described by Livak and Schmittgen (Livak and Schmittgen, 2001).

Parameters MDA (μmol\g protein) NO (mmol/L) TAC (μmol/L)

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3.12 ± 0.18a 0.346 ± 0.007a

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8.37 ± 0.31b 0.18 ± 0.004b

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3.2. DNA fragmentation percentage

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The effect of DM-induced DNA damage was evaluated by measuring the level of genomic DNA fragmentation percentage using the DPA assay (Table 2). Compared to the 1st group, DM induced marked increases in the DNA fragmentation percentage. The 3rd group, treated with GTE, showed a significant reduction in the DNA fragmentation 56.5%. No significant differences in DNA fragmentation were detected between the 1st and 4th groups.

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6.72 ± 0.39c 0.288 ± 0.009c

3.24 ± 0.42a 2.28 ± 0.89a 0.38 ± 0.004a

Values are expressed as the mean ± S.E. Different superscript letters in the same row indicate significant differences (p b 0.05).

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As shown in Fig. 1, a marked DNA laddering pattern was detected in 276 DM intoxicated group. Whereas, the 3rd group treated by both GTE and 277

4th Group c

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DM is known to induce a broad spectrum of toxicological effects and biochemical dysfunctions. These effects probably occur through the generation of ROS, causing damage to the various membranous components of the cell. MDA concentration is considered as an important indicator of LPO that indirectly reflects the oxidative degeneration of polyunsaturated fatty acids (PUFA). In the current study, the oral administration of albino rats to DM lead to a significant increase in the MDA level compared to control (Table 1). In the same consequence, NO, a gaseous, highly reactive free radical that can freely diffuse across biological membranes was also significantly elevated (Table 1). On the other hand, the TAC, the quick effective quantitative indicator for the overall antioxidant capacity against ROS overproduction, was significantly reduced after DM intoxication compared to the control group. Administration of GTE caused a significant reduction in the elevated MDA by 60.14% and NO by 19.7%, whereas, a significant elevation in the TAC of 60% was detected (Table 1). Non-significant changes were detected for oxidative parameters (MDA, NO and TAC) between the control group and the 4th group.

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2.7.2. Evaluation of P53 and COX2 immunostaining The quantitative immunoreactivity of P53 was evaluated in nuclear P53 stained cells. COX2 stained with dark brown color in the cytoplasm of the cell was considered an apoptotic cell. The expressions of P53 and COX2 immunostaining were examined in neurons in different areas of the brain tissue. The immunostained section was randomly counted in 10 microscopic fields under high-power field (X400) microscope. In each field, positive cells and total cell number were recorded. Percentage of positive stained cells (%) was calculated. It was performed by Lica Q image analysis system.

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Fig. 1. Electrophoretic migration of DNA isolated from brain tissue of different experimental groups on 1.5% agarose gel electrophoresis. Lane (1) 1st group; lane (2) 2nd group; lane (3) 3rd group; lane (4) 4th group. M: 100 bp DNA marker.

Please cite this article as: Ogaly, H.A., et al., Influence of green tea extract on oxidative damage and apoptosis induced by deltamethrin in rat brain, Neurotoxicol Teratol (2015), http://dx.doi.org/10.1016/j.ntt.2015.05.005

Tail moment

14.6 ± 1.76a 22.43 ± 3.06b 13.1 ± 1.6c 10.8 ± 2.9a

7.21 ± 0.65a 17.5 ± 2.44b 14.5 ± 2.99c 11.5 ± 1.7a

1.01 ± 0.24a 3.92 ± 0.65b 1.89 ± 0.11c 1.26 ± 0.1a

Values are expressed as the mean ± S.E. Different superscript letters in the same column indicate significant differences (p b 0.05). n = number of rats.

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DM showed a notable decrease in the DNA laddering. As far as the 1st and 4th groups, intact high molecular weight genomic DNA was detected.

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Comet assay is considered as a well-validated technique for measuring DNA damage in individual cells. Comet assay is a sensitive technique used to assess DNA fragmentation which is a typical component of toxic DNA damage and apoptosis. According to our results, DM significantly induced DNA damage compared to control group in rat's brain. The evidence for DNA damage is the significant elevation in the comet parameters, presented as tail length (μm), tail DNA (%) and tail moment. The tail moment is the main indicative parameter used for DNA damage (Table 3). Moreover, a comet-like tail implies the presence of a damaged DNA strand. The length of the tail increases with the extent of DNA damage as observed in the 2nd group (Fig. 2B). A small comet head and a large broom like tail were observed in DM intoxicated rats (Fig. 2B). On the other hand, co-administration of GTE (3rd group) deteriorated the effect of DM through significant reduction of the tail length, damaged DNA % and tail moment (Table 3), as well as reducing the intensity of comet tail (Fig. 2C). Intact head without tail was observed in both control and 4th group (Fig. 2A and D).

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3.5. Histopathological findings

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Histopathological examination of the brain tissue of 1st, control group revealed normal histological finding (Fig. 3A). The 2nd group treated with DM showed clear apoptotic and degenerative changes in the form of central chromatolysis, neuronal swelling and lysis of cell

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3.7. Gene expression The expression response of some apoptotic genes to DM intoxication was analyzed using qRT-PCR in rat brains and confirmed by immunohistochemistry at the protein level. TP53 is a tumor suppressor gene located on the short arm of chromosome 17 and its product is a DNAbinding phosphor-nucleoprotein. P53 protein is a pivotal factor that regulates the apoptosis. The P53 is inactive in normal cells unless cells are exposed to various stress signals; those signals activated the P53 through the post-translational modifications. TP53 was overexpressed after DM intoxication, reaching 9.48-fold, as observed in the 2nd group. The treatment of rats with GTE resulted in a 50.9% reduction in the expression level as observed in 3rd group (Fig. 6A). Cyclooxygenase

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Figs. 4 and 5 summarized the results of Immunohistochemical evaluation of P53 and COX2 expression in the different experimental groups. In P53 immunostaining positive cells, only cases in which the cells' nuclei became stained a dark brown color were considered a positive response (Fig. 4). COX2 immunoreactivity was characteristically cytoplasmic. The cytoplasm was stained dark brown color (Fig. 5). The percentage of immunopositive cells for P53 and COX2 in the neurons of the 2nd and 3rd groups was significantly elevated than the 1st and the 4th groups (Figs. 4E and 5E). The 3rd group showed significant reduction in the percentage of P53 immunopositive stained cells compared to the 2nd group (Fig. 4E). In the same sequence, a significant decrease in the percentage of COX2 immunopositive cells was detected in the 3rd group treated with GTE plus DM (Fig. 5E) in comparing to DM treated group. Non-significant differences in percentage of stained cells were detected between the 1st and 4th groups.

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3.6. Immunohistochemical analysis of P53 and COX2 proteins

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membrane as detected in many neurons. Some neuron appeared shrunken deeply basophilic with pyknotic or lysed nuclei (Fig. 3C). Vacuolation of neuropil with multifocal areas of gliosis was also observed. Most of cerebellar purkinje cells were degenerated (Fig. 3D) and some of them were necrosed with neurophagia. The 3rd group, treated with GTE showed few numbers of apoptotic, degenerated and necrosed neurons (Fig. 3E) in brain cortex. Cerebellar purkinje cells showed individual cell necrosis (Fig. 3F). The 4th group exhibited normal histological appearance (Fig. 3B).

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Table 3 Tail length, tail intensity, and tail moment measured with comet assay in brain cells of rats treated with DM and protective influence of GTE.

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H.A. Ogaly et al. / Neurotoxicology and Teratology xxx (2015) xxx–xxx

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Fig. 2. Apoptotic DNA damage detected by comet assay. (A1–A3) 1st group showed intact head without tail, (B1–B3) 2nd group showed a bright comet tailing, (C1–C3) 3rd group showed the protective influence of GTE and (D1–D3) 4th group showing intact head without tail.

Please cite this article as: Ogaly, H.A., et al., Influence of green tea extract on oxidative damage and apoptosis induced by deltamethrin in rat brain, Neurotoxicol Teratol (2015), http://dx.doi.org/10.1016/j.ntt.2015.05.005

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Fig. 3. Histopathological changes in the brain of the different groups (H and E X 400). A. Cerebrum of 1st control group showing normal histological picture; B. Cerebellum of the 4th GTE treated group showing normal purkinje cells (arrow); C. Cerebrum of 2nd DM treated group showing apoptosis, degeneration and necrosis of neurons (arrow) with gliosis and marked vacuolation of brain neuropil; D. Cerebellum of 2nd DM treated group showing necrosis of multiple purkinje cells (arrow); E. Cerebrum of the 3rd DM >E treated group showing few numbers of degenerated and necrosed neuron (arrow); F. Cerebellum of 3rd DM& GTE treated group showing of individual purkinje cell necrosis (arrow).

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Free radicals are an attractive explanation for the toxicity of numerous xenobiotics (e.g., pesticides). ROS are continuously produced inside the mammalian body (Li et al., 2011). Oxidative stress occurs as a consequence of the imbalance between the production of free radicals and the antioxidative process (Li et al., 2011). In fact, oxidative damage due to excessive production of ROS has been associated with defective organ dysfunction (Mossa et al., 2013). This oxidative damage has been considered an important etiological factor that is implicated in several chronic diseases, such as neurodegenerative disease (Hogg, 1998). The brain is considered highly vulnerable to oxidative stress because it consumes a high level of oxygen, contains high levels of PUFA and has low levels of antioxidant enzymes (Somani et al., 1996). DM accumulation in tissue like the brain is associated with the induction of oxidative stress and production of ROS, altering antioxidant defense mechanisms and enhancing LPO production (Abdel-Daim et al., 2014). In the current study, we aimed to elucidate the neurotoxic and apoptotic mechanisms that contribute to the DM effects in rats as well as pay attention to the neuroprotective influence of GTE. LPO, as evidenced by an increased level of MDA, is a biomarker for oxidative damage that

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(COX)-1 and -2 are key enzymes that catalyze the rate-limiting step in prostaglandin biosynthesis. COX2, is an inducible isoform, rapidly induced by growth factors, tumor promoters, oncogenes, toxin and carcinogens. According to our results DM induced the expression level of COX2 up to 3.20-fold (Fig. 6B). Oral administration of GTE attenuated its expression level by 67.1%, as detected in the 3rd group.

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has been suggested to contribute to the molecular mechanisms of DM toxicity (Abdel-Daim et al., 2014). The MDA level in DM-intoxicated rats was significantly elevated (Table 1), suggesting that there is an increased production of oxygen free radicals. The current findings are in agreement with those reported in Abdel-Daim et al. (2013); and Chargui et al. (2012). Highly reactive oxygen metabolites, especially hydroxyl radicals, act on the PUFA of phospholipid of membranes, leading to the production of unstable lipid peroxides, which tend to decompose into products like MDA (Esterbauer et al., 1991). NO plays a potent role in neuronal tissue damage and mitochondrial dysfunction (Ljubisavljevic et al., 2012). DM increases the activities of nitric oxide synthase (NOS) enzyme, initiating its protein expression and leading to overproduction of NO (Wu and Liu, 1999), which is in agreement with our findings (Table 1). The accumulation of DM in body systems increases ROS production and leads to depletion of antioxidant parameters, which was monitored by a significant reduction in the TAC (Table 1). The reduction in the antioxidant parameters in response to DM intoxication was previously reported in Abdou and Abdel-Daim (2014); and Ben Halima et al. (2014). Owing to the lipophilic nature of brain tissue, DM may have accumulated, resulting in excessive production of ROS and tissue damage (Abdel-Daim et al., 2013). There is a massive evidence of a direct involvement of the cellular redox status in the activation and the functioning of apoptosis (Oikawa et al., 2007). DNA fragmentation is considered a marker and typical characteristic feature of apoptosis (Li et al., 2009). According to our findings, the oral administration of DM significantly elevated the DNA fragmentation percentage (Table 2), and induced marked DNA laddering (Fig. 1) which are considered as a biochemical hallmark of apoptosis. We used the

Please cite this article as: Ogaly, H.A., et al., Influence of green tea extract on oxidative damage and apoptosis induced by deltamethrin in rat brain, Neurotoxicol Teratol (2015), http://dx.doi.org/10.1016/j.ntt.2015.05.005

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comet assay to confirm the results of DNA fragmentation. The comet assay is a famous technique in the field of genetic toxicology (Tice et al., 2002) which can detect different lesions of DNA damage i.e. single strand breakage, DNA cross-links and incomplete excision repair events at cellular level (Tice et al., 2002). This highly sensitive method can evaluate the level of DNA damage which affects the cell integrity accordingly causing cell death (Sreekumaran et al., 2005). In this study, the neurotoxic effect of DM assessed by the comet assay revealed that DM significantly induced DNA damage manifested by the elevated tail moment and extensive bright comet tail compared to control group (Fig. 2). Taken together, the results of DNA damage are consistent with data reported in Hossain and Richardson (2011). All those results were confirmed by pathological finding detected in neural cells (Fig. 3). DM initiates the apoptotic cascade through cell depolarization by prolonging the opening of voltage-gated Na+ channels (Cao et al., 2011). However, most recent studies demonstrated that DM at low dose can affect the development of neurons (Richardson et al., 2015). The ability of DM to cause apoptosis may be due to its potential to initiate a series of cell death signaling events, which finally lead to DNA fragmentation (Hossain and Richardson, 2011). In oxidative stress, P53 protein is activated through multiple post-translational events, including phosphorylation (Mihara et al., 2003). Activated P53 triggers a number of signaling pathways, leading to cell cycle arrest and apoptosis (Wu and Liu, 1999). These data could explain the over expression of the pre-apoptotic gene TP53 (Fig. 6A) detected in our study. These results are consistent with the immunohistochemistry finding (Fig. 4). The massive number of P53 immunostained cells reflected the overexpression of P53 protein in the DM intoxicated group (Fig. 4B). The upregulation of both P53

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Fig. 4. Representative P53 immunohistochemistry in the brain tissue of different experimental groups. A. 1st group (control) showing very weak P53 immunostaining reaction; B. 2nd group (DM-treated) showing strong nuclear immunopositive reaction in numerous cells (arrows); C. 3rd group (DM + GTE) showing reduction in the number of P53 immunostained cells (arrow); D. 4th group (GTE-treated) showing very weak immunostaining reaction; E. The bar chart represents immunopositive cells expressed as % of the total cell count. Values with different superscripts are significantly different (p b 0.05).

protein and mRNA after pesticide intoxication was previously reported by Chougule et al. (2013); and Galal et al. (2014). In the CNS, the apoptotic gene (COX2) is expressed under normal conditions (Minghetti, 2004). Moreover, the regulation of the COX2 gene is mediated through the TP53 gene (Wu et al., 2000). COX is a key regulatory enzyme in the biosynthesis of prostaglandins. Overexpression of COX2 is aroused to be both a marker and an effector of neural damage (Strauss, 2008). A synergistic interaction between NO and prostaglandin has been reported (Sheng et al., 1997). NOS selectively bind to COX2 and activate prostaglandin formation (Tian et al., 2008). The binding of NOS to COX2 directly elicits its activation, causing overexpression (Kim et al., 2005). We detected overexpression of COX2 with DM toxicity at both mRNA (Fig. 6B) and protein levels (Fig. 5B). The overproduction of NO through DM toxicity may be the dominant cause for COX2 overexpression. Because oxidative damage is considered a central mechanism of DM toxicity, the use of antioxidants to counteract ROS production is the cornerstone in alleviating the effects of such a hazard. The neuroprotective potential of medicinal plants against pesticide-induced neurotoxicity remains an area requiring extensive scientific research. Substantial attention has been paid to green tea in both scientific and consumer communities because of its benefit against a variety of health disorders, including neurological diseases (Zaveri, 2006). Although GTE is water soluble but there is evidence that its polyphenol metabolites have access to the brain. Studies with radioactively labeled EGCG demonstrated its incorporation into the brain tissue after oral administration (Suganuma et al., 1998; Nakagawa and Miyazawa, 1997). The use of GTE is effective in attenuating and repairing the damage sustained by insecticide exposure (Korany and Ezzat, 2011; Heikal et al., 2013).

Please cite this article as: Ogaly, H.A., et al., Influence of green tea extract on oxidative damage and apoptosis induced by deltamethrin in rat brain, Neurotoxicol Teratol (2015), http://dx.doi.org/10.1016/j.ntt.2015.05.005

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According to our results, DM intoxication is attenuated with GTE supplementation. GTE significantly enhances the antioxidant status, as evidenced by the reduction in the MDA and NO content as well as preserved TAC (Table 1). Those findings are in agreement with those reported by Korany and Ezzat (Korany and Ezzat, 2011) and Heikal et al. (Heikal et al., 2013). The reducing influence of GTE on MDA content was in harmony with the data reported by Elseweidy et al. (Elseweidy et al., 2009). The epicatechines can react with superoxide radical to form the corresponding semiquinones (Wang et al., 2003). In addition, they may chelate metal ions, which in turn, inhibit generation of hydroxyl radicals (Azam et al., 2004). The inhibitory effect of EGCG on

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NO production is thought to be through its suppressing action on NOS expression and subsequent NO production (Kim et al., 2010). Additionally the polyphenols could induce the expression of antioxidant enzymes (Cabrera et al., 2006), leading to an elevation in the TAC as detected in our study (Table 1). Moreover, dietary supplementation of GTE appeared to be a good method for counteracting the apoptotic toxicity induced by DM through the significant reduction in DNA damage observed through reduction of DNA fragmentation (Table 2), laddering assay (Fig. 1), comet assay parameters (Fig. 2, Table 3) and suppression of P53 and COX2 at both mRNA and protein expression levels (Figs. 4, 5 & 6). The inhibitory influence of GTE on oxidative DNA damage was

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Fig. 5. Representative COX2 immunohistochemistry in the brain tissue of different experimental groups. A. 1st group (control) showing little immunoreactivity; B. 2nd group (DM-treated) showing intense immunopositive staining (arrow) for the cytoplasmic COX2; C. 3rd group (DM + GTE) showing reduced immunostaining reaction (arrow) in the neurons; D. 4th group (GTE-treated) showing very weak immunostaining reaction; E. The bar chart represents immunopositive cell expressed as % of the total cell count. Values with different superscripts are significantly different (p b 0.05).

Fig. 6. Real-time PCR quantitation of mRNA expression level of TP53 (6A), COX2 (6B): 1st group (I), 2nd group (II), 3rd group (III) and 4th group (IV). Values represent fold changes in mRNA level over the control group. Values are expressed as mean ± S.E. Different superscript letters are significantly different (p b 0.05).

Please cite this article as: Ogaly, H.A., et al., Influence of green tea extract on oxidative damage and apoptosis induced by deltamethrin in rat brain, Neurotoxicol Teratol (2015), http://dx.doi.org/10.1016/j.ntt.2015.05.005

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“The authors declare that they have no competing interests to disclose.”

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Prof Dr. Abdel Azim participated in the study design, Dr. Marwa Ibrahim performed the molecular genetic studies, and Dr. Hanan Abdelsalam evaluated the biochemical parameters and helped to draft the manuscript. Dr Mona Khames evaluated the biochemical parameters and drafted the manuscript. Dr Reham Mohamad performed the pathological and immunohistochemical analysis. All authors read and approved the final manuscript.

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The Transparencydocument associated with this article can be found, in the online version.

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References

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In conclusion, the treatment of rats with GTE attenuated the oxidative damage and apoptotic influence of DM through reducing the DNA fragmentation and expression level of the apoptotic genes TP53 and COX2. This study demonstrated that GTE may have a potential neuroprotective influence against DM intoxication.

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proceed by the polyphenol compounds present in GTE which can inhibit the formation of DNA oxidative lesions such as 8-hydroxydeoxyguanosine (Zaveri, 2006; Tang et al., 2008). In the same line with our results, Katiyar et al. (Katiyar et al., 2001) reported that EGCG inhibited the phosphorylation of P53 after treatment with oxidant as H2O2. The inhibitory effect of EGCG on COX2 expression in brain tissue was reported by Wu et al. (Wu et al., 2012) through inhibition of activator protein 1(AP1) that regulates the promoters of various inflammatory genes (such as COX2 and NOS) (Huang et al., 1997). In the same sequence, Surh et al. (Surh et al., 2001) have shown that EGCG down-regulates the expression of COX2 and NOS by suppressing NF-κB activity (nuclear factor-kappa B). Regulation of both AP-1 and NF-κB could explain the anticancer effects of EGCG. Green tea polyphenols have the ability to influence the intracellular Ca2+ in a dose-dependant manner. EGCG at low concentrations results in a relatively limited increase in calcium ions, which would be compensated for by the calcium-buffering mechanisms of cells (Feng et al., 2010).

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Influence of green tea extract on oxidative damage and apoptosis induced by deltamethrin in rat brain.

In the present study, we investigated the protective effect of an aqueous extract of green tea leaves (GTE) against neurotoxicity and oxidative damage...
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