Role of melatonin in mitigating nonylphenol-induced toxicity in frontal cortex and hippocampus of rat brain

Accepted Manuscript Role of melatonin in mitigating nonylphenol-induced toxicity in frontal cortex and hippocampus of rat brain Heena Tabassum, Mohammad Ashafaq, Suhel Parvez, Sheikh Raisuddin PII:

S0197-0186(16)30112-7

DOI:

10.1016/j.neuint.2016.12.010

Reference:

NCI 3967

To appear in:

Neurochemistry International

Received Date: 21 May 2016 Revised Date:

5 December 2016

Accepted Date: 20 December 2016

Please cite this article as: Tabassum, H., Ashafaq, M., Parvez, S., Raisuddin, S., Role of melatonin in mitigating nonylphenol-induced toxicity in frontal cortex and hippocampus of rat brain, Neurochemistry International (2017), doi: 10.1016/j.neuint.2016.12.010. 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.

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Role of melatonin in mitigating nonylphenol-induced toxicity in frontal cortex and hippocampus of rat brain

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Heena Tabassum, Mohammad Ashafaq, Suhel Parvez and Sheikh Raisuddin*

Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University),

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New Delhi 110 062, India

*Author for correspondence: Prof. S. Raisuddin; Tel: +91 11 26059688x5478; Fax: +91 11 26059663 E-mail address: [email protected]

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Abstract Nonylphenol (NP), an environmental endocrine disruptor mimics estrogen and is a potential toxicant both under in vitro and in vivo conditions. In this study, the effect of melatonin on NP-

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induced neurotoxicity and cognitive alteration was investigated in adult male Wistar rats. Melatonin supplementation has been known to protect cells from neurotoxic injury. The animals were divided into three groups namely, control (vehicle) which received olive oil orally and

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treated rats received NP (25 mg/kg, per os) thrice a week for 45 days while the third group i.e., NP + melatonin, animals were co-administered melatonin (10 mg/kg, i.p.) along with NP. On the

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46th day, rats were assessed for anxiety, motor co-ordination, grip strength and cognitive performance using Morris water maze test and then sacrificed for biochemical and histopathological assays in brain tissues. Melatonin improved the behavioral performance in NP exposed group. The results showed that NP significantly decreased the activity of acetylcholine

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esterase (AchE), monoamine oxidase (MAO) and Na+/K+-ATPase, in rat brain tissue along with other enzymes of antioxidant milieu. The outcome of the study shows that NP, like other persistent endocrine disrupting pollutants, creates a potential risk of cognitive, neurochemical

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and histopathological perturbations as a result of environmental exposure. Taken together, our

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study demonstrates that melatonin is protective against NP-induced neurotoxicity.

Key Words: Nonylphenol, Melatonin, Neurobehavioral toxicity, Rats, Hippocampus, Brain, Cortex

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1. Introduction Most endocrine disrupting chemicals (EDCs) are manufactured intentionally as a raw material for plastic packaging, cosmetics, detergents or paints, and as additives for lubricants,

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while a certain percentage of them are unwanted by-products and wastes (Mao et. al., 2010). Humans are under continuous threat of exposure to many environmental xenobiotics and associated health hazards including EDCs. EDCs toxicity can potentially affect several organ

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systems including the pathophysiological role in central nervous system (CNS) and other neurological disorders. This peculiar ability to alter the neural transmission and the formation of

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neural networks, the EDCs are sometimes also referred to as neural-disrupting chemicals (Kakeyama et. al., 2001). Certain EDCs like bisphenol A (BPA) and diethylstilbestrol (DES) have been shown to adversely affect synaptic plasticity (Ogiue-Ikeda et. al., 2008). Nonylphenol (NP) is one such example of a widely spread and uncontrolled distributed

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environmental non-ionic surfactant. NP is a lipophilic, ubiquitous persistent contaminant in both wildlife including aquatic environment and humans. The primary use of NP is as an intermediate in the manufacture of its ethoxylates. They are widely used extensively in industrial applications

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as well as household and personal care products, including many plastics that have been in commerce for over 50 years. Products containing NP come from textile processing, pulp and

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paper processing, paints, resins and protective coatings, oil and gas recovery, steel manufacturing, pest control products and power generation sectors. NP’s unique chemical structure makes it resistant to physical, chemical and biological degradation which may result in its longer retention in the environment. Its lipophilic nature can also lead to its cellular and tissue bioaccumulation (Mao et. al., 2010). It is classified as EDC because of its structural similarity to

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the endogenous estrogens, e.g., 17-β-estradiol, and its ability to mimic or block hormonal effects (Jie et. al., 2013). Few others reports have appeared about the negative effects of NP on CNS along with

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reproductive and immune systems. It can also have neurodevelopment changes in both animals and human beings. Some evidence exists on NP-induced CNS neurotoxicity (Jie et al., 2016). The neurotoxicity of NP could be due to oxidative stress via activation of mitochondrial

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apoptosis pathway and inflammatory signaling pathway, which influence the expression of apoptosis genes and inflammatory mediators. Zhang et al. (2008) have reported that inducible

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nitric oxide synthase and cyclooxygenase-2 enzymes, which cause inflammation, are increased in mice brains due to chronic NP exposure leading to neurotoxicity. Uncontrolled chronic inflammation of the central nervous system can lead to neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases. However, the mechanisms behind the NP neurotoxic and

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cognition impairing ability still needs detailed investigation. Analyzing behavioral change is a way to assess any abnormality in the neural function. The NP exposure has been correlated to the disruptive cognitive function and behavioral responses in fish, reptiles, and birds because the

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gonadal hormones have a direct regulatory effect on CNS development. Recently, reports have indicated potential CNS neurotoxicity induced by NP in various aquatic and rodent models (Jie

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et. al., 2013). NP can penetrate blood-brain barrier (BBB) and has the potential to structurally mimic an endogenous hormone, estrogen or in some cases, block the effects of the same (Doerge et. al., 2002; Litwa et. al., 2014). Recent studies have focused on the possible capacity of natural compounds and phytochemicals extracted from fruits, vegetables and beverages displaying protective abilities in various animal models of neurological disorders (Wu et. al., 2010). Reduced glutathione (GSH)

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is one of the endogenous antioxidants and non-specific hydroxyl radical scavengers present in the brain. In addition, there are some exogenous antioxidants available in cells like flavonoids and vitamins which can also be supplemented if required (Valko et. al., 2007). N-Acetyl-5-

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methoxytryptamine also known as melatonin is one such lipophilic hormone that is mainly produced and secreted at night by the pineal gland. Research studies have explored the several physiological properties of melatonin such as circadian rhythms regulation, free radical

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scavenging, improving immunity, analgesic and neuroprotection (Reiter et. al., 2014). Generally, melatonin exerts both direct and indirect antioxidant protective actions both under in vivo and in

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vitro conditions (Reiter et. al., 2003). It easily crosses morpho-physiological barriers like BBB, intracellular and sub-cellular barriers acting as a free radical scavenger (Reiter et. al., 2009). The present study aimed to investigate the neurotoxic effects of chronic NP treatment on oxidative stress and cognitive alterations in male Wistar rats. Additionally, studies on protective action of

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melatonin co-administration against NP induced oxidative alterations in the brain tissue and

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associated neurobehavioral changes in exposed animals were also carried out.

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2. Methodology 2.1.Chemicals Acetylthiocholine iodide (ATC), benzylaminehydrochloride (BAHC), bovine serum albumin

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(BSA), 5,50-dithiobis (2-nitrobenzoic acid) (DTNB), oxidized glutathione (GSSG), reduced glutathione (GSH), reduced NADP(H), thiobarbituric acid (TBA) and xanthine were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). 1-amino-2-naphthol-4- sulphonic acid

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(ANSA), butylated hydroxyl taulene (BHT), 1- chloro-2,4-dinitrobenzene (CDNB), 2,4dinitrophenylhydrazine (DNPH), epinephrine, EDTA, orthophosphoric acid (OPA), perchloric

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acid (PCA), sulphosalicylic acid, sodium azide and trichloroacetic acid (TCA) were purchased from Merck Limited (Mumbai, India). p-NFκB, p65 polyclonal and β-Actin mouse monoclonal antibody was obtained from Santa cruz Biotechnology, USA. Monoclonal GFAP antibody was purchased from Chemichon International, Temecula, CA and anti-rabbit IgG was purchased

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from Jackson Immuno Research Laboratories Inc., West Groove, PA, USA. Nonylphenol (NP) was obtained from Sigma–Aldrich chemicals Pvt. Ltd., USA. 2.2.Animals

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The experimental protocol was approved by the Institutional Animal Ethics Committee (173/GO/Re/S/2000 CPCSEA). Male Wistar rats weighing 150-200 g, aged 5-6 weeks were

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maintained under standard conditions in an animal house (Central Animal House Facility, Hamdard University, New Delhi). Protocols were approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals. Animals were housed at room temperature with 12 h light and 12 h dark cycle. Food and drinking water was provided ad libitum. The animals were acclimatized for one week in the laboratory to adapt to the local environment and

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handling. During the maintenance period, each animal was taken out from the cage daily to record body weight, rectal temperatures using clinical digital thermometer. 2.3.Experimental design

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NP was suspended in corn oil and administered via oral route in rats (25 mg/kg b.wt.) for45 days. Neuroprotective agent, melatonin (10 mg/kg b.wt. suspended in distill water was administered via i.p. route. The animals were evaluated for neurobehavioral alterations

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employing several parameters at the end of the exposure regime. In addition to these parameters mentioned below, specific neurochemical, histopathological and immunohistochemical analysis

NP. 2.4.Neurobehavioral Paradigms 2.4.1. Morris Water Maze

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were carried out for corroborating cognitive impairment results with probable neurotoxicity of

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The Morris water maze procedure was employed to assess cognitive function. The learning and memory capabilities of the rats were evaluated using water maze task (Rowe et. al., 2007). The spatial learning and memory of animals were tested in a an apparatus, which consisted of a

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circular water tank (132 cm diameter, 60 cm height) filled 40 cm with water(25 ± 2 º C). A nontoxic paint was used to render the water opaque. The pool was divided into four equal quadrants,

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labeled north, south, east, and west. An escape platform (10 cm indiameter) was located 2 cm below the water surface in a constant position in one of four imaginary quadrants of the pool. The animal could use only distal visual cues (colored posters, pictures and a curtain) from within the testing room to locate the submerged platform. Latencies to locate the hidden platform during training and test trials (see procedures below) were recorded and analyzed using a computerbased tracking system (ANY-maze Video Tracking Software, USA).Before the training started,

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rats were allowed to swim freely in the pool for 60 s without platform. The animals were given 10 trials over 3 consecutive days (5 training trials on the first day followed by 4 trials on the second day and a single test trial on the last day at 20–30 min inter-trial interval) with the

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platform submerged. Trial duration was maximally 120 s; if the platform was not located the animal was guided to and had to remain on the platform for 20 s. Animals were placed into the pool facing the side wall. After each training trial, the rat was dried with a towel and allowed to

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remain in a cage. The time spent in the target quadrant indicated the degree of memory consolidation which had taken place after learning. The time and track taken to reach the

up to a cutoff time of 120 s. 2.4.2. Elevated Plus Maze Test

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platform was recorded by an overhead video camera. Latency to reach the platform was recorded

For elevated plus maze test, on the46thday animals were placed on the junction of the open

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and closed arms, facing the open arm and left on the maze for the next 5 min. Time spent in the closed and open arms were noted down while the animals were exploring the maze (Walf and Frye, 2007). Proper care was taken to avoid any sudden noise during the recording.

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2.4.3. Rotarod Test

To assess motor coordination, the activity of rats were evaluated on the rotarod unit (Omni

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Rotor, Omnitech Electronics, Inc., Columbus, OH, USA) consists of a rotating rod of 75mm diameter, which was divided into four compartments to permit the testing of four rats at a time. The time each rat remained on the rotating bar was recorded for three trials for each rat, at5 min interval and a maximum trial length of 180 s per trial. The apparatus automatically records the time in 0.1 s till the rats fall off the rotating shaft to the floor. The speed was set at 10 cycles and

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cut off time was 180 s. Data were presented as mean time on the rotating bar over the three test trials.

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2.4.4. Grip Strength

Grip strength was analyzed on six scale system (0 to 5): 0 – falls off; 1 – hangs onto string by two forepaws; 2 – as for 1 but attempts to climb on string; 3 – as for 1 plus one or both hind

2.5.Biochemical Analysis 2.5.1. Homogenate Preparation

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successive trials was taken for each animal.

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limbs; 4 – as for 1 plus tail wrapped around string and 5 – escape. The highest reading of three

The frontal cortex and hippocampal regions of brain tissue were homogenized in 0.1 M phosphate buffer (pH- 7.4) to obtain 10% homogenate using a Potter- Elevehjam homogenizer

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giving 6-8 strokes at medium speed keeping the sample under ice. 2.5.2. Post Mitochondrial Supernatant (PMS) Preparation

Homogenate was subjected to differential centrifugation in refrigerated centrifuge at

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temperature of 4°C. It was centrifuged at 10,000 g for 20 minutes. The resulting pellet is the primary mitochondrial pellet and the supernatant is 10% post mitochondrial supernatant. PMS

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was used for the estimation of various neurochemical analysis. 2.5.3. Determination of Lipid Peroxidation (LPO) LPO was measured using, 0.25 ml of homogenate prepared from cerebellum and cerebral cortex was mixed with 10 mM BHT, OPA(1%) and TBA (0.67%) were added and mixture was incubated at 90 ºC for 45 min (Tabassum et. al., 2007). The absorbance was measured at 535 nm.

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LPO was expressed as µmol TBARS formed/h/g tissue based on the molar extinction coefficient of 1.56 × 105M-1cm-1. 2.5.4. Determination of Protein Carbonyl (PC) Content

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PC content was quantified by using DNPH (Floor et. al., 1998). DNPH reacts with protein carbonyls to produce the corresponding hydrazone. The supernatant of cerebellum and cerebral cortex (0.5 mL) was reacted with 10 mM DNPH in 2 M HCl for 1 h at room temperature and

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precipitated with 40% TCA. The pelleted protein was washed thrice by resuspension in ethanol/ethyl acetate (1:1) mixture. Proteins were then solubilized in 6 M guanidine

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hydrochloride, formic acid (50%) and centrifuged at 1600 x g for 5 min to remove any trace of insoluble material. The carbonyl content was measured spectrophotometrically at 340nm. The results were expressed as nmol DNPH incorporated/mg protein based on the molar extinction coefficient of 2.1 × 104M-1cm-1.

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2.5.5. Estimation of Non-Protein Bound Thiol (NP-SH)

NP-SH levels were determined in samples (Govil et. al., 2012).The molar extinction coefficient of 13,100 M-1cm-1 at 412 nm was used for the measurement of NP-SH content. The

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values were expressed as mmoles NP-SH/g tissue, respectively. 2.5.6. Reduced Glutathione (GSH) Content

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GSH content was estimated with DTNB as substrate (Chaudhary and Parvez, 2012). The reaction is based on the fact that the thiol group of GSH reacts with the –SH reagent (DTNB) to form thionitrobenzoic acid. The GSH content was expressed as micromoles of GSH per gram tissue

2.5.7. Glutathione-S-Transferase (GST) Activity

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For determining GST activity, the conjugation of CDNB with GSH forming a colored conjugate glutathione 2,4-dinitrobenzene was monitored kinetically (Naseem and Parvez, 2014). For GST activity measurement, the reaction mixture contained 0.1 M sodium phosphate buffer

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(pH 7.4), 10 mM GSH, 10 mM CDNB, and 0.2 mL PMS. The enzyme activity was calculated as

extinction coefficient of 9.6×103M-1cm-1at 340 nm. 2.5.8. Glutathione Peroxidase (GPx) Activity

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nanomoles of CDNB conjugate formed per minute per milligram protein using a molar

GPx activity assay consisted of 0.1 M phosphate buffer (pH 7.4), 1 mM EDTA, 1 mM

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sodiumazide, 1mM GSH, 0.2 mM NADPH, 0.25 mM H2O2, and 0.1 mL PMS prepared from 10 % homogenate (Naseem and Parvez, 2014). Oxidation of NADPH was recorded spectrophotometrically at 340 nm. The enzyme activity was calculated as nanomoles of NADPH oxidized per minute per milligram of protein using a molar extinction coefficient of 6.22× 103 Mcm-1.

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2.5.9. Glutathione Reductase (GR) Activity

GR activity assay system consisted 0.1 M phosphate buffer (pH 7.4), 0.5 mM EDTA, 1 mM

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GSSG, 0.1 mM NADPH, and0.3 mL supernatant in a total volume of 2.0 mL PMS(Tabassum et. al., 2007). The enzyme activity was quantified by measuring the disappearance of NADPH at

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340 nm and was calculated as nanomoles of NADPH oxidized per minute per milligram protein using a molar extinction coefficient of 6.22×103 M-1cm-1. 2.5.10. Catalase (CAT) Activity CAT activity was assayed by the method based on the disappearance of H2O2 (Naseem and Parvez, 2014). The reaction volume contained 0.1 M sodium phosphate buffer (pH 7.4), 0.05 M H2O2, and 0.05 mL supernatant prepared from 10% homogenate. Change in absorbance was

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recorded kinetically at 240 nm. CAT activity was calculated in terms of µmol H2O2 consumed/min/mg protein using a molar extinction coefficient of 39.6 M-1cm-1. 2.5.11. Superoxide Dismutase (SOD) activity

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SOD activity assay was based on the ability of SOD to inhibit the auto-oxidation of epinephrine at alkaline pH (Chaudhary and Parvez, 2012). The assay mixture contained 50 mM glycine buffer (pH 10.4), supernatant of prefrontal cortex and hippocampus (prepared in glycine

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buffer), and epinephrine. SOD activity was measured kinetically at 480 nm indirectly by the oxidized product of epinephrine, i.e., adrenochrome using a molar extinction coefficient of 4020

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M-1cm-1.

2.5.12. Estimation of Xanthine Oxidase (XO)

The activity of XO was assayed by utilizing uric acid (Chaudhary and Parvez, 2012). There action mixture consisted of 0.2mL PMS supernatant, which was incubated for 5 min at 37 °C

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with 0.1 M phosphate buffer (pH 7.4). Then 0.15mM xanthine was added to the reaction mixtureand kept at 37 °C for 20 min, which was followed by the addition of 10% PCA and doubledistilled water in a total volume of 4 mL. The mixture was then centrifuged at 1,500 x g

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for 10min and the OD was taken at 290 nm. The enzyme activity was calculated as nmoles of uric acid formed/min/mg protein, using a molar extinction coefficient of 12,200 M-1cm-1.

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2.6.Neurotoxicity Markers

2.6.1. Acetylcholinesterase (AchE) Activity AchE was estimated by using the artificial substrate ATC, which is broken down in the presence of AChE to release thiocholine, which reacts with DTNB to form thionitrobenzoic acid (Chaudhary and Parvez, 2012). The reaction volume contained 0.1 M sodium phosphate buffer (pH 7.4), 10 mM DTNB, ATC, and 0.4 mL brain supernatant. The absorbance was measured at

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412 nm. The enzyme activity was calculated as nmol ATC hydrolysed/min/mg protein using a

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molar extinction coefficient of 1.36 ×104 M-1cm-1.

2.6.2. Na+, K+-ATPase Activity

Na+, K+-ATPase activity is measured as the release of inorganic phosphate (Pi) (Chaudhary

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and Parvez et. al., 2012). The supernatant of rat brain regions was prepared in0.2 M Tris-HCl buffer (pH 7.4). The reaction mixture for the Na+,K+-ATPase assay contained0.1 M MgCl2, 1 M

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NaCl, 0.2 M KCl, and 0.2 M Tris–HCl buffer (pH 7.4). The mixture was incubated at room temperature for 5 min, and then 0.025 M ATP was added to the supernatant to start the reaction. The mixture was again incubated at 37 ºC for 15 min, and 10% TCA was added to both the reaction mixtures to stop the reaction. Centrifugation was carried out at 1500 xg for 10 min. The

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pellet was discarded and the supernatant, distilled water, ammoniummolybdate, and ANSA were taken to make a final volume of 5 mL. The mixture was incubated at room temperature for 30 min, and the OD was taken at 660 nm. The activity was measured as µg Pi liberated/min/mg

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protein.

2.6.3. Monoamine Oxidase (MAO) Activity

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MAO activity was measured based on oxidation of BAHC to benzaldehyde. The reaction mixture consisted 0.1 M phosphate buffer (pH 7.4), distilled water, 0.1 M BAHC, 0.2 mL supernatant, which was incubated for 30 min at room temperature (Chaudhary and Parvez, 2012). Then 10% PCA was added to the reaction mixture and then centrifuged at 1500g for 10 min and the OD was taken at 280 nm. The enzyme activity was calculated as nmol BAHC hydrolysed/min/mg protein using a molar extinction coefficient of7.6925 M-1cm-1.

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2.7.Determination of Neurotransmitter Levels Estimation of dopamine in the brain PFC was done using HPLC-ECD (Vishnoi et. al., 2015). Chromatographic analyses were performed at room temperature. The data were acquired and

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processed in Empower Pro Operating System. For the sample preparation, the isolated PFC was homogenized using a homogenizer in ice-cold 0.1 M PCA and centrifuged at10,000 × g for 30 min at 4 ºC using a cooling centrifuge, and supernatant was filtered through a0.2-µm membrane

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and was used for HPLC analysis of dopamine. The filtered supernatant was injected to HPLC column. The mobile phase comprised sodium acetate (0.02 M), EDTA (0.2mM), methanol

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(16%), di-n-butyl amine (0.01%) and heptane sulfonic acid (0.055%), at pH 3.92adjusted with phosphoric acid, filtered through a 0.2-µm membrane and degassed on a sonicator. Flow rate of mobile phase was kept at 1 ml/min. Dopamine peaks were identified by comparing their retention time in the sample and its concentration was estimated according to area under curve

2.8.Western Blot Analysis

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using straight line equation y=mx+c. Dopamine concentration was represented as ng/mg protein.

Tissues were homogenized in chilled Lysis Buffer (20mM TrisHCl, pH 7.5; 150mMNaCl;

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1mM EDTA, pH 8; 0.1% NP40; 0.1% SDS with protease and phosphatase inhibitors added freshly), centrifuged at 4°C for 20 min at 10,000 x g. Protein estimation was done using Bradford

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reagent (Biorad) and equal amount of protein (40 µg/ lane) was loaded on 10%-12%SDSPolyacrylamide gels further transferred onto a PVDF membrane (IMMOBILON, Millipore). 5% Non- fat dry milk (Biorad) was used for blocking and blots were incubated overnight at 4°C with anti-phosphor NFKB (1:250) and anti-B- Actin (1:5000) was used as loading control. Horseradish peroxidase-conjugated secondary antibodies were used to develop the blots and

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bands were visualized using Supersignal chemiluminiscent substrate from Pierce (ThermoPierce). 2.9. Immunohistochemistry for GFAP

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At the end of the treatment regime animals were anesthetized with chloral hydrate (400mg/kg, i.p.) and perfused transcardially with 0.9% sodium chloride at 4 °C, followed by 4%paraformaldehyde in 0.1 M phosphate-buffer (pH 7.4). The brain was removed, kept in the

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same fixative for overnight at 4 ºC and immersed in 0.1 M PBS. The tissues were kept in final sucrose solution till sectioning. The fixed tissues were embedded in OCT compound

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(polyvinylglycol and water) and frozen at -20 °C. Coronal sections of 12 µm thicknesses were cut on a cryostat (Leica, Germany) and collected on gelatin coated slides and immersed in wash buffer(sodium phosphate 0.1 M, sodium chloride 0.5 M, Triton X-100, sodium azide) pH 7.4 for 20min. After a pre-incubation for 1 h in blocking solution (10% normal goat serum, 0.3%

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TritonX-100 in PBS), sections were incubated overnight at 4ºC with primary antibody; rabbit anti-GFAP (anti-glial fibrillary acidic protein; 1:200 dilution Chemicon International, Temecula, CA) diluted in a solution of 0.3% triton X-100 in PBS. The slides were washed with PBS to

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remove the unbound antibody and sections were incubated with 1:500 dilutions of goat anti rabbit IgG secondary antibody for 1 h at room temperature. Finally, the sections were mounted

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on gelatin-coated slides, air dried and cover slipped. Omission of primary or secondary antibody served as controls. 2.10.

Histopathology

Brain tissues were isolated from control and treated rats. Physiological saline solution (0.75% NaCl) was used to rinse and clean the tissues. They were fixed in aqueous Bouin’ssolution for 48 h processed through graded series of alcohols, cleared in xylene and embedded in paraffin wax.

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Sections were cut at 4-6 µm thickness with the help of 820-Spencer rotator microtome, stained with hematoxylin -eosin (dissolved in 70% alcohol). 2.11.

Protein Determination

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The protein content was determined in PMS of brain tissue by the method of (Bradford,

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1976) using BSA as a standard.

Statistical Analysis

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Data analysis was performed using Graph Pad prism5 software (Graph Pad Software Inc., San Diego, CA, USA), Student’s t test and one way ANOVA with Tukey’s Post hoc test were applied to calculate significance and represented as means ± SE. Values of p<0.05 were

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considered as significant. Image J software (NIH) was used to analyze the blots.

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Results There were no significant differences in body weight of rats from NP and NP + Melatonin groups when compared with the control group.

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Neurobehavioral Paradigms

The Morris water maze test was used to reveal the effects of NP on spatial learning and memory. The latencies to find the escape platform were significantly (p<0.01) higher in NP

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(25mg/kg b. wt.) treated rats when compared to in the control groups and significantly (p<0.05) improved with melatonin pre-treatment (NP + MEL) when compared to NP alone group

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(Fig.1A). Melatonin pre-treatment helped the rats to find the platform easily when compared to NP treated rats (Fig. 1B-D).Rats that received NP treatment significantly (p˂0.01) entered less and spent less time in the open arms as compared to control group (Fig. 2B). Thus the rats with NP exposure spent significantly (p˂0.01) more time in the closed arms as compared to control

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group (Fig. 2A). Pre-treating the animals with melatonin improved the exploration activity in comparison to NP treated group (p˂0.05) in both closed as well as open arms (Fig. 2 A and B). Significant differences were also observed in the number of entries made by rats from NP +

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MEL group when compared with NP group alone (Fig. 2D). No significant effect was observed on the number of closed arm entries on the EPM was observed (Fig. 2C).Fig. 3A shows no

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significant change in muscle coordination in NP group compared to control group. Also, no significant alteration was observed in NP + MEL group as compared to NP group. The grip strength was significantly decreased (p˂0.001) in NP group as compared to control group rats. The mean score in melatonin pre-treated NP group (NP+MEL) was significantly high (p˂0.05) as compared to the NP exposed group (Fig. 3B).

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Effects on oxidative stress Level of Rat Brain As a marker of lipid peroxidation, the level of TBARS was examined in frontal cortex and hippocampal regions of rat brain. The TBARS level increased in the group treated with NP when

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compared to controls (p< 0.001) in both the regions, but in NP combined with melatonin pretreated rats, the contents of TBARS were significantly lower (p< 0.001) than that of NP alone administered animals (Fig. 4 A and B). Fig. 4 C and D represents the effect of NP on PC in

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frontal cortex and hippocampus of rat brain. The PC content increased in the group treated with NP when compared to controls (p<0.001) in both the regions, but in NP combined with

that of NP-alone administered animals.

Effect on thiol status

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melatonin pre-treated rats, the level of PC formation were significantly prevented (p< 0.01) than

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NP exposure caused a significant decrease in GSH (p<0.001) and NP-SH (p<0.001) content in the frontal cortex and hippocampus of rat brain (Fig. 4 E and G). The NP + Melatonin treated group showed a significant rise in GSH and NP-SH levels (p<0.01) in both the brain regions

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compared to NP-treated animals (Fig. 4F and H).

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NP inhibits glutathione metabolizing enzymes activity NP exposure has shown a significant decrease in the activity of GST (p<0.001) in the frontal cortex and hippocampus of rat brain tissue when compared with control which was significantly improved by pretreatment with melatonin followed by NP exposure in both regions (p<0.01) (Fig. 5A and B). NP caused a significant (p<0.001) depletion in the activity of GR in comparison to control group. NP exposure along with melatonin pre-treatment (10 mg/kg bodyweight)

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restored the enzyme activity significantly (p<0.001), when compared to NP, treated group alone (Fig. 5C and D). The GPx activity was also markedly depleted (p<0.001) on exposure with NP and significantly (p<0.01) rise in the NP+MEL group in both frontal cortex and hippocampus

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(Fig. 5E and F).

NP alters enzymatic antioxidant defences and elicits the activity of XO

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In SOD determination of frontal cortex and hippocampus region of brain, there was a significant decrease in SOD activity in NP exposed groups (P<0.001) as compared to the control

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(Fig.6A and B). In NP combined with melatonin pre-treated rats, the level of SOD activity significantly increased (p<0.01) than that of NP-alone administered animals. There was a significant increase in CAT activity (p<0.001) with NP treatment as compared to the controls (Fig. 6C and D). The NP + Melatonin treated group showed a significant decrease in CAT

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activity (p<0.001) in both the brain regions compared to NP-treated animals. Fig. 6 E and F represents XO activity in frontal cortex and hippocampal preparation of rat brain markedly (p<0.01) increased in NP exposure when compared to the control and significantly (p<0.01)

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animals.

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decreased in the NP + Melatonin treated group in both the brain regions compared to NP-treated

NP modulates neurotoxicity biomarkers Fig. 7 A and B represents the activity of MAO in the frontal cortex and hippocampus prepared from brain tissue of rat. There was a significant decrease (p<0.001) in MAO activity in NP treatment group. Also, pretreatment with melatonin followed by NP significantly elevated the MAO activity in comparison to NP treated animals (p<0.01). Fig. 7 C and D shows the activity

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of AChE in the rat brain frontal cortex and hippocampus. NP administration significantly decreased (p<0.001) the AchE activity in frontal cortex and hippocampus when compared with control animals. NP treatment along with melatonin pre-treatment (10 mg/kg body weight)

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restored the enzyme activity significantly (p<0.01) when compared to NP treated group alone. In Na+/K+ ATPase determination there was a significant decrease (p<0.001) in the enzyme activity for the NP group as compared to the control which was significantly improved by melatonin

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Dopamine content in rat brain

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pretreatment (p < 0.01). (Fig. 7 E and F).

A significant increase at value of p<0.001 in the levels of dopamine was observed in the brain tissue sample of NP exposed animals in comparison to the control group which was

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significantly lowered (p<0.001) by melatonin co-administration (Fig. 8).

Western Blot

Fig. 9 shows the Western blot analysis in brain tissue obtained from control, NP-exposed and

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NP + MEL treated rats with anti- NF-kB p65 antibody. A significant increase in NF-kBp65levels (p<0.05) in frontal cortex from NP treated group compared to control one was measured, while

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NF-kBp65 protein level was only partially reversed in NP + MEL treated group in comparison to NP group.

Immunohistochemistry

The activation of astrocyte upregulation is associated with neuronal cell death. GFAP expression was found to be remarkably high NP treated group (Fig. 10B and E. A noticeable

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reduction in GFAP expression was observed in melatonin co-treated group as compared to NP group. Expression of GFAP was seen to be very scarce in control animals (Fig. 10C and F).

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Histopathology

Light microscope examination showed that the brain of animals in the control group has a characteristic normal appearance (Fig. 11A and C). There were lots of hyperchromatic cells

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forming spongioform degeneration in the brain of rats which were exposed to NP. Also presence of numerous vacuolated spaces was observed (Fig. 11B and E). Furthermore, melatonin co-

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administrated reduced spongiform degeneration and neuronal loss in the brain tissue significantly in comparison to the NP treated animals (Fig. 11C and F). Histological alterations (pyknotic nuclei) have been shown by arrow in the representative photographs based on previous work (Aydogan et al., 2008). The pyknotic nuclei have been defined as darkly stained punctuate nuclei

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and those nuclei that were fragmented were counted as single nucleus.

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4. Discussion Estrogenic EDCs are capable of disturbing the hormone mediated CNS events causing developmental, morphological and physiological alterations including cognitive function

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(Silbergeld et al., 2002). In the present study, we have evaluated the role of melatonin supplementation as a potential new prophylactic anti-oxidative and cognitive modulatory agent in neurotoxicity produced by NP exposure in the rat model. The proposed mechanisms

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underlying melatonin induced neuroprotection include elevating anti-oxidative defense enzyme activity, scavenging free radicals, inhibiting LPO, increasing glial cell-derived neurotrophic

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factors and stimulating enzymes which supply GSH and down regulate pro-oxidant enzymes (Lin et al., 2009). a. Cognitive alterations

EDCs may render nervous system more susceptible to damaging events by affecting

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hormonal balance (Schantz et al., 2001). Functional deficits are common neurologic sequel in animal models of chemically induced neurotoxicity. In line with above studies, we observed the alterations in the motor coordination skills, grip strength, anxiety like behavior and latency to

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reach platform in spatial navigation task for cognitive ability analysis in the NP exposed group in comparison to the controls. Also, the effect of melatonin co-treatment was studied to check for

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any kind of reversal in these alterations. Estrogens also have an impact on particular brain regions such as the hippocampus and cerebral cortex (Norbury et al., 2003). EDCs such as pesticides, bisphenol A and NP can bind to ERs due to their ability to mimic endogenous estrogen (Laws et al., 2000). Because hormone mediated events play a key role in CNS functions, there is speculation that some of the anxiety, motor and cognitive alterations might arise from exposure to environmental chemicals such as

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NP. Results from the water-maze experiments revealed that NP at a higher dose could impair the ability of spatial learning and memory as given in terms of latency to reach the platform. Similar results were obtained in a study on male mice exposed to NP ((Mao et al., 2010).

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In the elevated plus maze experiments, a significant increase in time spent in the closed arms was seen in NP exposed animals indicating decreased exploration and increased anxiety which was alleviated with melatonin co-treatment. The elevated plus maze is a validated and frequently

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used model for assessing anxiety in rodents (Walf and Frye 2007). BPA possessing the similar estrogenic activity as NP is known to consistently increase anxiety behavior in rats as measured

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by theelevated plus and open-field (Diaz et al., 2013). It is well recognized that the ovarian hormones alter behavioral indices of anxiety in male and female rats (Walf and Frye 2007). EDCs can disrupt the organization and function of dopaminergic pathways throughout the brain, resulting in a wide range of behavioural effects including elevated impulsivity (Jones et al.,

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2008). There was no significant difference in the motor coordination between the controls and NP treated animals tested on Rota-rod apparatus but a significant reduction in grip strength was observed with NP administration. It has been reported that neurotoxic compounds can alter grip

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strength associated with locomotor abnormalities in rats (Yamasaki et al., 2012). A number of in vitro studies have examined the neurodegenerative and CNS effects of NP (Jie et al.,

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2013).Oxidative damage plays an important role in the pathogenesis of various CNS disorders and neurobehavioral impairments and NP has been associated with oxidative stress recently (Mao et al., 2010).

NP exposure results in increased free radicals generation leading to oxidant stress in frontal cortex and hippocampus that have command over emotional, motor and learning activities (Grossberg, 2009). Oxidative damage of brain tissue by free radicals may cause cognitive

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problems such as depression, anxiety, and memory loss (Glade, 2010). Melatonin pretreatment in animals showed significant improvement in behavioral outcomes of our study. Melatonin has lipophilic nature so it is able to rapidly cross the BBB (Reiter, 1997). Melatonin possesses strong

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free radical scavenging and neuroprotection properties and also regulates the antioxidant enzymes activity (Carocci et al., 2014). Melatonin also improves cognitive function and neurogenesis (Luchettiet al., 2010). The improvement in memory and locomotor activity

pathogenesis of NP-induced cognitive decline.

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b. Neurochemical Alterations: Role of oxidative stress

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following pretreatment with melatonin underlies the significance of oxidative stress in the

The brain is very sensitive to oxidative damage because of the high unsaturated lipid content and extensive oxidative metabolism due to xenobiotic exposure including EDCs (Adogyan et al. 2008; Shibata and Kobayashi, 2008). Furthermore, free radicals have vital role in

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neurobehavioral deficit in experimental models through oxidative stress. Therefore, the poor neurobehavioral outcome in NP group rats might be attributed to oxidative stress–induced free radicals. Earlier studies have shown an improvement in various behavioral outputs as a result of

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antioxidant supplementation. Melatonin provides defence at two levels; firstly, as a direct free radical scavenger and secondly, indirectly inducing the antioxidant enzymes activity (Zhang et

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al., 2014).

Imbalance between pro-oxidants and antioxidants ratio leads to generation of ROS resulting in oxidative stress. Enhanced LPO may lead to neuronal damage by causing oxidative deterioration of the cellular macromolecules and enhances neurodegeneration worsening cognitive function (Negre-Salvayre, 2010; Glade, 2010). We observed that NP exposure significantly elevated the level of TBARS in both frontal cortex and hippocampal regions of rat

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brain. LPO has been hypothesized to be a major mechanism of cell damage by free radicals. The obtained data revealed that the significant increase in the level of LPO may be due to its poor antioxidant defense or the inactivation of antioxidant enzymes. Melatonin co-administration

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significantly decreased the level of TBARS formation in NP exposed animals. This can be attributed to the fact that melatonin possess antioxidative properties and reduces oxidative damage in the central nervous system as it is able to cross the BBB. This suggests affective

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pharmacological role of melatonin against free radical damage in brain (Feng and Zhang, 2005). We also investigated the possible role of NP in provoking protein oxidation, as confirmed by

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a marked increase of PC formation. It is presumed that excess reactive species causes oxidation of proteins (Shao et al., 2012). Because proteins govern the most biological functions in cells, their oxidation can lead to diverse functional consequences (Hong et al., 2014). This was ameliorated by melatonin treatment along with NP exposure. The protective effects of melatonin

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against membrane protein damage induced by oxidative stress may be a consequence of its free radical scavenging properties (Fuentes-Broto et al., 2010). It has been widely recognized that thiols plays an integral role in oxidative physiological

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homeostasis (Presnell et al., 2013). NP-SH level was significantly decreased in frontal cortex and hippocampus, which supports the finding of a significant decrease in GSH level in the brain

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tissue as it constitutes more than 90% of NP-SH pool. NP significantly reduced the total content of GSH which was improved in the group co-treated with melatonin owing to its anti-oxidative properties. Melatonin also elicits intracellular GSH content by acting on gamma-glutamyl cysteine synthetase (Winiarska, et al., 2006), the rate limiting enzyme in GSH synthesis, as well as recycling of GSH in cell (Manchester et al., 2015).

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There was a significant decrease in the activities of antioxidant enzymes of brain following exposure to NP pointing towards its neurotoxic potential. GST activity in frontal cortex and hippocampus regions of rat brain was significantly reduced. A marked inactivation of brain GST

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activity of NP-exposed frontal cortex and hippocampus indicates the insufficient availability of its substrate, GSH. The compensatory biotransformation activity can also be attributed to the decreased activity of GST. ROS has been known to decline the detoxification system produced

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by GST (Mao et al., 2010). In this study, melatonin exhibits a preventive effect against NP

GST activity (Meki and Hussein, 2001).

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induced oxidative stress through its role in the scavenging of free radicals and stimulation of

The activity of GR was significantly reduced in frontal cortex and hippocampus. The activity of GR also indicates the state of GSH synthesis in the cells and oxidative injury. The present study indicates reduction in GR activity on NP exposure which is improved by melatonin co-

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administration. It may be because melatonin is known to stimulate gamma-glutamyl cysteine synthase thereby increasing glutathione level, and it promotes the activity of GR which converts GSSG back to its reduced form, GSH (Rodriguez et al., 2004).

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GPx plays a major role in the detoxification of H2O2 and other lipid peroxides via the glutathione redox cycle (Duracková, 2010). The results obtained show a decreased GPx activity

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resulting due to H2O2 accumulation in the brain which in turn inactivates SOD. Supplementation of melatonin reduced the depletion of enzyme activity in comparison to NP-treated rat group probably due to its ability to stimulate the GPx in neural tissue and antioxidant activity mediating the transformation of GSH to its oxidized form along with conversion of H2O2 to H2O resulting in lowered OH-. Increase in GPx activity was accompanied by the significant increase in the concentration of melatonin in neural tissues (Barlow-Walden et al., 1995).

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ROS exerts depressant effects on antioxidant defense mechanisms including inhibition of SOD and CAT activity. Estrogen like chemicals can generate hydroxyl radicals (Obata and Kubota, 2000). In the present investigation the activity of both SOD and CAT was diminished in

the cell by its free scavenging mechanism (Rodriguez et al., 2004).

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frontal cortex and hippocampus. Melatonin appears to reduce the overall toxic environment in

In our study, the activity of XO, an endogenous pro-oxidant that produces superoxide was

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found to be significantly enhanced after exposure to NP. XO produces uric acid and other ROS like superoxide anion, H2O2 and OH-as by-products in reaction during the catabolism of purines

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(Nishino et al., 2005). Increased activity of XO to dehydrogenase form increases production of free radicals and LPO leading to cell damage. In our result, a significant decrease in XO activity in animals supplemented with melatonin along with NP treatment was seen. Melatonin plays a role in the redox enzyme activity modulation within cells (Rodriguez et al., 2004).

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Analysis of the degree of neurotoxicity can be measured in terms of alteration in activity of specific biomarker enzymes such as AChE, Na+,K+-ATPase and MAO. AChE is a cholinergic enzyme, which is very important in the synthesis and metabolism of Ach (Santos et al., 2012).

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The cholinergic system is involved in several physiological processes, including synaptic plasticity, learning and memory (Weinberger et al., 2006). In our study, we observed a

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significant decrease of the expression of AChE activity. The activity of AChE depends largely on the biophysical features of membrane. Oxidative stress decreases the fluidity of membrane lipid bilayer, thus affecting its normal functions (Goi et al., 2005). Secondary, neuronal damage and neuropsychiatric disorders such as anxiety and memory impairment may result from cholinergic neuronal overstimulation, generation of massive amounts of free radicals, cellular toxicity and inflammation (Ahmed et al., 2013). Melatonin supplementation clearly alleviated

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AChE variations in brain, suggesting its potent protective effects in regulating cholinergic functions. We, therefore, examined if NP-inhibited Na+, K+-ATPase activity in the exposed animals. This ubiquitous protein is abundantly found in brain areas and has vital role in

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regulating resting cell membrane potential, neural activity, transport of amino acids and glucose, and regulation of cell pH and volume neural excitability (Ribeiro et al., 2007). Impairment of Na+, K+- ATPase activity by free radical generation and LPO might lead to neural dysfunction

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(Lees et al., 1990; Viani et al., 1991). Also, dopamine can inhibit Na+, K+-ATPase activity (Bertorello et al., 1990). The present study showed that NP caused a significant decrease in the

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expression of MAO in frontal cortex and hippocampus. MAO is also a brain specific enzyme which plays an important role in the metabolism of. MAO activity in the brain is involved in the catabolism of several neurotransmitters such as dopamine, noradrenaline, and serotonin. Increased production of hydroxyl radicals may result in decreased MAO activity (Soto-Otero et

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al., 2001). Melatonin significantly improved the activities of both Na+, K+-ATPase and MAO in brain regions of NP treated animals which could be an effect of its antioxidative properties. NP treatment significantly increased level of neurotransmitter dopamine in the brain tissue of

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exposed rats. The dopaminergic system and its activity seem to be a primary target for environmental neurotoxicants, including pesticides, neuroendocrine disrupters, and heavy metals

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in various neurological adverse reactions. For example, BPA exposure has been reported to increase hyperactivity, by modifying dopaminergic transmission at multiple steps (Ishido et al., 2007). Considering the notion that estrogen regulates the production of monoamines (Sotomayor-Zárate et al., 2011), and that NP has an estrogenic activity, exposure to NP might disrupt the level of this amine in the brain, as shown in the present study. The elevated level of dopamine after treatment with NP implies that NP could enhance the DA synthesis or decrease

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its degradation; this was confirmed by the observed decrease in MAO activity following treatment with NP. The present findings can be supported by previous ones that revealed the ability of melatonin to reduce dopamine levels in multiple regions of rat brain. Melatonin and

(Ahmed et al., 2013). c. Western Blot and Immunohistochemical studies

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dopamine antagonized each other, whereas elevated levels of one induced low levels of the other

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In the present study western blot analysis showed that NP exposure lead to (NF)-kB activation. Estrogenic EDC such as BPA has been reported to activate (NF)-kB by inducing ROS

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production and apoptosis (Kovacic, 2010). ROS production may trigger an inflammatory/ survival response, via the activation of NF-κB (Chen et al., 2011). However, melatonin cotreatment could only partially reverse the situation because depending on the cellular context, melatonin leads to the modulation of the nuclear translocation of NF-κB, resulting in cell death

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or protection, respectively (Franco and Markus, 2014). Enhanced expression of GFAP is a biomarker of gliosis that represent sensitive and specific indices of toxicant- and disease-induced neural damage (O'Callaghan and Sriram 2005). GFAP expression may be influenced by age,

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neuronal damage, and sex steroid hormones (Gómora-Arrati et al., 2010). Microglia are thought to be the first line of defense in the CNS, and they are rapidly activated even in response to

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minor pathologic changes in the brain. It is particularly interesting that oxygen free radicals have the capacity to initiate the destruction of cell membranes by inducing LPO. It is evident from our study that microglial activation occurs primarily in the vulnerable brain regions following NPinduced toxicity in hippocampal and frontal cortical region, where evidence of astrogliosis and LPO were also noted. The administration of melatonin significantly reduced the GFAP

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expression in the hippocampal and cortical region. Our results suggest that melatonin administration in NP exposed group significantly reduces tissue damage. Histopathological examination showed that there are no morphological changes of the brain

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in the control animals. The frontal cortex and hippocampal regions appeared to be normal. No spongiform damage and neuronal eosinophilia was found. There were no astrocytic or oligodendroglial

changes

observed.

In

NP

treated

group,

marked

presence

of

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hyperchromaticcells, neuronal eosinophilia, nuclear pyknosis and neuronal karyorrhexis were observed. Similar changes have been reported by Adogyan et al. (2008). These harmful effects

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were toned down by melatonin pre-treatment, thereby pointing towards neuroprotective action of melatonin. 5. Conclusion

In conclusion, our study has demonstrated the neurotoxic actions and behavioral impairment

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produced by NP exposure in adult male rats and the protective role of melatonin in ameliorating the negative effects of NP in rat brain. We presume that NP induced oxidative stress contributes to toxicological perturbation in the rat brain which may ultimately be responsible for the

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disruption of cognitive functions. The precise cellular mechanisms involved will require further study like other several along with our present results summarized here, melatonin was found to

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have an essentially neuroprotective effect against NP-induced neurotoxicity. This being the case, its potential preventive application to forestall EDCs harmful effect should be further considered.

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Acknowledgements: Dr. Heena Tabassum is grateful to Department of Science and Technology, Government of India, for financial grant to support the study (DST Cognitive Science Initiative Program, sanction no. SR/CSI/PDF-76/2012). Dr. Mohammad Ashafaq is grateful to Indian

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Council of Medical Research for a Research Associateship (F.No. 82/2011 PHA/BMS). The Grant (no. F. 30-1/2013(SA-II)/RA-2012-14-GE-WES-2400), received as Research Award

Dr. Suhel Parvez, is thank fully acknowledged.

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(2012-14) from the University Grants Commission (UGC), New Delhi, Government of India to

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Competing interests: The authors declare no competing interests related to this work.

Author contributions:

Conceived and designed the experiments: H.T., S.P. and S.R.; Performed the experiments: H.T.

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and M.A.; Analyzed the data: H.T., S.P. and S.R..; Wrote the paper: H.T. and S.R.

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References Ahmed, M.A., Ahmed, H.I., El-Morsy, E.M., 2013. Melatonin protects against diazinon induced neurobehavioral changes in rats. Neurochem Res. 38, 2227-36. Aydogan, M., Korkmaz, A., Barlas, N., Kolankaya, D., 2008. The effect of vitamin C on

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bisphenol A, nonylphenol and octylphenol induced brain damages of male rats. Toxicology 249, 35–39.

Barlow-Walden, L.R., Reiter, R.J., Abe, M., Pablos, M., Menendez-Pelaez, A., Chen, L.D., Poeggeler, B., 1995. Melatonin stimulates brain glutathione peroxidase activity. Neurochem

SC

Int.26, 497-502.

Bertorello, A.M., Hopfield, J.F., Aperia, A., Greengard, P., 1990. Inhibition by dopamine of

synergism. Nature 347, 386-8.

M AN U

(Na(+)+K+)ATPase activity in neostriatal neurons through D1 and D2 dopamine receptor

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem72, 248-54. Carocci, A., Catalano, A., Sinicropi, M.S., 2014. Melatonergic drugs in development. ClinPharmacol. 6, 127-37.

TE D

Chaudhary, S., Parvez, S., 2012. An in vitro approach to assess the neurotoxicity of valproic acid-induced oxidative stress in cerebellum and cerebral cortex of young rats. Neuroscience 225, 258-68.

Chen, A.C., Arany, P.R., Huang, Y.Y., Tomkinson, E.M., Sharma, S.K., Kharkwal, G.B., et. al.,

EP

2011. Low-level laser therapy activates NF-kB via generation of reactive oxygen species in mouse embryonic fibroblasts. PLoS One 6, e22453.

AC C

Diaz, W.S., Villafane, J.J., Juliano, N., Bowman, R.E., 2013. Adolescent exposure to BisphenolA increases anxiety and sucrose preference but impairs spatial memory in rats independent of sex. Brain Res. 1529, 56–65. Doerge, D.R., Twaddle, N.C., Churchwell, M.I., Chang, H.C., Newbold, R.R, Delclos, K.B., 2002. Mass spectrometric determination of p-nonylphenol metabolism and disposition following oral administration to Sprague-Dawley rats. Reprod. Toxicol.16, 45-56. Duracková, Z., 2010. Some current insights into oxidative stress. Physiol. Res.59, 459-69. Feng, Z., Zhang, J.T., 2005. Long-term melatonin or 17beta-estradiol supplementation alleviates oxidative stress in ovariectomized adult rats. Free Radic. Biol. Med. 39, 195–204.

ACCEPTED MANUSCRIPT

Floor, E., Wetzel, M.G., 1998. Increased protein oxidation in human substantia nigra pars compacta in comparison with basal ganglia and prefrontal cortex measured with an improved dinitrophenylhydrazine assay. J. Neurochem.70, 268–275.

survival of mixed cerebellar cell culture. PLoS One 9, e106332.

RI PT

Franco, D.G., Markus, R.P., 2014. The cellular state determines the effect of melatonin on the

Fuentes-Broto, L., Miana-Mena, F.J., Piedrafita, E., Berzosa, C., Martínez-Ballarín, E., GarcíaGil, F.A., et. al., 2010. Melatonin Protects Against Taurolithocholic-Induced Oxidative Stress in Rat Liver. J. Cell Biochem. 110, 1219–25.

SC

Glade, M.J., 2010. Oxidative stress and cognitive longevity. Nutrition 26, 595.

Goi, G., Cazzola, R., Tringali, C., Massaccesi, L., Volpe, S.R., Rondanelli, M., et. al., 2005.

M AN U

Erythrocyte membrane alterations during ageing affect beta-D-glucuronidase and neutral sialidase in elderly healthy subjects. Exp. Gerontol.40, 219-25.

Gómora-Arrati, P., González-Arenas, A., Balandrán-Ruiz, M.A., Mendoza-Magaña, M.L., González-Flores, O., Camacho-Arroyo, I., 2010. Changes in the content of GFAP in the rat brain during pregnancy and the beginning of lactation. Neurosci. Lett. 484, 197-200. Govil, N., Chaudhary, S., Waseem, M., Parvez, S., 2012. Postnuclear supernatant: an in vitro

146, 402–409.

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model for assessing cadmium-induced neurotoxicity. Biological Trace Element Research

Grossberg, S., 2009. Cortical and subcortical predictive dynamics and learning during perception, cognition, emotion and action. Philos. Trans. R. Soc. Lond. B. Biol. Sci.364,

EP

1223-34.

Hong, I.S., Lee, H.Y., Kim, H.P., 2014. Anti-oxidativeeffectsofRooibostea(Aspalathuslinearis)

AC C

on immobilization-inducedoxidativestressinratbrain. PLoS One 9, e87061. Ishido, M., Yonemoto, J., Morita, M., 2007. Mesencephalic neurodegeneration in the orally administered bisphenol A-caused hyperactive rats. Toxicol. Lett. 173, 66–72. Jie, X., Jianmei, L., Zheng, F., Lei, G., Biao, Z., Jie, Y., 2013. Neurotoxic effects of nonylphenol: a review. The Central Europ. J. Med. 125, 61–70. Jie, Y., Xuefeng, Y., Mengxue, Y., Xuesong, Y., Jing, Y., Yin, T., Jie, X., 2016. Mechanism of nonylphenol-induced neurotoxicity in F1 rats during sexual maturity. Wien Klin. Wochenschr. 128, 426-34.

ACCEPTED MANUSCRIPT

Jones, D.C., Miller, G.W., 2008. The effects of environmental neurotoxicants on the dopaminergic system: A possible role in drug addiction. Biochem. Pharmacol.76, 569-81. Kakeyama, M., Sone, H., Tohyama, C., 2001. Changes in expression of NMDA receptor subunit mRNA by perinatal exposure to dioxin. Neuroreport 12, 4009–12.

transfer and oxidative stress. Med Hypotheses 75, 1-4.

RI PT

Kovacic, P., 2010. How safe is bisphenol A? Fundamentals of toxicity: metabolism, electron

Laws, S.C., Carey, S.A., Ferrell, J.M., Bodman, G.J., Cooper, R.L., 2000. Estrogenic activity of octylphenol, nonylphenol, bisphenol A and methoxychlor in rats. Toxicol. Sci.54, 154-67.

SC

Lees, G.J., Lehmann, A., Sandberg, M., Hamberg, H., 1990. The neurotoxicity of ouabain, a sodium-potassium ATPase inhibitor, in the rat hippocampus. Neurosci Lett.120, 159-62.

M AN U

Lin, A.M., Feng, S.F., Chao, P.L., Yang, C.H., 2009.Melatonin inhibits arsenite-induced peripheral neurotoxicity. J. Pineal. Res.46, 64-70.

Litwa, E., Rzemieniec, J., Wnuk, A., Lason, W., Krzeptowski, W., Kajta, M., 2014. Apoptotic and neurotoxic actions of 4-para-nonylphenol are accompanied by activation of retinoid X receptor and impairment of classical estrogen receptor signaling. J. Steroid Biochem. Mol. Biol. 144, 334–347.

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Luchetti, F., Canonico, B., Betti, M., Arcangeletti, M., Pilolli, F., Piroddi, M., 2010. Melatonin signalling and cell protection function. The FASEB J. 24, 3603-24. Manchester, L.C., Coto-Montes, A., Boga, J.A., Andersen, L.P., Zhou, Z., Galano, A., Vriend, J., Tan, D.X., Reiter, R.J., 2015. Melatonin: an ancient molecule that makes oxygen

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metabolically tolerable. J Pineal Res. 59, 403-19.Mao, Z., Zheng, Y.L., Zhang, Y.Q., 2010. Behavioral impairment and oxidative damage induced by chronic application of nonylphenol.

AC C

Int. J. Mol. Sci.12, 114–27.

Meki, A.R., Hussein, A.A., 2001. Melatonin reduces oxidative stress induced by ochratoxinA in rat liver and kidney. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 130, 305-13. Mu, S., Ou Yang, L., Liu, B., Zhu, Y., Li, K., Zhan, M., Liu, Z., Jia, Y., Lei, W., 2011. Protective effect of melatonin on 3-NP induced striatal interneuron injury in rats. Neurochem. Int.59, 224-34. Naseem, M., Parvez, S., 2014. Hesperidin restores experimentally induced neurotoxicity in Wistar rats. Toxicol. Mech. Methods24, 512-9.

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Negre-Salvayre, A., Auge, N., Ayala, V., Basaga, H., Boada, J., Brenke, R., et. al., 2010. Pathological aspects of lipid peroxidation. Free Radic. Res. 44, 1125-71. Nishino, T., Okamoto, K., Kawaguchi, Y., Hori, H., Matsumura, T., Eger, B.T., et. al., 2005. Mechanism of the conversion of xanthine dehydrogenase to xanthine oxidase: identification

RI PT

of the two cysteine disulfide bonds and crystal structure of a non-convertible rat liver xanthine dehydrogenase mutant. J. Biol. Chem. 280, 24888-894.

Norbury, R., Cutter, W.J., Compton, J., Robertson, D.M., Craig, M., Whitehead, M., Murphy,

Gerontology 38, 109–117.

SC

D.G., 2003. The neuroprotective effects of estrogen on the aging brain. Experimental

Obata, T., Kubota, S., 2000. Formation of hydroxy radicals by environmental estrogen-like

M AN U

chemicals in rat striatum. Neurosci. Lett. 296, 41–44.

O'Callaghan, J.P., Sriram, K., 2005. Glial fibrillary acidic protein and related glial proteins as biomarkers of neurotoxicity. Expert Opin. Drug Saf. 4, 433-42.

Ogiue-Ikeda, M., Tanabe, N., Mukai, H., Hojo, Y., Murakami, G., Tsurugizawa, T., Takata Kimoto, T., Kawato, S., 2008. Rapid modulation of synaptic plasticity by estrogens as well as endocrine disrupters in hippocampal neurons. Brain Res. Rev. 57, 363–75.

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Presnell, C.E., Bhatti, G., Numan, L.S., Lerche, M., Alkhateeb, S.K., Ghalib, M., et. al., 2013. Computational insights into the role of glutathione in oxidative stress. Curr. Neuro. Vasc. Res. 10, 185-94.

Reiter, R., Tang, L., Garcia, J.J., Munoz-Hoyos, A., 1997. Pharmacological actions of melatonin

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in oxygen radical pathophysiology. Life Sci. 60, 2255–71. Reiter, R.J., Tan, D.X., Korkmaz, A., 2009. The circadian melatonin rhythm and its modulation:

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possible impact on hypertension. J. Hypertens. Suppl. 27, S17-S20. Reiter, R.J., Tan, D.X., Mayo, J.C., Sainz, R.M., Leon, J., Czarnocki, Z., 2003. Melatonin as an antioxidant: biochemical mechanisms and pathophysiological implications in humans. Acta. Biochim. Pol. 50, 1129-46. Reiter, R.J., Tan, D.X., Galano, A., 2014. Melatonin: exceeding expectations. Physiology (Bethesda) 29, 325-33. Ribeiro, C.A., Balestro, F., Grando, V., Wajner, M., 2007. Isovaleric acid reduces Na, K ATPase activity in synaptic membranes from cerebral cortex of young rats. Cell Mol. Neurobiol. 27, 529 –40

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Rodriguez, C., Mayo, J.C., Sainz, R.M., Antolín, I., Herrera, F., Martín, V., Reiter, R.J., 2004. Regulation of antioxidant enzymes: a significant role for melatonin.J. Pineal Res. 36, 1-9. Rowe, W.B., Blalock, E.M., Chen, K.C., Kadish, I., Wang, D., Barrett, J.E., et. al. 2007. Hippocampal expression analyses reveal selective association of immediate-early,

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neuroenergetic, and myelinogenic pathways with cognitive impairment in aged rats. J. Neurosci. 27, 3098-110.

Santos, D., Milatovic, D., Andrade, V., Batoreu, M.C., Aschner, M., Marreilha dos Santos, A.P., 2012. The inhibitory effect of manganese on acetylcholinesterase activity enhances oxidative

SC

stress and neuroinflammation in the rat brain Toxicology. Toxicology 292, 90–98.

Schantz, S.L., Widholm, J.J., 2001. Cognitive effects of endocrine-disrupting chemicals in

M AN U

animals. Environ. Health Perspect109, 1197-206.

Shao, D., Oka, S., Brady, C.D., Haendeler, J., Eaton, P., Sadoshima, J., 2012. Redox modification of cell signaling in the cardiovascular system. J. Mol. Cell. Cardiol. 52, 550-8. Shibata, N., Kobayashi, M., 2008. The role for oxidative stress in neurodegenerative diseases. Brain Nerve 60, 157-70.

Silbergeld, E.K., Flaws, J.A., Brown, K.M., 2002. Organizational and activational effects of

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estrogenic endocrine disrupting chemicals. Cad. Saude. Publica18, 495-504. Sotomayor-Zárate, R., Tiszavari, M., Cruz, G., Lara, H.E., 2011. Neonatal exposure to single doses of estradiol or testosterone programs ovarian follicular development-modified hypothalamic neurotransmitters and causes polycystic ovary during adulthood in the rat.

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Fertil. Steril. 96, 1490–96.

Soto-Otero, R., Méndez-Alvarez, E., Hermida-Ameijeiras, A., Sánchez-Sellero, I., Cruz-

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Landeira, A., Lamas, M.L., 2001. Inhibition of brain monoamine oxidase activity by the generation of hydroxyl radicals Potential implications in relation to oxidative stress. Life Sciences 69, 879–89.

Tabassum, H., Parvez, S., Rehman, H., Banerjee, B.D., Raisuddin, S., 2007. Catechin as an antioxidant in liver mitochondrial toxicity: inhibition of tamoxifen-induced protein oxidation and lipid peroxidation. J. Biochem. Mol. Toxicol. 21, 110-17. Valko, M., Leibfritz, D., Moncol, J., Cronin, M.T., Mazur, M., Telser, J., 2007. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol. 39, 44-84.

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Viani, P., Cervato, G., Fiorilli, A., Cestaro, B., 1991. Age-related differences in synaptosomal peroxidative damage and membrane properties. J. Neurochem. 56, 253-58. Vishnoi, S., Raisuddin, S., Parvez, S., 2015. Modulatory effects of an NMDAR partial agonist in MK-801-induced memory impairment. Neuroscience 311, 22-33.

behavior in rodents. Nat. Protoc. 2, 322-8.

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Walf, A.A., Frye, C.A., 2007. The use of the elevated plus maze as an assay of anxiety-related

Weinberger, N.M., 2006. Food for thought: honeybee foraging, memory, and acetylcholine. Sci. STKE. 336, 23.

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Winiarska, K., Fraczyk, T., Melinska, D., Drozak, J., Bryla, J., 2006. Melatonin attenuates diabetes-induced oxidative stress in rabbits. J. Pineal. Res. 40, 168–76.

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Wu, P.F., Zhang, Z., Wang, F., Chen, J.G., 2010. Natural compounds from traditional medicinal herbs in the treatment of cerebral ischemia/reperfusion injury. Acta. Pharmacol. Sin. 31, 1523-31.

Yamasaki, K., Okuda, H., Haruhiko Kikkawa, H., 2012. Endocrine-mediated effects of a benzophenone-related chemical, 2,3,4,4’-Tetrahydroxybenzophenone, based on uterotrophic assay, hershberger assay, and subacute oral toxicity study. J. Clinic. Toxicol. 2, 129.

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Zhang, H.M., Zhang, Y., 2014. Melatonin: a well-documented antioxidant with conditional pro-

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oxidant actions. J. Pineal Res. 57, 131-146.

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Legends to Figures Fig. 1 (A) The Morris water maze test of NP treated Wistar rats (25mg/kg, oral) or vehicle (olive oil) and NP+ MEL group (10mg/kg, i.p.) (n=8). All values are expressed as means ± S.E.M.

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Each rat was subjected to total of nine training trials and latencies to platform on tenth or test trial; **p<0.01 NP vs. vehicle control; #p<0.05 NP vs. NP+MEL. (B-D) Representative tracks for

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the control, NP and NP + MEL rats during Morris water maze testing respectively.

Fig. 2 Time spent in (A) closed arms and (B) open arms in the elevated plus maze paradigm.

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Number of (C) closed arms entries and (D) open arms time in the elevated plus maze test. *p<0.01, NP vs. Control; #p<0.05, NP + MEL vs. NP (n = 8/group). All values are expressed as means ± S.E.M (n=8).

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Fig. 3 (A) Effect of NP treatment on muscular coordination skills in rats. No significant difference was observed in time spent on rotarod by NP treated animals in comparison to controls. Values are expressed as mean ± S.E.M of eight animals. (B) Effect of melatonin

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pretreatment on grip strength test in NP exposed rats. The grip strength was decreased significantly in NP treated group animals as compared to control group. Pre-treating the animals

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with melatonin followed by NP exposure (NP+MEL) has protected motor deficit as compared to NP group. Values are expressed as mean ± S.E.M of 8 animals. ***p˂0.001, NP vs. control, #p˂0.05,

NP+MEL vs. NP.

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Fig. 4 The effect of NP and MEL on (A-B) TBARS level; (C-D) PC content (E-F) NP-SH level and (G-H) GSH level in frontal cortex and hippocampal regions of the rat brain. Values are

the NP treatment. Values are expressed as mean ± S.E.M. (n=8).

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expressed as mean ± S.E.M (n=8). ***p< 0.001 vs. the vehicle control; ##p< 0.01, ###p< 0.001 vs.

Fig. 5 The effect of NP and MEL on (A-B) GST; (C-D) GR and (E-F) GPx activities in frontal

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cortex and hippocampal regions of the rat brain. GST activity was measured as nmol CDNB conjugate formed/min/mg protein. GR and GPx activities were measured as nmol NADPH

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oxidized/min/mg protein respectively. Values are expressed as mean ± S.E.M (n=8). ***p<0.001 vs. the vehicle control; ##p<0.01, ###p<0.001 vs. the NP treatment. Values are expressed as mean ± S.E.M. (n=8).

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Fig. 6 The effect of NP and MEL on (A-B) SOD; (C-D) CAT and (E-F) XO activities in frontal cortex and hippocampal regions of the rat brain. SOD, CAT and XO activities were measured as nmol (-) epinephrine protected from oxidation/ min/mg protein, µmolH2O2consumed/min/mg

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protein, and nmol uric acid formed/min/mg protein respectively. Values are expressed as mean ± S.E.M (n=8). **p<0.01, ***p<0.001 vs. the vehicle control;

##p<0.01, ###p<0.001

vs. the NP

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treatment. Values are expressed as mean ± S.E.M. (n=8).

Fig. 7 The effect of NP and MEL on (A) MAO (B) AchE and (C) Na+, K+-ATPase activity in frontal cortex and hippocampal regions of rat brain. AchE, MAO and Na+, K+-ATPase activities were

measured

as

µmol

BAHC

hydrolysed/min/mg

protein,

nmol

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hydrolysed/min/mgprotein and µg Pi liberated/min/mg protein respectively. Values are expressed

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as mean ± S.E.M (n=8). ***p<0.001 vs. the vehicle control;

##p<0.01

vs. the NP treatment.

Values are expressed as mean ± S.E.M. (n=8).

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Fig. 8 NP exposure increases dopamine levels (expressed as ng dopamine per mg total protein in the sample) in NP treated rat brain. Mean ± S.E.M. are shown. Sample size (n = 8). Significant differences compared with control value ***p<0.001. Significant differences with melatonin co-

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treatment were indicated by ###p< 0.001, when compared with NP group.

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Fig. 9 Western blotting detection of NF-kBp65 protein 907 expression in brain tissue of control and treated rats. *p<0.05 vs control. β-actin was used as an internal control. All values are expressed as mean ± S.E.M. The immunoblot exhibited in the figure is representative of four

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experiments.

Fig. 10 The photomicrograph shows representative frontal cortex and hippocampal brain sections respectively of (A and D) Control, (B and E) NP and (C and F) NP + MEL stained for GFAP.

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Profound expression of the GFAP was observed in the NP group as compared to control group. While Mel administration remarkably decreased the expression of GFAP as compared to NP

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alone group.

Fig. 11 Melatonin treatment protects histological alterations in frontal cortex and hippocampal brain sections. Representative photomicrographs show the sections with H&E staining. (A andD) Arrow indicate the normal neuron in control group (B and E) Arrow indicate the vacuolated

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region and degenerated neuron in NP group (C and F) while NP + MEL group treated with Mel

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shows normalized neuron staining. Magnification = 20X.

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Highlights The neurotoxic effect of nonylphenol was evaluated in a rodent model.

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Neuroprotective effects of melatonin was investigated.

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Melatonin improved the behavioral performance against nonylphenol exposure.

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Melatonin was able to inhibit ROS generation.

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Our results emphasize the potential use of melatonin as a nutraceutical.

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