Melatonin Ameliorates Neuropharmacological and

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Alterations Induced by Subchronic Exposure to Arsenic in Wistar Rats ... group II received As [sodium (meta) arsenite; NaAsO2] at 10 mg/kg bw (p.o.) for a period of 56 days. ... melatonin can overcome the sub-chronic As-induced oxidative and nitrosative .... Experimental rats belonging to various treatment groups were.

Biological Trace Element Research https://doi.org/10.1007/s12011-018-1537-1

Melatonin Ameliorates Neuropharmacological and Neurobiochemical Alterations Induced by Subchronic Exposure to Arsenic in Wistar Rats Prasada Ningappa Durappanavar 1 & Prakash Nadoor 2

&

Prashantkumar Waghe 3 & B. H. Pavithra 2 & G. M. Jayaramu 4

Received: 18 June 2018 / Accepted: 25 September 2018 # Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract An experimental study was conducted in Wistar rats to characterize the arsenic (BAs^)-induced alterations in neurobiochemistry in brain and its impact on neuropharmacological activities with or without the melatonin (MLT) as an antioxidant given exogenously. Male Wistar rats were randomly divided in to four groups of six each. Group I served as untreated control, while group II received As [sodium (meta) arsenite; NaAsO2] at 10 mg/kg bw (p.o.) for a period of 56 days. Experimental rats in group III received treatment similar to group II but in addition received MLT at 10 mg/kg bw (p.o.) from day 32 onwards. Rats in group IV received MLT alone from day 32 onwards similar to group III. Sub-chronic exposure to As (group II) significantly reduced both voluntary locomotor and forced motor activities and melatonin supplementation (group III) showed a significant improvement in motor activities, when subjected to test on day 42 or 56. Rats exposed to As showed a significant increase in anxiety level and a marginal nonsignificant reduction in pain latency. Sub-chronic administration of As induced (group II) significant increase in the levels of thiobarbituric acid reactive substance (TBARS) called malondialdehyde (MDA) in the brain tissue (5.55 ± 0.57 nmol g−1), and their levels were significantly reduced by MLT supplementation (group III 3.96 ± 0.15 nmol g−1). The increase in 3-nitrotyrosine (3-NT) levels in As-exposed rats indicated nitrosative stress due to the formation of peroxynitrite (ONOO−). However, exogenously given MLT significantly reduced the 3-NT formation as well as prostaglandin (PGE2) levels in the brain. Similarly, MLT administration have suppressed the release of pro-inflammatory cytokines (viz., IL-1β, IL-6, and TNF-α) and amyloid-β1–40 (Aβ) deposition in the brain tissues of experimental rats. To conclude, exogenous administration of melatonin can overcome the sub-chronic As-induced oxidative and nitrosative stress in the CNS, suppressed pro-inflammatory cytokines, and restored certain disturbed neuropharmacological activities in Wistar rats. Keywords Arsenic (As) . Neuropharmacology . Melatonin . Oxidative stress . Wistar rats

Introduction Environmental contamination of arsenic (BAs^), especially in drinking water, is a global issue. As is a naturally occurring element that exists ubiquitously in the environment in both * Prakash Nadoor [email protected] Prasada Ningappa Durappanavar [email protected]

organic and inorganic forms. It is the 20th most abundant element, comprising of about 500,000th’s of 1 % of the Earth’s crust [1] and is a component of more than 245 minerals. Groundwater contamination of As is a global problem [2], and it has gained its importance worldwide due to its toxic 1

Department of Veterinary Pharmacology and Toxicology; Karnataka Veterinary, Animal and Fisheries Sciences University; Veterinary College, Vinobanagar, Shivamogga, Karnataka 577 204, India

2

Department of Veterinary Pharmacology and Toxicology; Karnataka Veterinary, Animal and Fisheries Sciences University, Veterinary College, Veterinary College, Hebbal, Bengaluru, Karnataka 560 024, India

3

Department of Veterinary Pharmacology and Toxicology Veterinary College, Nandinagar, Bidar, Karnataka 585401, India

4

Department of Veterinary Pathology, Karnataka Veterinary, Animal and Fisheries Sciences University, Veterinary College, Vinobanagar, Shivamogga, Karnataka 577 204, India

Prashantkumar Waghe [email protected] B. H. Pavithra [email protected] G. M. Jayaramu [email protected]

Durappanavar et al.

effects on human and animal health consequent to chronic intake through contaminated food or water [3]. The current maximum permissible level of As in drinking water is 10 μg/L according to the World Health Organization [4] and the US Environment Protection Agency [5]. However, as per Bureau of Indian Standards (BIS), the permissible limit of As in drinking water is 50 μg/L [6]. Reports of As contamination of drinking water are available from more than 30 countries in the world [2], including South Asian countries like India and Bangladesh [7]. Arsenic in drinking water exists mainly in inorganic form, and it is more harmful than in the food, such as grains and vegetables [8]. Various mechanisms have been proposed to elucidate the cellular and molecular mediated As neurotoxicity, viz., (i) apoptosis in cerebral neurons by activating protein kinase (p53, MAPK) and JNK3 pathways [9]; (ii) formation of ROS through Fenton reactions resulting in cell damage and impairment in signal transduction [10]; and (iii) activation of nuclear factor kappa B (NF-kB) and activator protein-1 leading to DNA damage and apoptosis [11]. Further, cascade mechanisms of free radical formation derived from the superoxide radical, combined with glutathione-depleting agents, increase the sensitivity of cells to arsenic toxicity [12]. It is envisaged that As induces oxidative stress by increased reactive oxygen species (ROS) and nitric oxide (NO) generation [13]; however, the mechanism of As-mediated production of free radicals is still incomplete [14]. Several studies have associated high level of arsenic exposure from drinking water with elevated risk of vascular diseases, including peripheral vascular disease, hypertension, ischemic heart disease, and carotid atherosclerosis [15]. Similarly, As has been implicated in neurodegenerative diseases leading to neurologic and cognitive disorders as well as behavioral changes in man [16] or experimental rats [17]. Flora et al. [18] reported that gallium arsenide (inorganic As)-induced increase in ROS causes apoptosis of neurons via mitochondrial driven pathways leading to impaired neurobehavior in experimental rats. Sub-acute exposure to sodium arsenite decreases the levels of neurotransmitters in various regions of the central nervous system (CNS) in experimental rats [19]. Further, alterations in neurotransmitters level leads to loss of memory and learning in rats and arsenicexposed groups showed spatial learning and memory significantly below the average in a dose-dependent manner for the controls [20]. Similarly, experimental sub-chronic exposure to As (10 mg/L) through drinking water enhanced anxiety-like behavior in normal mice while it augmented depression-like behavior in following reserpine pretreatment [21]. Melatonin (N-acetyl-5-methoxyindolamine) is a neurohormone produced by pineal gland and its physiological actions, viz., control of circadian rhythms, sleep induction, regulation of seasonal reproduction, food intake, and immune enhancement [22], were well established [23, 24].

The broad spectrums of activity of melatonin [25] or its metabolite were well recognized [26]. The antioxidant properties of melatonin have been attributed to its capacity to act as a direct free radical scavenger [27], stimulate antioxidant enzymes [28], stimulate synthesis of glutathione [29], or protect and augment the activities of other antioxidants [30]. Many antioxidants are reported to ameliorate the oxidative stress induced by arsenical compounds, viz., α-lipoic acid, glutathione, selenium, zinc, N-acetylcysteine, methionine, cysteine, α-tocopherol, and ascorbic acid (vitamin-C) [31], green tea, garlic, and vitamin C [32]. However, Pieri et al. [33] observed that melatonin, a lipophilic antioxidant, was twofold active than vitamin E in terms of oxygen radical absorbing capacity in vitro. In addition, melatonin was found to be superior to vitamin E [34], vitamin C [35], and L-carnitine [36] in reducing the experimentally induced oxidative stress. Experimental studies have shown that melatonin can reduce the lipid peroxidation and oxidative stress induced by certain heavy metals or their congeners in the CNS [37] or the vital organs elsewhere in the body [38]. Further, in vitro studies using neuronal or other appropriate cell lines have explicitly shown that melatonin can (i) prevent the cobaltinduced oxidative stress, cytotoxicity, and amyloid-β (Aβ) release [39]; (ii) diminish sodium arsenite-induced elevation of cyclooxygenase-II (COX2) and expression of inducible nitric oxide synthase (iNOS) [40]; and (iii) attenuate the toxininduced inflammatory stress and NO production [41]. However, a comprehensive investigation to characterize the neurobiochemical alterations in the brain vis-à-vis neuropharmacological paradigms following sub-chronic exposure to As in the presence or absence of exogenous melatonin administration is lacking. Hence, the current study was undertaken with the objectives to study the effect of As [sodium (meta) arsenite; NaAsO2] on redox homeostasis in the central nervous system (CNS) and to assess the antioxidative property of melatonin in overcoming As-induced deleterious effects in the CNS following sub-chronic administration in Wistar rats.

Materials and Methods Chemicals, Reagents, and Assay Kits Sodium (meta) arsenite (NaAsO2; MW 129.91) and melatonin (C17H19CIN2S; MW 355.33) procured commercially from Sigma-Aldrich, USA, and Merck KGaA, Germany, respectively.

Experimental Animals Male Wistar rats (N = 36) of 6–7 weeks of age and weighing ~ 280–300 g were obtained from the Central Animal Facility, Indian Institute of Science, Bengaluru. Animals were housed

Melatonin Ameliorates Neuropharmacological and Neurobiochemical Alterations Induced by Subchronic Exposure...

in small animal facility, Veterinary College, Vinobanagar, Shivamogga (Reg. No. 1838/GO/ ReBi/S/15/CPCSEA) under polypropylene cages and standard management practices and were provided with 12-h light/dark cycle. Animals were fed with standard rodent chow (Amrut®, Ms. Pranav Agro Industries Ltd., Maharashtra State) and given free access to water (reverse osmosed) ad libitum. Prior approval of the Institutional Animal Ethics Committee (IAEC) was obtained, vide No. VCS/IAEC/005/2015-16 dated 23.07.2016 to carry out the current investigation as per the guidelines of the Committee for Prevention of Cruelty and Supervision of Experiment on Animals (CPCSEA), New Delhi.

Experimental Design After 1 week of acclimatization, the experimental rats were randomly divided into four groups as detailed below: group I (n = 6)—served as untreated control and had received only As-free drinking water throughout the experimental period ad libitum; group II (n = 6)—received sodium (meta) arsenite (NaAsO2) at 10 mg/kg given as oral gavage [rat feeding needle (14G, bent); Orchid Scientifics, Mumbai] daily during morning hours for a period of 56 days; group III (n = 6)—all the animals in this group received treatment similar to group II for a period of 56 days but in addition to it administered (per os) with melatonin 10 mg/kg daily from day 32 onwards till the end of experimental period (day 56); group IV (n = 6)— received only drinking water ad libitum as per group I but also received melatonin during morning hours from day 32 onwards up to till the end of the experiment. The remaining 12 rats were maintained under standard laboratory conditions similar to untreated control, and these rats were later to serve as standard reference (drug) control for chlorpromazine hydrochloride, diazepam hydrochloride, or acetyl salicylic acid each time utilizing six animals after giving appropriate washout period while carrying out studies on neuropharmacological paradigms.

Assessment of Voluntary Motor Activity The spontaneous motor activity, a measure of exploratory behavior of experimental animals, was measured using actophotometer (Photoactometer, INCO®, Haryana, India) for a period of 5 min, according to Kulkarni [42]. Experimental rats belonging to various treatment groups were individually placed in the activity chamber after 30 min, after the routine treatments. Each animal was subjected to an adaptation period of 5 min in the actophotometer. The counting was started after the adaptation period. Chlorpromazine HCl 3 mg/kg (i.p.; ~ 0.5 ml) was used to serve as reference drug (positive control) while all other experimental animals in the respective group received 0.5 ml (i.p.) distilled water. During the observation period, the beam of light falling on photocell was cut-off by the movement of animal inside the activity cage, and the counts were recorded automatically as the cumulative counts of spontaneous motor activity over a period of 5 min. Decrease in counts was regarded as a central nervous system depressant activity and vice versa. Results were expressed as counts per 5 min.

Assessment of Involuntary Motor Activity The forced locomotor activity, a measure of strength, and coordinated movement of experimental rats were studied by using rotarod apparatus as described by Dunham and Miya [43]. The test was carried out using a four-compartment rotarod apparatus (INCO®, Haryana, India). Each animal was placed on a horizontal rotating rod having a diameter of 80 mm. revolving at the rate of 25 rpm. Diazepam hydrochloride (1.0 mg/kg; i.p.) was administered to six rats to serve as positive control and subjected to rotarod 30 min after dosing. Four rats at a time were placed separately in each compartment of rotating rod, and Bfall-off^ time was noted through the digital display when animals loosen the grip and fall from the rotating rod.

Measure of Anxiety General Observation All the animals were observed for general clinical signs if any during the course of study period twice daily.

Neuropharmacological Studies The neuropharmacological activity in experimental animals with respect to voluntary motor activity (locomotor activity), involuntary motor activity (forced locomotor), and anxiety levels were studied on day 42 as well as on day 56. The analgesic activity was measured just before scarification of the experimental animals at term.

The anxiety level in experimental animals was measured on day 42 or day 56 using an elevated plus maze (EPM) system as described by Pellow et al. [44]. The EPM apparatus (model no. 520, INCO®, Haryana, India) consisted of two open arms (30 cm in length × 8 cm in breadth) and two closed arms (30 cm length × 8 cm breadth) with black plexiglass wall of 15 cm height were extended from a central platform of size 5 × 5 cm. The apparatus has the facility to digitally record the time spent by the rat in each arm through the sensors fitted at different positions of EPM. The test for assessing anxiety in the EPM was set for a period of 5 min. The experimental rats were placed on the central platform of the EPM facing an open arm and time spent in each arm were noted from the digital

Durappanavar et al.

recorder. The number of entries to each arm was also observed. The % time spent in open arm was calculated using the formula % time spent in open arm = time spent in open arm/[time spent in open + closed arm] × 100, while the % time spent in closed arm was derived from time spent in closed arm/[time spent in closed + open arm] × 100. The rats which served as positive control (diazepam hydrochloride; 1.0 mg/kg; i.p.) for the previous test were also subjected to EPM 1 h later to serve anxiolytic reference control.

concentration: 100 μmol/l) and the reaction mixture was incubated at 37 °C for 90 min. The NBT reduction was stopped by adding 1 ml of 0.5 N HCl. The amount of O2·−generated was quantified spectrophotometrically by measuring the absorbance of blue formazan at 540 nm against blank. The superoxide radical anion in the samples was measured as amount of NBT reduced (quantity of formazan), and it was calculated and expressed as picomol/min/mg protein.

Assessment of Peroxidative Damage Analgesic Activity The analgesic activities in experimental rats were measured by Eddy’s hot plate (INCO®, Haryana, India) technique as per the method described by Turner [45]. After presetting the hot plate temperature (52 ± 2 °C), analgesic activity was measured at 0, 30, 60, or 90 min. Time to assess the reaction time (s) by rats was taken as the criteria for assessing analgesic activity. Analgesic activity in six rats administered with acetyl salicylic acid (100 mg/kg per bw; p.o.) was measured at aforesaid time intervals to serve as positive control.

Brain peroxidative damage was assessed by evaluating lipid peroxidation (LPO) in terms of malondialdehyde (MDA) production as described by Paula et al. [47]. In brief, 1 ml of brain homogenate was mixed with 1 ml of 2% thiobarbituric acid, 1 ml of 25% HCl, and 90 μl butylated hydroxytolune. The mixture was kept in boiling water bath for 10 min at 95 °C and then centrifuged. The supernatant was transferred to a quartz cuvette, and the absorbance was read at 535 nm. Results have been expressed as nmol MDA formed/g of tissue.

Determination of SOD Activity Terminal Body Weight, Scarification At term (day 56), the terminal body weight of experimental animals were taken, and later, they were off-fed overnight but had free access to drinking water ad libitum. On day 57, soon after subjecting the experimental animals for assay of analgesic activity, they were sacrificed by cervical dislocation and subjected to further studies.

Preparation of Brain Tissue Homogenate Part of the brain tissue (500 mg) from each of the experimental rat was minced with scissors, transferred into a 10-ml glass tissue homogenizer (Borosil®, Mumbai), and homogenized by using 5 ml (tenfold) extraction buffer (ice-cold 50 mM potassium phosphate buffer, pH 7.4; containing a protease inhibitor cocktail) at 3–5 °C. The homogenate was then centrifuged at 10,000×g for 10 min, and the supernatant was stored at − 80 °C until assayed for enzymatic or nonenzymatic oxidative biomarkers.

Superoxide dismutase (SOD) activity in brain was determined by the procedure of Madesh and Balasubramanian [48]. Briefly, the reaction mixture contained 0.65 ml of PBS (pH 7.4), 30 μl of 3-(4-5 dimethyl thiazol 2-yl) 2,5-diphenyl tetrazolium bromide (1.25 mM), 75 μl of pyrogallol (100 μM), and 10 μl supernatant of brain homogenate (10%). The mixture was incubated at room temperature for 5 min, and the reaction was stopped by adding 0.75 ml of dimethyl sulfoxide. The absorbance was read at 570 nm, and the activity was expressed as unit/mg protein.

Determination of Catalase Activity

Neurobiochemical Parameters

Catalase activity in the brain homogenate was assayed by the spectrophotometric method of Aebi [49]. In brief, 1.99 ml of phosphate buffer (50 mM, pH 7.0) and 10 μl supernatant of homogenate (10%) were taken in a cuvette. Reaction was started by adding 1 ml of H2O2 (10 mM), and the absorbance was recorded at every 30 s for 3 min at 240 nm against water blank. The activity was expressed as mmol H2O2 utilized/min/ mg protein.

Estimation of Superoxide Radical Anion (O2·−) Formation

Determination of GR Activity

Superoxide radical anion generation was estimated indirectly in terms of formazan (blue color) formed due to the reduction of nitroblue tetrazolium (NBT) as an index of superoxide anion generation and measured (formazan) by using spectrophotometer at a wavelength of 540 nm [46]. To 900 μl of tissue homogenate, 100 μl of 1 mM NBT was added (final NBT

GR activity in brain was measured following the method of Goldberg and Spooner [50]. The 3 ml of reaction mixture contained 2.6 ml of PBS (0.12 M, pH 7.2), 0.1 ml of EDTA (15 mM), and 0.1 ml of oxidized glutathione (GSSG; 65.3 mM). To this, 10 μl supernatant of brain homogenate (10%) was added, and the volume was made up to 2.95 ml

Melatonin Ameliorates Neuropharmacological and Neurobiochemical Alterations Induced by Subchronic Exposure...

with distilled water. After incubation at room temperature for 5 min, 0.05 ml of NADPH (9.6 mM) was added. Decrease in absorbance/min was recorded immediately at 340 nm for 3 min. Control was run without GSSG. The activity of GR was expressed as μmol NADPH oxidized to NADP/mg protein/min.

Determination of Reduced GSH Content Glutathione (GSH) content was measured in the brain homogenate by the method of Sedlak and Lindsay [51]. Briefly, 1 ml supernatant of homogenate, 0.8 ml of water, and 0.2 ml of 50% trichloroacetic acid solution were added and incubated at room temperature for 15 min. This mixture was centrifuged at 3000 rpm for 15 min, and 0.4 ml of the supernatant was taken. To it, 0.8 ml of 1 M Tris buffer (pH 8.0) was added followed by 0.2 ml of 5,5′-dithiobis-2-nitrobenzoic acid reagent (0.01 M). The absorbance was read at 412 nm within 5 min. Reagent blank had no sample, and sample blank was without DNTB. The level of GSH was expressed as mmol of GSH/g of wet tissue.

Determination of GPx Activity Glutathione peroxidase (GPx) activity was determined by the method of Paglia and Valentine [52]. The reaction mixture contained 2.48 ml of phosphate buffer (50 mM, pH 7.0, containing 5 mM EDTA), 0.1 ml of NADPH (8.4 mM), 0.1 ml of GSH (150 mM), 0.1 ml of sodium azide (112.5 mM), 4.6 U glutathione reductase (Type III; Sigma Chemicals, USA), and 10 μl supernatant of brain homogenate. The reaction was initiated by adding 0.1 ml of H2O2 (2.2 mM) to the mixture. The change in absorbance was read at 340 nm for 3 min. The enzyme activity was expressed as μmol of NADPH oxidized to NADP/mg protein/min.

ELISA The level of 3-nitrotyrosine (3-NT), prostaglandin E2 (PGE2), amyloid β1–40 (Aβ1–40) assay (Elabscience® Biotechnology Inc., Wuhan, China), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) (RayBiotech Inc., USA) in brain homogenate were measured using enzyme-linked immunosorbent assay (ELISA) kits as per the manufacturer’s standard protocol.

Protein Estimation The protein content in the supernatant of brain homogenates was determined by following the method described by Lowry et al. [53] using bovine serum albumin as a standard.

Statistical Analysis The values obtained from the various experiments were expressed as mean + SE with Bn^ equal to number of animals or samples. Data obtained were statistically subjected to oneway analysis of variance (ANOVA) followed by Duncan’s post hoc multiple comparison test using SPSS statistic software (IBM® SPSS® statistic software, version 20.0.0, 2011, Armonk, NY, USA). Difference was considered significant at p < 0.05 or lower. Graphical presentation of the data was carried out by using GraphPad Prism software program (GraphPad® software Inc., version 7.0; San Diego, CA, USA).

Results The current study was carried out with an aim to characterize the effect inorganic As on neurobiochemical and neuropharmacological activities in Wistar rats following sub-chronic exposure and to examine the potential role of melatonin (MLT), a broad spectrum antioxidant under such circumstances. Sub-chronic exposure to As did not show any signs of illness or mortality. However, upon close clinical examination, there was a mild degree of rough hair coat in rats which received ‘As’ was observed. It was observed that As-treated rats showed a noticeable negative trend in growth over other experimental groups from the second week onwards. However, the bw of MLT-supplemented rats was similar to that of untreated control.

Neuropharmacological Studies Voluntary Motor Activity The data pertaining to voluntary locomotor activity in different experimental group of rats recorded on day 42 or day 56 is presented in Fig. 1. There was a significant (p < 0.05) reduction in voluntary locomotor activity on both occasions in Astreated rats when compared to either untreated control (group I) or MLT-supplemented rats (group III) (Fig. 1). The voluntary locomotor activity (scores/5 min) on day 42 and day 56 were 86.0 ± 4.24 and 63.0 ± 1.53, respectively, in As-treated rats while the corresponding values in MLT supplemented rats (group III) were 135.0 ± 2.92 and 92.17 ± 2.30, and these values recorded on both occasion differ significantly (p < 0.05). Further, the depressed voluntary locomotor activity recorded on day 56 in As-treated rats were statistically comparable to chlorpromazine hydrochloride, a standard reference drug (positive control) employed. Thus, MLT supplementation was able to overcome the depressed voluntary locomotor activity induced by As. Although restoration of locomotor

Durappanavar et al. Day 42

Locomotor activty (scores/ 5 min)

250

Day 56

a

200

a

a

150

a

b* b#

c*

100

c#

c#

d*

50 0

C

0.67 and 3.50 ± 0.22, respectively. Further, reduced involuntary locomotor activity in As-treated rats on day 56 was statistically comparable to diazepam hydrochloride, a standard reference drug (positive control). Thus, the MLT supplementation significantly (p < 0.05) restored the depressed forced locomotor activity induced by As. Anxiety Level

CPZ As As+MLT Experimental group(s)

MLT

Fig. 1 Mean (± SE) voluntary locomotor activity (scores for 5 min) measured on day 42 or day 56 in different groups of experimental rats (n = 6 each). Note: Bars bearing dissimilar alphabets between various experimental groups on day 42 or day 56 vary significantly; Bars bearing dissimilar symbols between corresponding experimental groups on day 42 and day 56 vary significantly; p < 0.05 [C = control, CPZ = chlorpromazine HCl, As = arsenic, As + MLT = arsenic + melatonin, MLT = melatonin]

activity in group III (As+MLT) was evident on both occasions (day 42 and day 56), but incomparable to either untreated control (group I) or MLT-alone-treated rats (group IV). Involuntary Motor Activity The forced locomotor activity (fall-off time in seconds) in Astreated rats (group II) on day 42 and day 56 were, respectively, 3.17 ± 0.31 and 1.67 ± 0.21 (Fig. 2). The corresponding forced locomotor activity in MLT-supplemented group were 4.50 ± Day 42

The anxiety level in experimental rats were measured individually on day 42 and day 56 by elevated plus maze as both number and % time spent or entries made in open or closed arm. The number of entries in open arm of the elevated plus maze in control and As-treated rats on day 42, respectively, were 1.33 ± 0.33 and 0.33 ± 0.21, while the corresponding values on day 56 were 1.67 ± 0.33 and 0.5 ± 0.22. Statistical analysis revealed a significant (p < 0.05) difference between the control and As-treated rats in terms of number of entries made in open arm on day 42 as well as day 56 (Table 1). Similarly, the time spent in open arm in control and Astreated rats were, respectively, 3.67 ± 0.99 s and 0.50 ± 0.34 s on day 42, while the corresponding values on day 56 were 6.17 ± 0.60 s and 0.67 ± 0.33 s. Thus, sub-chronic exposure to As enhanced the anxiety level (Fig. 3a, b). However, when the data was analyzed in comparison with the untreated (group I) or positive control (diazepam hydrochloride) in terms of percent number of entries or time spent in open or closed arm of elevated plus maze indicated that MLT supplementation (group III) had a marginal nonsignificant (p < 0.05) influence in decreasing the anxiety level in rats. Analgesic Activity

Day 56 8

Fall-off time (Seconds)

7

c

d d

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c

5 4

b

b*

3

a

2

a#

a

1 0

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DZP

As

As+MLT

MLT

Experimental group (s)

Fig. 2 Mean (± SE) forced locomotor activity (fall-off time in seconds) measured on rotarod on day 42 or day 56 in different groups of experimental rats (n = 6 each). Note: Bars bearing dissimilar alphabets between various experimental groups on day 42 or day 56 vary significantly. Bars bearing dissimilar symbols between corresponding experimental groups on day 42 and day 56 vary significantly; p < 0.05 [C = control, As = arsenic, DZP = diazepam HCl, As + MLT = arsenic + melatonin, MLT = melatonin]

Assay for analgesic activity in experimental animals carried out at term (day 57) revealed that sub-chronic exposure to As (group II) resulted in marginal nonsignificant (p > 0.05) decline in pain latency at all the time intervals tested (30, 60, and 90 min.). Interestingly, it was observed that MLT supplementation (group IV) significantly (p < 0.05) enhanced pain latency at 60 min compared to As-treated rats (group II). Although MLT supplementation in group III rats (As+MLT) did not significantly (p > 0.05) increase the pain latency, there was a noticeable enhancement in paw withdrawal time (s) in all the time intervals tested (Fig. 4).

Neurobiochemical Parameters Superoxide Radical Generation Superoxide radical generation measured as pmol of NBT reduced/min/mg protein indicated a significant (p < 0.05) increase in levels of superoxide radical in As-treated rats (group II) as compared to untreated control (group I). MLT

Melatonin Ameliorates Neuropharmacological and Neurobiochemical Alterations Induced by Subchronic Exposure... Table 1

Mean (± SE) number and percent of entries in open or closed arm in different groups of experimental rats measured on day 42 or day 56 Day 42

Day 56

Treatment Number of open Number of arm entries closed arm entries

% open arm entries

% closed arm Number of open Number of entries arm entries closed arm entries

% open arm entries

% closed arm entries

C DZP

1.33 ± 0.33b 4.00 ± 0.37c

19.54 ± 5.20a 44.56 ± 1.77b

80.46 ± 5.20a 1.67 ± 0.33bc 55.44 ± 1.77b 4.50 ± 0.34d

28.45 ± 4.56a 42.88 ± 1.61a

71.55 ± 4.56a 57.12 ± 1.61a

As

0.33 ± 0.21a

1.17 ± 0.17a

13.89 ± 9.04a

86.11 ± 9.04a 0.5 ± 0.22a

2.83 ± 0.31b 3.67 ± 0.33b

26.94 ± 2.17a 73.06 ± 2.17a 1.33 ± 0.21b 28.68 ± 2.90a* 71.35 ± 2.9a 2.33 ± 0.21c#

5.33 ± 0.42c 5.00 ± 0.52c

As+ MLT 1.00 ± 0.00ab MLT 1.50 ± 0.22b*

4.17 ± 0.48c 6.00 ± 0.45d 1.17 ± 0.17a

22.22 ± 10.24a 77.78 ± 10.24a

2.83 ± 0.31b 3.50 ± 0.22bc

32.22 ± 4.59a 67.78 ± 4.59a 39.92 ± 2.56a# 60.08 ± 2.56a

Data were analyzed by one-way ANOVA followed by Duncan’s multiple comparison post hoc test. Values bearing dissimilar alphabets within a column vary significantly. Values bearing different symbols within corresponding experimental group and parameter observed in between day 42 and day 56 vary significantly; n = 6 each; p < 0.05 [C = control, DZP = diazepam HCl, As = arsenic, As + MLT = arsenic + melatonin, MLT = melatonin]

administration (group III) exogenously significantly (p < 0.05) reduced the superoxide radical generation (Fig. 5a). Time spent in open arm (%)

a

c*

7

Day 42

Assessment of Nitrosative Stress and Oxidative Damage

Day 56

6 5

3-NT Assay

c#

4 3 2

b

b

b

ab

1

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0

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DZP

a As

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a

As+MLT

MLT

Experimental group (s)

Evaluation of nitrosative stress levels in brain tissue of experimental rats revealed that sub-chronic administration of As significantly (p < 0.05) increased the 3-NT levels when compared to untreated control. The nitrosative stress exerted by As was found significantly (p < 0.05) less following MLT supplementation (group III) (Fig. 5b). Further, the 3-NT levels in rats which received MLT alone did not had any influence on the basal level as recorded in untreated control group of rats.

Day 42

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Day 56 b

LPO Levels b

Sub-chronic As administration significantly (p < 0.05) elevated the LPO levels when compared to untreated control. MLTsupplemented group (As+MLT) showed a significant (p < 0.05) reduction in LPO level when compared to Astreated rats (Fig. 5c). Further, the LPO levels in rats which received MLT alone did not influence the basal level as recorded in the untreated control.

90 80 70 60

Enzymatic Antioxidants

50 C

DZP

As

As+MLT

MLT

Experimental group (s)

Fig. 3 Mean (± SE) time spent in open arm (%) (a) and closed arm (%) (b) of elevated plus maze in different groups of experimental rats on day 42 or 56 (n = 6 each) Note: Bars bearing dissimilar alphabets within day 42 or day 56 vary significantly while bars bearing dissimilar symbols vary significantly between day 42 and day 56; p < 0.05 [C = control, DZP = diazepam HCl, As = arsenic, As + MLT = arsenic + melatonin, MLT = melatonin]

SOD Activity Mean (± SE) SOD activity in the brain tissue of rats belonging to control and As-treated group were respectively 0.16 ± 0.004 and 0.12 ± 0.003 U mg−1 protein. And these values were significantly (p < 0.05) different. MLT supplementation (As+MLT) significantly (p < 0.05) improved the SOD activity

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Fig. 4 Mean (± SE) reaction time (s) in Eddy’s hot plate by different groups of experimental rats (note: acetyl salicylic acid given as 100 mg/kg: p.o. served as positive control) (n = 6 each). Note: Bars bearing dissimilar alphabets between various experimental groups within given tested time interval vary significantly. Bars bearing

dissimilar symbols on corresponding experimental groups between different time intervals vary significantly; p < 0.05 [C = control, ASA = acetyl salicylic acid, As = arsenic, As + MLT = arsenic + melatonin, MLT = melatonin]

(0.14 ± 0.003 U mg−1 protein); however, MLT-alone-treated rats (group IV) did not showed any deviation (p > 0.05) in enzyme activity (0.15 ± 0.003 U/mg protein) when compared to control (Fig. 6a).

0.01 μmol of NADPH oxidized/mg/min). However MLT supplementation in As-treated rats (As+MLT) showed a marginal recovery of GPx activity (0.19 ± 0.01 μmol of NADPH oxidized/mg/min) in the brain tissue (Fig. 7b).

CAT Activity

Nonenzymatic Antioxidant The catalase (CAT) activity was significantly (p < 0.05) reduced in As-treated rats (2.08 ± 0.14; mmol of H2O2 utilized/ min/mg protein) when compared to untreated control (Fig. 6b) (2.87 ± 0.20 mmol of H2O2 utilized/min/mg protein). MLT supplementation in As-treated rats (group III) showed a nonsignificant (p > 0.05) marginal increase in CAT activity (2.44 ± 0.08 mmol of H2O2 utilized/min/mg protein). GR The glutathione reductase (GR) activity in the brain tissue of untreated control and As-treated rats were 71.66 ± 2.22 and 57.84 ± 2.55 μmol of NADPH oxidized/mg/min, respectively. Statistical comparison between these two groups indicated that As induced a significant (p < 0.05) decline in glutathione reductase activity (Fig. 7a). However, MLT supplementation in As-treated rats (group III) showed significant (p < 0.05) recovery in the glutathione reductase activity (67.8 ± 3.01 μmol of NADPH oxidized/mg/min). GPx The GPx activity in the brain tissue of As-treated rats (0.17 ± 0.01 μmol of NADPH oxidized/mg/min) was significantly (p < 0.05) reduced when compared to untreated control (0.23 ±

GSH The GSH content of brain tissue of untreated control and Astreated rats were 0.55 ± 0.003 and 0.44 ± 0.021 mmol/g, respectively. And these values differ significantly (p < 0.05). The As-induced depletion in reduced glutathione levels in brain tissue were significantly (p < 0.05) elevated (0.48 ± 0.01 mmol/g) in MLT-supplemented group (group III) (Fig. 7c).

Pro-Inflammatory Mediators IL-1β Effect on the IL-1β production in the brain tissue of various experimental groups of rats is shown in Fig. 8a. The IL-1β level in the control group of rats was 126.34 ± 11.70 pg/mg tissue (Fig. 8a). This level was not significantly (p > 0.05) altered by MLT alone treatment. As treatment (182.41 ± 11.18 pg/mg) caused significant increased production of IL1β, while MLT treatment significantly (p < 0.05) reduced its production back to levels as observed in the untreated control group of rats (131.84 ± 19.60 pg/mg).

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Fig. 6 Mean (± SE) superoxide dismutase (SOD) activity (unit/mg protein) (a) and catalase (CAT) activity (mmol of H2O2 utilized/min/mg protein) (b) in brain tissue homogenate in different groups of experimental rats (n = 6 each). Note: Bars bearing dissimilar alphabets vary significantly between experimental groups; p < 0.05 [C = control, As = arsenic, As + MLT = arsenic + melatonin, MLT = melatonin]

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mg. As treatment (463.85 ± 27.64 pg/mg) (Fig. 8b) caused significant (p < 0.05) increase in production of IL-6, while MLT alone treatment significantly (p < 0.05) reduce its production (256.19 ± 5.60 pg/mg). However, MLT treatment (group III) significantly inhibited As-mediated increase and reduced its level similar to control group of rats (349.34 ± 9.69 pg/mg).

MLT

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Fig. 5 Mean (± SE) superoxide radical activity (pmol of NBT reduced/ min/mg/protein) (a), 3-nitrotyrosine (3-NT) levels (ng/mg protein) (b), and levels of MDA formed (nmol MDA/g tissue) (c) in brain tissue in different groups of experimental rats (n = 6 each). Note: Bars bearing dissimilar alphabets vary significantly between experimental groups; p < 0.05 [C = control, As = arsenic, As + MLT = arsenic + melatonin, MLT = melatonin]

IL-6 The IL-6 levels in brain tissue of rats are tabulated in Fig. 8b. The IL-6 level in control group of rat was 375.75 ± 22.84 pg/

TNF-α Assay The TNF-α production in brain of various experimental groups of rats is summarized in Fig. 8c. The level of its production in control (17.66 ± 4.99 pg/mg) and MLTalone-treated (29.56 ± 2.82 pg/mg) group of rats were comparable (p > 0.05). Rats treated with As exhibited a significant (p < 0.05) increase in its production (73.58 ± 7.72 pg/mg). MLT treatment (group III) significantly (p < 0.05) inhibited As-mediated increase in TNF-α levels (49.89 ± 8.92 pg/mg) (Fig. 8c).

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The Aβ1–40 levels in the brain tissue of As-treated rats was significantly (p < 0.05) increased (37.94 ± 1.65 pg/mg) than the untreated control (32.03 ± 1.43 pg/mg). The Aβ1–40 level in As-treated rats was significantly decreased following MLT supplementation (30.02 ± 1.41 pg/mg) which was statistically comparable to untreated control. Further, MLT-alone-treated rats showed a significant (p < 0.05) decrease in Aβ1–40 levels when compared to untreated control (23.70 ± 2.36 pg/mg) (Fig. 9).

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Fig. 7 Mean (± SE) glutathione reductase (GR) activity (μmol of NADPH oxidized to NADP/mg/min) (a), reduced glutathione (mmol GSH/g tissue) (b), and glutathione peroxidase (GPx) activity (μmol of NADPH oxidized to NADP/mg/min) (c) in brain tissue homogenate in different groups of experimental rats (n = 6 each). Note: Bars bearing dissimilar alphabets vary significantly between experimental groups; p < 0.05 [C = control, As = arsenic, As + MLT = arsenic + melatonin, MLT = melatonin]

PGE2 Assay The PGE2 levels in the brain tissue of As-treated rats was significantly (p < 0.05) increased (21.87 ± 1.13 pg/mg protein) when compared to untreated control (8.06 ± 2.83 pg/mg protein) (Fig. 8d). While MLT supplementation in As-treated (group III) rats showed a significant (p < 0.05) decrease in levels of PGE2 (16.66 ± 1.22 pg/mg protein) in brain tissue.

In the present study, sub-chronic administration of sodium (meta) arsenite (As) induced a significant decline in body weight when assessed as percent change over initial weight of rats. Experimental studies in rats have previously shown decreased growth rate [54] or negative bw gain at terminal phase of the experiment [55]. The body weight of rats that received MLT supplementation following As treatment (group III) was not significantly different from that of the untreated control, while rats which received MLT alone relatively gained body weight as compared to other groups of experimental rats. Shagirtha et al. [56] also observed that melatonin treatment has prevented the loss of body weight incurred in rats experimentally exposed to cadmium. Melatonin (alone) supplementation in the present study might have augmented the normal antioxidant defense system(s) in the body leading to body weight gain in relative terms. Rats in group II administered with As showed a significant decrease in voluntary as well as involuntary motor activity. Further, the decrease in both the motor activities occurred in a time-dependent manner (day 42 and day 56). Exposure to sodium arsenite was reported to cause impairment in locomotor activity, grip strength, and performance in rotarod [57]. Hence, our results are comparable to these reported observations in experimental rats and such disturbance in motor activity can be due to the motor deficit or asymmetry due to the involvement of dopaminergic system as it plays an integral role in motor physiology [58]. Further, studies of Schwarz et al. [59] demonstrated that dopamine could directly control movement by manipulating somatic motor neuron behavior and skeletal muscle tone through D1 receptors. Therefore, the As-induced disturbance in motor coordination in the current study could be due to disturbance in dopamine levels (central) which controls movement by modulation of higherorder motor centers like basal ganglia or directly affecting motor neuron functions. Supplementation of MLT in As-treated rats in the present study (group III) showed a significant recovery in voluntary

Melatonin Ameliorates Neuropharmacological and Neurobiochemical Alterations Induced by Subchronic Exposure...

a

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Fig. 8 Interleukin-1beta (IL-1β) (a), interleukin-6 (IL-6) (b), tumor necrosis factor-α (TNF-α) (c), prostaglandin E2 (PGE2) (d) levels (pg/mg protein) in brain tissue homogenate in different groups of experimental rats (n = 6 each). Note: IL-1β, IL-6, TNF-α, and PGE2 were measured with ELISA and recorded at absorbance 450 nm and data are expressed as mean (± SE) pg/mg protein. Bars bearing dissimilar alphabets vary significantly; p < 0.05 [C = control, As = arsenic, As + MLT = arsenic + melatonin, MLT = melatonin]

(1-40)

and involuntary motor activity in rats. Melatonin was reported to reverse the manganese chloride induced decreased spontaneous motor activity and rotarod performance in a concentration-dependent manner [60]. Thus, restoration of motor activity due to MLT supplementation observed in the present study (group III) might be due to its neuroprotection against dopaminergic neurodegeneration [60]. In addition, MLT might have enhanced the survival of dopaminergic neurons in substantia nigra and dorsolateral striatum as opined by Kim et al. [61]. Elevated plus maze has been widely used as a tool in the investigation of psychological and neurochemical

Fig. 9 Amyloid-β1–40 (Aβ1–40) levels (pg/mg protein) in brain tissue homogenate in different groups of experimental rats (n = 6 each). Note: Aβ1–40 was measured with ELISA and recorded at absorbance 450 nm, and data are expressed as mean (± SE) pg mg−1 protein. Bars bearing dissimilar alphabets vary significantly; p < 0.05 [C = control, As = arsenic, As + MLT = arsenic + melatonin, MLT = melatonin]

basis for anxiety. Sub-chronic As administration elevated anxiety levels significantly in the present study (group II) when rats were subjected (day 42 and day 56) to elevated plus maze. The increase in anxiety level may be due to single or combinations of alterations in levels of central neurotransmitters like nor-epinephrine, epinephrine, dopamine, γ-aminobutyric acid, and serotonin [62]. Alternatively, exposure to As increases caspase-3 activity followed by apoptosis of neurons in cortex and hippocampus [18] or death of hippocampal neurons lead to development of anxiety in rats [63]. Chang et al. [21] also observed increased anxiety-like behavior in mice due to sub-chronic exposure to arsenic trioxide as measured by elevated plus maze and open field tests. Additionally, Aktar et al. [64] reported increased anxiety in mice when exposed to lead and arsenic either individually or their combination. In our study, the anxiety levels were monitored in the various experimental group of rats during the daytime (9:30 am–12:00 noon), and it was observed that MLT did not significantly (p > 0.05) alleviated the anxiety level (group III) when compared to As-treated rats (group II) when evaluated on both occasions (day 42 and day 56). A marginal nonsignificant (p > 0.05) influence of MLT in reversing the As-induced anxiety levels in the present study may be due to diurnal variation in anxiolytic activity of MLT [65] or influence of sleep cycle in rats [66]. The anxiolytic property of MLT was due to its ability to enhance central GABAergic transmission by modulation GABA receptor [67], although the levels of various neurotransmitters in the CNS of experimental rats were

Durappanavar et al.

not quantified in the present study. However, it can be hypothesized that only a marginal reduction in anxiety levels by MLT supplementation may be attributable to complexity in mechanism involved in As induced anxiety [18, 63]. Eddy’s hot plate test revealed that rats treated subchronically to As showed only a marginal nonsignificant (p > 0.05) decline in pain latency in all the tested intervals of time at term (day 57). The impact of As exposure (through drinking water or i.p.) on analgesic activity in rats were assayed previously either by using tail flick model [68] or lipopolysaccharide-induced pyrexia/inflammatory model [69]. Thus, a nonsignificant marginal effect of Asinduced decreased pain latency may be attributed to differences in dose, route of exposure, or even methodologies adopted to assess the impact. In the current study, MLT supplementation (group III) showed a nonsignificant increase in pain latency in As-treated rats. However, MLTalone-supplemented rats (group IV) showed a significant increase in pain latency at 60 min over As-treated rats (group II). MLT was reported to cause dose-dependent antinociception in hot water tail flick model after a dose of 30, 60, or 120 mg/kg (i.p.) and antinociception lasted for more than 100 min in rats given MLT at a dose rate of 120 mg/kg [70]. Thus, antinociception effects of MLT may also be dependent on its dose and route of administration. Nevertheless, MLT-supplemented (group IV) rats showed a significant increase in pain latency (at 60 min) as compared to As-treated rats (group II), indicating its antinociception value, although it did not restored the Asinduced decrease in pain latency (group III) significantly. Sub-chronic As treatment (group II) significantly enhanced the generation of O2·− as compared to untreated control. It was also observed that As exposure (group II) not only significantly reduced the enzymatic antioxidant parameters assayed (viz., SOD, CAT, GR, GPx) but also depleted the tissue (brain) GSH. Further, sub-chronic As treatment also significantly increased the 3-NT levels in the brain tissue. Hence, in addition to oxidative injury, As treatment also resulted in peroxynitrite (ONOO−)-mediated nitrosation (nitrosative stress) of free- or protein-bound tyrosine. The present study explicitly demonstrated that sub-chronic administration of As induces oxidative stress, thus leading to significant increase in LPO levels in the brain. It is interesting to note that although sub-chronic As treatment induces generation of reactive oxygen or nitrogen species (ROS/RNS) in various experimental studies [71], the mechanism of being As-mediated production of free radicals is still incomplete [14]. The reduced antioxidant enzyme level could be attributable to increased utilization enzymes for maintenance of redox homeostasis in the brain, or alternatively, it may be due to direct binding of arsenicals to dithiol targets [72]. The

observation made in the present study was in accordance with the results of El-Demerash et al. [73]. Garcia-Chavez et al. [74] observed a positive quadratic relationship between TBARS (MDA) levels and the concentration of arsenicals in the nervous system. Similarly, increased As levels in brain along with significant increase in levels of LPO formed, decrease in activities of SOD, CAT, and GPx, and tissue GSH [57, 73]. It is important to note that metabolism of arsenic involves its methylation to monomethylarsonic acid [MMA (III)] and finally to dimethylarsenic acid [DMA (V)] by a methyl donor S-adenomethylmethionine catalyzed by methyltransferase in the presence of GSH. Further, GSH is also utilized for further reduction reactions from MMA V to MMA III and DMA V to DMA III. Therefore, a significant (p < 0.05) depletion of GSH content in brain tissue of As-treated rats in the present study invariably occurred on account of its involvement in metabolism cycle of arsenicals. The formation of 3-NT represents a specific peroxynitrite (ONOO−)-mediated protein modification; thus, detection of nitrotyrosine in proteins is considered as a biomarker for endogenous ONOO− activity [75]. Ding et al. [76] demonstrated formation of 3-NT in a concentration- as well as timedependent manner when human keratinocytes were exposed to trivalent arsenite (As+3). Similarly, 3-NT formation was significantly higher within the atherosclerotic plaque of in nominate artery in As-treated mice [77]. In the present study, MLT supplementation in As-treated rats (group III) showed a significant reduction in O2·− generated. Additionally, a significant recovery in the activities of SOD and GR and the levels of tissue GSH and a nonsignificant marginal improvement in activities of CAT and GPx were recorded. Further, significant improvement in redox balance in the brain was indicated through the reduced LPO (3.96 ± 0.15 nmol of MDA/g) or 3-NT levels in experimental rats (group III). Melatonin was known to possess a broad spectrum antioxidant effect [25] through direct scavenging of free radicals [27], chemical interaction with NO [78], stimulate GSH production [29] or by chelating heavy metals [79]. MLT treatment reversed the elevated levels of MDA and 4hydroxylalkyls and also reduced levels of CAT, GPx, SOD, GR, and GSH in rat brain due to oxidative stress induced by lead acetate [80] or fluoride [81]. In a similar way, Rao and coworkers [82] observed reversal of neuronal changes in cerebral cortex, cerebellum, and brain stem of adult male rats induced by mercuric chloride by MLT supplementation. Bharthi et al. [83] reported that buffalo pineal epiphyseal proteins can significantly counteract the As-induced decreased activities of SOD, CAT, GPx, GR, tissue GSH, and increased LPO. In the present study, there was only a marginal improvement in CAT activity following MLT supplementation. CAT is an iron (Fe)-containing enzyme. Therefore, there is a possibility that As treatment might be due to chelating of iron leading

Melatonin Ameliorates Neuropharmacological and Neurobiochemical Alterations Induced by Subchronic Exposure...

to relatively a less improvement in recovery following MLT supplementation when compared to other enzymatic antioxidant defense system(s). Huang et al. [84] reported that melatonin pretreatment significantly depresses the levels of nNOS, eNOS, iNOS, nitrotyrosine, and caspase-3 protein expression in hippocampus of hypobaric hypoxic rat model. Additionally, Song et al. [41] opined that melatonin inhibits NO production. Therefore, MLT treatment in the present study was able to significantly reduce the 3-NT levels in brain tissue of rats treated with As (group III). Sub-chronic As treatment (group II) significantly increased the levels of pro-inflammatory mediators, viz., IL-1β, IL-6, TNF-α, and PGE2 in brain tissue of rats. Zaazaa et al. [85] and Vijayakaran et al. [86] also observed a significant increase in levels of IL-1β, TNF-α, and PGE2 in rat brain tissue following As exposure. Increased production of inflammatory cytokines in the brain following sub-chronic exposure to As might be due to activation of ERK, JNK, p38, and NF-κB pathways, which govern the expression of variety of pro-inflammatory and cytotoxic genes [87]. It is interesting to note that MLT supplementation significantly (p < 0.05) attenuated the production of the inflammatory mediators induced by As (group III). In an in vitro study, MLT at micromolar concentration inhibited the activation of NF-kB pathway (prototypical pro-inflammatory signaling pathway) induced by TNF-α or ionizing radiation [88]. Studies have shown that MLT significantly attenuated the fluoride-induced increase in the TNF-α level in the rat brain [89]. In support of this Rosales-Corral et al. [90] reported that MLT treatment can significantly reduce the IL-1β, IL-6, and TNF-α levels during the inflammatory response induced by fibrillar Aβ. Alternatively, the decreased production of inflammatory mediators in MLT-supplemented rats (group III) may also be due to a coordinated attenuation of As-induced increases in heat shock protein-70, heme oxygenase-1, as well as phosphorylation of p38 mitogen-activated protein kinase and elevations in cyclooxygenase II and iNOS expression [40]. Pro-inflammatory cascade in brain induces the production of Aβ protein which is not only toxic to neurons but also further upregulate/induces the release of inflammatory mediators from microglial cells. This Aβ deposition in brain tissue has association with the progression of neurodegenerative pathology-like Alzheimer’s disease [91]. Sub-chronic As-exposed group (group II) rats showed a significant increase in levels of Aβ production in brain tissue of rats, while rats which received melatonin supplementation (group III) showed a significant reduction. While, MLT-alone-treated group (group IV) showed a significant difference in Aβ deposition compared to untreated control rats. Koropatnick et al. [92] reported that increase in amyloid precursor protein degradation products in rat brain after 6 days of single subcutaneous administration of arsenite. Further, MLT treatment

significantly attenuated a cobalt-induced Aβ release in SHSY5Y neuroblastoma cells [39]. In short, the present study explicitly demonstrated that subchronic administration of sodium (meta) arsenite (As) in Wistar rats lead to marked neurobiochemical changes in the brain and induced oxidative and nitrosative stress. Further, alterations in redox balance in the brain triggered the release of neuroinflammatory cytokines/mediators, which in turn favored Aβ, a protein normally deposited during neurogenerative diseases of CNS. Thus, sub-chronic administration of As mimicking the epidemiological relevant concentration of arsenic can elicit neurodegenerative changes and disturbed the motor activities and anxiety levels in Wistar rats. Secondly, exogenous administration of melatonin proved beneficial in restoring the redox homeostasis in the CNS and much of the neuropharmacological activities in the present experimental study. Acknowledgments The authors are thankful to the Dean, Veterinary College, Shivamogga 577 204, Karnataka, India, for necessary facilities to conduct the study.

Compliance with Ethical Standards Prior approval of the Institutional Animal Ethics Committee (IAEC) was obtained, vide No. VCS/IAEC/005/2015-16 dated 23.07.2016 to carry out the current investigation as per the guidelines of the Committee for Prevention of Cruelty and Supervision of Experiment on Animals (CPCSEA), New Delhi. Conflict of Interest The authors declare that they have no conflicts of interest

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