Zinc oxide nanoparticles mediated cytotoxicity, mitochondrial membrane potential and level of antioxidants in presence of melatonin

Accepted Manuscript Title: Zinc oxide nanoparticles mediated cytotoxicity, mitochondrial membrane potential and level of antioxidants in presence of melatonin Authors: Sruthi S., N. Millot, Mohanan PV. PII: DOI: Reference:

S0141-8130(17)30117-4 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.05.088 BIOMAC 7577

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

9-1-2017 31-3-2017 16-5-2017

Please cite this article as: Sruthi S., N.Millot, Mohanan PV., Zinc oxide nanoparticles mediated cytotoxicity, mitochondrial membrane potential and level of antioxidants in presence of melatonin, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.05.088 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.

Zinc oxide nanoparticles mediated cytotoxicity, mitochondrial membrane potential and level of antioxidants in presence of melatonin Sruthi S1, Millot N2, Mohanan PV1* 1

Toxicology Division, Biomedical Technology Wing

Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram - 695 012, Kerala, India

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Département Nanosciences, Laboratoire Interdisciplinaire Carnot de Bourgogne, UMR 6303

CNRS/Université de Bourgogne Franche-Comté 9 av. A. Savary, BP 47 870, 21 078 DIJON, France *Corresponding Author: [email protected] or [email protected] Phone: 91-471-2520266, Fax: 91-471-2341814 Graphical abstract

Zinc oxide nanoparticles induced toxicity on glial cells in presence of pharmacological concentration of melatonin 1

Highlights    

Synthesis and characterization of ZnONPs cytotoxicity and detection of ROS detection of Nitric oxide production Antioxidant assays

Abstract Zinc oxide nanoparticles (ZnO NPs) are widely used in a variety of products and are currently being investigated for biomedical applications. However, they have the potential to interact with macromolecules like proteins, lipids and DNA within the cells which makes the safe biomedical application difficult. The toxicity of the ZnO NP is mainly attributed reactive oxygen species (ROS) generation. Different strategies like iron doping, polymer coating and external supply of antioxidants have been evaluated to minimize the toxic potential of ZnO NPs. Melatonin is a hormone secreted by the pineal gland with great antioxidant properties. The melatonin is known to protect cells from ROS inducing external agents like lipopolysaccharides. In the present study, the protective effect of melatonin on ZnO NPs mediated toxicity was evaluated using C6 glial cells. The Cytotoxicity, mitochondrial membrane potential and free radical formation were measured to study the effect of melatonin. Antioxidant assays were done on mice brain slices, incubated with melatonin and ZnO NPs. The results of the study reveal that, instead of imparting a protective effect, the melatonin pre-treatment enhanced the toxicity of ZnO NPs. Melatonin increased antioxidant enzymes in brain slices. Keywords : ZnO NPs; Melatonin; ROS; Glial cells; antioxidant molecules 1. Introduction ZnO NPs are semiconductor nanoparticles with unique properties, which are already exploited for industrial and biomedical applications. These nanoparticles are already present in commercial products like cosmetics and daily care products. The ZnO NPs are reported to have preferential toxicity for the cancer cells. Moreover, these particles are investigated for drug delivery applications. However, the high toxicity exhibited by these nanoparticles makes it difficult for the safe biomedical application of ZnO NPs.

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The toxicity of ZnO NPs is attributed to its particle dissolution and ability to generate ROS. ROS generation can further lead to numerous secondary effects like alteration in mitochondrial membrane potential, endoplasmic reticulum stress and induction of apoptotic genes (1). Cells have endogenous antioxidant mechanisms to mitigate the ROS generated inside the cells. But during oxidative stress, the cells antioxidant machinery fails to alleviate ROS, leading to ROS mediated cell and tissue damage (2). The ZnO NPs are found to interfere with the antioxidant machinery of the cells and thereby inducing oxidative stress (3). A number of strategies are adapted to reduce ROS generated by the nanoparticles which include polymer coating (4), metal doping (5) and the addition of antioxidants (6). Antioxidants are mostly amino acid and protein derivatives that inhibit the oxidation of the other molecules. These molecules scavenge the free radicals generated by the oxidation reactions and thereby undergoing oxidation themselves. There are many naturally and artificially synthesized molecules which exhibit antioxidant properties. Organisms have its own antioxidant protective mechanism which protects the cells and tissue from reactive oxygen species generated as a metabolic by-product. One such molecule is melatonin, a hormone commonly found in animals, plants, fungi and bacteria. In higher forms of organisms, it is involved in the regulation of daynight cycle. In lower forms it mainly acts as an antioxidant (7, 8). In humans, this hormone is secreted by the pineal glands which control the sleep-wake cycles. Apart from its role in the maintenance of circadian rhythm, its antioxidant and anti-inflammatory effect thought to have a positive impact on clinical conditions like cancer, autism, gall bladder stone and infectious diseases (9-12). Melatonin brings about its action either by acting directly as an antioxidant or by stimulating the antioxidant enzymes of the cells. The protective action of melatonin against ROS producing agents has already been reported (13, 14). Melatonin pre-treatment at µM range ameliorates β-amyloid induced oxidative stress and apoptosis in C6 glial cells. Hence it is

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hypothesised that the melatonin can scavenge the ROS induced by the nanoparticle there by protect the cells against ZnO NPs mediated toxicity. In the present study, effect of melatonin on the ZnO NPs induced ROS and nitric oxide release was analysed in vitro using C6 cell lines. The effect of melatonin on the antioxidant machinery was analysed using mice brain homogenate. Completely characterised ZnO NPs were used for the study. 2. Materials and methods 2.1 Chemicals and reagents Phosphate Buffered Saline (PBS) and bovine serum albumin (BSA) were purchased from Gibco (Grand Island NY, USA). Melatonin, thiobarbituric acid (TBA), reduced glutathione (GSH), oxidized glutathione (GSSG) and dithio-bis-2-nitrobenzoic acid (DTNB) were purchased from Sigma Chemicals Co. Ltd. (St. Louis, MO USA). Ethylene triamine tetra acetic acid (EDTA), disodium hydrogen phosphate (Na2HPO4) and sodium dihydrogen phosphate (NaH2PO4) were obtained from (Merck, Germany). All other chemicals were purchased locally from India and were of analytical grade. 2.2 ZnO NPs synthesis ZnO NPs were synthesized by wet precipitation method using zinc nitrate (Zn (NO3)2) and sodium hydroxide (NaOH) as precursor. In brief, an aqueous solution of zinc nitrate (14.02 g) and sodium hydroxide (3.24 g) were prepared each in 100 ml of distilled water. The solution containing NaOH was heated to 55°C and the temperature was maintained throughout the synthesis process. The solution of Zn (NO3)2 was added dropwise for 40 min to the heated NaOH solution with constant stirring at 800 rpm. The reaction was kept undisturbed for 2 h. The precipitated ZnO NPs were washed five times with deionized water followed by absolute ethanol (3 times). Freeze dried samples were characterized using several techniques.

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2.3 ZnO NPs characterization SEM imaging accompanied by EDX analysis was performed with scanning electronic Microscope JEOL JSM6400F equipped by Oxford Instruments EDS analyzer. The sample was prepared by evaporating a diluted suspension of NPs in ethanol solution on a carbon-coated silicon substrate. Transmission Electron Microscopy (TEM) characterizations were performed using a JEOL JEM2100F microscope operating at 200 kV (point to point resolution of 0.19 nm). carbon-coated copper grid (carbon Film – 300mesh, EMS, Hatfield, PA). Specific surface area (SSA) of nanoparticles was analyzed by Brunauer, Emmett and Teller theory (BET), with the help of a Micromeritics Tristar II apparatus. Samples were outgassed in situ under a pressure of 20 mTorr and at 100°C. The measurements were performed at liquid N2 temperature with N2 adsorbing gas. Powder X-Ray Diffraction (XRD) patterns were collected using a D8 Advance diffractometer with Cu Kα1 (λα1 = 1.5406 Å). The phase identification was done by comparison of the diffraction patterns with the reference cards of the ICDD Powder Diffraction File. The data analysis was carried out with Topas® software. Rietveld method was used to obtain lattice parameters and mean crystallite size. A Discovery Thermo-Gravimetric Analysis (TGA) TA Instrument with an air flow rate of 25 mL.min1

and a temperature ramp of 5 °C/min from 25 °C to 800 °C was used to analyze powders.

X-ray Photoelectron Spectroscopy (XPS) measurements were collected with a PHI 5000 Versaprobe instrument equipped with a Al Kα monochromated radiation (EKα(Al)=1486.7 eV with a 200 µm diameter spot size). Powders were pressed on an indium sheet in order to immobilize NPs during measurements. Data were analyzed with CasaXPS processing and MultiPak software. A neutralization process has been applied in order to avoid a charge accumulation on the surface

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of samples. The carbon C1s peak at 284.5 eV was used as reference. A Shirley background was subtracted and Gauss (70%) - Lorentz (30%) profile was used. Full Width at Half Maximum (FWHM) were fixed between 1.5 and 2.0. Quantitative analysis has been realized with the help of MultiPak software. ζ-potential and Dynamic Light Scattering (DLS) measurements were performed at 25°C with a Malvern Nano ZS instrument supplied by DTS Nano V7.02 software. Prior to measurement, suspensions were diluted in an aqueous solution containing NaCl (10-2 M). Samples were filtered (0.45 µm filter) in order to remove eventual pollutant or large agglomerates. DLS curves were derived from intensity calculations. 2.4 Cell culture and particle treatment C6 cells were grown in monolayer to 70-80% confluency in a cell culture flask containing DMEM F12 supplemented with 10% FBS, 1% antibiotic- antimycotic solution, 1% glutamax and 1mM sodium pyruvate. Freshly prepared stock solutions of melatonin (100mM) were used in all the experiments. Cells were treated with melatonin 1h prior to ZnO NPs exposure. Before each experiment, the suspension of ZnO NPs (in water) was sonicated for 1h and working stocks were prepared in complete cell culture medium. 2.5 Cell viability- MTT assay MTT assay was carried out to determine the dose response of melatonin, ZnO NPs and for the combination treatment of melatonin and ZnO NPs. The concentration ranges of melatonin included 1, 10, 100, 200, 400, 800 and 1600µM. The ZnO NPs concentrations were 5, 10, 20, 40 and 80µg/ml. Briefly, cells were seeded at a density of 1x104 cells/well and kept overnight for attachment. They were incubated with different concentrations of melatonin or ZnO NPs or with a combination of both for 24h. Cells were then incubated with MTT (100μl/well) for 3h. The

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formazan crystals formed were solubilized using DMSO and absorbance was measured at 540nm using multiwell plate reader (Bio-TekWinooski, USA) (15). 2.6 ROS generation- DCF-DA assay Cells were pre-treated with melatonin (100 μM) and later with ZnO NPs (5, 20, 40 μg/ml) for 4h. The cells were harvested by trypsinisation and centrifugation. The pellets were resuspended in DCFDA working solution (0.1μM) for 30 min at 37°C. After washing with PBS, the pellets were resuspended in PBS and were immediately analyzed using flow cytometry (BD FACSAria™, BD BioSciences, San Jose, CA, USA) at an excitation-emission wavelengths of 485/530nm. 2.7 Nitric oxide production - Griess test Cells were grown in a 96 well plate using the complete medium. Melatonin pre-treated cells were treated with ZnO NPs for 4h. The supernatant was collected and centrifuged at 300g for 5min. The supernatant was mixed with Griess reagent in 1:1 ratio and incubated at 25°C for 10min. The absorbance was read at 540nm using multiwell plate reader (Bio-Tek, Winooski, USA). 2.8 Membrane integrity- Propidium Iodide (PI) staining The cells were treated with melatonin (100μM) and ZnO NPs (5, 20 and 40μg/ml) for 24h. Trypsinized cells were centrifuged at 3000rpm for 5min and resuspended in 500 μl PBS containing PI (0.5μl/ml). The cells were analyzed by fluorescence microscopy (Axio Scope.A1, Carl Zeiss, Germany). 100 random fields were counted under fluorescence microscope for PI positive cells. 2.9 Mitochondrial membrane potential – JC1 staining

Mitochondrial membrane potential (MMP) was analyzed by JC1 (5, 5′, 6, 6′-tetrachloro1,1′,3,3′-tetraethylbenzimi- dazolylcarbocyanine iodide). JC1 is a lipophilic cationic dye used to detect mitochondrial membrane potential. In brief, cells were seeded in 6 well plates at a seeding density of 1X106cells/well. Once confluent, cells were treated with 20 7

and 80 µg/ml of ZnO NPs for 6h with or without melatonin pre-treatment. Non-treated cells and cells exposed to melatonin alone were taken as control. At the end of the exposure, 1µM JC-1 was added to each well and incubated at room temperature for 20min. The cells after washing were observed under fluorescence microscope (Axio Scope.A1, Carl Zeiss, Germany) using a green and a red filter and photographs were taken. 2.10

Antioxidant assays

2.10.1 Preparation of brain homogenate Healthy Swiss albino mice weighing 15 to 20g were obtained with approval from the institute animal ethics committee and euthanized by cervical dislocation. The brains were immediately isolated and washed in cold phosphate buffered saline (PBS) and immediately placed in an ice bath. The brains were cut into small slices (200mg/ml of the brain in 0.1M phosphate buffer, pH 7.4.) and divided into two groups. The first group was treated with three different concentrations of ZnO NPs (5, 20 and 40μg/ml), the second group was treated with melatonin (100μM) for 1h followed by varying concentrations of ZnO NPs for 3h. After particle incubation, the cells were subjected to centrifugation at 3500 rpm for 10min at 4°C. The supernatant obtained were maintained in an ice bath and used to estimate the following parameters. 2.10.2 Total protein Total protein in mice brain homogenate was estimated following the method of Lowry et al., using bovine serum albumin (BSA) as standard (16). 2.10.3 Reduced glutathione (GSH)

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The level of GSH in the brain homogenate was determined by the method of Moron et al., with slight modification. Here the DTNB (5, 5’-dithiobis- (2- nitrobenzoic acid) reacts with GSH to form GSH-TNB that absorb at 412 nm. The change in absorbance gives the GSH concentration in the reaction sample. The amount of GSH was expressed as nmol/mg protein. (17). 2.10.4 Lipid peroxidation (LPO) The degree of lipid peroxidation in the homogenate was determined by adapting the protocol described by Okhawa et al., here the malondialdehyde (MDA) generated as a result of lipid peroxidation react with TBA to form a colored product. This colored product is measured spectrophotometrically at 532 nm. (18). 2.10.5 Glutathione peroxidase (GPx) GPx activity in the homogenate was estimated following the method of Rotruck et al. The GSH remaining after the enzyme-catalysed reaction complexes with DTNB in the reaction mixture to form a colored product. This has an absorption maximum at 412nm. GPx activity was expressed as μg of GSH consumed /min/mg protein. (19). 2.10.6 Glutathione reductase (GR) GR activity in liver homogenate was determined by measuring the reduction of GSSG in the presence of NADPH as described by Mize and Langdon (20). Briefly, this assay measures the rate of NADPH oxidation to NADP+, which is accompanied by a decrease in absorbance at 340nm. Thus, one GR unit is defined as the reduction of one μM of GSSG per min at 25° C and pH 7.6. 2.10.7 Superoxide dismutase assay (SOD) SOD in the brain homogenate was assessed using modified pyrogallol autoxidation method initially described by Marklund and Marklund (21). The colorimetric measurement is done at

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420nm. The SOD activity is expressed as units/mg protein. One unit of SOD activity was defined as the amount required for inhibiting pyrogallol autoxidation by 50% per min. 2.11

Statistical Analysis

All the samples were run in triplicates. Values are expressed as mean ± SD. Statistical differences between the control and experimental values were compared by students’t-test. For comparisons, p<0.05 was considered significant. 3. Results 3.1 ZnO NPs synthesis and characterisation The nanoparticles were characterized for their physicochemical properties (Fig.1). The size of the particle was around 50-100 nm as revealed by TEM analysis with an elongated shape. The SEM analysis showed both individual particles and confirmed their elongated morphology and aggregates, with flower shaped morphology. The SSA was 37.1± 0.6 m²/g for the sample. The colloidal stability was analyzed by DLS. The hydrodynamic diameter was 377.7± 8.5nm with a PDI of 0.43±0.03. The ζ-potential value at the physiological pH was around +30 mV with an isoelectric point around 9. Crystallographic pattern measured for the sample confirmed zincite crystal structure of ZnO NPs (ICDD card 01-070-8070, space group P63mc). The line broadening of diffraction peaks indicated nanosized particles. The crystal size of the particles calculated from Le Bail method was 45nm. The lattice parameters calculated for the samples were a=3.25Å and c=5.21Å; c/a was calculated to be 1.60, which is comparable to the ideal value for a hexagonal cell. Very few traces of impurities were revealed in XRD pattern (less than 1%), however, the strain value calculated for the samples were 0.20 suggesting few crystal defects.

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The chemical investigations on the particles were done using TGA and XPS. TGA is a technique which analyses the components based on its degradation with temperature. There was a major weight loss at 250°C which is thought to be due to the surface chemisorbed water (5% in mass) (22) beyond which no weight loss was evident. Above, at about 340°C, carbonates may be eliminated (1% in mass). In the XPS, O(1s) spectrum has been fitted taking into account the Zn-O, Zn-OH, O=C groups. Peak positions are in good agreement with the results reported earlier (2224). The contribution at 532.5 eV corresponds to the surface adsorbed carbonates (O=C groups), that at 530.7 eV to hydroxides (Zn-OH groups). These results are in good agreement with those obtained via TGA analysis. Zn (2p3/2) spectrum showed two separate peaks due to Zn-O and Zn-OH contributions, in agreement with the fit of the O(1s) spectrum. . 3.2 Cell viability Cell viability was determined from the MTT data (Fig.2). The MTT data on melatonin showed no reduction in percentage viability up to 100μM concentration, which started declining thereafter. The percentage viability was 99.8, 97.2, 71.9, 41.7, and 41.7 for 100, 200, 400, 800 and 1600μM respectively. Cells treated with ZnO NPs showed a decline in cell viability at and above 20µg/ml concentrations. In order to see if the nanoparticle and melatonin combination had any unique effect on the cells than that of either of the particle alone, two separate MTT experiments were carried out. In the first experiment, the cells were pre-treated with a range of melatonin concentrations (1, 10, 100 and 200µM) and then with a single nontoxic concentration of ZnO NPs (5µg/ml). A statistically significant reduction in cell viability was observed in the co-treatment group compared to melatonin alone at 100µM and above. In the second MTT, the cells were pretreated with 100µM melatonin and later with 5, 20, 40 and 80µg/ml of ZnO NPs. Here in all treatment group showed a higher cell death for co-treatment compared to ZnO NPs alone. Based on these MTT results 100µM of melatonin was taken for the rest of the studies.

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3.3 ROS generation ROS generated by ZnO NPs in the presence of the known antioxidant, melatonin was analyzed by FACS (Fig.3). Data showed a slight increase in ROS in melatonin pre-incubated samples when compared to samples without melatonin treatment. The increase in ROS was dependent on the ZnO NPs dose. 3.4 Nitric oxide production Nitric oxide release as detected by Griess reagent did not show any nitric oxide release for melatonin pre-treated samples in comparison with its counterpart treated with ZnO NPs alone. However, zinc oxide nanoparticle treated cells showed a slight increase in NO production in a concentration dependent manner (Fig.4). 3.5 Membrane integrity In order to analyze the extend of membrane damage, the samples were treated with PI. Melatonin pre-treatment induced more membrane damage than the one which was treated with ZnO NPs alone (Fig.5). The cytotoxicity showed a dose dependence on the concentration of ZnO NPs used. 3.6 Mitochondrial membrane potential Mitochondrial membrane potential was assessed by JC1 staining. Melatonin pre-incubated cells exposed to different concentrations of ZnO NPs did not show any significant change in the mitochondrial potential with respect to the untreated sample. However, in comparison with the control group, a slight reduction in red fluorescence could be observed for the samples treated with higher concentration of ZnO NPs, irrespective of melatonin pre-incubation (Fig.6). 3.7 Antioxidant assay Antioxidant assays were carried out on brain slices incubated with melatonin and ZnO NPs (Fig7). 3.7.1 LPO

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There was a statistically significant decrease (p> 0.05) in lipid peroxidation for tissue samples pretreated with melatonin. 3.7.2 GSH At lower concentration of ZnO NPs (5µg/ml), a statistically significant increase (p> 0.05) in GSH concentration was observed for tissue samples pre-treated with melatonin. But at higher concentrations of ZnO NPs (20 and 40μg/ml), the cells did not show any significant increase in the GSH level in the melatonin-treated group. 3.7.3 GPx A statistically significant increase (p>0.05) in the GPx level could be observed in tissue samples pre-treated with melatonin. However, at 40µg/ml of ZnO NPS, this effect was not evident. 3.7.4 GR Even though not statistically significant, the GR showed an increase when samples were pretreated with melatonin. 3.7.5 SOD SOD level was high in the case of melatonin-treated groups. However, statistically significant increase (p>0.05) was evident only at a lower concentration of ZnO NPs (5µg/ml). At higher concentrations both melatonin pre-treated and ZnO NPs alone groups showed similar values. 4. Discussion Antioxidant capacity of melatonin is already reported by different groups (25, 26). Melatonin upregulates the expression of antioxidant enzymes like SOD, GPx and GR. It is also reported to

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increase the level of GSH in presence of ROS and oxidative stress (27). In the present study, the effect of melatonin on the ZnO NP induced oxidative stress was evaluated. The nanoparticles were characterized by different techniques to understand their physicochemical nature. The particle size estimated from the TEM and SEM analyses showed an average range of 50 nm. The crystal size calculated from XRD was comparable to this value, proving that nanoparticles are monocristalline in nature. However, hydrodynamic diameter measured by DLS was around 380nm, which indicates the particle agglomeration in water with 10-2M, as suggested thanks to TEM and SEM observations. Weight loss at a single temperature as revealed by TGA, indicated the purity of the sample, which was further confirmed by XRD and XPS. Hydroxyls and carbonates highlighted via XPS and TGA analyses are commonly adsorbed in a large amount at the surface of metal oxides nanoparticles (22, 23). Some hydroxides nanoparticles Zn(OH)2 may also be obtained and not detected via XRD due to their amorphous nature. The cellular interaction of the nanoparticles and melatonin were studied using C6 glial cells. In the present study, cells were pre-treated with melatonin 1h prior to the ZnO NPs exposure. In order to understand the dose-response relationship, separate MTT assays were carried out for ZnO NPs and melatonin. To see whether nanoparticle treatment in melatonin pre-treated cells modulates the cytotoxicity, two separate MTT experiments were carried out. In one set the cells were preincubated with varying concentrations of melatonin and later with a nontoxic concentration of ZnO NPs. In the second experiment, the cells were pre-incubated with nontoxic concentrations of melatonin and later with varying concentrations of ZnO NPs. The nontoxic concentration of ZnO NPs (5µg/ml) and melatonin (100µM) were chosen here in order to see if both the particle in combination interacts differently with the cells in comparison with either of the particles alone. The results indicate an elevated cytotoxicity in cells exposed to both melatonin and ZnO NPs than

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either of the particle alone. The toxicity of ZnO NPs is attributed to its ability to induce reactive oxygen species (28) and melatonin on the other hand is a known antioxidant. The protective effect of melatonin on the ROS induced cytotoxicity of C6 cells has been reported (29, 30). In contrary to these results the present study, melatonin did not impart any protective effect on the ZnO NPs mediated cytotoxicity in C6 cells. On the other hand melatonin pre-treatment was enhancing the ZnO NPs mediated toxicity. To see whether melatonin has any effect on the ZnO NPs induced an inflammatory response, Griess assay was carried out. The assays results indicated decreased nitric oxide production in case of melatonin-treated groups in comparison to samples without melatonin treatment. The zinc ions can activate nitric oxide synthase and result in the elevation of nitric oxide release in the cells (31). The melatonin has the ability to regulate the nitric oxide release. Here we could observe a slight reduction in nitric oxide production when the cell was pre- treated with melatonin. Melatonin exerts its antioxidant effect by different mechanisms. It can either directly act as an antioxidant by scavenging the ROS (27) or by upregulating the expression of endogenous antioxidant enzymes of the cells (32). In the present study, the ZnO NPs induced ROS production in presence of melatonin was assessed using fluorescence probe DCFH-DA. Zinc oxide nanoparticles are very potent in inducing oxidative stress, which is one of the major reasons for its toxicity (33). To see the role of ROS in the loss of cell viability, cells were pre-incubated with 100µM melatonin and then with ZnO NPs. Though not significant, the FACS data indicated an increase in fluorescence for melatonin pre-treated cells which was not evident in melatonin treated control cells. The data from PI staining was in accordance with the ROS data, with more number of PI positive cells in melatonin treated group compared to ZnO NPs alone indicating elevated membrane damage and ROS. A reduced amount of ROS has been reported in cells due to surface adsorbed medium proteins on ZnO NPs after 24h exposure (34). Hence the present

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work assumes that the low level of ROS detected in the sample might be due to the adsorption of medium proteins on the surface of ZnO NPs. Even though the exact mechanism is not known, the antiproliferative action of melatonin at the pharmacological concentration on androgen sensitive tumours has been proposed by several groups. Martin et al., reported inhibition of cell growth in C6 cell line up on incubation with melatonin at mM concentrations. This work proposed that the antiprolifertive effect is due to the inhibition of ERK1/2 (35). Rodriguez et al., mention that some cancer cells are not sensitive to melatonin at physiological concentration, however at pharmacological concentration they reduce cell proliferation (36). C6 glioma is an oestrogen dependent tumour cell. González et al., reported that the antiproliferative activity of the melatonin is due to its capacity to inhibit aromatase, an enzyme which catalyses the conversion of androgen to oestrogen (37). The current results are in concordance with the above mentioned findings, where we observed an increase in cytotoxicity in melatonin pre-treated samples, though the cytotoxic effects observed in aforementioned studies were at millimolar concentrations. ROS mediated mitochondrial damage has been reported in human platelets, treated with melatonin (38). However, there were no marked changes in mitochondrial potential in melatonin preincubated samples in comparison to ZnO NPs alone groups. It is likely that increased ROS production in melatonin treated samples is coming from the mitochondria as mitochondria are a major contributor of cellular ROS production. It has been reported that the apoptotic effect of melatonin on cancer cells are mainly through oxidative stress imparted by the mitochondrial electron transport chain (39, 40). Unaltered MMP in the present study further supports this finding.

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The cell death analysis by PI staining indicated an increase in the number of cells with damaged membrane when pre-treated with melatonin. However, the melatonin treated control cells behaved similar to untreated control. Hence, from the study, it is assumed that the ROS generated by the ZnO NPs along with ROS production from the increased mitochondrial oxidative phosphorylation might be contributing to the increased membrane damage in melatonin pretreated cells (39). This comes as an additional blow to the cell population which undergoes apoptosis much faster than the ZnO NPs alone treated cells. However melatonin treated control cells do not exhibit theses response probably due to inherent antioxidant machinery of cells in action. Additionally, melatonin can activate antioxidant molecules in cells to reduce ROS generation which further explains the reduced ROS production and cytotoxicity in melatonin treated control cells. In order to see if the increased ROS in melatonin pre-treated cells was due to some inherent mechanism of the cancerous cells, or if it is also evident in normal tissue, the brain slices were incubated with melatonin prior to ZnO NPs treatment for 1h. It has been reported that the brain slices can better respond and regulate the melatonin than in vivo system (41). There was an increase in the antioxidant enzymes in melatonin pre-treated groups, compares to ZnO NPs alone. The results are in concordance with the above mentioned finding where melatonin at 100 µM concentration excreted antioxidant properties in brain slices following N-methyl-D-aspartate insult. The GSH depletion was also high in melatonin pre-treated sample compared to ZnO NPs alone group. The lipid peroxidation was significantly reduced in melatonin-treated groups after 3h of incubation with ZnO NPs. The result is in concordance with the previously published results of Teixeira et al., which demonstrate the ability of melatonin to protect against lipid peroxidation induced by free radicals (42). Antioxidant enzymes were found to rise in various organs of the rat when melatonin was administrated to attenuate the effect of fluoride-induced oxidative stress

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(43). However, in the present study, these enhanced antioxidant enzyme levels were evident only at lower concentrations of ZnO NPs. It is assumed that at high concentration of ZnO NPs the protective effect of the melatonin is taken over by the oxidative stress induced by the ZnO NPs. 5. Conclusion Zinc oxide nanoparticle toxicity is mainly attributed to particle dissolution and its ability to generate ROS. The treatment of melatonin, which is known to have a protective role against oxidative stress induced by external agents like lipopolysaccharides, appears to be ineffective against the oxidative stress induced by ZnO NPs. The present study indicates that the incubation of melatonin pre-treated cells with ZnO NPs increase the cytotoxicity in C6 cells. This may probably due to the mitochondria mediated ROS generation and antiproliferative effect induced by melatonin which makes the cell more susceptible to ZnO NPs mediated cell damage induced by the ZnO NPs. However the molecular mechanism underlying the enhanced toxicity has to be explored in detail. There was an increase in the antioxidant enzymes when brain slices were preincubated with melatonin, suggesting a selective protective effect on normal tissues. Though the study could not prove the protective effect of melatonin on ZnO NPs induced toxicity, it will be worth studying, if the melatonin - ZnO NPs combination can be used for cancer treatment. 6. Acknowledgement The authors thank the Director and the Head, Bio Medical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram for providing the infrastructure. Sruthi S thanks, University Grants Commission, New Delhi and Indo-French Centre for the Promotion of Advanced Research, New Delhi for the Junior Research Fellowship and the Raman-Charpak Fellowship. The authors would like to thank Dr. Frédéric Herbst (SEM), Dr. Nicolas

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Geoffroy (XRD), Dr. Olivier Heintz (XPS), Dr. Julien Boudon (TGA), Dr. Rémi Chassagnon (TEM) for their help during nanoparticles characterization. 7. Conflict of interest The authors declare no conflict of interest. 8. Reference 1. Rim K-T, Song S-W, Kim H-Y. Oxidative DNA damage from nanoparticle exposure and its application to workers' health: a literature review. Safety and health at work. 2013;4(4):177-86. 2. Fulda S, Gorman AM, Hori O, Samali A. Cellular Stress Responses: Cell Survival and Cell Death. International Journal of Cell Biology. 2010;2010:23. 3. Wang J, Deng X, Zhang F, Chen D, Ding W. ZnO nanoparticle-induced oxidative stress triggers apoptosis by activating JNK signaling pathway in cultured primary astrocytes. Nanoscale research letters. 2014;9(1):1-12. 4. Zhang Y, Kohler N, Zhang M. Surface modification of superparamagnetic magnetite nanoparticles and their intracellular uptake. Biomaterials. 2002 4//;23(7):1553-61. 5. Xia T, Zhao Y, Sager T, George S, Pokhrel S, Li N, et al. Decreased dissolution of ZnO by iron doping yields nanoparticles with reduced toxicity in the rodent lung and zebrafish embryos. ACS nano. 2011;5(2):1223-35. 6. Sharma V, Anderson D, Dhawan A. Zinc oxide nanoparticles induce oxidative DNA damage and ROS-triggered mitochondria mediated apoptosis in human liver cells (HepG2). Apoptosis. 2012;17(8):852-70. 7. Poeggeler B, Saarela S, Reiter RJ, Tan D-X, Chen L-D, Manchester LC, et al. Melatonin—A Highly Potent Endogenous Radical Scavenger and Electron Donor: New Aspects of the Oxidation Chemistry of this Indole Accessed in vitroa. Annals of the New York Academy of Sciences. 1994;738(1):419-20. 8. Tan D-X, Chen L, Poeggeler B, Manchester L, Reiter R. Melatonin: a potent, endogenous hydroxyl radical scavenger. Endocr j. 1993;1(4):57-60. 9. Maestroni GJ. The immunotherapeutic potential of melatonin. Expert opinion on investigational drugs. 2001;10(3):467-76. 10. Koppisetti S, Jenigiri B, Terron MP, Tengattini S, Tamura H, Flores LJ, et al. Reactive oxygen species and the hypomotility of the gall bladder as targets for the treatment of gallstones with melatonin: a review. Digestive diseases and sciences. 2008;53(10):2592-603.

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Fig.1. Characterisation data for ZnO NPs. (a) TEM micrograph (b) SEM micrograph (c) and (d) XPS analysis of oxygen and zinc respectively (e) XRD data NPs (λ=1.5406 Å, ICDD file 01-070-8070) (f) TGA analysis under air (5°C/min).

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Fig. 2. Cell viability - MTT assay (a) MTT results for ZnO NPs. (b) MTT assay for melatonin. (c) MTT for cells pre-treated with varrying concentrations of melatonin which was treated with 5µg/ml of ZnO NPs. (d) MTT results of cells treated with varrying concentrations of ZnO NPs, which are pre incubated with 100µM melatonin. Values are expressed in percentage with respect to control. The data represents mean ±SD of three independent experiments. Asterisks in the graph (a) and (b) indicate statistically significant difference in comparison with control group. Asterisks in the graph (c) and (d) indicate statistically significant difference among ZnO NPs treatment with and without melatonin pre-treatment (*P<0.05)

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Fig 3. ROS generation- DCFH-DA assay. The dot plots in red colour indicate the cells with increased fluorescence intensity, with respect to control. (a) untreated cells. (b) melatonin (100µM) treated cells. (c) (e) and (g) cells treated with 5,20 and 40µg/ml of ZnO NPs. (d) (f) and (h) melatonin preincubated cells treated with 5,20 and 40µg/ml of ZnO NPs. (i) positive control (0.02% H2O2). (j) graph showing the percentage of cells in showing DCF fluorescence. Blue bars in the control represents untreated cells and red bar indicate cells treated with melatonin (100µM). The data represents mean ±SD of three independent experiments. Asterisks in the graph indicates statistically significant difference among ZnO NPs treatment with and without melatonin pretreatment (*P<0.05).

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Fig. 4. Nitric oxide release- Griess reagent assay; blue bars in the control represents untreated cells and red bar indicate cells treated with melatonin (100µM). The data represents mean ±SD of three independent experiments. kjh

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Fig.5. Cell membrane integrity- PI staining. The cells were counted under fluorescence microscope. The Micrograph (a)- untreated cells, (b) to (d)- cells treated with 5, 20 and 40µg/ml ZnO NPs, e- cells treated with 100µM melatonin, (f) to (h)- cells treated with (100µM) melatonin and ZnO NPs of 5, 20 and 40µg/ml of ZnO NPs respectively. (i)- graphs showing cells which are positive for PI. Blue bar in the control represent cells treated with 100µM of melatonin and red bar, the untreated cells. The data represents mean ±SD of three independent experiments.

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Fig.6. Mitochondrial membrane potential- JC1 staining. (a) and (d) Representative images of untreated control cells and cells treated with 100µM melatonin respectively. The images (b) and (c) represents cells treated with ZnO NPs concentrations 20µg/ml and 80µg/ml respectively. The (e) and (f) are representative images of melatonin pre-incubated cells treated with 20µg/ml and 80µg/ml of ZnO NPs respectively

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Fig.7. The antioxidant assay results of brain slices incubated with melatonin and ZnO NPs for 3h. The data represents mean ±SD of three independent experiments. Asterisks in the graph indicate statistically significant difference among ZnO NPs treatment with and without melatonin pretreatment (*P<0.05)

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