# I. Introduction uO-NPs are widely used in gas sensors, catalysts, high temperature conductors, solar energy converters and antimicrobial agents owing to their high temperature conductivity, electron correlation effects, antimicrobial activity and special physicochemical properties in various fields (Chang et al., 2012;Huang et al., 2010). Indeed, as it is well known, nanoparticles exist as contaminants in water, air and food products as outputs of natural phenomena or due to the high increase in the anthropogenic activity (Ahamed et al., 2013;Elsaesser et al., 2011;Kim et al., 2010). CuO-NPs caused changes in different organs like lung, kidney, renal tubular, liver, spleen, gastrointestinal tract and stomach tissue (Barceloux, 1999;Cho et al., 2012;Lei et al., 2008;Manna et al., 2012). Acute death, abnormalities in the embryo and gill damage were observed in Zebra fish exposed to CuO-NPs (Griffitt et al., 2007;Yeo et al., 2009). The toxicity studies of CuO-NPs have been focused more generally on the pulmonary system and to a lesser extent on skin, breast, intestine and liver (Ahamed et Perreault et al., 2012). Therefore, it was aimed to evaluate the toxicity and possible mechanism of action of CuO-NPs in neuroblastoma cells following their cellular uptake potential. # II. Materials and Methods Chemicals: Eagle's minimum essential medium (EMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS, 10X), antibiotic solutions and ethylene diamine tetraacetic acid (EDTA) were purchased from Multicell Wisent (Quebec, Canada). Triton X-100 and MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) were purchased from Biomatik (Ontario, Canada). GSH, 8-OHdG, MDA and PC ELISA kits were purchased from Yehua Biological Technology Co., Ltd. (Shanghai, China). Annexin V-FITC apoptosis detection kit with PI and dye reagents for protein assay were obtained from Exbio (Vestec, Czech Republic) and Biorad (Munich, Germany), respectively. All other chemicals were obtained from Merck (NJ, USA). CuO-NPs were obtained from Sigma Chemical Co. Ltd. (St. Louis, MO, USA). The CuO-NPs suspensions in milli-Q water and cell culture medium with 10% FBS, were measured by Transmission Electron Microscopy (TEM) (Jem-2100 HR, Jeol, USA) (Abudayyak et al. 2016;2016a). The average diameter was calculated by measuring over 100 particles in random fields of TEM view. Copper release into cell medium: Copper release from CuO-NPs into the cell culture medium was determined using the Inductively Coupled Plasma Mass Spectrometry (ICP-MS) (Thermo Elemental X series 2, USA) method (Abudayyak et al. 2016;2016a). The released amount of copper was analyzed by ICP-MS. Cu content of the cell culture medium was also measured. Cell culture conditions: Human neuronal cell line (SH-SY5Y) was obtained from the American Type Culture Collection (CRL-2266?, ATCC, VA, USA). The cells were incubated in EMEM medium supplemented with FBS 10% and antibiotics at 5% CO 2 , 90% humidity and 37°C for 24 h (60-80% confluence). Cell densities were in the range from 1× 10 5 to 1×10 7 cells/mL for all assays (Abudayyak et al. 2016;2016a). Exposure occurred for 24 h. Cellular uptake and morphology examinations: It was evaluated by ICP-MS and TEM (Abudayyak et al. 2016;2016a). The cells were washed several times with equal volumes of PBS and cell culture medium with 10% FBS and counted via Luna cell counter (Virginia, USA) following exposure to two different concentrations of the particle suspension (2.5 and 25 µg/mL). Ultra-thin sections (50-60 nm) were cut by an ultra-microtome (Reichert UM 3, Austria). Sections were analyzed and photographed using a TEM (Jeol-1011, Tokyo, Japan) with attached digital camera (Olympus-Veleta TEM Camera, Tokyo, Japan). Cytotoxicity assays: Cytotoxic activities of CuO-NPs on SH-SY5Y cells were determined by MTT and NRU assays based on different cellular mechanisms (Abudayyak et al. 2016;2016a;Repetto et al., 2008;Van Meerloo et al., 2011). Optical density (OD) values were read at 590 and 540 nm for MTT and NRU, respectively, using a microplate spectrophotometer system (Epoch, Germany). In every assay, unexposed cells were served as a negative control. The inhibition of enzyme activity was calculated as compared to a negative control. The half-maximal inhibitory concentration (IC 50 ) was then expressed as the concentration of the sample causing a 50% inhibition of enzyme activity in cells. The CuO-NP concentrations were 2.5-60 µg/mL in the cytotoxicity assays. Genotoxicity assay: Genotoxic activities of CuO-NPs were determined by comet assay (Abudayyak et al. 2016;2016a;Collins et al., 2004;Speit et al., 1999). Hydrogen peroxide (H 2 O 2 ) (100 µM) and PBS were used as positive and negative controls, respectively. The number of DNA breaks was scored under a fluorescent microscope (Olympus BX53, Olympus, Tokyo, Japan) at 400X magnification using an automated image analysis system (Comet Assay IV, Perceptive Instruments, Suffolk, UK). DNA damage to individual cells was expressed as a percentage of DNA in the comet tail (tail intensity %). The CuO-NP concentrations were 5-50 µg/mL in the comet assay. Oxidative damage assays: The oxidative damage potentials of CuO-NPs were measured by human GSH, MDA, 8-OHdG, or PC ELISA kits with different endpoints according to the manufacturer's instructions. The OD value was read at 450 nm using a microplate spectrophotometer system. In every assay, the unexposed cells served as a negative control. The protein amount in 10 6 cells was measured according to Bradford (1976). Results were expressed as µmol, µmol, µg, and µg per g protein for GSH, MDA, 8-OHdG, and PC, respectively, using a standard calibration curve. The CuO-NP concentrations were 5-25 µg/mL in the oxidative damage assays. Apoptosis assay: The cellular apoptosis or necrosis was determined by Annexin V-FITC apoptosis detection kit with PI (Abudayyak et al. 2016;2016a). In every assay, the untreated cells served as a negative control. The results were expressed as a percentage of the total cell amount. The CuO-NP concentrations were 10-80 µg/mL in the apoptosis assay. Statistical analysis: The assays were done in triplicate and repeated four times. Data were expressed as mean±standard deviation (SD). Significant differences between untreated and treated cells were calculated by one-way ANOVA Dunnett t-test using SPSS version 17.0 for Windows. p values of less than 0.05 were considered significant. # III. Results and Discussion Particle size and distribution: According to the X-ray diffraction results supplied by the manufacturer (Sigma Chemical Co. Ltd., USA), the surface area of CuO-NPs was 29 m 2 /g (Figure 1). The average size was observed to be 34.9 nm with a narrow size distribution (ranging from 16.7-64.2 nm) after suspending in water. When suspending in the culture medium, the size of the particles was found to be slightly agglomerated and/or aggregated with 38.8 nm (ranging from 18.8-73.8 nm) (Figure 2). The copper ion release of CuO-NPs was evaluated in the cell culture medium. Although the concentration was 3.1 ± 0.322 µg/mL, which represented 15.5% of the nanoparticles, in the CuO-NPs cell culture suspension, there was no observed copper ions in the cell culture medium. Based on that, the observed toxicological endpoints and morphological changes were mainly due to CuO-NPs. Cellular uptake: ICP-MS revealed that the particles were taken up by SH-SY5Y cells in the range of 0.390-0.917 µg/10 5 cells in concentration dependent manner following exposure to CuO-NPs at 5-25 µg/mL concentrations (Table 1). Some researchers reported iron oxide and two different types of titanium dioxide nanoparticles to enter SH-SY5Y cells in concentration dependent manner (Kilic et al., 2016;Valdiglesias et al., 2013). Cellular morphology by TEM: The particles were observed in the cytoplasmic vacuoles. Mitochondria were visible in few of the cells exposed to both 2.5 and 10 µg/mL CuO-NPs. Some cells exposed to 2.5 µg/mL CuO-NPs revealed nuclear fragmentation. The electronlucent cytoplasmic vacuoles lead to complete disruption of the cytoplasm in few of the cells (Figure 3). respectively. The reduction in cell viability was concentration-dependent (Figure 4). The CuO-NPs were found to cause cytotoxic effects to HaCaT keratinocytes, BALB3T3 embryonic fibroblasts ( (Akhtar et al., 2016). Also, it could be via disruption of cell membrane integrity (Cronholm et al., 2011). However, there was no study about genotoxicity on SH-SY5Y cells. Oxidative damage: The oxidative damage potential of CuO-NPs was evaluated by measuring cellular levels of GSH, MDA, 8-OHdG, and PC (Table 2). CuO-NPs induced oxidative damage resulting in significant decrease in the GSH levels (?46.1%). Although an increase on the levels of MDA (?1.33 fold) was observed it was not significant. On the other hand, the levels of PC and 8-OHdG protein and DNA oxidative damage biomarkers did not change. In previous studies, it was observed that CuO-NPs induced oxidative damage in HaCaT keratinocytes (Alarifi et al., 2013) (Piret et al., 2012;Siddiqui et al., 2013). The reduction in cell viability observed could be due to an increase in oxidative stress after CuO-NPs exposure. Apoptosis: Death in SH-SY5Y cells was significantly induced by CuO-NPs, with a maximum percentage of 73.4 and 40.0% for apoptosis and necrosis, respectively. According to our results, apoptosis was seen to be the main pathway for cell death in the SH-SY5Y cell line. At the highest exposure concentration (40 µg/mL), the apoptosis percentage was 79.2% of the dead cells (Figure 6). The previous studies showed CuO-NPs could induce apoptosis in the following cells: MCF7 breast cancer (Laha et al., 2014) 2013) observed CuO-NPs induced apoptosis via a decrease in mitochondrial membrane potential with a concomitant increase in the gene expression ratio of Bax/Bcl2, up-regulation of p53 tumour suppressor and caspase-3 apoptotic genes. Also, the researchers showed apoptosis could be induced by reduction of BAD phosphorylation and an increase in cleaved caspase-3 products (Laha et al., 2014). An et al. (2012) indicated that the apoptosis and cognitive impairment could be via increased cleaved caspase-3 levels on hippocampal CA1 neuron in rats. # IV. Conclusion Generally, the studies about Cu based nanoparticles and CuO-NPs were focused on the pulmonary system. However, very few researchers were concerned about the possible toxicity over other systems. In the present study, it was observed that CuO-NPs taken up by the neuronal cells could produce cytotoxic, genotoxic, and apoptotic effects, as well as oxidative damage in the neuronal cells in vitro. Their commercial and industrial applications should be carefully evaluated because of their potential hazardous effects on human health. Further in vivo studies are needed to fully understand the toxicity mechanisms of CuO-NPs. # V. Acknowledgement This work was supported by the Research Fund of Istanbul University (Project No: 52253). Dr. M. Abudayyak carried out cell culture and exposure conditions, the toxicological assays and the particle characterisation. Prof. Dr. G. Özhan participate the toxicological assays and carried out the evaluation of the results. Dr. E. Guzel carried out the uptake and morphological changes in the cells. All authors wrote, read and approved the manuscript. Also, the authors declare there is no conflict of interest. All experiments were done in triplicate and each assay was repeated four times. # Volume XVI Issue III Version I The results were expressed as the mean cell death (%) compared to negative control (unexposed cell). ![Cytotoxicity: IC 50 values of CuO-NPs were 25.49±2.06 and 7.27±0.843 µg/mL by MTT and NRU assay, Volume XVI Issue III Version I](image-2.png "") ![, HepG2 (Siddiqui et al., 2013), and Caco-2 cells (Piret et al., 2012). In rats, CuO-NPs induced apoptosis via increased cleaved caspase-3 levels (An et al., 2012). Siddiqui et al. (](image-3.png "") ![G, del Peso A, Zurita JL 2008. Neutral red uptake assay for the estimation of cell viability/ cytotoxicity. Nature Protocols 3: 1125-1131. 30. Siddiqui M, Alhadlaq HA, Ahmad J, et al. 2013. Copper oxide nanoparticles induced mitochondria mediated apoptosis in human hepatocarcinoma cells. PloS One 8(8): e69534. 31. Speit G, Hartmann A. 1999. The comet assay (single-cell gel test): A sensitive genotoxicity test for the detection of DNA damage and repair. DNA Repair Protocols 113: 203-212. 32. Sun J, Wang S, Zhao D, et al. 2011. Cytotoxicity, permeability, and inflammation of metal oxide nanoparticles in human cardiac microvascular endothelial cells. Cell Biol. Toxicol. 27(5): 333-342. 33. Valdiglesias V, Costa C, Sharma V, et al. 2013.](image-4.png "") 1![Figure 1 : The X-ray diffraction analysis of CuO-NPs.](image-5.png "Figure 1 :") 2![Figure 2 : The TEM images and size distributions of CuO-NPs after dissolution in water (a) and cell culture medium (b).](image-6.png "Figure 2 :") 3![Figure 3 : TEM observations of SH-SY5Y cells after exposure to CuO-NPs. (a) cells exposed to CuO-NPs at 2.5 µg/mL; (b) cells exposed to CuO-NPs at 10 µg/mL; (c) unexposed cell (negative control).](image-7.png "Figure 3 :") 4![Figure 4 : Effects of CuO-NPs on SH-SY5Y cell viability.](image-8.png "Figure 4 :") © 2 016 Global Journals Inc. (US)Copper (II) Oxide Nanoparticles Induce High Toxicity in Human Neuronal Cell © 2016 Global Journals Inc. (US)Copper (II) Oxide Nanoparticles Induce High Toxicity in Human Neuronal Cell All experiments were done in triplicate and each assay was repeated four times. The results were presented as percentages of the total cell amount. ## Necrosis Apoptosis Cu content of the negative control (unexposed cell) was also measured. Every assay was repeated four times. The results were expressed as mean ± SD. The protein amount calculated for 4x10 4 cells in every assay according to Bradford (1976). The results were expressed as µmol, µmol, µg and µg per g protein for GSH, MDA, 8-OHdG and PC, respectively, using standard calibration curve. *p ?0.05 were selected as the levels of significance. * Nickel oxide nanoparticles exert cytotoxicity via oxidative stress and induce apoptotic response in human liver cells (HepG2) MAhamed DAli HAAlhadlaq Chemosphere 93 2013 * In vitro toxicological evaluation of cobalt ferrite nanoparticles MAbudayyak TAlt?ncekic GÖzhan Doi: 10.1007/s 12011-016-0803-3 Biol. Trace Element Res 2016 * Copper (II) oxide nanoparticles induced nephrotoxicity in vitro conditions MAbudayyak EEGuzel GÖzhan 10.1089/aivt.2016.0008 Appl. Vitro Toxicol 2016a * Genotoxic potential of copper oxide nanoparticles in human lung epithelial cells MAhamed MASiddiqui MJAkhtar Biochem. Biophys. Res. Commun 396 2010 * Protective effect of sulphoraphane against oxidative stress mediated toxicity induced by CuO nanoparticles in mouse embryonic fibroblasts BALB 3T3 MJAkhtar MAhamed MFareed J. Toxicol. Sci 37 1 2012 * Dose-dependent genotoxicity of copper oxide nanoparticles stimulated by reactive oxygen species in human lung epithelial cells MJAkhtar SKumar HAAlhadlaq Toxicol. Ind. Health 32 5 2016 * Cytotoxicity and genotoxicity of copper oxide nanoparticles in human skin keratinocytes cells SAlarifi DAli AVerma Int. J. Toxicol 32 4 2013 * Cognitive impairment in rats induced by nano-CuO and its possible mechanisms LAn SLiu ZYang Toxicol. Lett 213 2 2012 * DGBarceloux Copper. J. Toxicol. Clin. Toxicol 37 1999 * A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding MMBradford Anal. Biochem 7 1976 * The toxic effects and mechanisms of CuO and ZnO nanoparticles YNChang MZhang LXia Materials 5 2012 * Differential cytotoxicity of metal oxide nanoparticles JChen JZhu H-HCho J. Exp. Nanosci 3 2008 * Differential pro-inflammatory effects of metal oxide nanoparticles and their soluble ions in vitro and in vivo; zinc and copper nanoparticles, but not their ions,recruit eosinophils to the lungs WSCho RDuffin CAPoland Nanotoxicol 6 2012 * The comet assay for DNA damage and repair principles, applications, and limitations ARCollins Mol. Biotechnol 26 2004 * Effect of sonication and serum proteins on copper release from copper nanoparticles and the toxicity towards lung epithelial cells PCronholm KMidander HLKarlsson Nanotoxicol 5 2 2011 * Interference of CuO nanoparticles with metal homeostasis in hepatocytes under sub-toxic conditions MCuillel MChevallet PCharbonnier Nanoscale 6 3 2014 * Toxicology of nanoparticles AElsaesser CVHoward Drug Deliv Rev 64 2011 Adv * Copper oxide nanoparticles induce oxidative stress and cytotoxicity in airway epithelial cells BFahmy SACormier Toxicol. in Vitro 23 7 2009 * Exposure to copper nanoparticles causes gill injury and acute lethality in zebrafish (Danio rerio) RJGriffitt RWeil KAHyndman Environ. Sci. Technol 41 2007 * Toxicity of transition metal oxide nanoparticles: Recent insights from in vitro studies YWHuang CHWu RSAronstam Materials 3 2010 * Copper oxide nanoparticles are highly toxic: a comparison between metal oxide nanoparticles and carbon nanotubes HLKarlsson PCronholm JGustafsson Chem. Res. Toxicol 21 9 2008 * In vitro toxicity evaluation of silica-coated iron oxide nanoparticles in human SHSY5Y neuronal cells GKilic CCosta NFernández-Bertólez Toxicol. Res 5 2016 * Comparative study of cytotoxicity, oxidative stress and genotoxicity induced by silica nanomaterials in human neuronal cell line YJKim MYu HOPark Mol. Cell. Toxicol 6 2010 * Interplay between autophagy and apoptosis mediated by copper oxide nanoparticles in human breast cancer cells MCF7 DLaha APramanik JMaity Biochim. Biophys. Acta 1840 2014 * Integrated metabolomic analysis of the nano-sized copper particle-induced hepatotoxicity and nephrotoxicity in rats: a rapid in vivo screening method for nanotoxicity RLei CWu BYang Toxicol. Appl. Pharmacol 232 2 2008 * Contribution of nano-copper particles to in vivo liver dysfunction and cellular damage: role of I?B?/NF-?B, MAPKs and mitochondrial signal PManna MGhosh JGhosh Nanotoxicol 6 2012 * Genotoxic effects of copper oxide nanoparticles in Neuro 2A cell cultures FPerreault SPMelegari CHDe Costa Sci. Total Environ 441 2012 * Copper (II) oxide nanoparticles penetrate into HepG2 cells, exert cytotoxicity via oxidative stress and induce pro-inflammatory response JPPiret DJacques JNAudinot Nanoscale 4 2012