Buse KAYHAN, Şeyma TAŞDEMİR, Pelin İLHAN ÇORUK, Cansu GÖRGÜN, Aylin ÜRKMEZ ŞENDEMİR, GÜLGÜN ŞENGÜL

Graphene oxide has a neuroprotective effect against glutamate-induced excitoxicity on B35 neuroblastoma cell line*

Objectives:Graphene is a quasi-two-dimensional material with unique electrical and chemical properties. In terms of biomedical applications of graphene, nervous system would be an ideal breakthrough model because neural cells are electroactive. Extreme glutamate concentrations cause excitotoxicity. In this study, we aimed to investigate if graphene can increasethe resistance to glutamate stress in B35 rat neuroblastoma cells as a cultured cell model for central nervous system neurons. Methods: B35 neuroblastoma cells were grown in DMEM-F12 growth medium containing 10% fetal bovine serum.Graphene oxide (GO) powder was coated onto glass slides with chitosan as a thin film. B35 cells were cultured on GO films.Cells cultivated on glass slides were used as controls. After 24 h of cell culture, L-glutamine induced excitotoxicity wasimposed on B35 cells. After 24 h of glutamate-induced stress, cell morphology was examined by scanning electronmicroscopy. Cell viability was measured with MTT assay.Results: The effects of glutamate stress on cell viability were visible as early as 1 h. The cell viability on GO films was higher than that on glass slides, and cells recovered from stress within 6 h on GO surfaces. After 24 h, viability on glass surfaceswas 54% lower than that on GO surfaces; these findings were supported with cell morphology observations.. Conclusion:The results of this study showed that GO has a protective role in reducing glutamate-induced excitotoxicity inB35 cell culture, indicating a potential use of GO for treatment of excitotoxicity induced neurodegenerative diseases.

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Zhou Y, Danbolt NC. Glutamate as a neurotransmitter in the healthy brain. J Neural Transm 2014;121:799-817.

Danbolt NC. Glutamate uptake. Prog Neurobiol 2001;65:1-105.

Kritis AA, Stamoula EG, Paniskaki KA, Vavilis TD. Researching glutamate- induced cytotoxicity in different cell lines: a compara- tive/collective analysis/study. Front Cell Neurosci 2015;17:9-91.

Otey CA, Boukhelifa M and Maness P. B35 neuroblastoma cells: an easily transfected, cultured cell model of central nervous system neurons. Methods Cell Biol 2003;71:287-304.

Hosseini M, Khabbaz H, Dezfoli AS, Ganjali MR, Dadmehr M. Selective recognition of Glutamate based on fluorescence enhancement of graphene quantum dot. Spectrochim Acta A Mol Biomol Spectrosc 2014;136PC:1962-6.

Michaels RL, Rothman SM. Glutamate neurotoxicity in vitro: antagonist pharmacology and intracellular calcium concentrations. J Neurosci 1990;10:283-92.

Murphy TH, Miyamoto M, Sastre A, Schnaar RL, Coyle, JT. Glutamate toxicity in a neuronal cell line involves inhibition of cys- tine transport leading to oxidative stress. Neuron 1989;2:1547-58.

Bannai S. Exchange of cystine and glutamate across plasma mem- brane of human fibroblasts. J Biol Chem 1986;261:2256-63.

Murphy TH, Schnaar RL, Coyle JT. Immature cortical neurons are uniquely sensitive to glutamate toxicity by inhibition of cystine uptake. FASEB J 1990;4:1624-33.

Oka A, Belliveau MJ, Rosenberg PA, Volpe JJ. Vulnerability of oligodendroglia to glutamate: pharmacology, mechanisms and pre- vention. J Neurosci 1993;13:1441-53.

Davis JB, Maher P. Protein kinase C activation inhibits glutamate- induced cytotoxicity in a neuronal cell line. Brain Res 1994;652:169- 73.

Kato S, Higashida H, Higuchi Y, Hatakenaka S, Negishi K. Sensitive and insensitive states of cultured glioma cells to gluta- mate damage. Brain Res 1984;303:365-73.

Kato S, Negishi K, Mawatari K, Kuo CH. A mechanism for gluta- mate toxicity in the C6 glioma cells involving inhibition of cystine uptake leading to glutathione depletion. Neuroscience 1992;48:903-14.

Miyamoto M, Murphy TH, Schnaar RL, Coyle JT. Antioxidants protect against glutamate-induced cytotoxicity in a neuronal cell line. J Pharmacol Exp Ther 1989;250:1132-40.

Schubert D, Kimura H, Maher P. Growth factors and vitamin E modify neuronal glutamate toxicity. Proc Natl Acad Sci 1992;89:8264-7.

Tong G, Jahr CE. Block of glutamate transporters potentiates postsynaptic excitation. Neuron 1994;13:1195-203.

Diamond JS, Jahr CE. Transporters buffer synaptically released glutamate on a submillisecond time scale. J Neurosci 1997;17:4672-87.

Rothstein JD, Dykes-Hoberg M, Pardo CA, Bristol LA, Jin L, Kuncl RW, Kanai Y, Hediger MA, Wang Y, Schielke JP, Welty DF. Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron 1996;16:675-86.

Tanaka K, Watase K, Manabe T, Yamada K, Watanabe M, Takahashi K, Iwama H, Nishikawa T, Ichihara N, Kikuchi T, Okuyama S, Kawashima N, Hori S, Takimoto M, Wada K. Epilepsy and exacerbation of brain injury in mice lacking the glu- tamate transporter GLT-1. Science 1997;276:1699-702.

Gegelashvili G, Robinson MB, Trotti D, Rauen T. Regulation of glutamate transporters in health and disease. Prog Brain Res 2001;132:267-86.

Choi DW. Glutamate receptors and the induction of excitotoxic neuronal death. Prog Brain Res 1994;100:47-51.

Maragakis NJ, Rothstein JD. Glutamate transporters in neurolog- ic disease. Arch Neurol 2001;58:365-70.

Rossi DJ, Oshima T, Attwell D. Glutamate release in severe brain ischaemia is mainly by reversed uptake. Nature 2000;403:316-21.

Dutuit M, Touret M, Szymocha R, Nehlig A, Belin MF, Didier- Bazès M. Decreased expression of glutamate transporters in genet- ic absence epilepsy rats before seizure occurrence. J Neurochem 2002;80:1029-38.

Masliah E, Alford M, DeTeresa R, Mallory M, Hansen L. Deficient glutamate transport is associated with neurodegenera- tion in Alzheimer's disease. Ann Neurol 1996;40:759-66.

Plaitakis A, Shashidharan P. Glutamate transport and metabolism in dopaminergic neurons of substantia nigra: implications for the pathogenesis of Parkinson's disease. J Neurol 2000;247:S2:II25- 35.

Rothstein JD, Martin LJ, Kuncl RW. Decreased glutamate trans- port by the brain and spinal cord in amyotrophic lateral sclerosis. N Engl J Med 1992;326:1464-8.

Gilmore JL, Yi X, Quan L, Kabanov AV. Novel nanomaterials for clinical neuroscience. J Neuroimmune Pharmacol 2008;3:83-94.

Kabanov AV, Gendelman HE. Nanomedicine in the diagnosis and therapy of neurodegenerative disorders. Prog Polym Sci 2007;32: 1054-82.

Subramanian A, Krishnan UM, Sethuraman S. Development of biomaterial scaffold for nerve tissue engineering: Biomaterial mediated neural regeneration. J Biomed Sci 2009;16:108.

Ryu S, Kim BY. Culture of Neural Cells and Stem Cells on Graphene. J Tissue Eng Regen Med 2013;2:39-46.

Park S, Mohanty N, Suk JW, Nagaraja A, An J, Piner RD, Cai W, Dreyer DR, Berry V, Ruoff RS. Biocompatible, robust free-stand- ing paper composed of a TWEEN/graphene composite. Adv Mater 2010;22:1736-40.

Agarwal S, Zhou X, Ye F, He Q, Chen GC, Soo J, Boey F, Zhang H, Chen P. Interfacing live cells with nanocarbon substrates. Langmuir 2010;26:2244-7.

Lv M, Zhang Y, Liang L, Wei M, Hu W, Li X, Huang Q. Effect of graphene oxide on undifferentiated and retinoic acid-differenti- ated SH-SY5Y cells line. Nanoscale 2012;4:3861-6.

Lee WC, Lim CH, Shi H, Tang LA, Wang Y, Lim CT, Loh KP. Origin of enhanced stem cell growth and differentiation on graphene and graphene oxide. ACS Nano 2011;5:7334-41.

Paredes JI, Villar-Rodil S, Martínez-Alonso A, Tascón JM. Graphene oxide dispersions in organic solvents. Langmuir 2008;24:10560-4.

Rinaudo M. Chitin and chitosan: Properties and applications. Prog Polym Sci 2006;31:603-32.

Yang X, Tu Y, Li L, Shang S, Tao XM. Well-dispersed chi- tosan/graphene oxide nanocomposites. ACS Appl Mater Interfaces 2010;2:1707-13.

Zhang Y, Ali SF, Dervishi E, Xu Y, Li Z, Casciano D, Biris AS. Cytotoxicity effects of graphene and single-wall carbon nanotubes in neural phaeochromocytoma-derived PC12 cells. ACS Nano 2010;4:3181-6.

Croslan DR, Schoell MC, Ford GD, Pulliam JV, Gates A, Clement CM, Harris AE, Ford BD. Neuroprotective effects of neuregulin-1 on B35 neuronal cells following ischemia. Brain Res 2008; 1210:39-47.

Li N, Zhang X, Song Q, Su R, Zhang Q, Kong T, Liu L, Jin G, Tang M, Cheng G. The promotion of neurite sprouting and out- growth of mouse hippocampal cells in culture by graphene sub- strates. Biomaterials 2011;32:9374-82.

Park SY, Park J, Sim SH, Sung MG, Kim KS, Hong BH, Hong S. Enhanced differentiation of human neural stem cells into neurons on graphene. Adv Mater 2011;23:H263-7

Weaver CL, Cui XT. Directed Neural Stem Cell Differentiation with a Functionalized Graphene Oxide Nanocomposite. Adv Healthc Mater 2015;4:1408-16.

Shah S, Yin PT, Uehara TM, Chueng ST, Yang L, Lee KB. Guiding stem cell differentiation into oligodendrocytes using graphene-nanofiber hybrid scaffolds. Adv Mater 2014;26:3673-80.

Wang Y, Lee WC, Manga KK, Ang PK, Lu J, Liu YP, Lim CT, Loh KP. Fluorinated graphene for promoting neuro-induction of stem cells. Adv Mater 2012; 24:4285-90.

Bae S, Kim H, Lee Y, Xu X, Park JS, Zheng Y, Balakrishnan J, Lei T, Kim HR, Song YI, Kim YJ, Kim KS, Ozyilmaz B, Ahn JH, Hong BH, Iijima S. Roll-to-roll production of 30-inch graphene films for transparent electrodes. Nat Nanotechnol 2010;5:574-8.

Depan D, Girase B, Shah JS, Misra RD. Structure-process-prop- erty relationship of the polar graphene oxide-mediated cellular response and stimulated growth of osteoblasts on hybrid chitosan network structure nanocomposite scaffolds. Acta Biomater 2011;7: 3432-45.

Zuo PP, Feng HF, Xu ZZ, Zhang LF, Zhang YL, Xia W, Zhang WQ. Fabrication of biocompatible and mechanically reinforced graphene oxide-chitosan nanocomposite films. Chem Cent J 2013; 7:39.

Pan Y, Wu T, Bao H, Li L. Green fabrication of chitosan films reinforced with parallel aligned graphene oxide. Carbohydrate Polymers 2011;83:1908-15.

Anderson CM, Swanson RA. Astrocyte glutamate transport: review of properties, regulation, and physiological functions. Glia 2000;32:1-14.

Greene LA, Tischler AS. Establishment of an or adrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor Proc Natl Acad Sci 1976;73:2424-8.

Pereira CM, Oliveira CR. Glutamate toxicity on a PC12 cell line involves glutathione (GSH) depletion and oxidative stress. Free Radic Biol 1997;23:637-47.

Pereira CF, Oliveira CR. Oxidative glutamate toxicity involves mitochondrial dysfunction and perturbation of intracellular Ca2+ homeostasis. Neurosci Res 2000;37:227-66.

Penugonda S, Mare S, Lutz P, Banks WA, Ercal N. Potentiation of lead- induced cell death in PC12 cells by glutamate: protection by N-acetylcysteineamide (NACA), a novel thiol antioxidant. Toxicol Appl Pharmacol 2006;216:197-205.

Pourzitaki C, Klagas I, Kaidoglou A, Tzimagiorgis G, Kritis A. Caspase dependent and independent cell death in naive and NGF treated PC12 cells after glutamate induced exitotoxicity. Epitheor Klin Farmakol Farmakokinet 2007;25:94-7.

Pourzitaki C, Kanellos G, Klagas I, Kritis A. Combined treatment of as partyl protease inhibitor and NMDA antagonist in Pc12 cells after glutamate excitotoxicity. Rev Clin Pharmacol Int Ed 2008;22: 304-7.

Pourzitaki C, Klagas I, Kanellos G, Kritis A. Calpaine mediated cell death in naive and NGF treated PC12 cells after glutamate induced exitotoxicity. Epitheor Klin Farmakol Farmakokinet 2009; 27:79-82.

Lu S, Lu C, Han Q, Li J, Du Z, Liao L, Zhao RC. Adipose-derived mesenchymal stem cells protect PC12 cells from glutamate excito- toxicity-induced apoptosis by upregulation of XIAP through PI3- K/Akt activation. Toxicology 2010;279:189-95.

Bal-Price A, Brown GC. Nitric-oxide-induced necrosis and apop- tosis in PC12 cells mediated by mitochondria. J Neurochem 2000; 75:1455-64.

Ma S, Liu H, Jiao H, Wang L, Chen L, Liang J, Zhao M, Zhang X. Neuroprotective effect of ginkgolide K on glutamate-induced cytotoxicity in PC 12 cells via inhibition of ROS generation and Ca (2+) influx. Neurotoxicology 2011;33:59-69.