Induction of cell death by graphene in Arabidopsis thaliana (Columbia ecotype) T87 cell suspensions.

The toxicity of graphene on suspensions of Arabidopsis thaliana (Columbia ecotype) T87 cells was investigated by examining the morphology, mitochondrial dysfunction, reactive oxygen species generation (ROS), and translocation of graphene as the toxicological endpoints. The cells were grown in Jouanneau and Péaud-Lenoel (JPL) media and exposed to graphene at concentrations 0-80 mg/L. Morphological changes were observed by scanning electron microscope and the adverse effects such as fragmented nuclei, membrane damage, mitochondrial dysfunction was observed with fluorescence microscopy by staining with Hoechst 33342/propidium iodide and succinate dehydrogenase (mitochondrial bioenergetic enzyme). Analysis of intracellular ROS by 2',7'-dichlorofluorescein diacetate demonstrated that graphene induced a 3.3-fold increase in ROS, suggesting that ROS are key mediators in the cell death signaling pathway. Transmission electron microscopy verified the translocation of graphene into cells and an endocytosis-like structure was observed which suggested graphene entering into the cells by endocytosis. In conclusion, our results show that graphene induced cell death in T87 cells through mitochondrial damage mediated by ROS.

[1]  Kan Wang,et al.  Single Walled Carbon Nanotubes Exhibit Dual-Phase Regulation to Exposed Arabidopsis Mesophyll Cells , 2010, Nanoscale research letters.

[2]  E. Marra,et al.  The apoptosis/necrosis transition in cerebellar granule cells depends on the mutual relationship of the antioxidant and the proteolytic systems which regulate ROS production and cytochrome c release en route to death , 2003, Journal of neurochemistry.

[3]  Gerard I. Evan,et al.  The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant , 2000, Nature Cell Biology.

[4]  Yongsheng Chen,et al.  Superparamagnetic graphene oxide–Fe3O4nanoparticles hybrid for controlled targeted drug carriers , 2009 .

[5]  F. Breusegem,et al.  Morphological classification of plant cell deaths , 2011, Cell Death and Differentiation.

[6]  Tetsuya Watanabe,et al.  Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death , 2005, Nature.

[7]  L. Zitvogel,et al.  Decoding Cell Death Signals in Inflammation and Immunity , 2010, Cell.

[8]  Zhuang Liu,et al.  Carbon nanotubes as intracellular transporters for proteins and DNA: an investigation of the uptake mechanism and pathway. , 2006, Angewandte Chemie.

[9]  C. Edelstein,et al.  Caspases and calpain are independent mediators of cisplatin-induced endothelial cell necrosis. , 2006, American journal of physiology. Renal physiology.

[10]  Stefan Vogt,et al.  Uptake and distribution of ultrasmall anatase TiO2 Alizarin red S nanoconjugates in Arabidopsis thaliana. , 2010, Nano letters.

[11]  S. Stankovich,et al.  Graphene-based composite materials , 2006, Nature.

[12]  Jannik C. Meyer,et al.  The structure of suspended graphene sheets , 2007, Nature.

[13]  Yanli Chang,et al.  In vitro toxicity evaluation of graphene oxide on A549 cells. , 2011, Toxicology letters.

[14]  Yang Deng,et al.  Interactions between engineered nanoparticles (ENPs) and plants: phytotoxicity, uptake and accumulation. , 2010, The Science of the total environment.

[15]  Maumita Bandyopadhyay,et al.  Multi-walled carbon nanotubes (MWCNT): induction of DNA damage in plant and mammalian cells. , 2011, Journal of hazardous materials.

[16]  J. Hickman,et al.  Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. , 1993, The EMBO journal.

[17]  Bunshi Fugetsu,et al.  Graphene phytotoxicity in the seedling stage of cabbage, tomato, red spinach, and lettuce , 2011 .

[18]  Yu-Chang Tsai,et al.  Developmental phytotoxicity of metal oxide nanoparticles to Arabidopsis thaliana , 2010, Environmental toxicology and chemistry.

[19]  A. Brambrink,et al.  Neuronal Death in Newborn Striatum after Hypoxia-Ischemia Is Necrosis and Evolves with Oxidative Stress , 2000, Neurobiology of Disease.

[20]  M. Redlak,et al.  Oxygen Radical Induced Gastric Mucosal Cell Death: Apoptosis or Necrosis? , 2008, Digestive Diseases and Sciences.

[21]  Eleonore Fröhlich,et al.  A Yeast Mutant Showing Diagnostic Markers of Early and Late Apoptosis , 1997, The Journal of cell biology.

[22]  R. Ruoff,et al.  The chemistry of graphene oxide. , 2010, Chemical Society reviews.

[23]  Zhuang Liu,et al.  Nano-graphene oxide for cellular imaging and drug delivery , 2008, Nano research.

[24]  C. Curie,et al.  A protocol for transient gene expression in Arabidopsis thaliana protoplasts isolated from cell suspension cultures , 1992 .

[25]  D. Bouchez,et al.  Mitochondrial succinic-semialdehyde dehydrogenase of the γ-aminobutyrate shunt is required to restrict levels of reactive oxygen intermediates in plants , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[26]  B. Fugetsu,et al.  Phytotoxicity of multi-walled carbon nanotubes on red spinach (Amaranthus tricolor L) and the role of ascorbic acid as an antioxidant. , 2012, Journal of hazardous materials.

[27]  R. R. Ariza,et al.  cDNA cloning, expression and functional characterization of an Arabidopsis thaliana homologue of the Escherichia coli DNA repair enzyme endonuclease III , 2000, Plant Molecular Biology.

[28]  Andre K. Geim,et al.  The rise of graphene. , 2007, Nature materials.

[29]  D. Green,et al.  The BCL-2 family reunion. , 2010, Molecular cell.

[30]  Y. Kim,et al.  Evaluation of CNT toxicity by comparison to tattoo ink , 2011 .

[31]  B. Nowack,et al.  Occurrence, behavior and effects of nanoparticles in the environment. , 2007, Environmental pollution.

[32]  B. Fugetsu,et al.  Studies on toxicity of multi-walled carbon nanotubes on suspension rice cells , 2009 .

[33]  Zhuang Liu,et al.  PEGylated nanographene oxide for delivery of water-insoluble cancer drugs. , 2008, Journal of the American Chemical Society.

[34]  A. Wyllie,et al.  Apoptosis: A Basic Biological Phenomenon with Wide-ranging Implications in Tissue Kinetics , 1972, British Journal of Cancer.

[35]  Huang-Hao Yang,et al.  Using graphene to protect DNA from cleavage during cellular delivery. , 2010, Chemical communications.

[36]  J. Franklin,et al.  Cytochrome c release precedes mitochondrial membrane potential loss in cerebellar granule neuron apoptosis: lack of mitochondrial swelling , 2002, Journal of neurochemistry.

[37]  Alberto Bianco,et al.  Graphene: safe or toxic? The two faces of the medal. , 2013, Angewandte Chemie.

[38]  B. Fugetsu,et al.  Studies on toxicity of multi-walled carbon nanotubes on Arabidopsis T87 suspension cells. , 2009, Journal of hazardous materials.

[39]  G. Kroemer,et al.  Role of the Mitochondrial Permeability Transition Pore in Apoptosis , 1997, Bioscience reports.

[40]  H. Hirt,et al.  Reactive oxygen species: metabolism, oxidative stress, and signal transduction. , 2004, Annual review of plant biology.

[41]  Ying Liu,et al.  The triggering of apoptosis in macrophages by pristine graphene through the MAPK and TGF-beta signaling pathways. , 2012, Biomaterials.

[42]  T. Xia,et al.  Toxic Potential of Materials at the Nanolevel , 2006, Science.

[43]  G. Peters,et al.  Maintenance of ATP favours apoptosis over necrosis triggered by benzamide riboside , 2002, Cell Death and Differentiation.

[44]  H. Uchimiya,et al.  Mitochondrial behaviour in the early stages of ROS stress leading to cell death in Arabidopsis thaliana. , 2005, Annals of botany.

[45]  J. Greenberg,et al.  Programmed cell death: a way of life for plants. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[46]  T. Casoli,et al.  Quantitative cytochemistry of succinic dehydrogenase activity in rat mitochondria. , 1998, Analytical and quantitative cytology and histology.

[47]  C. Foyer,et al.  Ascorbic Acid Deficiency Activates Cell Death and Disease Resistance Responses in Arabidopsis1 , 2005, Plant Physiology.

[48]  A. Ganguli,et al.  Enhanced functionalization of Mn2O3@SiO2 core-shell nanostructures , 2011, Nanoscale research letters.

[49]  M. Prato,et al.  Translocation of bioactive peptides across cell membranes by carbon nanotubes. , 2004, Chemical communications.

[50]  N. Yao,et al.  Induction of programmed cell death in Arabidopsis and rice by single-wall carbon nanotubes. , 2010, American journal of botany.

[51]  Yingjin Yuan,et al.  Differentiation of apoptotic and necrotic cells in suspension cultures of Taxus cuspidata by the combined use of fluorescent dying and histochemical staining methods , 2004, Biotechnology Letters.

[52]  Agnes B Kane,et al.  Biological interactions of graphene-family nanomaterials: an interdisciplinary review. , 2012, Chemical research in toxicology.

[53]  Zhijun Zhang,et al.  Functional graphene oxide as a nanocarrier for controlled loading and targeted delivery of mixed anticancer drugs. , 2010, Small.

[54]  Yuliang Zhao,et al.  Nanotoxicology: Are carbon nanotubes safe? , 2008, Nature nanotechnology.

[55]  Xingfa Gao,et al.  Unraveling Stress‐Induced Toxicity Properties of Graphene Oxide and the Underlying Mechanism , 2012, Advanced materials.

[56]  Jiali Zhang,et al.  Biocompatibility of Graphene Oxide , 2010, Nanoscale research letters.

[57]  J. T. Chou,et al.  Succinate dehydrogenase and mitochondria in the hair cells in the organ of corti of mature and old shaker—1 mice , 1987, The Journal of Laryngology & Otology.

[58]  Filip Braet,et al.  Carbon nanomaterials in biosensors: should you use nanotubes or graphene? , 2010, Angewandte Chemie.

[59]  SUPARNA DUTTASINHA,et al.  Graphene: Status and Prospects , 2009, Science.