In planta genotoxicity of nZVI: influence of colloidal stability on uptake, DNA damage, oxidative stress and cell death

Nanoremediation of soil, ground and surface water using nanoscale zerovalent iron particles (nZVI) has facilitated their direct environmental exposure posing ecotoxicological concerns. Numerous studies elucidate their phytotoxicity in terms of growth and their fate within the plant system. However, their potential genotoxicity and cytotoxicity mechanisms are not known in plants. This study encompasses the physico-chemical characterisation of two forms of nZVI (nZVI-1 and nZVI-2) with different surface chemistries and their influence on uptake, root morphology, DNA damage, oxidative stress and cell death in Allium cepa roots after 24 h. To our knowledge, this is the first report on the cyto-genotoxicity of nZVI in plants. The adsorption of nZVI on root surfaces caused root tip, epidermal and root hair damage as assessed by Scanning Electron Microscopy. nZVI-1, due to its colloidal destabilisation (low zeta potential, conductivity and high polydispersity index), smaller size and high uptake imparted enhanced DNA damage, chromosome/nuclear aberrations (CAs/NAs) and micronuclei formation compared to nZVI-2. Although nZVI-2 exhibited high zeta potential and conductivity, its higher dissolution and substantial uptake induced genotoxicity. nZVI incited the generation of reactive oxygen species (ROS) (hydrogen peroxide, superoxide and hydroxyl radicals) leading to membrane lipid peroxidation, electrolyte leakage and mitochondrial depolarisation. The inactivation of catalase and insignificant glutathione levels marked the onset of oxidative stress. Increased superoxide dismutase and guaiacol peroxidase enzyme activities, and proline content indicated the activation of antioxidant defence machinery to alleviate ROS. Moreover, ROS-mediated apoptotic and necrotic cell death occurred in both nZVI-1 and nZVI-2-treated roots. Our results open up further possibilities in the environmental safety appraisal of bare and modified nZVI in correlation with their physico-chemical characters.

[1]  N. Tuteja,et al.  Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. , 2010, Plant physiology and biochemistry : PPB.

[2]  Gen-xuan Wang,et al.  Calcium‐Mediated Mitochondrial Permeability Transition Involved in Hydrogen Peroxide‐Induced Apoptosis in Tobacco Protoplasts , 2006 .

[3]  M. Faisal,et al.  Changes in photosynthetic activity, pigment composition, electrolyte leakage, lipid peroxidation, and antioxidant enzymes during ex vitro establishment of micropropagated Rauvolfia tetraphylla plantlets , 2009, Plant Cell, Tissue and Organ Culture (PCTOC).

[4]  Armand Masion,et al.  Relation between the redox state of iron-based nanoparticles and their cytotoxicity toward Escherichia coli. , 2008, Environmental science & technology.

[5]  C. Clapp,et al.  Mechanisms of plant growth stimulation by humic substances: The role of organo-iron complexes , 2004 .

[6]  V. Rotello,et al.  Effect of Surface Charge on the Uptake and Distribution of Gold Nanoparticles in Four Plant Species , 2012, Environmental science & technology.

[7]  Yen-Ping Peng,et al.  Nano zerovalent iron particles induce pulmonary and cardiovascular toxicity in an in vitro human co-culture model , 2016, Nanotoxicology.

[8]  A. Samsudin,et al.  Development of Nano-Zero Valent Iron for the Remediation of Contaminated Water , 2012 .

[9]  Warren C W Chan,et al.  Nanoparticle-mediated cellular response is size-dependent. , 2008, Nature nanotechnology.

[10]  A. Sánchez-Moreiras,et al.  The natural compound trans-chalcone induces programmed cell death in Arabidopsis thaliana roots. , 2012, Plant, cell & environment.

[11]  U. Epa,et al.  Office of Solid Waste and Emergency Response , 2002 .

[12]  A. Mukherjee,et al.  Sensitivity of Allium cepa and Vicia faba towards cadmium toxicity , 2014 .

[13]  E. Ábrahám,et al.  Methods for determination of proline in plants. , 2010, Methods in molecular biology.

[14]  Roberto Rosal,et al.  A Colloidal Singularity Reveals the Crucial Role of Colloidal Stability for Nanomaterials In-Vitro Toxicity Testing: nZVI-Microalgae Colloidal System as a Case Study , 2014, PloS one.

[15]  J. Sedlák,et al.  Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. , 1968, Analytical biochemistry.

[16]  B. Mueller‐Roeber,et al.  ROS-mediated abiotic stress-induced programmed cell death in plants , 2015, Front. Plant Sci..

[17]  I. D. Teare,et al.  Rapid determination of free proline for water-stress studies , 1973, Plant and Soil.

[18]  Yang Deng,et al.  Phytotoxicity and uptake of nanoscale zero-valent iron (nZVI) by two plant species. , 2013, The Science of the total environment.

[19]  Maumita Bandyopadhyay,et al.  Genotoxicity of titanium dioxide (TiO2) nanoparticles at two trophic levels: plant and human lymphocytes. , 2010, Chemosphere.

[20]  N. Chandrasekaran,et al.  A comparative study with biologically and chemically synthesized nZVI: applications in Cr (VI) removal and ecotoxicity assessment using indigenous microorganisms from chromium-contaminated site , 2016, Environmental Science and Pollution Research.

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

[22]  S. McNeil.  Characterization of Nanoparticles Intended for Drug Delivery , 2011, Methods in Molecular Biology.

[23]  Awadhesh N Jha,et al.  Stabilization of engineered zero-valent nanoiron with Na-acrylic copolymer enhances spermiotoxicity. , 2011, Environmental science & technology.

[24]  A. Mukherjee,et al.  Effects of ZnO nanoparticles in plants: Cytotoxicity, genotoxicity, deregulation of antioxidant defenses, and cell-cycle arrest. , 2016, Mutation research. Genetic toxicology and environmental mutagenesis.

[25]  M. A. Marin-Morales,et al.  Allium cepa test in environmental monitoring: a review on its application. , 2009, Mutation research.

[26]  Warren C W Chan,et al.  Understanding and controlling the interaction of nanomaterials with proteins in a physiological environment. , 2012, Chemical Society reviews.

[27]  Maumita Bandyopadhyay,et al.  MWCNT uptake in Allium cepa root cells induces cytotoxic and genotoxic responses and results in DNA hyper-methylation. , 2015, Mutation research.

[28]  G. Speit,et al.  The low molecular weight DNA diffusion assay as an indicator of cytotoxicity for the in vitro comet assay. , 2014, Mutagenesis.

[29]  N. Mock,et al.  An improved method for monitoring cell death in cell suspension and leaf disc assays using evans blue , 1994, Plant Cell, Tissue and Organ Culture.

[30]  T. Begley,et al.  Oral ingestion of silver nanoparticles induces genomic instability and DNA damage in multiple tissues , 2015, Nanotoxicology.

[31]  Bernd Nowack,et al.  Application of nanoscale zero valent iron (NZVI) for groundwater remediation in Europe , 2012, Environmental Science and Pollution Research.

[32]  Miroslav Mashlan,et al.  Multimodal action and selective toxicity of zerovalent iron nanoparticles against cyanobacteria. , 2012, Environmental science & technology.

[33]  N. Chandrasekaran,et al.  In Vivo Genotoxicity Assessment of Titanium Dioxide Nanoparticles by Allium cepa Root Tip Assay at High Exposure Concentrations , 2014, PloS one.

[34]  Pratim Biswas,et al.  Characterization of size, surface charge, and agglomeration state of nanoparticle dispersions for toxicological studies , 2009 .

[35]  N. Chandrasekaran,et al.  In vivo nanotoxicity assays in plant models. , 2012, Methods in molecular biology.

[36]  Sedigheh Mehrabian,et al.  Antimicrobial and Genotoxicity Effects of Zero-valent Iron Nanoparticles , 2014, Jundishapur journal of microbiology.

[37]  Da Xing,et al.  Implication of reactive oxygen species and mitochondrial dysfunction in the early stages of plant programmed cell death induced by ultraviolet-C overexposure , 2008, Planta.

[38]  Tanapon Phenrat,et al.  Partial oxidation ("aging") and surface modification decrease the toxicity of nanosized zerovalent iron. , 2009, Environmental science & technology.

[39]  S-H A Y D E N.  Mechanistic Toxicity Assessment of Nanomaterials by Whole-Cell-Array Stress Genes Expression Analysis , 2010 .

[40]  M. Plewa,et al.  Evaluation of the nuclear DNA Diffusion Assay to detect apoptosis and necrosis. , 2005, Mutation research.

[41]  B. P. Klein,et al.  Effects of Naturally Occurring Antioxidants on Peroxidase Activity of Vegetable Extracts , 1990 .

[42]  A. Mukherjee,et al.  Enhanced Cr(VI) Removal by Nanozerovalent Iron-Immobilized Alginate Beads in the Presence of a Biofilm in a Continuous-Flow Reactor , 2016 .

[43]  G. Nienhaus,et al.  Engineered nanoparticles interacting with cells: size matters , 2014, Journal of Nanobiotechnology.

[44]  I. Fridovich,et al.  Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. , 1971, Analytical biochemistry.

[45]  M. Zhang,et al.  Stimulation of Peanut Seedling Development and Growth by Zero-Valent Iron Nanoparticles at Low Concentrations , 2015, PloS one.

[46]  R. Sethi,et al.  Nanoscale zerovalent iron particles for groundwater remediation: a review , 2014 .

[47]  N. Pietrasiak,et al.  Iron Oxide and Titanium Dioxide Nanoparticle Effects on Plant Performance and Root Associated Microbes , 2015, International journal of molecular sciences.

[48]  S. Dwivedi,et al.  Cobalt oxide nanoparticles aggravate DNA damage and cell death in eggplant via mitochondrial swelling and NO signaling pathway , 2016, Biological Research.

[49]  R. Miller.  Nitric-perchloric Acid Wet Digestion In An Open Vessel , 1997 .

[50]  Q. Saquib,et al.  Phytotoxic hazards of NiO-nanoparticles in tomato: a study on mechanism of cell death. , 2013, Journal of hazardous materials.

[51]  Qi Chen,et al.  Rapid magnetic removal of aqueous heavy metals and their relevant mechanisms using nanoscale zero valent iron (nZVI) particles. , 2013, Water research.

[52]  K. Yagi,et al.  Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. , 1979, Analytical biochemistry.

[53]  Pei-Jen Chen,et al.  Stabilization or oxidation of nanoscale zerovalent iron at environmentally relevant exposure changes bioavailability and toxicity in medaka fish. , 2012, Environmental science & technology.

[54]  A. Mukherjee,et al.  Evaluation of genotoxicity and oxidative stress of aluminium oxide nanoparticles and its bulk form in Allium cepa , 2016, The Nucleus.

[55]  R. Sunkar.  Plant stress tolerance : methods and protocols , 2010 .

[56]  T. Schwarzacher.  Preparation and Fluorescent Analysis of Plant Metaphase Chromosomes. , 2016, Methods in molecular biology.

[57]  A. Patri,et al.  Zeta potential measurement. , 2011, Methods in molecular biology.

[58]  Xuan Li,et al.  Aggregation kinetics and dissolution of coated silver nanoparticles. , 2012, Langmuir : the ACS journal of surfaces and colloids.

[59]  Guadalupe de la Rosa,et al.  Evidence of the differential biotransformation and genotoxicity of ZnO and CeO2 nanoparticles on soybean (Glycine max) plants. , 2010, Environmental science & technology.

[60]  M. A. Marin-Morales,et al.  Action mechanisms of petroleum hydrocarbons present in waters impacted by an oil spill on the genetic material of Allium cepa root cells. , 2008, Aquatic toxicology.

[61]  C. R I S T I N,et al.  Oxidative Stress Induced by Zero-Valent Iron Nanoparticles and Fe ( II ) in Human Bronchial Epithelial Cells , 2009 .

[62]  Cheng Lei,et al.  Toxicity of iron-based nanoparticles to green algae: Effects of particle size, crystal phase, oxidation state and environmental aging. , 2016, Environmental pollution.

[63]  C. Ciniglia,et al.  ACRIDINE ORANGE/ETHIDIUM BROMIDE DOUBLE STAINING TEST: A SIMPLE IN-VITRO ASSAY TO DETECT APOPTOSIS INDUCED BY PHENOLIC COMPOUNDS IN PLANT CELLS , 2010 .

[64]  Navid B. Saleh,et al.  Aggregation and sedimentation of aqueous nanoscale zerovalent iron dispersions. , 2007, Environmental science & technology.

[65]  S. Lutts,et al.  Changes in plant response to NaCl during development of rice (Oryza sativa L.) varieties differing in salinity resistance , 1995 .

[66]  B. B. Panda,et al.  Aluminium-induced DNA damage and adaptive response to genotoxic stress in plant cells are mediated through reactive oxygen intermediates. , 2010, Mutagenesis.

[67]  Jose R Peralta-Videa,et al.  Interaction of nanoparticles with edible plants and their possible implications in the food chain. , 2011, Journal of agricultural and food chemistry.

[68]  Maumita Bandyopadhyay,et al.  In vitro and in vivo genotoxicity of silver nanoparticles. , 2012, Mutation research.

[69]  G Koppen,et al.  The alkaline comet test on plant cells: a new genotoxicity test for DNA strand breaks in Vicia faba root cells. , 1996, Mutation research.

[70]  Gareth J.S. Jenkins,et al.  Potential toxicity of superparamagnetic iron oxide nanoparticles (SPION) , 2010, Nano reviews.

[71]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[72]  Jae-hwan Kim,et al.  Exposure of iron nanoparticles to Arabidopsis thaliana enhances root elongation by triggering cell wall loosening. , 2014, Environmental science & technology.

[73]  Dongye Zhao,et al.  Higher concentrations of nanoscale zero-valent iron (nZVI) in soil induced rice chlorosis due to inhibited active iron transportation. , 2016, Environmental pollution.

[74]  F. Loreto,et al.  Isoprene produced by leaves protects the photosynthetic apparatus against ozone damage, quenches ozone products, and reduces lipid peroxidation of cellular membranes. , 2001, Plant physiology.

[75]  A. Pruden,et al.  Toxicity of engineered nanomaterials and their transformation products following wastewater treatment on A549 human lung epithelial cells , 2014, Toxicology reports.