TiO2 nanoparticles induce oxidative DNA damage and apoptosis in human liver cells

Abstract Titanium dioxide nanoparticles (TiO2 NPs), widely used in consumer products, paints, pharmaceutical preparations and so on, have been shown to induce cytotoxicity, genotoxicity and carcinogenic responses in vitro and in vivo. The present study revealed that TiO2 NPs induce significant (p < 0.05) oxidative DNA damage by the Fpg-Comet assay even at 1 µg/ml concentration. A corresponding increase in the micronucleus frequency was also observed. This could be attributed to the reduced glutathione levels with concomitant increase in lipid peroxidation and reactive oxygen species generation. Furthermore, immunoblot analysis revealed an increased expression of p53, BAX, Cyto-c, Apaf-1, caspase-9 and caspase-3 and decreased the level of Bcl-2 thereby indicating that apoptosis induced by TiO2 NPs occurs via the caspase-dependent pathway. This study systematically shows that TiO2 NPs induce DNA damage and cause apoptosis in HepG2 cells even at very low concentrations. Hence the use of such nanoparticles should be carefully monitored.

[1]  K. Jan,et al.  Ultrafine titanium dioxide particles in the absence of photoactivation can induce oxidative damage to human bronchial epithelial cells. , 2005, Toxicology.

[2]  Ritesh K Shukla,et al.  ROS-mediated genotoxicity induced by titanium dioxide nanoparticles in human epidermal cells. , 2011, Toxicology in vitro : an international journal published in association with BIBRA.

[3]  M. Fenech.  The in vitro micronucleus technique. , 2000, Mutation research.

[4]  Alok Dhawan,et al.  Cellular uptake and mutagenic potential of metal oxide nanoparticles in bacterial cells. , 2011, Chemosphere.

[5]  B. Lau,et al.  An automated micro-fluorometric assay for monitoring oxidative burst activity of phagocytes. , 1993, Journal of immunological methods.

[6]  A. Pandey,et al.  Stable metal oxide nanoparticle formulation for toxicity studies. , 2011, Journal of biomedical nanotechnology.

[7]  C. Franceschi,et al.  JC‐1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess ΔΨ changes in intact cells: implications for studies on mitochondrial functionality during apoptosis , 1997, FEBS letters.

[8]  R. Tice,et al.  A simple technique for quantitation of low levels of DNA damage in individual cells. , 1988, Experimental cell research.

[9]  J. Gearhart,et al.  In vitro toxicity of nanoparticles in BRL 3A rat liver cells. , 2005, Toxicology in vitro : an international journal published in association with BIBRA.

[10]  Benjamin Gilbert,et al.  Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties. , 2008, ACS nano.

[11]  A. Collins,et al.  Oxidative damage to DNA: do we have a reliable biomarker? , 1996, Environmental health perspectives.

[12]  S. Wong,et al.  Evaluating cytotoxicity and cellular uptake from the presence of variously processed TiO2 nanostructured morphologies. , 2010, Chemical research in toxicology.

[13]  Christopher J. Rhodes,et al.  Role of oxygen radicals in DNA damage and cancer incidence , 2004, Molecular and Cellular Biochemistry.

[14]  K. Hensley,et al.  Reactive oxygen species, cell signaling, and cell injury. , 2000, Free radical biology & medicine.

[15]  B. Sanderson,et al.  Cyto- and genotoxicity of ultrafine TiO2 particles in cultured human lymphoblastoid cells. , 2007, Mutation research.

[16]  Alok Dhawan,et al.  Nanomaterials: A challenge for toxicologists , 2009 .

[17]  Sonja Boland,et al.  Carbon black and titanium dioxide nanoparticles elicit distinct apoptotic pathways in bronchial epithelial cells , 2010, Particle and Fibre Toxicology.

[18]  Y. Ibuki,et al.  Simple and easy method to evaluate uptake potential of nanoparticles in mammalian cells using a flow cytometric light scatter analysis. , 2007, Environmental science & technology.

[19]  E Borenfreund,et al.  Toxicity determined in vitro by morphological alterations and neutral red absorption. , 1985, Toxicology letters.

[20]  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.

[21]  R. Tice,et al.  Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing , 2000, Environmental and molecular mutagenesis.

[22]  Vicki Stone,et al.  Identification of the mechanisms that drive the toxicity of TiO2 particulates: the contribution of physicochemical characteristics , 2009, Particle and Fibre Toxicology.

[23]  T. Dragani,et al.  Libri Ricevuti: IARC Monographs on the Evaluation of Carcinogenic Risks to Humans , 1992 .

[24]  A. Pandey,et al.  Comet assay responses in human lymphocytes are not influenced by the menstrual cycle: a study in healthy Indian females. , 2005, Mutation research.

[25]  G. Ellman,et al.  Tissue sulfhydryl groups. , 1959, Archives of biochemistry and biophysics.

[26]  Tao Zhang,et al.  Oxidative stress and apoptosis induced by nanosized titanium dioxide in PC12 cells. , 2010, Toxicology.

[27]  Alok Dhawan,et al.  Zinc oxide nanoparticle induced genotoxicity in primary human epidermal keratinocytes. , 2011, Journal of nanoscience and nanotechnology.

[28]  A. Zuckerman,et al.  IARC Monographs on the Evaluation of Carcinogenic Risks to Humans , 1995, IARC monographs on the evaluation of carcinogenic risks to humans.

[29]  Ritesh K Shukla,et al.  DNA damaging potential of zinc oxide nanoparticles in human epidermal cells. , 2009, Toxicology letters.

[30]  E. Hwang,et al.  Dinstinct ROS and biochemical profiles in cells undergoing DNA damage-induced senescence and apoptosis , 2005, Mechanisms of Ageing and Development.

[31]  Vincent Castranova,et al.  Iron oxide nanoparticles induce human microvascular endothelial cell permeability through reactive oxygen species production and microtubule remodeling , 2009, Particle and Fibre Toxicology.

[32]  Ashutosh Kumar,et al.  Titanium dioxide nanoparticles induce oxidative stress-mediated apoptosis in human keratinocyte cells. , 2011, Journal of biomedical nanotechnology.

[33]  Keith T Palmer,et al.  IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Volume 98: Painting, Firefighting and Shiftwork. International Agency for Research on Cancer , 2011 .

[34]  M. Reigosa,et al.  Comparative study of the cytotoxic and genotoxic effects of titanium oxide and aluminium oxide nanoparticles in Chinese hamster ovary (CHO-K1) cells. , 2010, Journal of hazardous materials.

[35]  Soo-Jin Choi,et al.  Comparative cytotoxicity of Al2O3, CeO2, TiO2 and ZnO nanoparticles to human lung cells. , 2010, Journal of nanoscience and nanotechnology.

[36]  Jinshun Zhao,et al.  Titanium Dioxide (TiO2) Nanoparticles Induce JB6 Cell Apoptosis Through Activation of the Caspase-8/Bid and Mitochondrial Pathways , 2009, Journal of toxicology and environmental health. Part A.

[37]  Z. Chai,et al.  Acute toxicity and biodistribution of different sized titanium dioxide particles in mice after oral administration. , 2007, Toxicology letters.

[38]  Robert H Schiestl,et al.  Titanium dioxide nanoparticles induce DNA damage and genetic instability in vivo in mice. , 2009, Cancer research.

[39]  T. Hei,et al.  Genotoxic responses to titanium dioxide nanoparticles and fullerene in gpt delta transgenic MEF cells , 2009, Particle and Fibre Toxicology.

[40]  T. Mosmann.  Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. , 1983, Journal of immunological methods.

[41]  H. Lindberg,et al.  Genotoxic effects of nanosized and fine TiO2 , 2009, Human & experimental toxicology.

[42]  Dragan Uskoković,et al.  DNA damage and alterations in expression of DNA damage responsive genes induced by TiO2 nanoparticles in human hepatoma HepG2 cells , 2011, Nanotoxicology.

[43]  Jongheop Yi,et al.  Oxidative stress and apoptosis induced by titanium dioxide nanoparticles in cultured BEAS-2B cells. , 2008, Toxicology letters.

[44]  Alok Dhawan,et al.  Toxicity assessment of nanomaterials: methods and challenges , 2010, Analytical and bioanalytical chemistry.

[45]  Guping Tang,et al.  In vivo acute toxicity of titanium dioxide nanoparticles to mice after intraperitioneal injection , 2009, Journal of applied toxicology : JAT.