Biological Trace Element Research

, Volume 149, Issue 1, pp 123–132 | Cite as

Titanium Dioxide Nanoparticles-Mediated In Vitro Cytotoxicity Does Not Induce Hsp70 and Grp78 Expression in Human Bronchial Epithelial A549 Cells

  • Sasitorn Aueviriyavit
  • Duangkamol Phummiratch
  • Kornphimol Kulthong
  • Rawiwan Maniratanachote


Titanium dioxide nanoparticles (TiO2NPs) are increasingly being used in various industrial applications including the production of paper, plastics, cosmetics and paints. With the increasing number of nano-related products, the concern of governments and the general public about the health and environmental risks, especially with regard to occupational and other environmental exposure, are gradually increasing. However, there is insufficient knowledge about the actual affects upon human health and the environment, as well as a lack of suitable biomarkers for assessing TiO2NP-induced cytotoxicity. Since the respiratory tract is likely to be the main exposure route of industrial workers to TiO2NPs, we investigated the cytotoxicity of the anatase and rutile crystalline forms of TiO2NPs in A549 cells, a human alveolar type II-like epithelial cell line. In addition, we evaluated the transcript and protein expression levels of two heat shock protein (HSP) members, Grp78 and Hsp70, to ascertain their suitability as biomarkers of TiO2NP-induced toxicity in the respiratory system. Ultrastructural observations confirmed the presence of TiO2NPs inside cells. In vitro exposure of A549 cells to the anatase or rutile forms of TiO2NPs led to cell death and induced intracellular ROS generation in a dose-dependent manner, as determined by the MTS and dichlorofluorescein (DCF) assays, respectively. In contrast, the transcript and protein expression levels of Hsp70 and Grp78 did not change within the same TiO2NPs dose range (25–500 μg/ml). Thus, whilst TiO2NPs can cause cytotoxicity in A549 cells, and thus potentially in respiratory cells, Hsp70 and Grp78 are not suitable biomarkers for evaluating the acute toxicological effects of TiO2NPs in the respiratory system.


TiO2 nanoparticles Cytotoxicity Heat shock protein Hsp70 Grp78 A549 cells 



This work was financial supported by the Research, Development and Engineering Fund through the National Nanotechnology, NSTDA, Thailand. We thank Dr. Robert Butcher for comments and for the English language review.

Supplementary material

12011_2012_9403_Fig8_ESM.jpg (63 kb)

(JPEG 62 kb)

12011_2012_9403_MOESM1_ESM.tif (133 kb)
High resolution image (TIFF 132 kb)


  1. 1.
    Kaida T, Kobayashi K, Adachi M, Suzaki F (2004) Optical characteristics of titanium oxide interference film and the film laminated with oxides and their applications for cosmetics. J Cosmet Sci 55:219–220PubMedGoogle Scholar
  2. 2.
    Allen NS, Edge M, Sandoval G, Verran J, Stratton J, Maltby J (2005) Photocatalytic coatings for environmental applications. Photochem Photobiol 81:279–290PubMedCrossRefGoogle Scholar
  3. 3.
    Higarashi NM, Jardim WE (2002) Remediation of pesticide contaminated soil using TiO2 mediated by solar light. Catal Today 76:201–207CrossRefGoogle Scholar
  4. 4.
    Konstantinou IK, Albanis TA (2004) TiO2-assisted photocatalytic degradation of azo dyes in aqueous solution: kinetic and mechanistic investigation-a review. Appl Catal B Environ 49:1–14CrossRefGoogle Scholar
  5. 5.
    Oberdöster G, Maynard A, Donaldson K, Castranova V, Fitzpatrick J, Ausman K, Carter J, Karn B, Kreyling W, Lai D, Olin S, Monteiro-Riviere N, Warheit D, Yang H (2005) Principles for characterizing the potential human health effects from exposure to nanomaterials: elements of a screening strategy. Particle Fibre Toxicol 2:2–8CrossRefGoogle Scholar
  6. 6.
    Oberdöster G, Oberdöster E, Oberdöster J (2005) Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect 113:823–839CrossRefGoogle Scholar
  7. 7.
    Simon-Deckers A, Gouget B, Mayne-L’Hermite M, Herlin-Boime N, Reynaud C, Carrière M (2008) In vitro investigation of oxide nanoparticle and carbon nanotube toxicity and intracellular accumulation in A549 human pneumocytes. Toxicology 253:137–146PubMedCrossRefGoogle Scholar
  8. 8.
    Bhattacharya K, Davoren M, Boertz J, Schins RPF, Hoffmann E, Dopp E (2009) Titanium dioxide nanoparticles induce oxidative stress and DNA-adduct formation but not DNA-breakage in human lung cells. Particle Fibre Toxicol 6:1–11CrossRefGoogle Scholar
  9. 9.
    Shi Y, Wang F, He J, Yadav S, Wang H (2010) Titanium dioxide nanoparticles cause apoptosis in BEAS-2B cells through the caspase 8/t-Bid-independent mitochondrial pathway. Toxicol Lett 196:21–27PubMedCrossRefGoogle Scholar
  10. 10.
    Geiser M, Casaulta M, Kupferschmid B, Schulz H, Semmler-Behnke M, Kreyling W (2008) The role of macrophages in the clearance of inhaled ultrafine titanium dioxide particles. Am J Respir Cell Mol Biol 28:371–376Google Scholar
  11. 11.
    Golli-Bennour EE, Bacha H (2011) Hsp70 expression as biomarkers of oxidative stress: mycotoxin’s exploration. Toxicology 287:1–7PubMedCrossRefGoogle Scholar
  12. 12.
    Hartl FU (1996) Molecular chaperones in cellular protein folding. Nature 381:571–579PubMedCrossRefGoogle Scholar
  13. 13.
    Schlesinger MJ (1990) Heat shock proteins. J Biol Chem 265:12111–12114PubMedGoogle Scholar
  14. 14.
    Gupta SC, Sharma A, Mishra M, Mishra RK, Chowdhuri DK (2010) Heat shock proteins in toxicology: how close and how far? Life Sci 86:377–384PubMedCrossRefGoogle Scholar
  15. 15.
    Lanneau D, Wettstein G, Bonniaud P, Garrido C (2010) Heat shock proteins: cell protection through protein triage. Sci World J 10:1543–1552CrossRefGoogle Scholar
  16. 16.
    Vos MJ, Hageman J, Carra S, Kampinga HH (2008) Structural and functional diversities between members of human HSPB, HSPH, HSPA, and DNAJ chaperone families. Biochemistry 47:7001–7011PubMedCrossRefGoogle Scholar
  17. 17.
    Ni M, Zhang Y, Lee AS (2011) Beyond the endoplasmic reticulum: atypical GRP78 in cell viability, signaling and therapeutic targeting. Biochem J 434:181–188PubMedCrossRefGoogle Scholar
  18. 18.
    Lee AS (2007) GRP78 induction in cancer: therapeutic and prognostic implications. Cancer Res 67:3476–3499Google Scholar
  19. 19.
    Suzuki H, Toyooka T, Ibuki Y (2007) Simple and easy method to evaluate uptake potential of nanoparticles in mammalian cells using a flow cytometric light scatter analysis. Environ Sci Technol 41:3018–3024PubMedCrossRefGoogle Scholar
  20. 20.
    Yoshida Y, Shimakawa S, Itoh N, Niki E (2007) Action of DCFH and BODIPY as a probe for radical oxidation in hydrophilic and lipophilic domain. Free Radic Res 37:861–72CrossRefGoogle Scholar
  21. 21.
    Maniratanachote R, Miami K, Katoh M, Nakajima M, Yokoi T (2005) Chaperone proteins involved in troglitazone-induced toxicity in human hepatoma cell lines. Toxicol Sci 83:293–302PubMedCrossRefGoogle Scholar
  22. 22.
    Lu TH, Su CC, Chen YW, Yang CY, Wu CC, Hung DZ, Chen CH, Cheng PW, Liu SH, Huang CF (2011) Arsenic induces pancreatic β-cell apoptosis via the oxidative stress-regulated mitochondria-dependent and endoplasmic reticulum stress-triggered signaling pathways. Toxicol Lett 201:15–26PubMedCrossRefGoogle Scholar
  23. 23.
    Ahamed M, Posgai R, Gorey TJ, Nielsen M, Hussain SM, Rowe JJ (2010) Silver nanoparticles induced heat shock protein 70, oxidative stress and apoptosis in Drosophila melanogaster. Toxicol Appl Pharmacol 242:263–269PubMedCrossRefGoogle Scholar
  24. 24.
    Liu F, Inageda K, Nishitai G, Matsuoka M (2006) Cadmium induces the expression of Grp78, an endoplasmic reticulum molecular chaperone, in LLC-PK1 renal epithelial cells. Environ Health Perspect 114:859–864PubMedCrossRefGoogle Scholar
  25. 25.
    Forti F, Salovaara S, Cetin Y, Bulgheroni A, Tessadri R, Jennings P, Pfaller W, Prieto P (2011) In vitro evaluation of the toxicity induced by nickel. Toxicol In Vitro 25:454–461PubMedCrossRefGoogle Scholar
  26. 26.
    Ahamed M, Siddiqui MA, Akhtar MJ, Ahmad I, Pant AB (2010) Genotoxic potential of copper oxide nanoparticles in human lung epithelial cells. Biochem Biophys Res Commun 396:578–583PubMedCrossRefGoogle Scholar
  27. 27.
    Timblin CR, Janssen YMW, Goldberg JL, Mossman BT (1998) Grp78, Hsp72/72 and CJUN stress protein levels in lung epithelial cells exposed to asbestos, cadmium, or H2O2. Free Radical Biol Med 24:632–642CrossRefGoogle Scholar
  28. 28.
    Rothen-Rutishauser B, Blank F, Mühlfeld C, Gehr P (2008) In vitro models of the human epithelial airway barrier to study the toxic potential of particulate matter. Expert Opin Drug Metab Toxicol 4:1075–1089PubMedCrossRefGoogle Scholar
  29. 29.
    Thio BJR, Zhou D, Keller AA (2011) Influence of natural organic matter on the aggregation and deposition of titanium dioxide nanoparticles. J Hazard Mater 189:556–63PubMedCrossRefGoogle Scholar
  30. 30.
    Allouni ZE, Cimpan MR, Hol PJ, Skodvin T, Gjerdet NP (2009) Agglomeration and sedimentation of TiO2 nanoparticles in cell culture medium. Colloids Surf B: Biointerf 68:83–87CrossRefGoogle Scholar
  31. 31.
    Singh S, Shi T, Duffin AC, Berlo DV, Hohr D, Fubini B, Martra G, Fenoglio BPJA, Schins RPF (2007) Endocytosis, oxidative stress and IL-8 expression in human lung epithelial cells upon treatment with fine and ultrafine TiO2: role of the specific surface area and of surface methylation of the particles. Toxicol Appl Pharmacol 222:141–151PubMedCrossRefGoogle Scholar
  32. 32.
    Srivastava RK, Rahman Q, Kashyap MP, Lohani M, Pant AB (2011) Ameliorative effects of dimetylthiourea and N-acetylcysteine on nanoparticles induced cyto-genotoxicity in human lung cancer cells-A549. PLoS One 6:1–12Google Scholar
  33. 33.
    Malhotra JD, Miao H, Zhang K, Wolfson A, Pennathur S, Pipe SW, Kaufman RJ (2008) Antioxidants reduce endoplasmic reticulum stress and improve protein secretion. Proc Natl Acad Sci U S A 105:18525–18530PubMedCrossRefGoogle Scholar
  34. 34.
    Park EJ, Yi J, Chung KH, Ryu DY, Choi J, Park K (2008) Oxidative stress and apoptosis induced by titanium dioxide nanoparticles in cultured BEAS-2B cells. Toxicol Lett 180:222–2229PubMedCrossRefGoogle Scholar
  35. 35.
    Hfaiedh N, Allagui AS, El Feki A, Gaubin Y, Murat JC, Soleilhavoup JP, Croute F (2005) Effects of nickel poisoning on expression pattern of the 72/73 and 94 kDa stress proteins in rat organs and in the COS-7, HepG2, and A549 cell lines. J Biochem Mol Toxicol 19:12–18PubMedCrossRefGoogle Scholar
  36. 36.
    Croute F, Beau B, Arrabit C, Gaubin Y, Delmas F, Murat JC, Soleilhavoup JP (2000) Environ Health Perspect 108:55–60PubMedCrossRefGoogle Scholar
  37. 37.
    NIOSH. Current intelligence bulletin 63: Occupational exposure to titanium dioxide. Department of Health and Human Services. Centers for Disease Control and Prevention. National Institute for Occupational Safety and Health. DHHS (NIOSH) Publication No. 2011–160Google Scholar
  38. 38.
    Sayes CM, Reed KL, Warheit DB (2007) Assessing toxicity of fine and nanoparticles: Comparing in vitro measurement to in vivo pulmonary toxicity profiles. Toxicol Sci 97:163–180PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Sasitorn Aueviriyavit
    • 1
  • Duangkamol Phummiratch
    • 1
  • Kornphimol Kulthong
    • 1
  • Rawiwan Maniratanachote
    • 1
  1. 1.National Nanotechnology Center, National Science and Technology Development AgencyKlong LuangThailand

Personalised recommendations