Ecotoxicology

, Volume 17, Issue 5, pp 410–420

Genotoxic and cytotoxic potential of titanium dioxide (TiO2) nanoparticles on fish cells in vitro

Article

Abstract

Intrinsic genotoxic and cytotoxic potential of titanium dioxide (TiO2) engineered nanoparticles (ENPs) were evaluated in a metabolically competent, established fish cell line derived from rainbow trout (Oncorhyncus mykiss) gonadal tissue (i.e. RTG-2 cells). Prior to evaluation of the toxic potential, mean size of the ENPs was determined using transmission electron microscopy (TEM). As a prerequisite, an extensive characterisation of the ENPs was carried out following sonication which enabled the synthesis of an efficient dosing strategy for the cells in which exposure in phosphate buffered saline (PBS) gave an optimal agglomeration effects compared to distilled water (H2O) and minimal essential media (MEM). Interaction of the ENPs with cells under scanning electron microscope (SEM) was also studied. The genotoxic and cytotoxic potential of the ENPs were determined either alone or in combination with ultraviolet radiation (i.e. UVA). Whilst genotoxic potential was determined by evaluating DNA strand breaks using single cell gel electrophoresis (SCGE) or the comet assay and induction of cytogenetic damage using cytokinesis-blocked micronucleus (MN) assay, cytotoxicity was determined by measuring the retention of supra vital stain, neutral red, by the lysosomes using the neutral red retention (NRR) assay. In addition, while performing the comet assay, lesion specific bacterial endonuclease, formamidopyrimidine DNA glycosylase (Fpg), which recognises oxidised purine bases, was used to determine oxidative DNA damage. The results suggested that the highest concentration of the ENPs (i.e. 50 μg ml−1) did not produce elevations in DNA damage over 4 h (comet assay), 24 h (modified comet assay) or 48 h (MN assay) exposures in the absence of UVA irradiation, although there was a significant reduction in lysosomal integrity over 24 h exposure (NRR assay). The induction of MN did not show any enhanced levels as a function of ENP concentration. A significantly increased level of strand breaks was observed in combination with UVA (3 kJ m−2). In general, the NRR assay suggested elevated levels of cytotoxicity when the UVA exposure was carried out with MEM compared to PBS, although both showed an increase when in combination with the highest concentration of ENPs (i.e. 50 μg ml−1). Overall, the study emphasises the need for adoption of an holistic approach while evaluating the potential toxic effects of ENPs in which appropriate measures should be taken to avoid agglomeration or aggregation to facilitate efficient cellular uptake to evaluate potential biological responses.

Keywords

Nanoparticles Genotoxicity Cytotoxicity Photo-genotoxicity RTG-2 cells Comet assay Micronucleus assay Neutral red retention assay 

References

  1. Adams LK, Lyon DY, Alvarez PJJ (2006) Comparative eco-toxicity of nanoscale TiO2, SiO2, and ZnO water suspensions. Water Res 40:3527–3532CrossRefGoogle Scholar
  2. Bols NC, Boliska SA, Dixon DG, Hodson PV, Kaiser KLE (1985) The use of fish cell cultures as an indication of contaminant toxicity to fish. Aquat Toxicol 6:147–155CrossRefGoogle Scholar
  3. Brezova V, Gabcova S, Dvoranova D, Stasko A (2005) Reactive oxygen species produced upon photoexcitation of sunscreens containing titanium dioxide (an EPR study). J Photochem Photobiol B 79:121–134CrossRefGoogle Scholar
  4. Cai R, Kubota Y, Shuin T, Sakai H, Hashimoto K, Fujishima A (1992) Induction of cytotoxicity by photoexcited TiO2 particles. Cancer Res 52:2346–2348Google Scholar
  5. Carp O, Huisman CL, Reller A (2004) Photoinduced reactivity of titanium dioxide. Prog Solid State Chem 32:33–177CrossRefGoogle Scholar
  6. Cho M, Chung H, Choi W, Yoon J (2004) Linear correlation between inactivation of E. coli and OH radical concentration in TiO2 photocatalytic disinfection. Water Res 38:1069–1077CrossRefGoogle Scholar
  7. Dixon DR, Pruski AM, Dixon LRJ, Jha AN (2002) Marine invertebrate eco-genotoxicology: a methodological overview. Mutagenesis 17:495–507CrossRefGoogle Scholar
  8. Dopp E, Schuler M, Schiffmann D, Eastmond DA (1997) Induction of micronuclei, hyperdiploidy and chromosomal breakage affecting the centricrpericentric regions of chromosomes 1 and 9 in human amniotic fluid cells after treatment with asbestos and ceramic fibers. Mutat Res 377:77–87Google Scholar
  9. Federici G, Shaw BJ, Handy RD (2007) Toxicity of titanium dioxide nanoparticles to rainbow trout (Oncorhynchus mykiss): Gill injury, oxidative stress, and other physiological effects. Aquat Toxicol 84:415–430CrossRefGoogle Scholar
  10. Fenech M (1993) The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human populations. Mutat Res-Fund Mol M 285:35–44CrossRefGoogle Scholar
  11. Fenech M (2000) The in vitro micronucleus technique. Mutat Res/Fund Mol M 455:81–95CrossRefGoogle Scholar
  12. Gulledge WP (2007) Letter to the editor. Mutat Res-Gen Tox En 634:241–242CrossRefGoogle Scholar
  13. Gurr JR, Wang ASS, Chen CH, Jan KY (2005) Ultrafine titanium dioxide particles in the absence of photoactivation can induce oxidative damage to human bronchial epithelial cells. Toxicology 213:66–73CrossRefGoogle Scholar
  14. Halliwell B, Gutteridge JMC (1999) Free radicals in biology and medicine, 3rd edn. Oxford University PressGoogle Scholar
  15. Handy RD, Kammer Fvd, Lead JR, Hassello M, Owen R, Crane M (2008) The ecotoxicology and chemistry of manufactured nanoparticles. Ecotoxicology 17:287–314CrossRefGoogle Scholar
  16. Hardman RA (2006) A toxicologic review of quantum dots: toxicity depends on physicochemical and environmental factors. Environ Health Perspect 114:165–72CrossRefGoogle Scholar
  17. Jha AN (2004) Genotoxicological studies in aquatic organisms: an overview. Mutat Res-Fund Mol M 552:1–17CrossRefGoogle Scholar
  18. Jha AN (2008) Ecotoxicological applications and significance of the comet assay. Mutagenesis 23:207–221 CrossRefGoogle Scholar
  19. Kocan RM, Landolt ML, Sabo KM (1982) Anaphase aberrations: a measure of genotoxicity in mutagen treated fish-cells. Environ Mutagen 4:181–189CrossRefGoogle Scholar
  20. Kumaravel TS, Jha AN (2006) Reliable Comet assay measurements for detecting DNA damage induced by ionising radiation and chemicals. Mutat Res-Gen Tox En 605:7–16CrossRefGoogle Scholar
  21. Liotta LA, Kohn EC (2001) The microenvironment of the tumour-host interface. Nature 411:375–379CrossRefGoogle Scholar
  22. Moore MN (2006) Do nanoparticles present ecotoxicological risks for the health of the aquatic environment? Environ Int 32:967–976CrossRefGoogle Scholar
  23. National Nanotechnology Initiatives (2006) What Is nanotechnology? http://www.nano.gov/html/facts/whatIsNano.html
  24. Neeves KB, Sawyer AJ, Foley CP, Saltzman WM, Olbricht WL (2007) Dilation and degradation of the brain extracellular matrix enhances penetration of infused polymer nanoparticles. Brain Res 1180:121–132CrossRefGoogle Scholar
  25. Oesterling E, Chopra N, Gavalas V, Arzuaga X, Lim EJ, Sultana R, Butterfield DA, Bachas L, Hennig B (2008) Alumina nanoparticles induce expression of endothelial cell adhesion molecules. Toxicol Lett. doi:10.1016/j.toxlet.2008.03.011
  26. Ogino C, Kanehira K, Sasai R, Sonezaki S, Shimizu N (2007) Recognition and effective degradation of 17[beta]-estradiol by anti-estradiol-antibody-immobilized TiO2 nanoparticles. J Biosci Bio Eng 104:339–342Google Scholar
  27. Owen R, Depledge M (2005) Nanotechnology and the environment: risks and rewards. Mar Pollut Bull 50:609–612CrossRefGoogle Scholar
  28. Rahman Q, Lohani M, Dopp E, Pemsel H, Jonas L, Weiss DG, Schiffmann D (2002) Evidence that ultrafine titanium dioxide induces micronuclei and apoptosis in syrian hamster embryo fibroblasts. Environ Health Perspect 110:797–800Google Scholar
  29. Raisuddin S, Jha AN (2004) Relative sensitivity of fish and mammalian cells to sodium arsenate and arsenite as determined by alkaline single-cell gel electrophoresis and cytokinesis-block micronucleus assay. Environ Mol Mutagen 44:83–89CrossRefGoogle Scholar
  30. Reeves JF, Davies SJ, Dodd NJF, Jha AN (2008) Hydroxyl radicals (OH) are associated with titanium dioxide (TiO2) nanoparticle-induced cytotoxicity and oxidative DNA damage in fish cells. Mutat Res-Fund Mol M 640:113–122CrossRefGoogle Scholar
  31. Schultz M, Lewald B, Kolpoth M, Rusche B, Lorenz K, Unruh E, Hansen P-D, Miltenburger H (1995) Fischzellinien in der toxikologischen Bewertung von Abwasserproben. Altex Altern Tierexp 12:188–195Google Scholar
  32. Serpone N, Dondi D, Albini A (2007) Inorganic and organic UV filters: their role and efficacy in sunscreens and suncare products. Inorg Chim Acta 360:794–802CrossRefGoogle Scholar
  33. Skubal LR, Meshkov NK, Rajh T, Thurnauer M (2002) Cadmium removal from water using thiolactic acid-modified titanium dioxide nanoparticles. J Photochem Photobiol C 148:393–397CrossRefGoogle Scholar
  34. Spielmann H, Balls M, Dupuis J, Pape WJ, Pechovitch G, de Silva O, Holzhutter HG, Clothier R, Desolle P, Gerberick F, Liebsch M, Lovell WW, Maurer T, Pfannenbecker U, Potthast JM, Csato M, Sladowski D, Steiling W, Brantom P (1998) The international EU/COLIPA in vitro phototoxicity validation study: results of phase II (blind trial). Part 1. The 3T3 NRU phototoxicity test. Toxicol in Vitro 12:305–327CrossRefGoogle Scholar
  35. Tran DN, Ota LC, Jacobson JD, Patton WC, Chan PJ (2007) Influence of nanoparticles on morphological differentiation of mouse embryonic stem cells. Fertil Steril 87:965–970CrossRefGoogle Scholar
  36. Uchino T, Tokunaga H, Ando M, Utsumi H (2002) Quantitative determination of OH radical generation and its cytotoxicity induced by TiO2-UVA treatment. Toxicol In Vitro 16:629–635CrossRefGoogle Scholar
  37. Wamer WG, Yin J-J, Wei RR (1997) Oxidative damage to nucleic acids photosensitized by titanium dioxide. Free Radical Bio Med 23:851–858CrossRefGoogle Scholar
  38. Wang RJ, Nixon BR (1978) Identification of hydrogen peroxide as a photoproduct toxic to human cells in tissue culture medium irradiated with daylight fluorescent light. In Vitro 14:715–22CrossRefGoogle Scholar
  39. Wang RJ, Ananthaswamy HN, Nixon BT, Hartman PS, Eisenstark A (1980) Induction of DNA single strand breaks in human cells by H2O2 formed in near UV (blacklight) irradiated medium. Radiat Res 82:269–278CrossRefGoogle Scholar
  40. Wang JJ, Sanderson BJS, Wang H (2007) Cyto- and genotoxicity of ultrafine TiO2 particles in cultured human lymphoblastoid cells. Mutat Res-Gen Tox En 628:99–106CrossRefGoogle Scholar
  41. Warheit DB, Hoke RA, Finlay C, Donner EM, Reed KL, Sayes CM (2007) Development of a base set of toxicity tests using ultrafine TiO2 particles as a component of nanoparticle risk management. Toxicol Lett 171:99–110CrossRefGoogle Scholar
  42. Wiesner MR, Lowry GV, Alvarez P, Dionysiou D, Biswas P (2006) Assessing the risks of manufactured nanomaterials. Environ Sci Technol 40:4336–4345CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  1. 1.Ecotoxicology and Stress Biology Research Centre, School of Biological SciencesUniversity of PlymouthPlymouthUK

Personalised recommendations