Titanium dioxide nanoparticles induce oxidative stress and DNA-adduct formation but not DNA-breakage in human lung cells

Abstract

Titanium dioxide (TiO2), also known as titanium (IV) oxide or anatase, is the naturally occurring oxide of titanium. It is also one of the most commercially used form. To date, no parameter has been set for the average ambient air concentration of TiO2 nanoparticles (NP) by any regulatory agency. Previously conducted studies had established these nanoparticles to be mainly non-cyto- and -genotoxic, although they had been found to generate free radicals both acellularly (specially through photocatalytic activity) and intracellularly. The present study determines the role of TiO2-NP (anatase, slashed circle < 100 nm) using several parameters such as cyto- and genotoxicity, DNA-adduct formation and generation of free radicals following its uptake by human lung cells in vitro. For comparison, iron containing nanoparticles (hematite, Fe2O3, slashed circle < 100 nm) were used. The results of this study showed that both types of NP were located in the cytosol near the nucleus. No particles were found inside the nucleus, in mitochondria or ribosomes. Human lung fibroblasts (IMR-90) were more sensitive regarding cyto- and genotoxic effects caused by the NP than human bronchial epithelial cells (BEAS-2B). In contrast to hematite NP, TiO2-NP did not induce DNA-breakage measured by the Comet-assay in both cell types. Generation of reactive oxygen species (ROS) was measured acellularly (without any photocatalytic activity) as well as intracellularly for both types of particles, however, the iron-containing NP needed special reducing conditions before pronounced radical generation. A high level of DNA adduct formation (8-OHdG) was observed in IMR-90 cells exposed to TiO2-NP, but not in cells exposed to hematite NP. Our study demonstrates different modes of action for TiO2- and Fe2O3-NP. Whereas TiO2-NP were able to generate elevated amounts of free radicals, which induced indirect genotoxicity mainly by DNA-adduct formation, Fe2O3-NP were clastogenic (induction of DNA-breakage) and required reducing conditions for radical formation.

Figure 1

Scanning electron microscopy images of (a) titanium dioxide and (b) hematite nanoparticles and EDX spectral analysis of particle surface composition.

Figure 2

BEAS-2B cell exposed to hematite-NP (10 μg/cm² for 48 h). Agglomerated and single particles were found near the nucleus (1), in the endoplasmic reticulum or lysosomes and surrounded by mitochondria (2). No particles were found within the nucleus or intracellular organelles such as mitochondria or ribosomes. Similarly, also the TiO₂-NP were located in the cytosol (mainly in lysosomes) without entering the nucleus.

Figure 3

IMR-90 cells exposed to titanium dioxide (a) and hematite (b) nanoparticles (exposure time: 24 h).

Figure 4

BEAS-2B cells exposed to titanium dioxide (a) and hematite nanoparticles (b) for 24 h.

Figure 5

Electron paramagnetic resonance measurement of acellular reactive oxygen species being generated by titanium dioxide and hematite nanoparticles.

Figure 6

Electron paramagnetic resonance measurement of acellular reactive oxygen species being generated by titanium dioxide and hematite nanoparticles a: treatment of particles with ascorbic acid and H₂O₂, b: treatment of particles with cell lysate and H₂O₂).

Figure 7

Intracellular radical measurement after exposure of BEAS-2B cells to TiO₂-NP (a) and hematite NP (b). Hematite NP induced a delayed effect (significant results after 12 h) compared to TiO₂ NP (significant results after 6 h exposure). Desferal treatment (concentration: 100 μM) of particle-exposed cultures induced a significant reduction of radical formation in TiO₂ but not in hematite exposed cells.

Figure 8

Relative values of 8-OHdG adduct formation in IMR-90 cells after treatment with titanium dioxide and hematite nanoparticles for 24 h. TiO₂-NP were found to generate 1 and 1.1 ng/ml of 8-OHdG adducts at the concentrations of 5 and 10 μg/cm², respectively, whereas hematite NP failed to induce any 8-OhdG adducts in IMR-90 cells.