Effects of Titanium Dioxide Nanoparticles on the Hprt Gene Mutations in V79 Hamster Cells

Abstract

The genotoxicity of anatase/rutile TiO₂ nanoparticles (TiO₂ NPs, NM105 at 3, 15 and 75 µg/cm²) was assessed with the mammalian in-vitro Hypoxanthine guanine phosphoribosyl transferase (Hprt) gene mutation test in Chinese hamster lung (V79) fibroblasts after 24 h exposure. Two dispersion procedures giving different size distribution and dispersion stability were used to investigate whether the effects of TiO₂ NPs depend on the state of agglomeration. TiO₂ NPs were fully characterised in the previous European FP7 projects NanoTEST and NanoREG2. Uptake of TiO₂ NPs was measured by transmission electron microscopy (TEM). TiO₂ NPs were found in cytoplasmic vesicles, as well as close to the nucleus. The internalisation of TiO₂ NPs did not depend on the state of agglomeration and dispersion used. The cytotoxicity of TiO₂ NPs was measured by determining both the relative growth activity (RGA) and the plating efficiency (PE). There were no substantial effects of exposure time (24, 48 and 72 h), although a tendency to lower RGA at longer exposure was observed. No significant difference in PE values and no increases in the Hprt gene mutant frequency were found in exposed relative to unexposed cultures in spite of evidence of uptake of NPs by cells.

Keywords: Hprt; V79 cells; genotoxicity; titanium dioxide nanoparticles.

Figure 1

Particle size distribution obtained by (NTA) of TiO₂ NPs using the two proposed dispersion procedures (DP1 and DP2) in culture medium at 0 and 24 h. The black line is the mean distribution and the red filling represent standard errors between captured videos.

Figure 2

Representative transmission electron microscopy (TEM) figures of titanium dioxide TiO₂ NM105 uptake by Chinese hamster lung fibroblast (V79-4) cells exposed to 3, 10 and 30 μg/cm² of TiO₂ NPs dispersed according to dispersion procedure 1 (DP1) and dispersion procedure 2 (DP2). DP1 3 μg/cm² (a–e), DP1 10 μg/cm² (f–i), DP1 30 μg/cm² (j,k), DP2 3 μg/cm² (l–o), DP2 10 μg/cm² (p–r), DP2 30 μg/cm² (s,t), Negative control untreated V79-4 cells (u). N = nucleus; C = cytoplasm; V = vesicle, M = mitochondrion.

Figure 3

(a) and (b). Cytotoxic effects measured as the relative growth activity (RGA %) on V79-4 cells exposed to TiO₂ NPs prepared using the two dispersion procedures (DP1 and DP2). Cells were treated with 5 concentrations (μg/cm²) of TiO₂ NPs for 24, 48 and 72 h, and the cell numbers were counted at each time point immediately, following trypan blue staining. There were no statistical significances between exposed and unexposed cultures. Cytotoxicity of MMS was not been observed (RGA = 70%). Data are expressed as the means ± SEM of two parallel experiments, according to the used procedures.

Figure 4

Cytotoxic effects of TiO₂ NPs measured by the plating efficiency (PE %) in V79-4 cells. Bars represent cytotoxicity relative to 100% of untreated cells Data are expressed as the means ± SEM of two parallel seedings for plating efficiency, according to the used procedure. No statistical significances between exposed and unexposed cultures were observed.

Figure 5

Induction of Hprt gene mutants after the exposure of V79-4 cells to different concentrations of TiO₂ NPs for 24 h. There were no statistical significances between exposed and unexposed cultures. Hprt gene mutant frequency in treated cells with positive control MMS (0.1 mM, 3 h), which gave 131.6 ± 2.30 Hprt gene mutants. This value is indicated as a red-dashed line. Data are expressed as the means ± SEM of two parallel seedings for mutation frequency MF1 and MF2, according to the used procedure.