Comparative cellular toxicity of titanium dioxide nanoparticles on human astrocyte and neuronal cells after acute and prolonged exposure

Although in the last few decades, titanium dioxide nanoparticles (TiO₂NPs) have attracted extensive interest due to their use in wide range of applications, their influences on human health are still quite uncertain and less known. Evidence exists indicating TiO₂NPs ability to enter the brain, thus representing a realistic risk factor for both chronic and accidental exposure with the consequent needs for more detailed investigation on CNS.A rapid and effective in vitro test strategy has been applied to determine the effects of TiO₂NPs anatase isoform, on human glial (D384) and neuronal (SH-SY5Y) cell lines. Toxicity was assessed at different levels: mitochondrial function (by MTT), membrane integrity and cell morphology (by calcein AM/PI staining) after acute exposure (4-24-48h) at doses from 1.5 to 250μg/ml as well as growth and cell proliferation (by clonogenic test) after prolonged exposure (7-10 days) at sub-toxic concentrations (from 0.05 to 31μg/ml). The cytotoxic effects of TiO₂NPs were compared with those caused by TiO₂ bulk counterpart treatment.Acute TiO₂NP exposure produced (i) dose- and time-dependent alterations of the mitochondrial function on D384 and SH-SY5Y cells starting at 31 and 15μg/ml doses, respectively, after 24h exposure. SH-SY5Y were slightly more sensitive than D384 cells; and (ii) cell membrane damage occurring at 125μg/ml after 24h exposure in both cerebral cells. Comparatively, the effects of TiO₂ bulk were less pronounced than those induced by nanoparticles in both cerebral cell lines.Prolonged exposure indicated that the proliferative capacity (colony size) was compromised at the extremely low TiO₂NP doses namely 1.5μg/ml and 0.1μg/ml for D384 and SH-SY5Y, respectively; cell sensitivity was still higher for SH-SY5Y compared to D384. Colony number decrease (15%) was also evidenced at ≥0.2μg/ml TiO₂NP dose. Whereas, TiO₂ bulk treatment affected cell morphology only.TiO₂ internalization in SH-SY5Y and D384 cells was appreciated using light microscopy.These findings indicated, that (i) human cerebral SH-SY5Y and D384 cell lines exposed to TiO₂NPs were affected not only after acute but even after prolonged exposure at particularly low doses (≥ 0.1μg/ml), (ii) these in vitro critical doses were comparable to literature brain Ti levels detected in lab animal intranasally administered with TiO₂NP and associated to neurotoxic effects.In summary, the applied cell-based screening platform seems to provide effective means to initial evaluation of TiO₂NP toxicity on CNS.