Titanium dioxide induces apoptotic cell death through reactive oxygen species-mediated Fas upregulation and Bax activation

Abstract

Background

Titanium dioxide (TiO₂) has been widely used in many areas, including biomedicine, cosmetics, and environmental engineering. Recently, it has become evident that some TiO₂ particles have a considerable cytotoxic effect in normal human cells. However, the molecular basis for the cytotoxicity of TiO₂ has yet to be defined.

Methods and results

In this study, we demonstrated that combined treatment with TiO₂ nanoparticles sized less than 100 nm and ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-dependent upregulation of Fas and conformational activation of Bax in normal human cells. Treatment with P25 TiO₂ nanoparticles with a hydrodynamic size distribution centered around 70 nm (TiO₂^P25–70) together with ultraviolet A irradiation-induced caspase-dependent apoptotic cell death, accompanied by transcriptional upregulation of the death receptor, Fas, and conformational activation of Bax. In line with these results, knockdown of either Fas or Bax with specific siRNA significantly inhibited TiO₂-induced apoptotic cell death. Moreover, inhibition of reactive oxygen species with an antioxidant, N-acetyl-L-cysteine, clearly suppressed upregulation of Fas, conformational activation of Bax, and subsequent apoptotic cell death in response to combination treatment using TiO₂^P25–70 and ultraviolet A irradiation.

Conclusion

These results indicate that sub-100 nm sized TiO₂ treatment under ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-mediated upregulation of the death receptor, Fas, and activation of the preapoptotic protein, Bax. Elucidating the molecular mechanisms by which nanosized particles induce activation of cell death signaling pathways would be critical for the development of prevention strategies to minimize the cytotoxicity of nanomaterials.

Keywords:

• TiO₂
• reactive oxygen species
• apoptotic cell death
• Fas upregulation
• Bax activation
• mitochondrial membrane potential loss
• caspase activation

Acknowledgment

This work was supported by the Korea Ministry of Environment and The Eco-Technopia 21 Project (091-091-081).

Disclosure

The authors report no conflicts of interest in this work.

Supplementary figures

Figure S1 Measurement of cell death by FACS analysis after propidium iodide staining. Chang cells were treated with TiO₂ nanoparticles of different hydrodynamic size distributions (TiO₂^P25–70, TiO₂^P25–130 and TiO₂^P25–300) at each concentration (0, 50, 100, 150 200, 400 ppm) for 24 hours, and cell death was measured by flow cytometry with propidium iodide staining. Treatment with a lower concentration of TiO₂ nanoparticles than 200 ppm did not cause significant cell death, while treatment with a higher concentration (200 and 400 ppm) resulted in dramatic cell death at any TiO₂ nanoparticle size.

Note: Error bars represent the mean ± standard deviation of triplicate samples.

Figure S2 Representative FACS plots for measurement of cell death receptor expression levels. FACS analysis reveals upregulation of Fas expression in Chang cells treated with TiO₂^P25–70 in combination with ultraviolet A irradiation, while single treatment with either TiO₂^P25–70 or ultraviolet A did not upregulate the level of Fas expression. Expression levels of other death receptors, ie, TNF, DR4, and DR5, were not altered in Chang cells by combined treatment with TiO₂^P25–70 and ultraviolet A irradiation.

Abbreviations: TNF, tumor necrosis factor; DR, death receptor; UVA, ultraviolet A.