The size-dependent apoptotic effect of titanium dioxide nanoparticles on endothelial cells by the intracellular pathway

Abstract

Concerns over the health risk of the widely distributed, commonly used titanium dioxide nanoparticles (nano-TiO₂ ) are increasing worldwide. Yet, up-to-now, our understanding in their potential effects on the cardiovascular system is very limited and the toxicological mechanisms are still unclear. In the present study, the CCK-8 assay was performed to determine the cytotoxicity of four sizes (10, 30, 50, and 100 nm) of anatase nano-TiO₂ on human umbilical vein endothelial cells (HUVECs) in culture, and the flow cytometry was employed to investigate the potential of these nano-TiO₂ to induce the apoptosis of HUVECs. The apoptotic pathway was also probed through the determination of the protein expression and activation of p53, Bax, Bcl-2, caspases-9, -7, -3, and PARP by western blot. The results showed that at the administrative levels (1, 5, 25 μg/mL), all the four sizes of nano-TiO₂ could significantly inhibit the viability of HUVECs and elicit significant apoptosis in them, compared with the negative control (P < .05, P < .01). Moreover, the apoptotic rates of HUVECs were increased respectively with the elevating levels and decreasing sizes of the administrative nano-TiO₂ , showing a clear dose- and size-dependent effect relationships. Interestingly, the increasing phosphorylation of p53, decreasing ratio of Bcl-2/Bax, and enhancing activation of the downstream proteins caspase-9, -7, -3, and PARP, were also observed with the decreasing sizes of the administrative nano-TiO₂ in the western blot, indicating that the intracellular approach of apoptosis, the p53-caspase pathway, is the major way of the nano-TiO₂ -mediated apoptosis in HUVECs in culture and that the size is an important parameter that may determine the potential of nano-TiO₂ to induce cellular response. In conclusion, these results suggested that high levels of nano-TiO₂ exposure may pose potential risks to human cardiovascular health by inducing cardiovascular EC apoptosis.

Keywords: apoptosis; endothelial cell (EC); p53; titanium dioxide nanoparticle (nano-TiO2).