**5. TiNSs-caused cell death and vacuole formation**

Before the culture experiments, the diameters of TiNSs in water and in culture media supplemented with 10% of FBS were measured using a Zetasizer. The results showed that the diameter of about 25 nm in water is consistent with the size estimated by TEM images, while that in the media was about 422 nm, indicating induced agglomeration of TiNSs in the culture media. In addition, TiNSs in water showed a zeta potential of −22.1 mV. To examine the effects of exposure to TiNSs on human immune cells, peripheral blood mononuclear cells (PBMCs) were prepared and cultured with various doses of TiNSs, or with asbestos as a control cytotoxic material, for 7 days. Exposure to asbestos caused an increase in annexin V (Anx)-positive apoptosis of cells when exposed to more than a dose of 1 μg/ml at day 2 after culture, whereas apoptosis was not observed in the culture exposed to TiNSs even at the maximum dose of 10 μg/ml. However, exposure to TiNSs caused dose-dependent apoptosis to the same degree as asbestos at day 7 of the culture. The effects of exposure to bulk TiO<sup>2</sup> particles and crystalline silica were compared with TiNSs, but they did not cause apoptosis of PBMCs. The cell death induced by exposure to TiNSs or asbestos, but not to TiO2 or silica, was also confirmed by measuring the sub-G1 population, apoptotic cells with a low DNA content, using flow cytometry. Interestingly, marked formation of vacuoles was observed in the culture of PBMCs exposed to TiNSs but not the other materials. It was confirmed by staining with fluorescence-labeled antibodies to CD14 that vacuole formation was present in monocytes but not in lymphocytes. Q-VD-OPh, a pan-caspase inhibitor, suppressed the increase in Anx+ cells induced by TiNSs as well as asbestos. The increase in apoptotic cells caused by TiNS exposure was also observed in the culture of isolated CD14+ monocytes as well as CD4+ lymphocytes. These findings indicate that TiNSs have the potential to cause caspase-dependent apoptosis in immune cells, particularly where monocytes show the formation of large vacuoles prior to apoptosis upon exposure to TiNSs.
