**8. Discussion**

Our study revealed the unique toxicity of TiNSs. This 2D nano-scaled material has the harmful potential to cause caspase-dependent apoptosis of immune cells to the same degree as asbestos. In particular, monocytes showed formation of marked vacuoles prior to cell death upon exposure to TiNSs, which were later found in the vacuoles and suggest the actual engulfment of TiNSs by monocytes. A consideration of these findings with the observation regarding co-localization of vacuoles with endosomal dextran indicates that engulfed TiNSs entered the endosomal pathway, leading to the formation of vacuoles in monocytes and subsequent cell death. As mentioned previously, TiNSs have a very large surface area per gram. **Figure 3** represents an illustration showing the large surface area of 2D nano-materials. Nano-scaled spheres with a diameter of 20 nm have a volume of 4189 nm3 and surface area of 1257 nm2 . Diamond-shaped nanosheets with diagonals of 20 and 30 nm, a depth of 1 nm and resulting volume of 300 nm3 have the same density as a nanosphere. The total volume of 14 nanosheets (4200 nm3 ) is almost equivalent to the volume of one nanosphere (4189 nm3 ), but those nanosheets result in a total surface area of 8400 nm2 as the sum of both sides, which is 6.68 times as large as the surface area of the nanosphere. Thus, nanosheets have an extremely large surface that probably enhances the chemical activities of titanium oxide, which might contribute to the toxicity of TiNSs. In addition, TiNSs are negatively charged on the surface due to the presence of the oxygen atom, which might cause cationic interference in endo-lysosomes that leads to the formation of vacuoles. Various kinds of stimulation such as oxidative stress disrupt the integrity of the lysosomal membrane and cause lysosomal-membrane permeabilization (LMP), which triggers cell death including caspase-dependent apoptosis [51, 52]. Autophagy is the machinery of the intracellular degradation process, which is also the part of the intracellular membrane system and is linked to the endolysosomal pathway [51, 53]. Furthermore, recent studies have been accumulating concerning a new type of cell death associated with large vacuoles, named methuosis, although it is thought

**Figure 3.** Illustration of a comparison of volume and surface area between nanosphere and nanosheet. A nanosphere with a diameter of 20 nm has a volume of 4189 nm3 and surface area 1257 nm2 . A diamond-shaped nanosheet with 20- and 30-nm diagonals and depth of 1 nm has the same density as a nanosphere. Fourteen nanosheets have almost the same volume as a nanosphere but possess a total surface area of 8400 nm2 as the sum of both sides, which are 6.68 times as large as the surface area of the nanosphere.

that this type of cell death does not require caspases [54, 55]. Some of the machineries mentioned above might be related to the cell death caused by TiNSs. TiNSs in the endosomal pathway might affect immune functions executed by cell surface receptors through interference of endosomal trafficking. Additionally, CD4<sup>+</sup> lymphocytes were also damaged by exposure to TiNSs, which suggests possible alteration of immune responses in a direct manner caused by TiNSs. Further investigation concerning these issues will contribute to a clarification of the toxic machinery of TiNSs and the immunotoxicological effects of TiNSs.
