**3. Studies concerning nano-scaled titanium dioxide particles**

alloy are used for various kinds of biomaterials such as artificial joints and dental implants, where generation of titanium oxide (titania) film is beneficial because of its bio-inertness [1–3]. Titanium oxide was therefore regarded as a harmless material. However, studies detailing the toxic effects of titanium oxide have been accumulating recently as shown in the next section. The International Agency for Research on Cancer (IARC) decided in 2010 to change its cat-

humans" to "Group 2B: Possibly carcinogenic to humans". This conclusion resulted from sufficient evidence in experimental animals and inadequate evidence from epidemiological studies. The carcinogenicity of titanium oxide was evaluated by examining the relationship between exposure to titanium oxide and the risk of lung cancer in two previously conducted case-control studies, which showed no detectable excess risk of lung cancer [4]. In contrast, two studies using animal experiments demonstrated elevated lung cancer in rats exposed to fine or ultra-

[5, 6]. Additionally, it is estimated that the total production of nano-TiO2

approximately 2.5 million metric tons (MT) per year in 2025 from 40,000 MT in 2006 in the US [7], which means we would become more exposed to nano-scaled materials of titanium oxide in the future, thereby motivating us to better clarify the toxicological effects of this material. The various forms of titanium oxide are known and include spheres, rods, needles and fibers, as well as sheets. Titanate nanosheets (TiNSs) are crystalline materials composed of titanium and oxygen with a very thin and flat structure representing 2D materials. TiNSs are expected to be valuable materials in industry for production of UV- or corrosion-resistant films, dielectric thin films and catalysts. Therefore, we recently examined the effects of exposure to TiNSs on human immune cells (manuscript of an original article under preparation). Here, we would like to review the progress of studies regarding the toxicity of titanium oxides, summarize our recent study concerning the toxicity of TiNSs and finally discuss the findings obtained from that study.

The following two studies form the basis for the decision to reappraise the carcinogenicity

caused tumors in comprising squamous cell carcinomas, adenocarcinomas and benign squamous cell tumors in female rats [6]. In addition, Schins et al. evaluated data in the literature

reactive oxygen species (ROS) and reactive nitrogen species (RNS) that were produced by a

rutile and anatase due to the difference in crystal structure. It has been reported that an anatase

with a rutile type of the material in an *in vitro* experiment with human fibroblasts and lung

production of 8-hydroxyl-2′-deoxyguanosine (8-OHdG), a DNA adduct, which contributed to the development of tumor, whereas the nanoparticles did not cause DNA breakage in human

showed higher production of ROS and more toxic characteristics compared

. In 1985, Lee et al. conducted *in vivo* experiments with rats and reported the occurrence of bronchioalveolar adenomas carcinomas and squamous cell carcinomas in a portion

) from "Group 3: Not classifiable as to carcinogenicity to

, which possesses a micro-scaled diameter [5].

involves a mechanism of genetic damage caused by

nano-scaled particles

is distinguished as

nanoparticles caused high

would reach

egorization of titanium dioxide (TiO2

**2. Toxicity of titanium dioxide materials**

of both sexes exposed by inhalation to fine TiO<sup>2</sup>

and reported that tumorigenesis by TiO2

The study by Heinrich et al. demonstrated that exposure to ultrafine TiO<sup>2</sup>

epithelial cells [9]. In addition, it has been demonstrated that TiO2

exposure-induced inflammatory response [8]. Naturally occurring TiO2

fine TiO<sup>2</sup>

164 Cytotoxicity

of TiO2

TiO2

type of nano-TiO2

Before examining TiNSs, previous studies regarding the toxicity of nano-scaled TiO2 materials should be reviewed. The toxicological effect of TiO<sup>2</sup> nanoparticles on skin and in dermal tissue is of interest to medical science and manufacturers because TiO2 is used as a physical photoprotective agent in sunscreen and as a whitening agent in cosmetics. It is unlikely that TiO2 nanoparticles easily reach dermal tissue. Experiments using human skin-transplanted mice show that TiO2 nanoparticles did not penetrate the barrier of an intact epidermis [14]. Sadrieh et al. [15] and Newman et al. [16] also demonstrated no significant penetration of TiO<sup>2</sup> nanoparticles through the epidermis. However, this does not eliminate concern regarding its toxicity on skin because an *in vitro* experiment using cell lines of keratinocytes, sebocytes, fibroblasts and melanocytes showed decreases in viable cells, an increase in apoptosis and decreases in the differentiation markers of those cells [14]. Moreover, several studies have shown actual penetration of TiO2 nanoparticles through skin. Bennat and Muller-Goymann demonstrated that TiO2 nanoparticles are able to pass through skin using an oil-in-water emulsion [17]. Another study demonstrated that TiO2 nanoparticles reached the deep area of the epidermis in the pig ear after topical skin exposure to the nanoparticles, some of which even reached the brain, whereas there was no penetration in an *in vitro* experiment using isolated porcine skin [18]. Once TiO2 nanoparticles pass through the epidermis due to the broken or unhealthy status of the epidermal barrier, they produce harmful effects on the tissue. A study utilizing *in vitro* experiments confirmed the phototoxicity of nano-sized TiO<sup>2</sup> in an experiment with human skin keratinocytes of HaCaT under irradiation of UVA, which is mainly dependent on the ROS production level [19]. Furthermore, a study investigating the mechanism of toxicity of TiO2 nanoparticles upon exposure to UVA found that exposure to TiO2 caused decreases in the mitochondrial membrane potential and ATP level and an increase in caspase 3 activity [20]. It has also been shown that subcutaneous injection with TiO2 nanoparticles promoted dermal sensitization induced by dinitrochlorobenzene (DNCB) [21]. Moreover, intradermal administration with TiO2 nanoparticles resulted in aggravated skin lesions such as those of atopic dermatitis related to mite antigen in NC/Nga mice [22]. These findings indicate that TiO<sup>2</sup> nanoparticles do not penetrate through the epidermis easily as long as the barrier is healthy, but these nanoparticles have the potential to cause toxic effects on dermal tissue when that barrier is broken. Pulmonary exposure to TiO<sup>2</sup> nanoparticles is expected to result in these toxic effects because a barrier comprising pulmonary tissue is not tough. In addition, alveolar macrophages are always ready to engulf particles in the region, leading to inflammatory responses. Sager et al. used F344 rats to examine the pulmonary response to intratracheal instillation of nano-sized ultrafine TiO<sup>2</sup> in comparison to fine TiO<sup>2</sup> particles. Administration with ultrafine TiO<sup>2</sup> caused higher levels of polymorphonuclear neutrophil (PMN) cell number, lactate dehydrogenase (LDH) activity, albumin and inflammatory cytokines compared with fine TiO<sup>2</sup> [23]. Interestingly, they also examined the amounts of TiO2 in trachea-bronchial and thymic lymph nodes at days 7 and 42 after administration and then measured alterations in those tissues during that period. Ultrafine TiO2 showed a faster decline of the remaining amount in trachea-bronchial nodes than fine TiO2 . In addition, although both ultrafine and fine TiO<sup>2</sup> were translocated to lymph nodes, the amount was higher for ultrafine TiO<sup>2</sup> than fine TiO<sup>2</sup> . There was also a striking difference in the amounts of lavagable and non-lavagable components between fine and ultrafine TiO<sup>2</sup> in the lungs. Eight-one percent of ultrafine TiO<sup>2</sup> was non-lavagable, suggesting migration to the interstitium, even at day 7 post-exposure, whereas 91% of fine TiO<sup>2</sup> was still lavagable at this stage. Shinohara et al. investigated pulmonary clearance kinetics of TiO2 nano- and submicron particles by intratracheal administration to male F344 rats [24], and results confirmed the translocation of administered TiO2 to the thoracic lymph nodes that increased in a timeand dose-dependent manner. van Ravenzwaay also observed that inhaled TiO2 nanoparticles reached the lungs and draining lymph nodes [25]. These findings indicate that it is easier for TiO2 nanoparticles to migrate from bronchoalveolar to interstitial tissue or be cleared by alveolar macrophages, leading to increased inflammatory responses, and that TiO<sup>2</sup> nanoparticles have a greater potential to influence the function of immune cells. Actually, several studies have reported alteration of immune functions following administration with TiO2 through the respiratory pathway. Chang et al. demonstrated that intratracheal instillation with TiO2 nanoparticles caused an increase in GATA-3 and decrease in T-bet mRNA levels, which are master genes for Th2 and Th1 cell development, respectively [26]. Mishra et al. examined the effect of administration with TiO<sup>2</sup> nanoparticles as an adjuvant in an experiment utilizing a murine asthma model. It was found that TiO2 nanoparticles augmented airway hyperresponsiveness, lung damage and a mixed Th2/Th1 dependent immune response, associated with increases in Stat3, Socs3, NF-κB, IL-6 and TNF-α [27]. The effect of intradermal administration with TiO2 nanoparticles as mentioned above should also be understood in relation to acquired immunity. NC/Nga mice treated with TiO2 nanoparticles showed over production of IL-4 in the skin, IgE and histamine levels in serum, as well as aggravation of skin lesions [22]. Studies using intraperitoneal administration of TiO2 nanoparticles are valuable for an understanding of the immunological influences of these particles. Larsen et al. showed that mice receiving intraperitoneal treatment with TiO2 nanoparticles and OVA and subsequently challenged with aerosols of OVA responded with high production of IgE and IgG1 antibodies specific for OVA in serum with increases in eosinophils, neutrophils and lymphocytes in bronchoalveolar lavage fluid (BALF), which suggests induction of a Th2-dominant immune response [28]. Moreover, Moon et al. demonstrated that intraperitoneal injection with TiO<sup>2</sup> nanoparticles results in the damaged development and proliferation of B and T cells, a decreased cytokine production by LPS-stimulated peritoneal macrophages and a decreased

percentage of NK cells in spleenocytes, leading to an increased tumor growth of implanted B16F10 melanoma cells [29]. Several *in vitro* studies also provide further information regard-

of immune functions compared with large particles. Therefore, we planned to examine the toxicity of nano-scaled 2D materials composed of titanium and oxygen (TiNSs) on human

Techniques to synthesize nano-scaled 2D materials have been studied recently. The basis of this field is derived from the development of methods for manipulating graphene, a carbon nanosheet with a thickness of one atom, which triggered the subsequent development of various 2D nanomaterials [31–33]. It is against this background that inorganic nanosheets have acquired greater interest because they have an ultrathin structure as well as a diversity of compounds and structures leading to various functions [34–36]. Oxide nanosheets are included in the group comprising inorganic nanosheets, and titanate nanosheets (TiNSs) represent a form of oxide nanosheets.

unique crystal structure of lepidocrocite, differing from anatase or rutile, which results in a shape having an ultralow thickness and high aspect ratio [34]. In the 1990s, Sasaki et al. first succeeded in delaminating layered titanate into single titanate nanosheets [37, 38], and TiNSs are now incorporated into useful applications such as photocatalysts, semiconductors and dielectric materials [39–42]. However, the following characteristics suggest possible harmful effects of TiNSs. First, it is noteworthy that TiNSs have a very large surface area per gram due to their ultralow thick-

together with the repeated linkage of the octahedron horizontally [34]. Such a large surface of TiNSs might enhance the toxic machinery of bulk titanium particles. Second, the large surface of TiNSs is known to be negatively charged due to oxygen atoms existing at the edges of the octahedron, and this suggests the possible influence of TiNSs through a cationic interaction. In addition to TiNSs delaminated from layered titanate, it has been reported that TiNSs with a small diamond shape and crystal structure of lepidocrocite can be synthesized in a bottom-up manner [43, 44], which allows TiNSs to be synthesized at a small scale. Dr. Yoshioka, one of our colleagues, has modified that method to synthesize TiNSs in our group. **Figure 1** shows images of TiNSs taken by transmission electron microscopy (TEM). The TiNSs showed a diamond shape with about 20- and 30-nm diagonals, which is almost the same as that reported in a previous study [43]. Additionally, the TiNSs showed the characteristic peaks of a lepidocrocite structure confirmed by X-ray diffraction analysis. Since it was verified that TiNSs could be synthesized, we therefore

ness, which is generated from the limited height of one and a half of a sideways TiO6

started to examine the effect of TiNSs on human immune cells using *in vitro* experiments.

 nanoparticles showed greater influence on the antigen presenting activity of monocytes and alveolar macrophages [30]. In addition, Moon et al. showed a reduction in lymphocyte proliferation induced by lipopolysaccharide (LPS) or concanavalin A (Con A) upon expo-

nanoparticles [29]. The overall results of these *in vivo* or *in vitro* studies indicate

particles have the potential to cause harmful outcomes if they enter

octahedron as the particles of TiO2

nanoparticles on immune cells. Munidasa et al. reported that

particles cause greater alteration

Toxicity of Titanate Nanosheets on Human Immune Cells

http://dx.doi.org/10.5772/intechopen.72234

167

, TiNSs have the

octahedron

ing the direct effects of TiO<sup>2</sup>

the body. In addition, it is also clear that nano-scaled TiO2

**4. The characteristics of titanate nanosheets (TiNSs)**

Although TiNSs are composed of a TiO6

TiO2

sure to TiO2

immune cells.

that nano-scaled TiO2

percentage of NK cells in spleenocytes, leading to an increased tumor growth of implanted B16F10 melanoma cells [29]. Several *in vitro* studies also provide further information regarding the direct effects of TiO<sup>2</sup> nanoparticles on immune cells. Munidasa et al. reported that TiO2 nanoparticles showed greater influence on the antigen presenting activity of monocytes and alveolar macrophages [30]. In addition, Moon et al. showed a reduction in lymphocyte proliferation induced by lipopolysaccharide (LPS) or concanavalin A (Con A) upon exposure to TiO2 nanoparticles [29]. The overall results of these *in vivo* or *in vitro* studies indicate that nano-scaled TiO2 particles have the potential to cause harmful outcomes if they enter the body. In addition, it is also clear that nano-scaled TiO2 particles cause greater alteration of immune functions compared with large particles. Therefore, we planned to examine the toxicity of nano-scaled 2D materials composed of titanium and oxygen (TiNSs) on human immune cells.
