**Toxicity of Titanate Nanosheets on Human Immune Cells Toxicity of Titanate Nanosheets on Human Immune Cells**

DOI: 10.5772/intechopen.72234

Yasumitsu Nishimura, Daisuke Yoshioka, Naoko Kumagai-Takei, Suni Lee, Hidenori Matsuzaki, Kei Yoshitome and Takemi Otsuki Yasumitsu Nishimura, Daisuke Yoshioka, Naoko Kumagai-Takei, Suni Lee, Hidenori Matsuzaki, Kei Yoshitome and Takemi Otsuki Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

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

#### **Abstract**

Titanium oxide is regarded as a bio-inert material, but studies concerning the toxic effects of titanium dioxide (TiO2 ), particularly nano-scaled TiO2 particles, have been accumulating that indicate nano-scaled TiO2 particles show more harm and cause greater alteration of immune functions compared with large particles. Inorganic nanosheets have been the focus of increasing interest because of their ultrathin structure, as well as diversity of compounds and structures leading to various functions. Oxide nanosheets are included in the group comprising inorganic nanosheets, and titanate nanosheets (TiNSs) represent a form of oxide nanosheets. We therefore examined the toxicity of nano-scaled 2D materials of TiNSs on human immune cells. Our study revealed that TiNSs have the potential to cause harm through caspase-dependent apoptosis of human peripheral blood mononuclear cells (PBMCs) to the same degree as asbestos. Furthermore, isolated monocytes developed marked vacuoles prior to cell death upon exposure to TiNSs, which were found in the vacuoles and indicated engulfment of TiNSs. A consideration of these findings with the co-localization of vacuoles with endocytosed fluorescence-labeled dextran indicates that TiNSs entered the endosomal pathway, leading to the formation of vacuoles in monocytes and subsequent cell death. TiNSs might therefore affect immune functions through interference of endo-lysosomal functions.

**Keywords:** titanate nanosheets, apoptosis, vacuole formation, endosome

### **1. Introduction**

Titanium oxide is used broadly in industrial production for ceramics, materials containing composite oxides and photocatalysts, and even as a food additive. In addition, titanium and its

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 categorization of titanium dioxide (TiO2 ) from "Group 3: Not classifiable as to carcinogenicity to 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 ultrafine TiO<sup>2</sup> [5, 6]. Additionally, it is estimated that the total production of nano-TiO2 would reach 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.

lung fibroblasts of IMR-90 [10]. Chen et al. demonstrated that single intratracheal instillation

ways associated with the cell cycle, apoptosis, chemokines and the complement system [11].

oxide, but the toxicity of nano-scaled materials needs to be examined further, particularly the

photoprotective agent in sunscreen and as a whitening agent in cosmetics. It is unlikely that

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

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

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

increase in caspase 3 activity [20]. It has also been shown that subcutaneous injection with

skin lesions such as those of atopic dermatitis related to mite antigen in NC/Nga mice [22].

ily as long as the barrier is healthy, but these nanoparticles have the potential to cause toxic

caused decreases in the mitochondrial membrane potential and ATP level and an

nanoparticles promoted dermal sensitization induced by dinitrochlorobenzene (DNCB)

nanoparticles easily reach dermal tissue. Experiments using human skin-transplanted

nanoparticles did not penetrate the barrier of an intact epidermis [14].

nanoparticles through skin. Bennat and Muller-Goymann

nanoparticles pass through the epidermis due to the

nanoparticles upon exposure to UVA found that exposure

nanoparticles do not penetrate through the epidermis eas-

nanoparticles are able to pass through skin using an oil-in-water

rods and dots [12]. Moreover, their study compared various kinds of TiO2

crucial role played by the size, crystal structure and composition of TiO2

tissue is of interest to medical science and manufacturers because TiO2

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

Before examining TiNSs, previous studies regarding the toxicity of nano-scaled TiO2

that the pulmonary effects caused by exposure to ultrafine TiO<sup>2</sup>

als should be reviewed. The toxicological effect of TiO<sup>2</sup>

emulsion [17]. Another study demonstrated that TiO2

[21]. Moreover, intradermal administration with TiO2

 nanoparticles increased expression of placenta growth factor (PIGF), CXCL1, CXCL5 and CCL3, and results were similar to those observed in an *in vitro* experiment with human THP-1 cells. Warheit et al. demonstrated the difference in effects resulting from exposure to nano-TiO<sup>2</sup>

nanoparticles caused altered expression of mouse lung-tissue genes involved in path-

[13]. These findings show evidence for the toxicity of titanium

and demonstrated

165

materi-

in regard to toxicity.

is used as a physical

differ based on the crystal struc-

Toxicity of Titanate Nanosheets on Human Immune Cells

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

nanoparticles on skin and in dermal

nanoparticles reached the deep area of

nanoparticles resulted in aggravated

with TiO2

ture and composition of TiO2

TiO2

TiO2

to TiO2

TiO2

mice show that TiO2

shown actual penetration of TiO2

isolated porcine skin [18]. Once TiO2

the mechanism of toxicity of TiO2

These findings indicate that TiO<sup>2</sup>

demonstrated that TiO2

## **2. Toxicity of titanium dioxide materials**

The following two studies form the basis for the decision to reappraise the carcinogenicity of TiO2 . 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 of both sexes exposed by inhalation to fine TiO<sup>2</sup> , which possesses a micro-scaled diameter [5]. The study by Heinrich et al. demonstrated that exposure to ultrafine TiO<sup>2</sup> nano-scaled particles 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 and reported that tumorigenesis by TiO2 involves a mechanism of genetic damage caused by reactive oxygen species (ROS) and reactive nitrogen species (RNS) that were produced by a TiO2 exposure-induced inflammatory response [8]. Naturally occurring TiO2 is distinguished as rutile and anatase due to the difference in crystal structure. It has been reported that an anatase type of nano-TiO2 showed higher production of ROS and more toxic characteristics compared with a rutile type of the material in an *in vitro* experiment with human fibroblasts and lung epithelial cells [9]. In addition, it has been demonstrated that TiO2 nanoparticles caused high 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 lung fibroblasts of IMR-90 [10]. Chen et al. demonstrated that single intratracheal instillation with TiO2 nanoparticles caused altered expression of mouse lung-tissue genes involved in pathways associated with the cell cycle, apoptosis, chemokines and the complement system [11]. TiO2 nanoparticles increased expression of placenta growth factor (PIGF), CXCL1, CXCL5 and CCL3, and results were similar to those observed in an *in vitro* experiment with human THP-1 cells. Warheit et al. demonstrated the difference in effects resulting from exposure to nano-TiO<sup>2</sup> rods and dots [12]. Moreover, their study compared various kinds of TiO2 and demonstrated that the pulmonary effects caused by exposure to ultrafine TiO<sup>2</sup> differ based on the crystal structure and composition of TiO2 [13]. These findings show evidence for the toxicity of titanium oxide, but the toxicity of nano-scaled materials needs to be examined further, particularly the crucial role played by the size, crystal structure and composition of TiO2 in regard to toxicity.
