Cyto- and genotoxicity of ultrafine TiO₂ particles in cultured human lymphoblastoid cells

Abstract

Titanium dioxide is frequently used in the production of paints, paper, plastics, welding rod-coating material, and cosmetics, because of its low toxicity. However, recent studies have shown that nano-sized or ultrafine TiO₂ (UF-TiO₂) (<100 nm in diameter) can generate pulmonary fibrosis and lung tumor in rats. Cytotoxicity induced by UF-TiO₂ in rat lung alveolar macrophages was also observed. This generates great concern about the possible adverse effects of UF-TiO₂ for humans. The cytotoxicity and genotoxicity of UF-TiO₂ were investigated using the methyl tetrazolium cytotoxicity (MTT) assay, the population growth assay, the apoptosis assay by flow cytometry, the cytokinesis block micronucleus (CBMN) assay, the comet assay, and the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene mutation assay. WIL2-NS cells were incubated for 6, 24 and 48 h with 0, 26, 65 and 130 μg/ml UF-TiO₂. Significant decreases in viability were seen in the MTT assay at higher doses; for example, 61, 7 and 2% relative viability at 130 μg/ml for 6, 24 and 48-h exposure (P < 0.01). A dose-dependent relationship was observed, while a time-dependent relationship was seen only at the highest dose (130 μg/ml) after exposure for 24 and 48 h. Treatment with 130 μg/ml UF-TiO₂ induced approximately 2.5-fold increases in the frequency of micronucleated binucleated cells (P < 0.01). In addition, a significant reduction in the cytokinesis block proliferation index was observed by the CBMN assay (P < 0.05). In the comet assay, treatment with 65 μg/ml UF-TiO₂ induced approximately 5-fold increases in olive tail moment (P < 0.05). In the HPRT mutation assay, treatment with 130 μg/ml UF-TiO₂ induced approximately 2.5-fold increases in the mutation frequency (P < 0.05). The results of this study indicate that UF-TiO₂ can cause genotoxicity and cytotoxicity in cultured human cells.

Introduction

Titanium dioxide (TiO₂) is a poorly soluble particulate (PSP) that has been widely used as a white pigment in the production of paints, paper, plastics, welding rod-coating material and food colorant. Nano-sized or ultrafine TiO₂ (UF-TiO₂) (<100 nm) is used increasingly in other industrial products, such as cosmetics and pharmaceuticals [1], [2], [3], [4]. Therefore, potential widespread exposure may occur during both manufacturing and use.

Coarse and fine (>100 nm in diameter) particles of TiO₂ were classified as biologically inert in both human and animals [5], [6], [7]. However, Garabrant et al. [8] reported that 50% of workers exposed to TiO₂ suffered from respiratory symptoms, accompanied by impairment of pulmonary function. Furthermore, Ahn et al. [9] showed that goblet cell hyperplasia and Muc 5ac expression were induced in rats after a single intratracheal instillation of TiO₂.

On the other hand, the cytotoxicity induced by TiO₂ was relevant to the size of particles [10]. There is evidence that UF-TiO₂ can cause inflammation, fibrosis, pulmonary damage and even DNA damage [11], [12], [13]. UF-TiO₂ might be able to enter the human stratum corneum and interact with the immune system [14], [15], [16], since UFP can be translocated to the subepithelium space to a greater extent than the fine particles [17]. An increase in the level of IL-8 was seen in human endothelial cells after exposure to UF-TiO₂ [18]. A significant decrease in the level of glutathione was observed in rat alveolar macrophage following exposure to UF-TiO₂ [11]. The authors suggested that induction of reactive oxygen species (ROS) might be responsible for this depletion. Oxidative DNA damage and increases in the level of cellular nitric oxide were observed in human bronchial epithelial cells after exposure to UF-TiO₂ [19]. There are potential multiple effects of UF-TiO₂, and the possible adverse effects of TiO₂ exposure need further clarification.

In the present study, we have investigated the toxicity of UF-TiO₂ particles. The cytotoxic and genotoxic effects of UF-TiO₂ were assayed in cultured WIL2-NS human lymphoblastoid cells.