Induction of sister chromatid exchanges and micronuclei by titanium dioxide in Chinese hamster ovary-K1 cells

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Abstract

Titanium dioxide (TiO2) has color properties of extreme whiteness and brightness, is relatively inexpensive, and is extensively used as a white pigment in a variety of materials. TiO2, an effective blocker of ultraviolet light, is frequently added to sunscreens and cosmetic creams. However, the genotoxicity of TiO2 remains to be controversial. In this report, we have demonstrated that TiO2 can be transported into Chinese hamster ovary-K1 (CHO-K1) cells. The effects of TiO2 on induction of sister chromatid exchanges (SCE) and micronuclei (MN) were then studied in these cells. The SCE frequency in CHO-K1 cells treated with TiO2 at a nonlethal dose range (0 to 5 μM) for 24 h was significantly and dose-dependently increased. By the conventional MN assay, TiO2 at the dose ranged from 0 to 20 μM slightly increased the MN frequency in CHO-K1 cells. However, in the cytokinesis-block MN assay, the number of MN per 1000 binucleated cells was significantly and dose-dependently enhanced in CHO-K1 cells treated TiO2 at the same dose range for 24 h. These results suggest that TiO2 is a potential genotoxic agent.

Introduction

Titanium is the ninth most common element in the earth's crust. Due to its extreme whiteness and brightness, titanium dioxide (TiO2) is widely used as a white pigment in a variety of materials [1]. The toxicology of TiO2 has been the subject of much investigation because of its widespread use in industry. In general, titanium and its compounds are not toxic. However, exposure to TiO2 dust may result in its deposit in lung tissues and induce mild lung fibrosis [2]. Numerous studies have shown that TiO2 exposure significantly increases the expression of macrophage inflammatory proteins [3]and production of hydroxyl radicals [4]. A recent report has shown that the elicited neutrophilic inflammation in rats exposed to poorly soluble particles including α-quartz, carbon black, and TiO2 is associated with increased hprt gene mutation in alveolar type II cells [5]. In addition, numerous reports have shown that a comparable amount of growth factor activity was secreted from macrophages following in-vitro or in-vivo administration of TiO2 particles 2, 6, 7, and exposure of animals to TiO2 at doses greater than 50 mg/kg transiently increased the release of tumor necrosis factor [8]. Although the pathological effects of TiO2 is not as profound as other mineral particles such as asbestos and silica, TiO2 is considered a nuisance dust 9, 10.

Numerous reports have shown that ultraviolet (UV) light from the sun can cause genetic damage and skin cancer. To protect themselves from these dangers, people often spread sunscreens on their skin before exposure to the sun. It has been demonstrated that cream vehicles containing TiO2 can effectively block light from 400–700 nm wavelengths 11, 12and can hence shield skin from erythema [13]. Due to its effective ability to block long-wave ultraviolet light, TiO2 has frequently been used as an important ingredient in sunscreens and cosmetic creams. However, the evidence for TiO2 genotoxicity remains to be further clarified. TiO2 was evaluated to be negative in inducing chromosomal aberrations, sister chromatic exchanges (SCEs), and micronuclei (MN) in Chinese hamster ovary (CHO) cells 14, 15. Karlsson et al. [16]have reported that condensate of smoke from TiO2/hexachloroethane dose-dependently induced mutations in Salmonella assays with strains TA98 and TA100. In the same report, TiO2 smoke condensate slightly but significantly inhibited erythropoietic activity, but did not induce micronuclei in mice. However, Shelby et al. [17]have demonstrated that TiO2 could significantly elevate MN frequency in mice by a 3-times daily exposure protocol.

Recently, the cytokinesis-block MN assay, a more sensitive method than the conventional MN assay, has increasingly been used for genotoxicity test of a variety of compounds [18]. Thus, we reexamined the effects of TiO2 on induction of SCEs and MN in CHO-K1 cells in this report.

Section snippets

Materials and cell culture

Titanium standard solution was purchased from Merck (Darmstadt, Germany); TiO2 (CAS No. 13463-67-7), Colcemid, Hoechst 33528 and cytochalasin B were purchased from Sigma (St. Louis, MO). CHO-K1 cells were cultured in McCoy's 5A medium supplemented with 0.22% sodium bicarbonate, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.03% l-glutamine and 10% fetal calf serum. The cultures were maintained at 37°C in a humidified atmosphere with 5% CO2.

Intracellular titanium accumulation

Logarithmically growing CHO-K1 cells were incubated

Results and discussion

According to atomic absorption analysis, titanium accumulated in CHO-K1 cells in a dose-dependent manner (Fig. 1). These results indicated that TiO2 could be taken by the cells at this low dose range. According to the estimation of Korte and Yasui [23], the size of CHO-K1 cells is 580 μm3. Therefore, a 24-h exposure to 5 μM TiO2 resulted in titanium accumulation to approximately 81.4 μM in CHO-K1 cells. However, according to the colony-forming assay, TiO2 at doses below 5 μM did not cause cell

Acknowledgements

The authors thank Mr. Douglas Platt for carefully reading this article. This work was supported by Academia Sinica and the National Science Council, Republic of China.

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