Comparative study of the cytotoxic and genotoxic effects of titanium oxide and aluminium oxide nanoparticles in Chinese hamster ovary (CHO-K1) cells

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Abstract

The aim of this study was to analyze the cytotoxicity and genotoxicity of titanium oxide (TiO2) and aluminium oxide (Al2O3) nanoparticles (NPs) on Chinese hamster ovary (CHO-K1) cells using neutral red (NR), mitochondrial activity (by MTT assay), sister chromatid exchange (SCE), micronucleus (MN) formation, and cell cycle kinetics techniques. Results showed a dose-related cytotoxic effect evidenced after 24 h by changes in lysosomal and mitochondrial dehydrogenase activity. Interestingly, transmission electronic microscopy (TEM) showed the formation of perinuclear vesicles in CHO-K1 cells after treatment with both NPs during 24 h but no NP was detected in the nuclei. Genotoxic effects were shown by MN frequencies which significantly increased at 0.5 and 1 μg/mL TiO2 and 0.5–10 μg/mL Al2O3. SCE frequencies were higher for cells treated with 1–5 μg/mL TiO2. The absence of metaphases evidenced cytotoxicity for higher concentrations of TiO2. No SCE induction was achieved after treatment with 1–25 μg/mL Al2O3. In conclusion, findings showed cytotoxic and genotoxic effects of TiO2 and Al2O3 NPs on CHO-K1 cells. Possible causes of controversial reports are discussed further on.

Introduction

Nanomaterials are designed structures of at least one dimension of 100 nm or less. These materials are present in a number of commercially available products including fillers, catalysts, cosmetics, and many industrial applications. TiO2 NPs are used for protection against UV ray exposure due to their high refractive index. Many sunscreens contain these NPs as well as surface coating products which are colorless and reflect and scatter UV rays more efficiently than larger particles [1], [2], [3]. Nanosized Al-containing particles are also used in industrial, domestic, and medical products, energetic systems (composite propellants), to replace lead primers in artillery, etc.

There is scarce information about the possible toxic effects of NPs on health and environment despite of their current use in novel technology. Since the risks of exposure to nanomaterials are still unknown, alarming speculations are put forward [4].

NP properties differ substantially from bulk material with similar composition, allowing them to exhibit unusual values of reactivity, conductivity, and optical sensitivity among other properties. These capabilities may yield harmful interactions with biological systems and the environment, with potentially toxic effects [5], [6], [7], [8], [9]. Thus, the evaluation of their toxicity is essential.

On the other hand, the toxic action of most biomaterial components in consumer goods has been so far evaluated at macroscale with well established toxicity assessment methods. However, those methods have not been frequently employed for materials at nanoscale dimension [8]. There are some few studies on the toxicological effects of in vitro exposure to this type of manufactured nanomaterials. In fact, the development of new toxicity assessment methods for NPs has been suggested [9].

The toxicity of several metal oxide NPs such as TiO2, ZnO, and Fe2O3 has been analyzed using different cell lines. A complete review about this issue has been recently reported [10]. Few studies on the toxicity of Al-containing particles have so far been published. Some articles report micronuclei increase [11], DNA damage detected by Comet assay [12] and chromosomal aberrations [13] caused by AlCl3 NPs. In the case of Al2O3 NPs, reports are even more limited. Rat bone marrow [14] and alveolar macrophages (NR8383) [15] were used. Lower toxicity of Al2O3 NPs than Al NPs was found.

CHO-K1 cells have also been used as a mammalian cell line model in several research studies. However there are controversial reports. Theogaraj et al. [16] and Warheit et al. [17] did not find TiO2 NPs to be genotoxic since they did not observe DNA damage frequency increase after the chromosomal aberration test in CHO cells. Conversely, Nakagawa et al. [18], Lu et al. [19], Uchino et al. [20], and Zhu et al. [21] found toxic effects of TiO2 on this cell line. However, in some cases NPs used were not characterized.

In an attempt to elucidate the causes of the apparently inconsistent results found for TiO2 NPs and to complement scarce previous information on Al2O3 NPs, several end points rigorously and carefully performed with previously characterized TiO2 and Al2O3 NPs were assayed in CHO-K1 cell line in order to analyze both cyto- and genotoxic effects. Following this purpose, cytotoxicity was determined and NPs concentration range free of cytotoxic effects was identified in CHO-K1 cells. Afterwards, the analysis of genotoxicity at this low concentration level (from 1 μg/mL up to the previously identified threshold value) was performed.

Section snippets

Chemicals

Titanium oxide NPs were purchased from Aldrich (Milwaukee, WI, USA) and aluminium oxide NPs from Sigma–Aldrich (St. Louis, MO, USA). We estimated mean particle diameters and the corresponding errors using transmission electron microscopic (TEM) images from commercially available nanoparticles.

Nanoparticle stock suspensions were prepared in phosphate buffered saline (PBS), vortexed for 10 min and stored at 4 °C in the dark. All other chemicals were of highest quality available.

Characterization of TiO2 and Al2O3 NPs

Shape, size

NPs characterization

Shape, diameter distribution, and specific surface area of TiO2 and Al2O3 NPs were experimentally obtained. TiO2 NPs shape is complex while that of Al2O3 is spherical. Hence, an estimation of the particle size distribution for the latter case was straightforward (i.e. measurement of diameters) while for TiO2 different options were available. Among them, we chose the largest length as characteristic dimension. Thus, average particle sizes for TiO2 and Al2O3 were 20 ± 7 nm and 28 ± 19 nm, calculated

Discussion

Nanotechnology development leads to the increasing use of nanomaterials. However, safety considerations such as the risk assessment of environment and human health have not paralleled industrial development, and consequently results are scarce [29]. In the present study, genotoxicity and cytotoxicity of TiO2 and Al2O3 NPs were evaluated in vitro in CHO-K1 cells, which are particularly sensitive to NPs.

It is worth noting that cellular responses elicited by TiO2 NPs are strongly dependent on NPs

Acknowledgements

This study was supported by: Agencia Nacional de Promoción Científica y Tecnológica (grant BID 1728 OC/AR PICT 0533225; PICT 05-32906; PAE 22771), Universidad Nacional de La Plata (grant 11-05I-129), and Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 6075). Dr. Arnal thanks Prof. F.Schüth at Max Planck Institut für Kohlenforschung for supporting nanoparticles characterization by TEM and Nitrogen Sorption.

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    Formerly at: Max Planck Institut für Kohlenforschung, Kaiser-Wihelm-Platz 1, 45470 Mülheim an der Ruhr, Germany.

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