Exposure to ZnO nanoparticles induces oxidative stress and cytotoxicity in human colon carcinoma cells

https://doi.org/10.1016/j.taap.2010.04.012Get rights and content

Abstract

Engineered nanoparticles offer great promise in many industrial and biomedical applications, however little information is available about gastrointestinal toxicity. The purpose of this study was to assess the cytotoxicity, oxidative stress, apoptosis and proinflammatory mediator release induced by ZnO nanoparticles on human colon carcinoma LoVo cells. The biological activity of these particles was related to their physico-chemical characteristics. The physico-chemical characteristics were evaluated by analytical electron microscopy. The cytotoxicity was determined by growth curves and water-soluble tetrazolium assay. The reactive oxygen species production, cellular glutathione content, changes of mitochondrial membrane potential and apoptosis cell death were quantified by flow cytometry. The inflammatory cytokines were evaluated by enzyme-linked immunoadsorbent assay. Treatment with ZnO (5 μg/cm2 corresponding to 11.5 μg/ml) for 24 h induced on LoVo cells a significant decrease of cell viability, H2O2/OHradical dot increase, O2radical dot and GSH decrease, depolarization of inner mitochondrial membranes, apoptosis and IL-8 release. Higher doses induced about 98% of cytotoxicity already after 24 h of treatment. The experimental data show that oxidative stress may be a key route in inducing the cytotoxicity of ZnO nanoparticles in colon carcinoma cells. Moreover, the study of the relationship between toxicological effects and physico-chemical characteristics of particles suggests that surface area does not play a primary role in the cytotoxicity.

Introduction

Engineered nanoparticles (NPs), defined as particles designed and produced to have at least one dimension lower than 100 nm, possess physico-chemical characteristics that make them desirable for commercial, medical, environmental and industrial applications. Nanomaterials are used in cosmetics, dental cements, electrodes for solar cells, pigments for paints, gas sensors and, not least, in the field of environmental remediation, demilitarization of chemical and biological warfare agents and photocatalytic decontamination. Promising developments of nanotechnologies in medical diagnostic and photodynamic therapy, food and packaging fields require health and environmental risk evaluation.

It is known that with decreasing particle size, the proportion of atoms on the surface is larger than the proportion of its volume, so small particles can become more responsive and have a biological activity different from the larger ones and not directly predicted by their bulk chemical constituents (Oberdörster et al., 2005b). For these reasons information about the safety and potential hazards of NPs is urgently needed.

One of the most important portals of entry for NPs is the gastrointestinal tract (GI) and NPs uptake by intestinal epithelium has been recently demonstrated (Hillyer and Albrecht, 2001).

Therefore, the evaluation of cytotoxic damage eventually induced by ingested NPs on intestinal epithelial cells is very attractive because they can impair the protective function of the GI. The resulting traslocation of NPs across the intestinal epithelium to the blood stream can turn out to their transport and uptake by organs such as the brain and the liver (Gopee et al., 2007). Today few data are available on the in vitro toxicity of NPs via ingestion exposure (Wang et al., 2008), while several in vivo and in vitro studies on pulmonary inflammation and oxidative stress of various inhaled NPs are published (Muller et al., 2008, Karlsson et al., 2008).

In light of the potential exposure and medical applications of zinc oxide nanoparticles (ZnO-NPs) (Chen and Zhang, 2006), we have chosen a human colon carcinoma cell line (LoVo) to evaluate the potential toxicity of ZnO-NPs in the GI. The effects induced by ZnO-NPs have been compared to those by “fine” titanium dioxide (TiO2) particles; these latter were selected as negative controls (Oberdörster et al., 2005a).

In particular, the effects of ZnO-NPs on LoVo cells, for short and long times, at low and high doses of exposure, were determined. High priority should be given to investigate the cumulative effects of repeated low doses of NPs, because of the great potential of nanomaterials for the biomedical applications.

Rare researches have been focusing on the relationship between the physico-chemical characteristics of NPs and their toxicological properties. In the scientific community it is believed that size distribution, agglomeration state, shape, surface area, crystal structure and chemical composition are important physico-chemical characteristics in determining the NPs toxic effects. In this respect we have firstly characterized both ZnO and TiO2 particles by analytical electron microscopy. Then, we tried to relate some physical parameters, such as structure, size distribution, agglomerate state and impurities in the elemental composition, to biological activity ensuing from NPs–cell interaction. In particular, cytotoxicity, oxidative stress and release of proinflammatory mediators were analyzed on human colon carcinoma cell line (LoVo).

The relationship between the physical properties and the toxicological effects of ZnO-NPs was evaluated expressing dose against mass, surface area and particle number.

Section snippets

Particle and sample preparation for characterization

ZnO-NPs and TiO2 particles were obtained from Sigma-Aldrich Company Ltd. (Gillingham, Dorset, UK). The nominal size of particles as purchased is 50–70 nm for ZnO-NP and about 1 µm for fine TiO2.

A few milligrams of particles were weighted with a Mettler H54 AR electric balance (precision 0.01 mg). The particles were suspended in the culture medium of cells to mimic their exposure with the biological system. The stock suspensions of ZnO-NPs and TiO2 particles were sonicated with a probe sonicator

Particle characterization

SEM and TEM observations (Figs. 1A and C, respectively) allowed us to recognize three different particle morphologies for the ZnO-NPs: a) spherules approximately ranging from 45 to 60 nm; b) particles with rod-like shapes up to 70 nm width and up to 170 nm length; c) aggregates ranging from 100 nm to 600 nm.

Fig. 1E shows the size distribution obtained for ZnO-NPs. The size distribution showed an average diameter equal to 196 nm. 34% of particles possessed dimensions below 100 nm, 29% were aggregates

Discussion

In this study we used a human colon carcinoma cell line (LoVo) as a model for investigating the interaction between ZnO-NPs and a hypothetical cell target, organ specific, and to evaluate the possible cytotoxic effect induced by such an interaction.

Our work showed a significant decrease in cell viability, remarkable morphological changes, apoptosis induction via ROS production and IL-8 release. It is well known that IL-8 is an important chemokine for innate immune responses and recruitment of

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