Neuronal cytotoxicity and genotoxicity induced by zinc oxide nanoparticles

Highlights

• Zinc oxide nanoparticles (ZnO NPs) are one of the most abundantly used nanomaterials.

• There is a significant lack of toxicological data for ZnO NPs on the nervous system.

• ZnO NP effects on SHSY5Y cells were studied under different exposure conditions.

• They induced dose- and time-dependent cytotoxicity and genotoxicity on neuronal cells.

• Free Zn^2 + ions released from ZnO NPs were not responsible for the viability decrease.

Abstract

Zinc oxide nanoparticles (ZnO NPs) are one of the most abundantly used nanomaterials in consumer products and biomedical applications. As a result, human exposure to these NPs is highly frequent and they have become an issue of concern to public health. Although toxicity of ZnO NPs has been extensively studied and they have been shown to affect many different cell types and animal systems, there is a significant lack of toxicological data for ZnO NPs on the nervous system, especially for human neuronal cells and tissues. In this study, the cytotoxic and genotoxic effects of ZnO NPs on human SHSY5Y neuronal cells were investigated under different exposure conditions. Results obtained by flow cytometry showed that ZnO NPs do not enter the neuronal cells, but their presence in the medium induced cytotoxicity, including viability decrease, apoptosis and cell cycle alterations, and genotoxicity, including micronuclei production, H2AX phosphorylation and DNA damage, both primary and oxidative, on human neuronal cells in a dose- and time-dependent manner. Free Zn^2 + ions released from the ZnO NPs were not responsible for the viability decrease, but their role on other types of cell damage cannot be ruled out. The results obtained in this work contribute to increase the knowledge on the genotoxic and cytotoxic potential of ZnO NPs in general, and specifically on human neuronal cells, but further investigations are required to understand the action mechanism underlying the cytotoxic and genotoxic effects observed.

Introduction

Zinc oxide nanoparticles (ZnO NPs) are one of the most abundantly used nanomaterials in consumer products and biomedical applications due to their specific properties, e.g. transparency, high isoelectric point, biocompatibility, and photocatalytic efficiency. They are widely employed in a variety of devices including cosmetics, toothpaste, sunscreens, fillings in medical materials, textiles, wall paints, and other building materials, and they can be also utilized in environmental remediation for elimination or degradation of pollutants in water or air (Qiang, 2001). Furthermore, ZnO NPs have promising applications in the medicine field since they have been proposed as a possible treatment for cancer and/or autoimmune diseases after being found to be selectively toxic towards potential disease-causing cells (Hanley et al., 2008, Premanathan et al., 2011, Akhtar et al., 2012), and they are being considered to be used in fabrication of nerve guidance channels for treatment of nerve injury (Seil and Webster, 2008). As a result of all these uses, human exposure to these NPs is highly frequent. They can enter the organism through different pathways (respiratory tract, digestive system and parenteral routes) and have shown a systemic distribution in in vivo studies (Vandebriel and De Jong, 2012), so they can potentially reach any organ or tissue and involve a risk for human health. Toxicity of ZnO NPs has been extensively studied and they have been shown to affect many different cell types and animal systems (Chiang et al., 2012, De Berardis et al., 2010, Osman et al., 2010, Sharma et al., 2012a, Sharma et al., 2012b, Wahab et al., 2011). Commonly, the toxicity of NPs is associated with their small size and high specific surface area and therefore, nanoforms are theoretically expected to be more toxic than their bulk counterparts (Xiong et al., 2011).

In recent years, there have been an increasing number of works reporting that different NPs can reach the brain and cause neurological injuries, being associated even with neurodegenerative diseases (Block et al., 2004, Hu and Gao, 2010, Peters et al., 2006). This translocation can happen both directly, through axonal transport from olfactory epithelium, or indirectly by passing to the bloodstream and crossing the blood brain barrier (Oberdörster et al., 2004). Similarly to other metal oxide NPs, it has been recently found that ZnO NPs reach the brain of experimental animals after oral (Lee et al., 2012) and inhalatory (Kao et al., 2012a) administration. Still, there is a significant lack of toxicological data for ZnO NPs on nervous system, especially for human neuronal cells and tissues.

In vitro data have shown that ZnO NPs induce cytotoxicity in mouse neuroblastoma Neuro-2A cells and neural stem cells (Deng et al., 2009, Jeng and Swanson, 2006), in rat RSC96 Schwann cells and primary neuronal cells (Chiang et al., 2012, Yin et al., 2012), and in human glioma cells (Ostrovsky et al., 2009). Besides, they were found to enhance the excitability of rat neurons by altering the ion channels (Zhao et al., 2009) and to decrease the adhesion of rat astroglial cells (Seil and Webster, 2008), although in this last case the cells were exposed to composite materials with ZnO NPs.

The only in vivo study describing neurological effects after ZnO NP exposure reported attenuation in spatial learning and memory ability by alteration of synaptic plasticity in rats after intraperitoneal administration (Han et al., 2011). Besides, morphological and histochemical changes in brains of rats fed with bulk ZnO were also described (Kozik et al., 1980).

Given the wide and frequent use of ZnO NPs in many fields closely related to human beings, their promising beneficial applications in medicine, and the scarce knowledge on their potential neurotoxic effects, the main objective of this work was to investigate the cytotoxic and genotoxic effects of ZnO NPs on human SHSY5Y neuronal cells and to explore the underlying mechanisms involved in these effects.