Evaluation of the neurotoxic effects of engineered nanomaterials in C57BL/6J mice in 28-day oral exposure studies

Highlights

• 28-day neurotoxicity study in mice with foodborne TiO₂ and Ag nanomaterial.

• Absence of neuroinflammation, changes in spontaneous alternation and anxiety-like behavior.

• Motor function changes in female mice post 28-days exposure to silver nanomaterials.

• Increased tyrosine kinase activity in cortex of silver nanomaterial exposed female mice.

• Persistent accumulation of silver in mouse brain up to 4 weeks post-exposure.

Abstract

In recent years, concerns have emerged about the potential neurotoxic effects of engineered nanomaterials (NMs). Titanium dioxide and silver are among the most widely used types of metallic NMs. We have investigated the effects of these NMs on behaviour and neuropathology in male and female C57BL/6J mice following 28-day oral exposure with or without a 14-day post-exposure recovery. The mice were fed ad libitum with food pellets dosed with 10 mg/g TiO₂, 2 mg/g polyvinylpyrrolidone-coated Ag or control pellets. Behaviour was evaluated by X-maze, open field, string suspension and rotarod tests. Histological alterations were analysed by immunohistochemistry and brain tissue homogenates were investigated for markers of oxidative stress, inflammation and blood-brain barrier disruption. Effects of the NMs on tyrosine and serine/threonine protein kinase activity in mouse brains were investigated by measuring kinase activity on peptide microarrays.

Markers of inflammation, oxidative stress and blood-brain barrier integrity were not significantly affected in the male and female mice following exposure to Ag or TiO₂. Both types of NMs also revealed no consistent significant treatment-related effects on anxiety and cognition. However, in the Ag NM exposed mice altered motor performance effects were observed by the rotarod test that differed between sexes. At 1-week post-exposure, a diminished performance in this test was observed exclusively in the female animals. Cortex tissues of female mice also showed a pronounced increase in tyrosine kinase activity following 28 days oral exposure to Ag NM. A subsequent Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) based toxicokinetic study in female mice revealed a rapid and persistent accumulation of Ag in various internal organs including liver, kidney, spleen and the brain up to 4 weeks post-exposure. In conclusion, our study demonstrated that subacute exposure to foodborne TiO₂ and Ag NMs does not cause substantial neuropathological changes in mice. However, the toxicokinetic and specific toxicodynamic findings indicate that long-term exposures to Ag NM can cause neurotoxicity, possibly in a sex-dependent manner.

Introduction

The widespread use of engineered metallic nanomaterials (NMs) in various consumer products has led to a growing concern about their adverse effects on human health. Depending on their physico-chemical properties (e.g. size, agglomeration, surface reactivity) and their route of exposure (e.g. inhalation, ingestion, dermal exposure, intravenous application), NMs may be absorbed into the body and distributed to different secondary target organs and tissues (Oberdörster et al., 2005). In recent years, there has been increasing interest in the investigation of the central nervous system (CNS) as target organ for NMs (Boyes et al., 2012; Boyes and van Thriel, 2020). Initial awareness of the potential adverse effects of nanosize particles on the CNS came with a study that demonstrated translocation of ultrafine particles into the brains of rats upon short-term inhalation exposure (Oberdörster et al., 2004). Since then, several rodent inhalation studies have provided support for the effects of nanoparticles on the brain and, thereby, revealed a mechanistic link to epidemiological studies that show an association between exposure to ambient air pollution particles and neurological diseases (reviewed by (Heusinkveld et al., 2016)). While inhalation represents the primary exposure route for ambient (nanosize) particles as well as for many bulk-manufactured NMs in occupational settings, oral exposure to NMs has also become a topic of increasing investigation. Concerns about the adverse health effects of ingested NMs, including neurotoxicity, have emerged with the growing number of applications in nanomedicine and, in particular, the food sector where NMs are used e.g. as food additives or in food packaging (Bouwmeester et al., 2009; Sohal et al., 2018).

For inhalation exposure, the translocation of NMs via the olfactory nerve into the brain has emerged as an important exposure route (Heusinkveld et al., 2016; Oberdörster et al., 2005). However, for ingested NMs, brain targeting of these particulate entities or their dissolved compounds requires the translocation from the gastrointestinal tract into the circulation and subsequent passage of the blood-brain barrier (BBB). Absorption from the gastrointestinal tract has been shown for various NMs, including silver (Ag) and titanium dioxide (TiO₂) (Geraets et al., 2014; Kreyling et al., 2017; Lee et al., 2019; Wang et al., 2007; Boudreau et al., 2016; Loeschner et al., 2011; van der Zande et al., 2012). Furthermore, crossing of the BBB and accumulation into the CNS has been suggested for Ag and TiO₂ in several studies that applied intravenous or oral administration (Kreyling et al., 2017; Lee et al., 2019; Fabian et al., 2008; Disdier et al., 2015; Loeschner et al., 2011; Recordati et al., 2015; van der Zande et al., 2012). Ag and TiO₂ have also been reported to disrupt the BBB integrity (Trickler et al., 2010; Brun et al., 2012; Xu et al., 2015).

The main mechanisms whereby NMs can cause neurotoxicity in the CNS are thought to be through the induction of oxidative stress and inflammation (Feng et al., 2015; Song et al., 2016; Boyes and van Thriel, 2020). Because of its high content of polyunsaturated fatty acids and low concentrations of antioxidants and antioxidant enzymes, the brain is more sensitive to oxidative stress than other tissues (Valko et al., 2007; Oberdörster et al., 2009; Islam, 2017). Indeed, increased levels of the lipid peroxidation marker malondialdehyde (MDA) and changes in the glutathione antioxidant defense system have been shown in the brain but not in the liver of rats after repeated oral gavage application of Ag NM (Skalska et al., 2016).

Despite the increasing number of neurotoxicological studies on metallic NMs, their role in neuroinflammation has not yet been well understood. It is suggested to result from their ability to activate microglia and astrocytes. Activation of these glial cell types results in secretion of mediators triggering neuronal repair or the release of proinflammatory cytokines and reactive oxygen species, resulting in neuroinflammation (Mayer et al., 2013). Increased expression of the glial fibrillary acidic protein (GFAP), which is expressed in astrocytes (Sofroniew and Vinters., 2010), and ionized calcium-binding adapter molecule 1 (IBA-1), which is expressed upon activation of microglia (Kovacs, 2017) are therefore recognised as important markers of neuroinflammation. In response to exposure to NMs, microglia cells have been shown to enhance the release of proinflammatory cytokines, whereas astrocytes prefer to increase the production of anti-inflammatory factors (Wu and Tang., 2017). The acute innate pro-inflammatory cytokines tumour necrosis factor α (TNF-α), interleukin 1β (IL-1β) and IL-6 are all regulated by mitogen activated protein kinase (MAPK) signalling pathway (Wu and Tang, 2017). These serine-threonine protein kinases regulate cellular activities including proliferation, differentiation, apoptosis or survival and inflammation (Kim and Choi, 2015). Tyrosine kinases represent another type of protein kinase that regulate the majority of cellular pathways as well. They can be subdivided into the receptor tyrosine kinases which have extra-cellular ligand-binding domains and the cytoplasmic (i.e. nonreceptor) tyrosine kinases (Shah et al., 2018). Altered activities of protein kinase signalling pathways have been implicated in the pathology of diverse human diseases including cancer and neurodegeneration (Dhillon et al., 2007; Rosenberger et al., 2016).

In this study, we investigated the potential neurotoxic effects of two different types of NMs in male and female C57BL/6J mice in a 28-day oral repeated exposure design including a 14-day post-exposure recovery period. We choose TiO₂ and Ag since these are widely used metal based NMs in numerous consumer applications and products (Vance et al., 2015; FAO/WHO, 2010). Oral exposure studies with NMs in rodents predominantly have used gavage as the method of their administration. However, while this bolus application procedure ensures a precise dosimetry, it poorly represents the way in which humans may typically be exposed. Accordingly, in our present study, we explored the effects of the TiO₂ and Ag NMs by their incorporation into the mouse feed pellets. The aim of our study was to explore the effects of repeated oral exposures to these NMs on neurobehaviour and expression of markers of oxidative stress, inflammation and blood-brain-barrier disruption, with specific evaluation of sex-specificity of treatment-related changes. Effects of the NMs on tyrosine and serine/threonine protein kinases activity were investigated using peptide microarrays.