Titanium dioxide nanoparticles promote oxidative stress, autophagy and reduce NLRP3 in primary rat astrocytes

https://doi.org/10.1016/j.cbi.2020.108966Get rights and content

Highlights

  • Titanium dioxide nanoparticles have strong oxidant properties.

  • Titanium dioxide nanoparticles induced oxidative stress and mitochondrial damage in rat astrocytes.

  • Titanium dioxide nanoparticles induced NF-κB translocation and autophagy.

  • Titanium dioxide nanoparticles decreased NLRP3 protein expression and inflammasome response.

Abstract

Titanium dioxide nanoparticles (TiO2-NPs) are widely used in the food industry, cosmetics, personal care and paints among others. Through occupational exposure and daily consumption, and because of their small size, TiO2-NPs can enter the body through different routes such as oral, dermal and inhalation, and accumulate in multiple organs including the brain. TiO2-NPs cause severe damage to many cell types, however their effects in the central nervous system remain largely unexplored. Therefore, in the present study we determined the cytotoxic effect of TiO2-NPs on rat astrocytes. We tested the oxidant properties of TiO2-NPs through DTT depletion, and measured oxidative stress-induced damage in mitochondria, through oxidation of 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) and loss of mitochondrial membrane potential (ΔΨm) with Mitotracker Green FM. We further examined oxidative stress-derived responses such as IκB-α degradation by Western Blot, NF-κB translocation by EMSA, autophagy induction by LC3-II levels, and expression of the inflammasome protein NLRP3. TiO2-NPs showed high oxidant properties and induced strong oxidative stress in astrocytes following their internalization, causing mitochondrial damage detected by ΔΨm loss. Responses against oxidative damage such as NF-κB translocation and autophagy were induced and NLRP3 protein expression was downregulated, indicating lower inflammasome-mediated responses in astrocytes. These results support TiO2-NPs cytotoxicity in astrocytes, cells that play key roles in neuronal homeostasis and their dysfunction can lead to neurological disorders including cognitive impairment and memory loss.

Introduction

Titanium dioxide nanoparticles (TiO2-NPs) are widely used in many industries such as food, plastics and cosmetics [1]. TiO2-NPs applications include sunscreen formulations [2], food whitening [3], chewing gums and toothpastes [4], among others. Europe authorized TiO2-NPs more than two decades ago [5], however numerous studies demonstrated TiO2-NPs-derived cytotoxicity in different cell types through multiple routes (oral, dermal, respiratory). After exposition, TiO2-NPs have contact with the organs and cells and are internalized by these last, damaging both their morphology and function. In spite of there are safety regulations about the production and consumption of TiO2-NPs in several countries, there is evidence of damage at cellular level in humans [6,7]. TiO2-NPs can enter the brain directly through the olfactory bulb after inhalation and deposit in the hippocampus. Several studies have shown that TiO2-NPs can also reach the brain after chronic oral consumption [8,9]. To reach the cerebral cells, TiO2-NPs have to cross the blood brain barrier (BBB), which is formed by endothelial cells. Some studies have shown endothelial cell leakiness as consequence of the interaction of nanoparticles with endothelial cells' adherens junction protein VE-cadherin. When TiO2-NPs interact with VE-cadherin, this last is phosphorylated. This phosphorylation affects the interaction between two molecules of VE-cadherin or between one molecule of VE-cadherin with p120 or β-catenin [10,11]. The rupture of the interaction between these molecules, could explain the pass of nanoparticles through the BBB. The presence of TiO2-NPs in the brain causes pathological changes [12], release of monoamine neurotransmitters [13] and altered gene expression affecting memory and learning [14]. Once inside the brain, TiO2-NPs can interact with astrocytes and other glial cells. Astrocytes not only provide structural and nutritional support but maintain neuronal function and homeostasis [15]; however, they can also play a role in neurodegenerative diseases such as Alzheimer's disease, Huntington's disease and Parkinson's disease [16,17].

TiO2-NPs cause cell death and morphological alterations in astrocytes [18] reducing their glutamate uptake and mitochondrial function [19]. We have previously shown TiO2-NPs-induced oxidative stress, lipid peroxidation and changes of mitochondrial membrane potential (ΔΨm) in C6 and U373 astrocytomas [20].

Death and survival pathways are redox-regulated. For example, nuclear translocation of NF-κB is associated with survival and death responses to oxidative stress [21]. NF-κB is over activated in brain cells during Alzheimer's disease [22], whereas TiO2-NPs induce NF-κB translocation in endothelial [23] and microglial cells [24], triggering inflammatory responses including NLRP3 inflammasome activity mediated by pro-caspase 1 and ASC pyroptosome. These proteins induce caspase 1 activation releasing IL-1β and IL-18 leading to pyroptosis [25]. Autophagy plays a key role in stress response and maintenance of organelle function. Autophagy is also a survival mechanism for cells exposed to oxidative stress [21]; however, can contribute to cell death in different pathologies [26].

The aim of this work was to evaluate the oxidant properties of TiO2-NPs in cell-free conditions. We also determined whether TiO2-NPs could be uptaken and induce oxidative stress, ΔΨm loss and specific cell survival responses such as NF-κB translocation and autophagy, as well as their relationship with NLRP3 protein expression in primary astrocytes cultures derived from neonatal rats.

Section snippets

Oxidant properties of TiO2-NPs

The dithiothreitol (DTT) assay was used as a quantitative measure of oxidation. TiO2-NPs oxidize DTT, the remaining non-oxidized DTT reacts with 5,5′-dithio-bis- [2-nitrobenzoic acid] (DTNB) forming 5-thio-2-nitrobenzoic acid (TNB) of yellow color, monitored by spectrophotometry. DTT depletion is proportional to the oxidative capacity of TiO2-NPs [27].

Glass tubes previously washed with acetone were used to remove any organic residue. The reaction was evaluated at 0, 15, 30 and 45 min by

Astrocytes were identified

Primary cell cultures from neonatal rat brains were characterized by GFAP protein expression as glial astrocyte-specific marker. Results showed around 98% of cells displaying positive GFAP staining (green fluorescence), therefore our cell population corresponds to glial astrocytes (Fig. 1S, supplementary material).

Oxidant properties of TiO2-NPs

We evaluated oxidant properties of TiO2-NPs in a cell-free DTT assay. After 15 min exposure, TiO2-NPs (116 μg/mL) oxidized DTT roughly to 50% compared with Vitamin K3 as positive

Discussion

The aim of this work was to determine the oxidant properties of TiO2-NPs in cell-free conditions, to evaluate their cellular uptake as well as the effects on oxidative stress and cell survival in response to the latter. Our results showed strong oxidant properties of TiO2-NPs, inducing oxidative stress related to mitochondrial alterations, NF-κB translocation and autophagy in normal astrocytes derived from neonatal rat brains. This model has widely used and it is considered as a better in vivo

Conclusion

TiO2-NPs have an important oxidant properties and their internalization in astrocytes caused damage associated to oxidative stress, triggering survival responses such as NF-κB translocation and autophagy. Indeed, these nanoparticles can be dangerous to human health in occupational exposure and commercial products. Further studies are required to fully understand cellular effects of TiO2-NPs.

Funding sources

We thank CONACyT for providing financial support to José Antonio Pérez-Arizti, graduate student from the Posgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México, grant number 29452, as well as to Zaira Colín-Val, graduate student from the Doctorado en Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana-Iztapalapa. Grant number 570169. Also we thank the Instituto Nacional de Cardiología “Ignacio Chávez” for the academic support.

CRediT authorship contribution statement

José Antonio Pérez-Arizti: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft. José Luis Ventura-Gallegos: Methodology, Investigation, Resources, Writing - review & editing, Visualization. Roberto Erasmo Galván Juárez: Methodology, Formal analysis, Visualization. María del Pilar Ramos-Godinez: Methodology, Formal analysis, Resources. Zaira Colín-Val: Methodology, Visualization. Rebeca López-Marure: Methodology, Resources, Writing - review & editing,

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We thank Dr. Arnulfo Albores Medina for the use of his laboratory to evaluate the oxidant activity of TiO2-NPs, and MSc. Felipe Alonso Massó Rojas for helping with confocal microscopy.

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