Neurotoxicity of Titanium Dioxide Nanoparticles: A Comprehensive Review

Xing Zhang; Yuanyuan Song; Hongyang Gong; Chunyan Wu; Binquan Wang; Wenxuan Chen; Jiawei Hu; Hanhui Xiang; Ke Zhang; Mingkuan Sun

doi:10.2147/IJN.S442801

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Review

Neurotoxicity of Titanium Dioxide Nanoparticles: A Comprehensive Review

Authors Zhang X, Song Y, Gong H, Wu C, Wang B, Chen W, Hu J, Xiang H, Zhang K, Sun M

Received 2 October 2023

Accepted for publication 25 November 2023

Published 5 December 2023 Volume 2023:18 Pages 7183—7204

DOI https://doi.org/10.2147/IJN.S442801

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 2

Editor who approved publication: Professor R.D.K. Misra

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Xing Zhang,^1,^* Yuanyuan Song,^1,^* Hongyang Gong,^1,^* Chunyan Wu,¹ Binquan Wang,¹ Wenxuan Chen,² Jiawei Hu,² Hanhui Xiang,² Ke Zhang,¹ Mingkuan Sun¹

¹Department of Toxicology, School of Public Health, Nanjing Medical University, Nanjing, Jiangsu, People’s Republic of China; ²The Second Clinical Medical School, Nanjing Medical University, Nanjing, Jiangsu, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Mingkuan Sun, Department of Toxicology, School of Public Health, Nanjing Medical University, No. 101 Longmian Avenue, Jiangning District, Nanjing, 211166, People’s Republic of China, Email [email protected]

Abstract: The increasing use of titanium dioxide nanoparticles (TiO₂ NPs) across various fields has led to a growing concern regarding their environmental contamination and inevitable human exposure. Consequently, significant research efforts have been directed toward understanding the effects of TiO₂ NPs on both humans and the environment. Notably, TiO₂ NPs exposure has been associated with multiple impairments of the nervous system. This review aims to provide an overview of the documented neurotoxic effects of TiO₂ NPs in different species and in vitro models. Following exposure, TiO₂ NPs can reach the brain, although the specific mechanism and quantity of particles that cross the blood-brain barrier (BBB) remain unclear. Exposure to TiO₂ NPs has been shown to induce oxidative stress, promote neuroinflammation, disrupt brain biochemistry, and ultimately impair neuronal function and structure. Subsequent neuronal damage may contribute to various behavioral disorders and play a significant role in the onset and progression of neurodevelopmental or neurodegenerative diseases. Moreover, the neurotoxic potential of TiO₂ NPs can be influenced by various factors, including exposure characteristics and the physicochemical properties of the TiO₂ NPs. However, a systematic comparison of the neurotoxic effects of TiO₂ NPs with different characteristics under various exposure conditions is still lacking. Additionally, our understanding of the underlying neurotoxic mechanisms exerted by TiO₂ NPs remains incomplete and fragmented. Given these knowledge gaps, it is imperative to further investigate the neurotoxic hazards and risks associated with exposure to TiO₂ NPs.

Keywords: TiO₂ NPs, neurotoxic effects, oxidative stress, neuronal damage, neurotoxic mechanisms

Graphical Abstract:

Introduction

Nanomaterials (NMs) are materials defined as having at least one dimension ranging from 1 to 100 nanometers (nm). Titanium dioxide nanoparticles (TiO₂ NPs) rank among the top five NMs used in consumer products, such as food additives, toys, cosmetics, electronic products, and pharmaceuticals (Figure 1).^1,² Consequently, the presence of TiO₂ NPs in air, water, soil, and other environmental media has gradually increased due to their widespread use.³ This growing application and contamination have made human and animal exposure to TiO₂ NPs unavoidable. Apart from skin exposure, inhalation, and oral exposure, other routes of exposure to TiO₂ NPs include intraperitoneal injection, subcutaneous injection, and intramuscular injection.⁴ Importantly, regardless of the route of exposure, TiO₂ NPs can ultimately enter the systemic circulation and translocate to various tissues and organs (Figure 2).⁴ As the accumulation of TiO₂ NPs in the body increases, the associated health hazards become more severe.⁵

Figure 1 Application of TiO₂ NPs.

Figure 2 TiO₂ NPs can enter the human body through different ways and accumulate in the body, posing a threat to human health.

Before the rise of nanotechnology, TiO₂ was widely used in the form of fine particles (FPs), which were considered as poor soluble and low toxicity particles.⁶ However, some studies have contradicted this view, such as lung tumors in rats exposed to high levels of TiO₂ FPs for two years.⁷ Furthermore, TiO₂ has been classified as a Group 2B carcinogen (possibly carcinogenic to humans) by the International Agency for Research on Cancer.⁸ Although the specific carcinogenicity of TiO₂ FPs is still debated, there is no doubt that TiO₂ FPs pose a health risk.⁹ Compared to TiO₂ FPs, TiO₂ NPs present stronger catalytic activity and bioactivity due to their nanoscale dimensions.⁹ Consequently, the toxicity of TiO₂ NPs cannot be solely inferred from the known toxicology of TiO₂ FPs, nor can it be determined using conventional methods.¹⁰ In recent years, extensive exploration of the effects of TiO₂ NPs exposure on human health also reflects the high concern about the safety of TiO₂ NPs. In addition to considering all relevant exposure scenarios and biological intermediate steps, understanding the final toxic outcome is critical for human health risk assessment.¹¹ Results from another child cohort study in China indicated that Ti can cross the placental barrier (PB) to harm fetuses that are extremely sensitive to environmental threats.¹² Several epidemiological studies have confirmed that Ti exposure increases the risk of adverse birth outcomes, including neural tube defects, preterm birth, fetal distress, and low birth weight.^13–15 Moreover, TiO₂ NP exposure can have detrimental effects on the health of the population beyond the fetus. Emerging epidemiological evidence suggests that higher levels of urinary or blood Ti are associated with an increased risk of various adverse health effects, including diabetes and cardiopulmonary disorders (Figure 2).^16–19 In recent years, laboratory studies on the toxicity of TiO₂ NPs have surpassed epidemiological studies. Common animal models such as mice, rats, zebrafish, and Drosophila have been used to study TiO₂ NPs. In vivo, studies have shown that TiO₂ NPs exposure may be linked to lung inflammation, pneumoconiosis, cardiovascular disease, reproductive toxicity, retinal impairments, etc.^20–24 In vitro, studies have also supported these toxic effects of TiO₂ NPs.^25–28 Given that nanoparticles can enter the brain, concerns regarding their neurotoxic effects, including those of TiO₂ NPs, have gained significant attention.²⁹

The entry of TiO₂ NPs into the brain mainly occurs through the blood-brain barrier (BBB), via absorption-mediated transversion or intranasal pathways.^30,31 However, the mechanisms by which nano-titanium dioxide penetrates and targets different brain regions remain unknown. The degree of TiO₂ NPs accumulation in each brain region closely correlates with the extent of neurotoxic effects. Common neurotoxic effects include behavior deficits, nervous system dysfunction, and structural changes induced by oxidative stress, autophagy, inflammation, or the activation of specific signaling pathways.³² Although emerging studies support the role of TiO₂ NPs exposure as an environmental risk factor for human health, conscientious and systematic investigations are scarce into the extent of TiO₂ NPs translocation to different brain regions and the resulting damage to the neuronal system in relation to particle dose and particle size. The lack of information on the neurotoxicity of TiO₂ NPs also complicates risk assessment following exposure. Therefore, this paper will mainly focus on current studies concerning the neurotoxicology of TiO₂ NPs, while also reviewing the molecular mechanisms underlying their neurotoxic effects to mitigate potential damage resulting from exposure.

Evidence from Epidemiological and Human Exposure Studies

In earlier years, population exposure to TiO₂ NPs was primarily investigated among occupational populations. Welding fumes, industrial waste combustion, and mineral mining can all result in environmental contamination by TiO₂ NPs, thereby increasing the exposure risk for workers.³³ Exposure to fumes from metal-inert gas soldering has been found to increase the risk of Parkinson’s disease (PD).³⁴ Although these fumes mainly consist of zinc, copper, and iron, Andujar et al discovered an excessive accumulation of Ti in the lung tissue sections of welders in 2014.³⁵ Industrial waste, pesticides, and automobile exhaust are common sources of environmental pollutants associated with neurotoxic effects.³⁶ Among various environmental pollutants, NPs can easily penetrate the BBB and induce neurotoxicity by activating innate immune responses in astrocytes, microglia, and neurons.³⁶ TiO₂ NPs are a major component among environmental pollutants, with up to 760 tons of TiO₂ NPs being released into the soil through sewage and sludge each year.^37,38 Currently, there is no direct evidence of neurotoxic effects caused by TiO₂ NPs exposure in mineral miners, but a previous study suggested a significantly increased inflammatory response in mineral miners exposed to TiO₂ NPs.³⁹ It is well known that the occurrence of inflammatory reactions in other organs is closely related to nervous system damage.⁴⁰ With the increasing application of TiO₂ NPs, concerns have also arisen regarding the neurotoxic effects of non-occupational populations exposed to TiO₂ NPs. A recent cohort study demonstrated that high levels of urinary Ti during pregnancy were significantly associated with impaired language development, suggesting that TiO₂ NPs might act as developmental neurotoxicants.⁴¹ Furthermore, elevated levels of Ti in maternal hair were also significantly associated with an increased risk of neural tube defects.⁴² However, epidemiological studies on the neurotoxic effects caused by TiO₂ NPs are still limited. Currently, laboratory studies are the main basis for evaluating the neurotoxicity of TiO₂ NPs.

Literature Search

To review the neurotoxic potential of TiO₂ NPs, a comprehensive literature search was conducted using the “Pubmed” database, covering articles from 1991 to September 29, 2023. The search utilized combinations of the following keywords: Titanium dioxide nanoparticles exposure; E171 exposure; Titanium dioxide nanoparticles neuron; Titanium dioxide nanoparticles brain; Titanium dioxide nanoparticles behavior; and Titanium dioxide nanoparticles neurotoxicology. Our search strategy involved an initial screening of all titles and abstracts, followed by a full-text review of the pertinent review articles. One hundred forty-seven papers were selected, including one hundred twenty-one research papers. Citations within twenty-six reviews were also screened for additional studies not identified in the electronic search; however, no additional research papers were found through these references. In the end, a total of one hundred and twenty-one research papers were selected, all of which included neurotoxicity endpoints in their experimental designs.

For experimental research, we identified in vitro or in vivo studies involving the administration of TiO₂ NPs. The researched organism groups mainly consisted of rodents, zebrafish, and cells. Fifty-five studies were conducted on rodents, twenty on zebrafish, and two on Caenorhabditis elegans (C. elegans), while one each on Pheretima hawayana, Tegillarca granosa, and Drosophila melanogaster. Additionally, forty-one studies used in vitro cells, including various animals and human-derived neuronal cells (Figure 3).

Figure 3 Study selection flow diagram. The flow chart illustrates the number of citations and resources that underwent screening, exclusion, and/or inclusion in the review.

Neurotoxic Effects of TiO₂ NPs in Rodents

The results of the literature search demonstrate that rodents were the most commonly used species in neurotoxicity studies of TiO₂ NPs, forming the basis of 55 articles. Among these, 23 articles focused on rat models, with the Wistar rat being the most commonly employed. The exposed TiO₂ NPs generally had a particle size of less than 20 nm, and the exposure doses ranged below 200 mg/kg (Table 1). Intratracheal instillation, intragastric administration, intravenous injection, and intraperitoneal injection are common methods of rat exposure to TiO₂ NPs. Pregnancy and lactation have been identified as critical periods of neurodevelopment and have been selected as exposure windows to toxicants in many neurotoxicological studies, including the neurotoxicity of TiO₂ NPs.^43–45 Exposure to TiO₂ NPs (10 nm, 100 mg/kg) during pregnancy or lactation has been found to impair memory and learning in Wistar offspring rats by reducing hippocampal cell proliferation.^44,46 Prenatal exposure to TiO₂ NPs (5 nm, 1 μg/μL) via subcutaneous injection has been shown to enhance depressive-like behavior in adult Sprague-Dawley (SD) rats.⁴⁷ Additionally, Engler-Chiurazzi et al conducted locomotion, learning, and anxiety tests on male adult SD rats and observed significant cognitive impairments in offspring resulting from inhalation exposure to TiO₂ NPs (170.9 ± 6.4 nm, 10.4 ± 0.4 mg/m³) during pregnancy.⁴⁸ In earlier studies, the roles of age and sex in early-life exposure at different developmental stages were often overlooked. However, a recent study demonstrated that female pups were more susceptible to adverse outcomes after early exposure to oral TiO₂ NPs (postnatal day, PND 2–5 or PND 7–10) compared to male pups, while male pups exhibited more severe motor deficits following exposure to nano-titanium dioxide during late lactation (PND 17–20).⁴⁵ The behavioral impairments resulting from TiO₂ NPs exposure during pregnancy and lactation involve multiple mechanisms, including oxidative damage, changes in neurotransmitter concentrations, and metabolic disturbances.^45,47 Furthermore, exposure to TiO₂ NPs during pregnancy has been associated with neuronal apoptosis, decreased neurogenesis, altered expression of brain-derived neurotrophic factor (BDNF), and impaired synaptic plasticity.^49–51

Table 1 Overview of Literature Investigating Neurotoxic Effects of TiO₂ NPs in Rats

Adolescence is a transitional period of physical and behavioral development between childhood and adulthood, and it represents a particularly vulnerable neurodevelopmental phase.^67,68 In a study by Cui et al exposure to TiO₂ NPs (5 nm, 20 mg/kg) during adolescence via intravenous injection induced anxiety-like behavior, cognitive impairment, neuroinflammation, and oxidative damage in the hippocampus.⁶³ Similar results were observed in another study using adult Wistar rats, where subacute exposure to 20 mg/kg TiO₂ NPs increased the anxious index.⁶¹ Furthermore, motor functional damage and spatial cognitive impairments were observed in adult Wistar rats exposed to TiO₂ NPs.^54,57,60 Certain regions of the mammalian brain continue to exhibit neurogenesis throughout adulthood, such as the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus.⁶⁹ Adult neurogenesis is a multiple-step process, and abnormalities at any stage can impair neurogenesis and brain function, leading to cognitive impairment and neurodegenerative diseases.⁷⁰ Exposure to TiO₂ NPs in adulthood also induces various neurotoxic effects, including mitochondrial dysfunction, oxidative stress, cell apoptosis, alterations in neuronal architecture, neuroinflammation, decreased neurogenesis, and electrophysiological alterations.^{52–54,56,58,59,64}

To date, slightly more neurotoxicology studies on TiO₂ NPs have been conducted using mice as animal models compared to rats, totaling 32 studies. Among them, 21 studies used ICR mice, 7 studies used C57BL/6, 2 studies used Albino mice, while BALB/c mice and Swiss Webster mice were used in 1 study each (Table 2). The exposure methods used in most of the neurotoxicological studies of TiO₂ NPs using mice as animal models mimic the actual pathways of human exposure to TiO₂ NPs. TiO₂ NPs can enter the human body via intentional or unintentional inhalation; therefore, intranasal administration was chosen as the exposure method in 10 studies. Intranasal drug administration is a promising method to bypass BBB, resulting in higher bioavailability and greater brain exposure compared to oral administration at the same dose.⁷¹ Several studies have shown that intranasal administration of TiO₂ NPs in mice resulted in various neurological damages, including hippocampal cell apoptosis, tissue necrosis, oxidative damage, neuroinflammation, imbalance of glutamate metabolism, and altered gene expression.^72–79 These structural and functional impairments in the nervous system are closely related to behavioral deficits. Ze et al found that exposure to TiO₂ NPs (208–330 nm, 2.5 mg/kg body weight, BW) for 90 consecutive days via intranasal administration damaged the hippocampus structure, decreased long-term potentiation, altered gene expression, and caused spatial recognition deficits.⁸⁰ Intragastric administration, a method commonly used to assess the effects of oral exposure in the hazard assessment of environmental toxicants, was employed in 13 neurotoxicological studies of TiO₂ NPs using mice as animal models.⁸¹ Pregnancy exposure to TiO₂ NPs (21 nm, 150 mg/kg) via intragastric administration caused the delayed appearance of neurobehavioral impairments in both dams and offspring, which may be related to disruption of the gut-brain axis.^82,83 Furthermore, exposure to low levels of TiO₂ NPs (6.5 nm, < 5 mg/kg) via intragastric administration during pregnancy and lactation retarded axonal and dendritic outgrowth, impaired cognitive ability, and increased hippocampal neurons apoptosis.^84–86 In addition to common impairments in the nervous system associated with TiO₂ NPs exposure, such as neuroinflammation, oxidative stress, and altered gene expression, Wang et al found that exposure to TiO₂ NPs (10 nm) increased the risk of PD.⁸⁷

Table 2 Overview of the Literature Investigating Neurotoxic Effects of TiO₂ NPs in Mice

Neurotoxic Effects of TiO₂ NPs in Zebrafish

Zebrafish (Danio rerio) is commonly used as in vivo model system for studying the toxicity of nanomaterials due to its low cost, rapid growth, and significant homology to humans.¹⁰⁵ A total of 20 studies have investigated the neurotoxic effects of TiO₂ NPs in zebrafish, with 11 of them examining co-exposure to other compounds (Table 3). Among the 11 studies, 8 studies selected the embryonic stage of zebrafish for TiO₂ NPs exposure, 2 studies selected adult zebrafish, and one study selected zebrafish larvae. The most commonly used dose of TiO₂ NPs in studies involving co-exposure to other compounds was 100 μg/L. So far, TiO₂ NPs have been shown to enhance Pb,^106,107 decabromodiphenyl oxide (BDE-209),¹⁰⁸ cypermethrin,¹⁰⁹ triphenyl phosphate,¹¹⁰ bisphenol A,^111,112 difenoconazole,¹¹³ tetracycline,¹¹⁴ and microcystin-LR¹¹⁵ -induced neurotoxicity. TiO₂ NPs mainly enhance the neurotoxicity of these compounds by increasing their bioconcentration and bioavailability in zebrafish. Interestingly, co-exposure with TiO₂ NPs did not alter pentachlorophenol-induced neurotoxicity.¹¹⁶

Table 3 Overview of Literature Investigating Neurotoxic Effects of TiO₂ NPs in Zebrafish

Exposure to TiO₂ NPs alone is also able to induce a variety of neurotoxic effects in zebrafish. The embryonic stage of zebrafish is the most commonly used exposure stage for TiO₂ NPs exposure models, which may be attributed to the incomplete development of the BBB during this period.¹²⁶ TiO₂ NPs exposure during the embryonic stage of zebrafish significantly alters motor behavior, social behavior, and spatial recognition memory.^117–120 In addition to behavioral impairments, TiO₂ NPs exposure causes oxidative stress, promotes neuronal proliferation, decreases motor neuron axon length, alters gene expression, and increases cell apoptosis.^120–123 Two study chose the adult stage of zebrafish for TiO₂ NPs exposure, and their results suggested that TiO₂ NPs exposure caused cognitive deficit, promoted neuroinflammation, and altered biochemical constituents of the brain.^124,125

Neurotoxic Effects of TiO₂ NPs in Other Animal Models

In vivo, studies investigating the potential neurotoxic effects of TiO₂ NPs exposure on animals other than rodents and zebrafish are relatively scarce. To date, only five studies investigated the neurotoxicity of TiO₂ NPs exposure in other animal models, namely C. elegans, Tegillarca granosa, Pheretima hawayana, and Drosophila melanogaster (Table 4).

Table 4 Overview of the Literature Investigating Neurotoxic Effects of TiO₂ NPs in Other Animal Models

The nervous system of the C. elegans model is structurally and functionally similar to that of mammals. Its small size, short life cycle, and high reproductive rate make C. elegans an advantageous model in neuroscience.¹³² Long-term exposure to TiO₂ NPs (10 nm) resulted in severe defects in the development of AVL and DVB neurons that control defecation in nematodes.¹²⁷ Neurons exposed to both anatase and rutile TiO₂ NPs exhibited shorter axon growth, which may be the reason for defective locomotion behavior in nematodes.¹²⁸

Drosophila melanogaster is an excellent animal model for evaluating the neurotoxicity of various NPs due to its low-cost, physiological similarities to humans, and well-known behavioral and developmental characteristics.^133,134 The results of a recent study indicated that chronic exposure to TiO₂ NPs (approximately 20 nm, 20 mg/kg) induced deficits in motor behavior by disrupting the development of the neuromuscular junction (NMJ) in Drosophila.¹²⁹

Tegillarca granosa and Pheretima hawayana are rarely studied in neurotoxicological research involving NPs. Tegillarca granosa inhabits intertidal mudflat, where the concentration of NPs is predicted to be higher than in other parts of the ocean.¹³⁵ Selecting Tegillarca granosa as an animal model provides better insights into the neurotoxicity of NPs in marine bivalve mollusks.¹³⁰ Exposure to TiO₂ NPs (200 nm) increased neurotransmitters concentrations, suppressed the activity of acetylcholinesterase (AChE), and decreased the expression of neurotransmitter-related genes, which may disrupt various physiological processes in Tegillarca granosa.¹³⁰ While TiO₂ NPs are increasingly being released into the soil, their effects on soil biota remain largely unknown.¹³⁶ The traditional sentinel species for soil toxicity testing is the earthworm, and Pheretima hawayana is a species of Egyptian earthworm. Exposure to TiO₂ NPs altered biochemical indices related to the function of the nervous system, such as inhibiting AChE, increasing antioxidant enzymes, and accumulating malondialdehyde (MDA).¹³¹

Neurotoxic Effects of TiO₂ NPs in vitro Models

In vitro models are widely used to assess neurotoxic effects on cellular functions.¹³⁷ Several studies have evaluated the neurotoxic effects of TiO₂ NPs using in vitro models. Primary hippocampal and cortical neurons are widely used in vitro models for neurotoxicology testing as they are easily polarized and form unique axons and dendrites. In addition, these models are used to study neuronal polarization, axon/dendrite morphology, synaptic formation, and central nervous system (CNS) functions.¹³⁸ Exposure to TiO₂ NPs impairs neuronal function, inhibits neuroblast proliferation, reduces cell viability, and increases cell apoptosis by promoting oxidative stress in both primary hippocampal and cortical neurons.^139–144 Furthermore, TiO₂ NPs inhibit neurite outgrowth of hippocampal neurons by interfering with glutamate metabolism and impairing N-methyl-D-aspartic acid (NMDA) receptor function.¹⁴⁵ According to some previous studies, the suppression of axonal development, dendritic development, and synapse development by TiO₂ NPs was associated with decreased expression of axon growth-related factors and inhibition of the Wnt/β-catenin and BDNF-TrkB pathways.^146–148 See Table 5 for details.

Table 5 Overview of the Literature on Neurotoxic Effects of TiO₂ NPs on Primary Neuron and Nerve Cell Lines

Rat pheochromocytoma (PC12) cell line and human SH-SY5Y neuroblastoma cell line have been used as models for neurotoxicity testing of TiO₂ NPs (Table 5). PC12 cell line shows morphological and functional differentiation similar to sympathetic neurons. PC12 cell line is a suitable model for studying the chemical disruption of neuronal differentiation, synthesis, storage, and release of neurotransmitters, function and regulation of ion channels, and the interaction of compounds with membrane-bound receptors.¹⁶⁰ A previous study revealed that treatment of PC12 cells with TiO₂ NPs (< 36 nm, < 200 μg/mL) decreased cell viability, increased cell apoptosis via oxidative stress, inhibited the neurite outgrowth, disturbed cell cycle, and disrupted the ubiquitin-proteasome system.^149–151 The human-derived SH-SY5Y cell line is preferred over the PC12 cell line as it avoids interspecific differences in chemical action.¹⁶¹ The SH-SY5Y cell line is an excellent model for studying toxicity on proliferating or differentiated cells because it can be maintained as neuroblasts or induced to differentiate into more neuron-like morphologies.¹⁶¹ TiO₂ NPs were shown to cause endoplasmic reticulum (ER) stress, autophagy, inhibition of cell proliferation, disturbance of the microtubule dynamics, and membrane damage in SH-SY5Y cells.^153–158 Several in vivo studies investigated the neurotoxic effects of TiO₂ NPs on mouse hippocampus. However, one in vitro study explored the neurotoxic effects of TiO₂ NPs on mouse hippocampal neuronal HT22 cells. The study revealed that TiO₂ NPs increased apoptosis of HT22 cells via oxidative stress- and calcium imbalance-mediated ER stress.¹⁵⁹

Acute or prolonged exposure to TiO₂ NPs is associated with toxic effects on neuronal and glial cells.¹⁶² Glial cells are critical cells of the nervous system, which serve as tissue-resident macrophages. Microglia are crucial regulators that influence nervous system development, maintenance of the neural environment, and response to injury and repair.¹⁶³ The immortalized mouse microglia cell line BV2 is often used as an alternative for primary microglia in cell experiments. Some previous studies showed that exposure of BV2 cells to TiO₂ NPs was associated with mitochondrial dysfunction and increased oxidative stress.^28,164 Astrocytes play a key role in innate and adaptive immune responses in CNS injury.¹⁶⁵ Due to advancements in cell culture technology, primary astrocytes have become a common primary cell model. Previous studies revealed that TiO₂ NPs induced mitochondria damage, oxidative stress, autophagy, neuroinflammation, and cell apoptosis in primary rat cortical astrocytes.^166–168 Other studies employed human glial cell lines as in vitro models for neurotoxicity studies to eliminate species differences. Some previous studies revealed that TiO₂ NPs inhibited cell proliferation, induced morphological changes, decreased immuno-location of F-actin fibers, and increased cell apoptosis in U374 cells.^169,170 Furthermore, several studies have investigated the neurotoxic effects of TiO₂ NPs in a co-culture of glial cells and other cells. For example, Yang et al showed that TiO₂ NPs stimulate the inflammatory reaction in brain microglia and damage neuron using a co-culture model of primary microglia and PC12 cell line.¹⁷¹ Similarly, TiO₂ NPs was shown to stimulate the inflammatory reaction in brain microglia and damage neurons in co-culture models of BV2 and N27 mesencephalic neurons, and BV2 and N2a neuroblastoma cells.^172,173 See Table 6 for details.

Table 6 Overview of the Literature on Neurotoxic Effects of TiO₂ NPs in Primary Glial Cells and Glial Cell Lines

Most in vivo and in vitro studies have evaluated the neurotoxic effects of TiO₂ NPs in the cortex, hippocampus, and cerebellum. However, to the best of our knowledge, no studies have evaluated the neurotoxic effects of TiO₂ NPs on other brain regions. The BBB is effective in protecting the brain from chemical damage. Therefore, there is a need to understand the effects of TiO₂ NP_S on the BBB. A previous study exploring the effects of TiO₂ NPs on an in vitro model of BBB established by co-culturing primary human brain microvascular endothelial cells (HBMECs) and primary human astrocytes, revealed that TiO₂ NPs increased the permeability of the BBB.³¹ Another study showed that acute or long-term exposure of an in vitro model of the BBB established by co-culturing primary rat endothelial cells and glial cells to TiO₂ NPs was associated with BBB dysfunction related to increased inflammatory response and altered expression of the ABC transporter.¹⁷⁴ Moreover, treatment of T98G human glioblastoma cells with TiO₂ NPs was associated with changes in the transcriptome, suggesting that exposure to TiO₂ NPs could compromise BBB integrity and cause neuroinflammation.¹⁷⁵ Furthermore, TiO₂ NPs can be internalized by dorsal root ganglion cells (DRG) and cause damage via apoptosis.^176,177 Yu et al showed an association between the toxic effects of TiO₂ NPs on olfactory bulb neuron cells and its pathogenicity to neurodegenerative diseases.¹⁷⁸ Furthermore, exposure to TiO₂ NPs was associated with varying degrees of cytotoxicity to the human cerebral endothelial cell line (HCECs), human neural stem cell line (hNSCs), and neuroectodermal stem cell line (1C11) models.^179–181 See Table 7 for details.

Table 7 Overview of the Literature on Neurotoxic Effects of TiO₂ NPs in Other Cells

Factors Influencing the Neurotoxic Potential of TiO₂ NPs

The neurotoxic effects of TiO₂ NPs are influenced by various factors. The exposure characteristics, such as exposure dose, method, duration, and species, can influence the toxic effects of TiO₂ NPs in vivo. A review of the literature showed that the exposure dose in vivo and in vitro experiments was larger than the actual exposure dose of the population. According to a previous study, the levels of TiO₂ NPs in air and water ranged from 0.7 to 16 μg/L.¹⁸² It is estimated that children have an intake of TiO₂ NPs of about 2–3 mg/kg/day, while adults have a TiO₂ NPs intake of about 1 mg/kg/day.² Human exposure to TiO₂ NPs is mainly through dietary intake and air inhalation. Although the exposure methods selected in animal studies attempted to mimic human exposure closely, there are some gaps. For example, the system for intranasal administration is simple compared to inhalation administration. Furthermore, intranasal administration is significantly affected by the inhalational dose.¹⁸³ The intranasal administration volumes in rodents at a given time should be limited to approximately 5 μL per nostril since volumes greater than this are likely to become wasted.^184,185 Furthermore, ingested TiO₂ NPs first interacts with the oral mucosa. However, intragastric administration does not interact with the oral mucosa and is thus associated with significant differences in absorption, bioavailability, and metabolism with implications for assumptions and models of toxicity kinetics.⁸¹ In addition, the exposure period and duration also influence the neurotoxic effects of TiO₂ NPs.^45,94 However, the exposure duration in experiments tends to be shorter than that in humans. Species differences are often unavoidable. Therefore, there is a need to conduct epidemiological studies exploring the neurotoxic effects of TiO₂ NPs on humans.

Furthermore, the physical and chemical properties of TiO₂ NPs can affect their neurotoxicity. Particle size is key. In general, small particles are more likely to be absorbed and thus exert toxic effects.¹⁸⁶ According to some previous studies, the neurotoxic effects of TiO₂ NPs depend on particle size.^60,168 The hydrodynamic diameter or secondary particle sizes of TiO₂ NPs are important with respect to neurotoxicity. While smaller NPs may seem more neurotoxic, they are also more likely to clump together and form aggregates.¹⁸⁶ Theoretically, the particle aggregation would increase the effective particle size thus reducing the neurotoxic potential. Several studies have used dynamic light scattering (DLS) to determine the effects of hydrodynamics or secondary particle size of TiO₂ NPs on neurotoxicity. However, no studies have explored the effect of aggregate particle size on the neurotoxicity of TiO₂ NPs. The zeta potential of TiO₂ NPs has also been investigated in most neurotoxicological studies. Since most cell membranes are negatively charged, the zeta potential affects the tendency of NPs to penetrate the membrane, with cationic particles generally exhibiting higher toxicity associated with cell wall damage.¹⁸⁷ Furthermore, the surface charge of the nanoparticles can determine the degree of aggregation.^122,162,164 However, further studies are needed to investigate whether the zeta potential affects the neurotoxicity of TiO₂ NPs. In addition, the toxicity of TiO₂ NPs is dependent on crystalline phases. The anatase form of TiO₂ NPs is more neurotoxic than that of rutile TiO₂ NPs and P25 TiO₂ NPs since anatase has a higher ability to induce oxidative stress.^152,166,188

Taken together, various factors can affect the neurotoxic potential of TiO₂ NPs, including physical and chemical properties of TiO₂ NPs, and exposure dose, exposure duration, exposed species. However, the specific effects of these factors on the neurotoxic effects of TiO₂ NPs still need to be systematically compared.

Reflections on Neurotoxicity Induced by TiO₂ NPs

Most studies to date have focused on rodents, and most experimental exposures used are not very realistic for human exposure. In addition, there is currently limited information on the levels of TiO₂ NPs in the environment, consumer goods, and food products. For humans, more accurate monitoring is needed to determine daily exposure levels, particle characteristics and exposure route, all of which affect the neurotoxic potential of TiO₂ NPs. Evaluating and availing data on TiO₂ NPs levels in different environmental media helps to reliably estimate human exposure and thus assess the risk of TiO₂ NPs. Furthermore, the degree of uptake through the digestive system, respiratory system, potential BBB crossing, and potential translocation to or even accumulation in nervous system should be further investigated. This information will indicate which route of exposure mitigation is most valuable for human health protection. However, apart from the recommended exposure limits (REL) established by the National Institute for Occupational Safety and Health (NIOSH), no other regulatory agencies have set occupational or environmental exposure limits for TiO₂ NPs.⁹ There are limitations in the monitoring methods of TiO₂ NPs. There is an urgent need to develop appropriate methods for reducing TiO₂ NPs in environmental media and food to prevent their potentially harmful health effects.¹⁸⁹

The specific mechanisms behind the neurotoxic effects of TiO₂ NPs have only been explored through animal and cell experiments. TiO₂ NPs increase the formation of reactive oxygen species (ROS) in the brain, thus inducing oxidative stress. Ze et al reported that TiO₂ NPs induced oxidative stress thus causing brain damage through overactivation of the p38-Nrf-2 signaling pathway.⁷⁸ Oxidative stress can induce neuroinflammation, thus further aggravating cell damage.^63,73,92 Cell damage, including structural and functional damage, is associated with increased onset and development of neurodevelopmental or neurodegenerative diseases, such as autism spectrum disorder (ASD) and PD.^87,100 Cell damage is also linked to behavioral deficits.^46,57 Abnormal motor ability could be caused by a decrease in the axon length of motor neurons.¹²⁰ In addition, changes in hippocampal synaptic plasticity could lead to decreased spatial recognition.⁸⁰ The development of axons, dendrites and synapses is regulated by various signaling pathways. TiO₂ NPs impair the growth of axons and dendrites through excessive activation of the ERK1/2/MAPK signaling pathway.⁸⁴ In addition, impairment of dendritic growth by TiO₂ NPs is also related to inhibition of the Wnt/β-catenin signaling pathway.¹⁴⁷ Moreover, suppression of the neuronal synaptic outgrowth by TiO₂ NPs is linked to the inhibition of the BDNF-TrkB signaling pathway.¹⁴⁸ Furthermore, the accumulation of TiO₂ NPs in the brain could cause alterations in brain biochemistry and changes in neurotransmitter levels, contributing to behavioral changes.^45,57,117 Although all of these studies confirm that TiO₂ NPs cause neurotoxic effects through different mechanisms, most of the evidence on the neurotoxic effects of TiO₂ NPs is fragmentary and is obtained from different species. Furthermore, few of these mechanism studies have explored whether the neurotoxic effects of TiO₂ NPs are mediated through synergistic interactions of multiple brain regions, organs, and systems. Whether TiO₂ NPs with different characteristics cause different degrees of toxic effects through different mechanisms remains be further explored. Extensive systematic studies are needed to fully elucidate the neurotoxic mechanisms of TiO₂ NPs, which will be helpful for the prevention and treatment of neurotoxic effects of TiO₂ NPs.

Conclusion

Animals and humans can be exposed to TiO₂ NPs through different exposure pathways, thus posing health hazards. At present, the neurotoxic effects of TiO₂ NPs have only been evaluated through animal models, including rats, mice, and zebrafish, and cell studies, including primary neurons, PC12, and SH-SY5Y cell lines. TiO₂ NPs can induce oxidative stress, promote neuroinflammation, alter brain biochemistry, or damage neurons. Neuronal damage can further lead to various behavioral disorders and is closely associated with increased onset and development of neurodevelopmental or neurodegenerative diseases. However, due to the lack of relevant epidemiological studies, whether TiO₂ NPs are linked to neurodevelopmental or neurodegenerative diseases in humans remains unknown. Furthermore, the neurotoxic potential of TiO₂ NPs can be affected by various factors. There is a need for researchers to understand the neurotoxic effects of TiO₂ NPs on humans and develop strategies for mitigating the effects of TiO₂ NPs on human health.

Abbreviations

NMs, nanomaterials; TiO₂ NPs, titanium dioxide nanoparticles; FPs, fine particles; PB, placental barrier; BBB, blood-brain barrier; C. elegans, Caenorhabditis elegans; SD rats, Sprague-Dawley rats; PND, postnatal day; BDNF, brain-derived neurotrophic factor; BW, body weight; BDE-209, decabromodiphenyl oxide; NMJ, neuromuscular junction; AchE, acetylcholinesterase; MDA, malondialdehyde; CNS, central nervous system; NMDA, N-methyl-D-aspartic acid; ER, endoplasmic reticulum; HBMECs, human brain microvascular endothelial cells; DRG, dorsal root ganglion; HCECs, human cerebral endothelial cell line; HNSCs, human neural stem cell line; 1C11, neuroectodermal stem cell line; DLS, Dynamic light scattering; REL, recommended exposure limit; NIOSH, National Institute for Occupational Safety and Health; ROS, reactive oxygen species; ASD, autism spectrum disorder; PD, Parkinson’s disease.

Acknowledgment

The authors would like to thank all the reviewers who participated in the review, as well as MJEditor (www.mjeditor.com) for providing English editing services during the preparation of this manuscript.

Funding

This work was supported by the Research Foundation for Talented Scholars, Nanjing Medical University (NMUR20210002).

Disclosure

The authors declare that they have no known competing interests.

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Neurotoxicity of Titanium Dioxide Nanoparticles: A Comprehensive Review

Introduction

Evidence from Epidemiological and Human Exposure Studies

Literature Search

Neurotoxic Effects of TiO2 NPs in Rodents

Neurotoxic Effects of TiO2 NPs in Zebrafish

Neurotoxic Effects of TiO2 NPs in Other Animal Models

Neurotoxic Effects of TiO2 NPs in vitro Models

Factors Influencing the Neurotoxic Potential of TiO2 NPs

Reflections on Neurotoxicity Induced by TiO2 NPs

Conclusion

Abbreviations

Acknowledgment

Funding

Disclosure

References

Neurotoxic Effects of TiO₂ NPs in Rodents

Neurotoxic Effects of TiO₂ NPs in Zebrafish

Neurotoxic Effects of TiO₂ NPs in Other Animal Models

Neurotoxic Effects of TiO₂ NPs in vitro Models

Factors Influencing the Neurotoxic Potential of TiO₂ NPs

Reflections on Neurotoxicity Induced by TiO₂ NPs