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Toxicity of Titanium Dioxide Nanoparticles Induced by Reactive Oxygen Species

Di Zhou, Shuo Han, Tenglong Yan, Changmao Long, Jiayu Xu, Pai Zheng, Zhangjian Chen, and Guang Jia 

Department of Occupational and Environmental Health Sciences, School of Public Health, Peking University, Beijing, China 

Correspondence: zhangjianchen@bjmu.edu.cn (Z.C.) 

Zhou D et al. Reactive Oxygen Species 8(23):267–275, 2019; ©2019 Cell Med Press

http://dx.doi.org/10.20455/ros.2019.857

(Received: June 19, 2019; Revised: July 3, 2019; Accepted: July 4, 2019) 

ABSTRACT | Titanium dioxide nanoparticles (TiO2 NPs) are one of the most widely used types of nanoparticles which can be used in food additives and drugs. However, the safety of TiO2 NPs is still controversial and the biological mechanism of TiO2 NPs-induced toxicity is not clear yet. We reviewed the research about toxicity of TiO2 NPs induced by reactive oxygen species (ROS) in vitro or in vivo. TiO2 NPs could induce significant increase of ROS and excessive free radicals, destroying redox balance. ROS-mediated oxidative stress leads to the occurrence of lipid and protein peroxidation, which in turn would induce autophagy, apoptosis, and necrosis of cells. Increased ROS production induced by TiO2 NPs was generally associated with inflammatory response, mitochondrial dysfunction, and genetic damage in vitro and in vivo. Although ROS may play an important role in TiO2 NPs-induced biological effects, the specific way it produces and its complex relationship with subsequent biological effects need to be further clarified. 

KEYWORDS | Nanomaterial; Oxidative stress; Reactive oxide species; Titanium dioxide nanoparticle; Toxicity 

ABBREVIATIONS | GSH, reduced glutathione; MDA, malondialdehyde; MPTP, mitochondrial permeability transition pore; 8-OHdG, 8-hydroxy-2′-deoxyguanosine; RIRR, ROS-induced ROS release; ROS, reactive oxygen species; TiO2 NPs, titanium dioxide nanoparticles 

CONTENTS 

  1. Introduction
  2. Oxidative Stress Induced by TiO2 NPs
  3. Lipid Peroxidation Induced by TiO2 NPs
  4. Protein Peroxidation Induced by TiO2 NPs
  5. Inflammatory Response Induced by TiO2 NPs
  6. Mitochondrial Dysfunction Induced by TiO2 NPs
  7. Genetic Damage Induced by TiO2 NPs
  8. Conclusion and Perspectives

1. Introduction 

Titanium dioxide nanoparticles (TiO2 NPs) are one of the most widely used types of nanoparticles in the world, with great application in food additives, medical treatments, water purification, cosmetics, sunscreens, paints, and coatings. Since 1969, the European Union (EU) has approved the application of food-grade titanium dioxide (E171) as a colorant in the food industry [1, 2]. Because of its high refractive index and uniform dispersion, E171 is used as a lightening and brightening agent in foods, including confectionery (chocolate, sweets, and chewing gum), white sauces and icing. The US Food and Drug Administration (FDA) has also approved the use of titanium dioxide as food additive, merely limiting the amount to not exceeding 1% of the total food mass. This is consistent with the China Food and Drug Administration (CFDA) standards (GB 2760-2014). On the basis of the expert review of the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1970, the EU did not establish an acceptable daily intake (ADI) for E171. 

Recent studies have shown that up to 36% of particles in E171 are nanoparticles [2]. It was reported that nanoparticles can be absorbed more easily and excreted more difficultly than traditional coarse particles [3]. In addition, previous studies showed that TiO2 NPs could penetrate the blood-brain barrier and placental barrier to accumulate in the central nervous system and fetus, and affect target organs [4]. With the rapid development of nanotechnology and the widespread application of TiO2 NPs, assessment of the toxicity and safety of TiO2 NPs has attracted much attention, but there is still considerable controversy [5]. In 2016, the European Food Safety Authority (EFSA) re-evaluated the safety of E171, concluding that the food additive E171 was not genotoxic and had a no observed adverse effect level (NOAEL) of 2,250 mg/kg body weight in rats. When calculating the mass of the nanoparticles contained in E171 at the NOAEL level with the upper mass concentration limit in E171 (3.2%) mentioned in the report, the NOAEL of the TiO2 NPs should be at least 72 mg/kg body weight [6]. However, this is in contradiction with some experiments in which significant genetic damage was observed in groups treated with TiO2 NPs at doses of less than 72 mg/kg body weight [7, 8]. The researchers reported that oral administration of E171 (10 mg/kg) for 7 days increased pre-cancerous colonic lesions in the rats [9], and E171 (5, 10, and 50 μg/cm2) increased the micronucleus frequency (1.9–3.6-fold) of the HCT116 cell line in vitro [1]. This makes the debate about the safety of TiO2 in foods more intense. 

In EU 872/2012, it was specified that if nanotechnology was applied to the food additive, EFSA should be noticed, and then a new approval and a new product label for the modifications is required [10]. However, there is no regulation to specify the mass limitation of nanoscale particles as an additive in foods. The US National Institute for Occupational Safety and Health (NIOSH) has supported this distinction by setting two separate occupational exposure limits for fine TiO2 particles and ultrafine TiO2 (< 100 nm). Thus, traditional knowledge on the safety of occupational and environmental exposure to TiO2 was challenged. Meanwhile, as TiO2 NPs was classified as a Class 2B carcinogen by the International Agency for Research on Cancer in 2006, further attention should be paid to the genotoxicity and carcinogenicity of TiO2 NPs. As such, re-evaluation of the safety of TiO2 NPs is warranted, especially for dietary exposure closely related to consumers. 

In this review, we focused on the toxicity of TiO2 NPs associated with ROS generation. Research on the ROS-mediated toxicity of TiO2 NPs in vitro or in vivo (via oral exposure) and the underlying mechanisms were summarized so as to provide insights for reducing their health risk.

2. Oxidative Stress Induced by TiO2 NPs 

Excessive free radicals and ROS could destroy redox balance and induce inflammation and mitochondrial dysfunction, leading to cellular apoptosis. Shukla et al.demonstrated that TiO2 NPs have a tendency to generate free hydroxyl radicals leading to oxidative stress, genotoxicity, and ultimately cell death in different cell lines [11, 12]. Subsequently, they also observed a dose-dependent increase in the generation of ROS (e.g., superoxide and H2O2) in the liver of mice exposed to TiO2 NPs orally for 14 consecutive days, leading to cellular stress [13]. A significant dose-dependent decrease in reduced glutathione (GSH) levels also suggested increased oxidative stress in the liver, which may lead to hepatic injury. Indeed, oxidative DNA damage caused by oxidative stress consequently initiated the expression of apoptotic proteins resulting in hepatic injury. 

The ability of TiO2 to produce ROS was reported to be closely related to the particle size, and the smaller the size, the stronger the ability. Xiong et al. found that compared with bulk TiO2, the quantities of hydroxyl radicals in the TiO2 NPs suspensions were much higher [14]. Hamzeh et al. also found that TiO2 NPs decreased cell viability in Chinese hamster lung fibroblast cells through ROS generation in a mass-based concentration- and size-dependent manner [15]. In addition, the photocatalytic activity of TiO2 NPs was considered to be the main basis for ROS production [16]. TiO2 NPs significantly exerted an oxidative effect on cells upon exposure to solar or ultraviolet (UV) irradiation [17]. However, recent studies also found obvious effects of TiO2 NPs under dark conditions on sensitive organisms such as Physarum polycephalum or Escherichia coli [18, 19]. TiO2 NPs induced oxidative stress in macroplasmodium of Physarum polycephalum under dark conditions, evidenced by the increased levels of ROS, 8-hydroxy-2′-deoxyguanosine (8-OHdG), and total soluble phenols (TSP) [19].

3. Lipid Peroxidation Induced by TiO2 NPs 

The generation of free radicals and ROS can lead to the occurrence of lipid peroxidation, which in turn would induce autophagy and apoptosis of cells. It had been shown that ROS generated by TiO2 NPs could induce lipid peroxidation by directly oxidizing the biological membranes or molecules [20]. A dose-dependent significant increase in the levels of malondialdehyde (MDA) was observed in TiO2 NPs-treated mouse liver cells. MDA is a typical biomarker of lipid peroxidation. Several types of damage including oxidation of sulfhydryl groups, formation of disulfides, peptide fragmentation, and modification of prosthetic groups or metal clusters are documented due to lipid peroxidation [13]. Meanwhile, the MDA content of zebrafish gills exposed to TiO2 NPs in illumination and dark was 217.2% and 174.3% of controls, respectively [14]. The level of hydroxyl radicals ascended with the increase of MDA, indicating that the occurrence of lipid peroxidation may be partly due to the generation of hydroxyl radicals. 

Increased lipid peroxidation may be closely related to cell autophagy, apoptosis, and necrosis. As the autolysosomes derived from autophagy fail to repair peroxide damage, an advanced process of apoptosis or necrosis will be activated [21, 22]. Hussain et al. found that lysosomal membrane damage and lipid peroxidation might contribute to TiO2 NPs -induced cell death [23]. Increased autophagy and apoptosis were also observed in cells exposure to other nanoparticles such as Ag or SiO2 [24]. Previous study had indicated that TiO2 NPs (5–150 mg/kg) induced apoptosis in the mouse splenocytes via mitochondria-mediated pathway and ROS accumulation [25]. A significant increase in cytochrome c, Bax, and caspase-3/-9 expression, and a significant decrease in Bcl-2 expression were detected in the TiO2 NPs-treated mouse spleen. Park et al. and Shi et al. also demonstrated a TiO2 NPs-induced, dose- and time-dependent decrease in cell viability, caspase-3 activation, and DNA condensation indicative of apoptosis in BEAS-2B bronchial epithelial cells, along with ROS production, GSH depletion, and increased heme oxygenase-1 [26, 27].

4. Protein Peroxidation Induced by TiO2 NPs 

A wide variety of pathways could be activated by oxidizing amino acid residues of proteins, including cell signaling proteins (NF-κB, MAPK, Keap1-Nrf2-ARE, and PI3K-Akt), ion channels and transporters (Ca2+ channels and mitochondrial permeability transition pore), and the protein kinase and the ubiquitination/proteasome system [28, 29]. It has been shown that peroxidation of signaling proteins induced by ROS accumulation from TiO2 NPs can disrupt ion channels and transporters and cause cellular dysfunction and apoptosis [30]. Additionally, ROS can influence various ion channels and transporters, including voltage-gated Ca2+ channels, mitochondrial Ca2+ uniporter, Ca2+ release-activated Ca2+ (CRAC), channels, as well as P2X, IP3R/RyR, and voltage-gated K+ channels. It was reported that TiO2 NPs-induced oscillations in the intracellular calcium concentration could be attributed to the oxidation of certain redox sensitive residues in the mitochondrial channels such as the mitochondrial permeability transition pore (MPTP) [31]. The activation of those residues can prolong the MPTP openings and release a ROS and Ca2+ burst. The phenomenon that ROS trigger MPTP-related ROS release has been termed “ROS-induced ROS release” (RIRR) [31].

5. Inflammatory Response Induced by TiO2 NPs 

Evidence accumulated over the past two decades has pointed to significant connections between inflammation and oxidative stress, with both processes contributing to fuel the other one, thereby establishing a vicious cycle able to perpetuate and propagate the inflammatory response [32]. With sub-chronic intragastric administration of TiO2 NPs (2.5–10 mg/kg) in mice, a significant increase in NF-κB, TNF-α, IL-1β/-6, and IFN-α and a significant decrease in I-κB were observed in the heart of mice. It indicated that the low-dose and long-term exposure to TiO2 NPs can cause a cardiac inflammatory response in mice [33]. Another long-term study indicated that the alteration of Th2 factor expression may be involved in the control of hepatic inflammation induced by chronic TiO2 NPs toxicity [34]. The study found a significantly upregulated expression of the inflammation-related genes including IL-4, IL-5, IL-12, IFN-γ, GATA3, GATA4, T-bet, STAT3, STAT6, Eotaxin, MCP-1, and MIP-2 in the liver of mice after oral gavage of TiO2 NPs for 6 months. Renal inflammation was also reported in mice after a chronic gavage administration of TiO2 NPs (1.25–5 mg/kg) for 9 months [35]. It was observed that pathways in the MAPKs family, including JUN, p38, and ERKs pathways were activated in the kidney [35]. In addition, the same researchers also found thymic inflammation in mice following exposure to TiO2 NPs [36]. These studies together suggested that chronic exposure to TiO2 NPs may disrupt immune cell homeostasis and induce systemic inflammatory response.

6. Mitochondrial Dysfunction Induced by TiO2 NPs 

It is well known that endogenous cellular ROS are mainly generated in the process of mitochondrial oxidative phosphorylation. Increased ROS production is generally associated with mitochondrial dysfunction. Many studies have pointed out that TiO2 NPs could induce mitochondrial dysfunction in vitro and in vivo [37–39]. After incubation of mitochondria isolated from rat lung tissue with TiO2 NPs, significant decreases in NADH levels and mitochondrial transmembrane potential (△Ψm) were observed [37]. In addition, the generation of ROS after exposure to TiO2 NPs was found to be mainly (46% out of 46.5%) caused by dysfunction of mitochondrial respiratory complexes [37]. Another in vitro experiment on the BV2 cell line confirmed that TiO2 NPs could interfere with respiratory chain complexes, and that a dysfunction of the respiratory complex could increase the generation of ROS [39]. The exogenous ROS can oxidize and consecutively open the MPTP mitochondrial ion channel. The consecutive opening of MPTP will alter the permeability of mitochondria, causing mitochondrial swelling which may interfere with mitochondrial electron transport chain (ETC) function. Damage to or interference with ETC (such as complexes Ⅰand Ⅲ) may increase the production and release of endogenous ROS, which may in turn exacerbate mitochondrial dysfunction. This is the way that RIRR functions, and might be the primary pathways for oxidative stress induced by TiO2 NPs [31]. TiO2 NPs also produced morphological changes, damage of mitochondria, and an increase in mitochondrial membrane potential in rat and human glial cells (C6 and U373) [38]. Meanwhile, Shi et al. found that TiO2 NPs cause apoptosis in BEAS-2B cells through the caspase 8/t-Bid-independent mitochondrial pathway [27]. Mitochondrial dysfunction may be able to serve as a sensitive biomarker for the toxicity of TiO2 NPs [40]. It could be detected even in the hippocampus of offspring after maternal exposure to TiO2 NPs in rats [41].

7. Genetic Damage Induced by TiO2NPs 

Oxidative stress and inflammatory responses were originally thought to be the mechanism by which oral exposure to the TiO2 NPs induced genetic toxicity. Trouiller et al. observed elevated expression of inflammatory cytokines such as TNF-α, IFN-γ, and IL-8 in the blood of mice after oral intake of TiO2 NPs at 100 mg/kg for 5 days [8]. They suggested that the genotoxicity in mice induced by TiO2 NPs may be mainly associated with the inflammation and/or oxidative stress, which was called a secondary genotoxic mechanism. Afterwards, several publications confirmed that the genotoxicity of most nanomaterials is likely to be associated with indirect consequences of inflammation and generation of oxidative species by inflammatory cells (neutrophils and macrophages) [7, 42–48]. Studies in vitro have also shown that TiO2 NPs can cause cytotoxicity and genotoxicity through oxidative stress in cells including human hepatocytes and epidermal cells [12, 49–51]. Our previous studies had found TiO2 NPs could induce genetic toxicity both in vitro and in vivo via oral exposure [52], as well as obvious inflammatory response evidenced by significantly increased white blood cell count and serum concentrations of IL-1α, IL-4, and TNF in rats after daily oral exposure to TiO2 NPs at 50 mg/kg for 90 days [53]. Inflammation induced by TiO2 NPs produced obvious oxidative stress, which was evidenced by decreased level of GSH and increased levels of glutathione disulfide (GSSG) and MDA in the liver tissue of TiO2 NPs-exposed rats. 

Oxidative DNA damage is a major marker of genotoxicity induced by TiO2 NPs. The DNA base pairs can be attacked by oxidants, of which guanine is the most sensitive and can be oxidized to 8-OHdG in most cases. It has been observed that TiO2 NPs could induce increased 8-OHdG in the liver of C57BL/6Jpun/pun mice [8]. In addition, 8-OHdG can mimic T functionally in the syn conformation and form a stable 8-oxoG(syn)•A(anti) base pair. Thus, the absence of 8-OHdG repair before replication can lead a G-to-T transversion mutation [54, 55]. The mutant frequency of 6TGRHGPRT, which is an early indicator of potential carcinogenicity, showed a significant 2.98-fold increase after TiO2 NPs exposure in mammalian lung fibroblasts cells (V79) [56]. TiO2 NPs can interfere with the redox-sensitive base excision repair enzyme, or regulate the gene for the repair of basic excision and thereby cause accumulation of genetic damage [57]. In comparison with the nuclear genome, the mitochondrial genome is more likely to be oxidized by ROS without the protection of histone and nuclear envelope. It was also reported that TiO2 NPs could induce single-strand breaks or double-strand cleavage mediated by ROS [58]. The Comet assay, which is often used to detect DNA double-strand breaks, was the most commonly used method for genotoxicity testing of nanomaterials and showed higher probability of positive results [59]. The formation of γ-H2AX foci, a biomarker of double-strand DNA breaks, also showed a significant dose-dependence positive relationship with TiO2 NPs concentration [6]. The increased micronucleus frequency, as an indicator of chromosome damage, was also reported. Increased micronucleus frequency was observed in the blood of marine fish Trachinotus carolinus 72 h after administration with TiO2 NPs (1.5 or 3.0 μg/g body weight) via a single intraperitoneal injection [60]. In mice administered with TiO2 NPs (40–1000 mg/kg body weight) by gavage for 7 days, a significant dose-dependent increase in the micronucleus frequency in the liver and kidney was observed [7].

8. Conclusion and Perspectives 

In conclusion, substantial evidence suggests a critical involvement of ROS in TiO2 NPs-induced toxicity (Figure 1). Nevertheless, studies on the toxicity of TiO2 NPs are still in their infancy. There are three areas that need further study. Firstly, the toxicity of TiO2 NPs can be influenced by various of physical and chemical characters, which complicates the risk assessment of TiO2 NPs. For example, the toxicity of TiO2 NPs depends on crystalline form, size, duration of exposure, surface properties, dosage, route of administration, and surface modification. The different characteristics of particles among studies could lead to contradictory conclusions [4–6, 61]. Secondly, the origin and location of TiO2 NPs- induced ROS were not yet clear. Therefore, the causal relationship between TiO2 NPs-induced ROS and many biological effects remain to be clarified. A few studies have shown that the toxicity of TiO2 NPs decreases significantly in case of co-administration with antioxidants. These related studies were helpful to clarify the causal relationship, but further studies are still needed. Lastly, current research on the toxicity of TiO2 NPs has been limited to animal experiments in vivo or cell experiments in vitro. Whether the same results will be observed in humans is still unknown. The well-designed epidemiological study is recommended to evaluate the effect of TiO2 NPs on health following a long-term, low-dose exposure.

FIGURE 1. The role of free radicals and ROS in the toxicity of TiO2 nanoparticles. As illustrated, formation of free radicals and ROS may be an upstream event leading to various forms of oxidative/inflammatory damage and consequent cell injury and death. 

ACKNOWLEDGMENTS 

This work was supported by the National Science and Technology Major Project of the Ministry of Science and Technology of China (2017YFC1600204) and National Natural Science Foundation of China (81703257). The authors declare no conflicts of interest. 

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