Cytotoxicity of TiO2 nanoparticles to mussel hemocytes and gill cells in vitro: Influence of synthesis method,crystalline structure,size and additive

Abstract

Increasing the production and applications of TiO₂ nanoparticles (NPs) has led to grow concerns about the consequences for the environment. In this study, we investigated the effects of a set of TiO₂ NPs on the viability of mussel hemocytes and gill cells using neutral red and thiazolyl tetrazolium bromide assays. For this, we compared the cytotoxicity of TiO₂ NPs (0.1–100?mg Ti/L) produced by different techniques: rutile NPs (60?nm) produced by milling and containing disodium laureth sulfosuccinate (DSLS), rutile NPs (10, 40 and 60?nm) produced by wet chemistry and anatase/rutile NPs (～100?nm) produced by plasma synthesis. The commercially available P25 anatase/rutile NPs (10–20?nm) were also tested. Exposures were performed in parallel with their respective bulk forms and the cytotoxicity of the additive DSLS was also tested. Z potential values in distilled water indicated different stabilities depending on the NP type and all NPs tested formed agglomerates/aggregates in cell culture media. In general, TiO₂ NPs showed a relatively low and dose-dependent toxicity for both cell models with the two assays tested. NPs produced by milling showed the highest effects, probably due to the toxicity of DSLS. Size-dependent toxicity was found for NPs produced by wet chemistry (10?nm?>?40?nm and 60?nm). All TiO₂ NPs tested were more toxic than bulk forms excepting for plasma produced ones, which were the least toxic TiO₂ tested. The mixture bulk anatase/rutile TiO₂ was more toxic than bulk rutile TiO₂. In conclusion, the toxicity of TiO₂ NPs varied with the mode of synthesis, crystalline structure and size of NPs and can also be influenced by the presence of additives in the suspensions.     

Nanoparticle size and combined toxicity of TiO2 and DSLS (surfactant) contribute to lysosomal responses in digestive cells of mussels exposed to TiO2 nanoparticles

The aim of this investigation was to understand the bioaccumulation, cell and tissue distribution and biological effects of disodium laureth sulfosuccinate (DSLS)-stabilised TiO₂ nanoparticles (NPs) in marine mussels, Mytilus galloprovincialis. Mussels were exposed in vivo to 0.1, 1 and 10?mg Ti/L either as TiO₂ NPs (60 and 180?nm) or bulk TiO₂, as well as to DSLS alone. A significant Ti accumulation was observed in mussels exposed to TiO₂ NPs, which were localised in endosomes, lysosomes and residual bodies of digestive cells, and in the lumen of digestive tubules, as demonstrated by ultrastructural observations and electron probe X-ray microanalysis. TiO₂ NPs of 60?nm were internalised within digestive cell lysosomes to a higher extent than TiO₂ NPs of 180?nm, as confirmed by the quantification of black silver deposits after autometallography. The latter were localised mainly forming large aggregates in the lumen of the gut. Consequently, lysosomal membrane stability (LMS) was significantly reduced upon exposure to both TiO₂ NPs although more markedly after exposure to TiO₂-60 NPs. Exposure to bulk TiO₂ and to DSLS also affected the stability of the lysosomal membrane. Thus, effects on the lysosomal membrane depended on the nanoparticle size and on the combined biological effects of TiO₂ and DSLS.     

Oral intake of added titanium dioxide and its nanofraction from food products,food supplements and toothpaste by the Dutch population

Titanium dioxide (TiO₂) is commonly applied to enhance the white colour and brightness of food products. TiO₂ is also used as white pigment in other products such as toothpaste. A small fraction of the pigment is known to be present as nanoparticles (NPs). Recent studies with TiO₂ NPs indicate that these particles can have toxic effects. In this paper, we aimed to estimate the oral intake of TiO₂ and its NPs from food, food supplements and toothpaste in the Dutch population aged 2 to over 70 years by combining data on food consumption and supplement intake with concentrations of Ti and TiO₂ NPs in food products and supplements. For children aged 2–6 years, additional intake via ingestion of toothpaste was estimated. The mean long-term intake to TiO₂ ranges from 0.06?mg/kg bw/day in elderly (70+), 0.17?mg/kg bw/day for 7–69-year-old people, to 0.67?mg/kg bw/day in children (2–6 year old). The estimated mean intake of TiO₂ NPs ranges from 0.19?μg/kg bw/day in elderly, 0.55?μg/kg bw/day for 7–69-year-old people, to 2.16?μg/kg bw/day in young children. Ninety-fifth percentile (P95) values are 0.74, 1.61 and 4.16?μg/kg bw/day, respectively. The products contributing most to the TiO₂ intake are toothpaste (in young children only), candy, coffee creamer, fine bakery wares and sauces. In a separate publication, the results are used to evaluate whether the presence of TiO₂ NPs in these products can pose a human health risk.     

Influence of silver and titanium dioxide nanoparticles on in vitro blood-brain barrier permeability

An in vitro blood-brain barrier (BBB) model being composed of co-culture with endothelial (bEnd.3) and astrocyte-like (ALT) cells was established to evaluate the toxicity and permeability of Ag nanoparticles (AgNPs; 8 nm) and TiO₂ nanoparticles (TiO₂NPs; 6 nm and 35 nm) in normal and inflammatory central nervous system. Lipopolysaccharide (LPS) was pre-treated to simulate the inflammatory responses. Both AgNPs and Ag ions can decrease transendothelial electrical resistance (TEER) value, and cause discontinuous tight junction proteins (claudin-5 and zonula occludens-1) of BBB. However, only the Ag ions induced inflammatory cytokines to release, and had less cell-to-cell permeability than AgNPs, which indicated that the toxicity of AgNPs was distinct from Ag ions. LPS itself disrupted BBB, while co-treatment with AgNPs and LPS dramatically enhanced the disruption and permeability coefficient. On the other hand, TiO₂NPs exposure increased BBB penetration by size, and disrupted tight junction proteins without size dependence, and many of TiO₂NPs accumulated in the endothelial cells were observed. This study provided the new insight of toxic potency of AgNPs and TiO₂NPs in BBB.     

Nanoparticle (NP) uptake by type I alveolar epithelial cells and their oxidant stress response

Abstract

Mammalian cells take up nanoparticles (NPs) and some NPs increase ROS. We used imaging and measure ROS in parallel to evaluate NP-cell interactions with type I-like alveolar epithelial cells exposed to NPs at 1.2 µg/cm². Titanium dioxide (Ti0₂), gold (Au), silver (Ag), and manganese (Mn) were internalized by R3-1 cells; copper (Cu) NPs were observed at the cell surface only. TiO₂ and Au did not increase cell death but Mn and Cu did, with surviving cells recovering after initial Cu exposure. Ag NPs caused 80% of R3-1 cells to lift off the slides within 1 h. Amplex Red was used to report H₂O₂ production after exposure to 0.4 µg/cm² TiO₂, Au, Cu, Mn and Ag. TiO₂, Au, and Ag caused no significant increase in H₂O₂ while Cu and Mn increased H₂O₂. NPs that give up electrons, increase ROS production and cause cell death in R3-1 cells.     

Quantitative evaluation of the pulmonary microdistribution of TiO2 nanoparticles using X‐ray fluorescence microscopy after intratracheal administration with a microsprayer in rats

The unevenness of pulmonary nanoparticle (NP) distribution, which hinders the establishment of an absolute dose–response relationship, has been described as one of the limitations of intratracheal administration techniques for toxicological assessment of inhaled NPs. Quantification of the NP microdistribution would facilitate the establishment of a concentration–response relationship in localized regions of the lung; however, such quantitative methods have not been reported. Here, we established a quantitative method for evaluating pulmonary TiO₂ NP microdistribution in rats using X‐ray fluorescence microscopy. Ti intensity in lung sections from rats intratracheally administered 10 mg kg^–1 TiO₂ NPs with a microsprayer was measured using X‐ray fluorescence with a 100 µm beam size. Ti reference samples were prepared by dropping different concentrations of Ti solutions on glass slide or lung sections of untreated rat. Ti intensity increased linearly with Ti content in the reference samples on both substrates. The detection limit of TiO₂ was estimated to be 6.3 ng mm^–2. The reproducibility was confirmed for measurements done in the short‐ (2 weeks) and long‐term (6 months). The quantitative results of TiO₂ NP microdistribution suggested that more TiO₂ NPs were distributed in the right caudal and accessory lobes, which are located downstream of the administration direction of the NP suspension, and the lower portion of each lobe. The detection rates of TiO₂ NPs were 16.6–25.0%, 5.19–15.6%, 28.6–39.2%, 21.4–38.7% and 10.6–23.2% for lung sections from the right cranial, middle, caudal, accessory and left lobes, respectively. Copyright © 2015 John Wiley & Sons, Ltd.     

Dietary exposure to titanium dioxide nanoparticles in rainbow trout, (Oncorhynchus mykiss): no effect on growth, but subtle biochemical disturbances in the brain

Our laboratory recently reported gut pathology following incidental ingestion of titanium dioxide nanoparticles (TiO₂ NPs) during aqueous exposures in trout, but there are almost no data on dietary exposure to TiO₂ NPs in fish. The aim of this experiment was to observe the sub-lethal effects of dietary exposure to TiO₂ NPs in juvenile rainbow trout (Oncorhynchus mykiss). Stock solutions of dispersed TiO₂ NPs were prepared by sonication without the use of solvents and applied to a commercial trout diet. Fish were exposed in triplicate to either, control (no added TiO₂), 10, or 100 mg kg⁻¹ TiO₂ NPs diets for 8 weeks followed by a 2 week recovery period where all fish were fed the control diet. TiO₂ NPs had no impact on growth or nutritional performance, and no major disturbances were observed in red or white blood cell counts, haematocrits, whole blood haemoglobin, or plasma Na⁺. Ti accumulation occurred in the gill, gut, liver, brain and spleen during dietary TiO₂ exposure. Notably, some of these organs, especially the brain, did not clear Ti after exposure. The brain also showed disturbances to Cu and Zn levels (statistically significant at weeks 4 and 6; ANOVA or Kruskal–Wallis, P < 0.05) and a 50% inhibition of Na⁺K⁺-ATPase activity during TiO₂ NP exposure. Na⁺K⁺-ATPase activity was unaffected in the gills and intestine. Total glutathione in the gills, intestine, liver and brain were not affected by dietary TiO₂ NPs, but thiobarbituric acid reactive substances (TBARS) showed up to 50% decreases in the gill and intestine. We conclude that TiO₂ NPs behave like other toxic dietary metals where growth rate and haematology can be protected during sub-lethal exposures, but in the case of TiO₂ NPs this may be at the expense of critical organs such as the brain and the spleen.     

Titanium dioxide nanoparticles prime a specific activation state of macrophages

Titanium dioxide nanoparticles (TiO₂ NPs) are widely used in foods, cosmetics, and medicine. Although the inhalation toxicity of TiO₂ NPs has been studied, the potential adverse effects of oral exposure of low-dose TiO₂ NPs are largely unclear. Herein, with macrophage cell lines, primary cells, and mouse models, we show that TiO₂ NPs prime macrophages into a specific activation state characterized by excessive inflammation and suppressed innate immune function. After a month of dietary exposure in mice or exposure in vitro to TiO₂ NPs (10 and 50?nm), the expressions of pro-inflammatory genes in macrophages were increased, and the expressions of anti-inflammatory genes were decreased. In addition, for macrophages exposed to TiO₂ NPs in vitro and in vivo, their chemotactic, phagocytic, and bactericidal activities were lower. This imbalance in the immune system could enhance the susceptibility to infections. In mice, after a month of dietary exposure to low doses of TiO₂ NPs, an aggravated septic shock occurred in response to lipopolysaccharide challenge, leading to elevated levels of inflammatory cytokines in serum and reduced overall survival. Moreover, TLR4-deficient mice and primary macrophages, or TLR4-independent stimuli, showed less response to TiO₂ NPs. These results demonstrate that TiO₂ NPs induce an abnormal state of macrophages characterized by excessive inflammation and suppressed innate immune function in a TLR4-dependent manner, which may suggest a potential health risk, particularly for those with additional complications, such as bacterial infections.     

Cytotoxic and inflammatory responses of TiO2 nanoparticles on human peripheral blood mononuclear cells

Titanium dioxide nanoparticles (TiO₂‐NPs) have been widely used in many applications. Owing to their nanoscale size, interactions between cells and NPs have been expansively investigated. With the health concerns raised regarding the adverse effects of these interactions, closer examination of whether TiO₂‐NPs can induce toxicity towards human cells is greatly needed. Therefore, in this study, we investigated the cytotoxicity of TiO₂‐NPs towards human blood cells (peripheral blood mononuclear cells [PBMCs]) in serum‐free medium, for which there is little information regarding the cytotoxic effects of TiO₂‐NPs. Our results provide evidence that PBMCs treated with TiO₂‐NPs (at concentrations ≥25 μg ml^?1) for 24 h significantly reduced cell viability and significantly increased production of toxic mediators such as reactive oxygen species and inflammatory response cytokines such as interleukin‐6 and tumor necrosis factor‐α (P < 0.05). Cell apoptosis induction also occurred at these concentrations. Significant expressions of cyclooxygenase‐2 and interleukin‐1β were also observed in PBMCs treated with TiO₂‐NPs at concentrations ≥125 μg ml^?1. Our data presented here clearly indicate that the concentration of TiO₂‐NPs (at size ~26.4 ± 1.2 nm) applied to human blood cells has a strong impact on cytotoxic induction. Copyright © 2016 John Wiley & Sons, Ltd.     

Mechanism of TiO2 nanoparticle‐induced neurotoxicity in zebrafish (Danio rerio)

Zebrafish (Danio rerio) has been used historically for evaluating the toxicity of environmental and aqueous toxicants, and there is an emerging literature reporting toxic effects of manufactured nanoparticles (NPs) in zebrafish embryos. Few researches, however, are focused on the neurotoxicity on adult zebrafish after subchronic exposure to TiO₂ NPs. This study was designed to evaluate the morphological changes, alterations of neurochemical contents, and expressions of memory behavior‐related genes in zebrafish brains caused by exposures to 5, 10, 20, and 40 μg/L TiO₂ NPs for 45 consecutive days. Our data indicated that spatial recognition memory and levels of norepinephrine, dopamine, and 5‐hydroxytryptamine were significantly decreased and NO levels were markedly elevated, and over proliferation of glial cells, neuron apoptosis, and TiO₂ NP aggregation were observed after low dose exposures of TiO₂ NPs. Furthermore, the low dose exposures of TiO₂ NPs significantly activated expressions of C‐fos, C‐jun, and BDNF genes, and suppressed expressions of p38, NGF, CREB, NR1, NR2ab, and GluR2 genes. These findings imply that low dose exposures of TiO₂ NPs may result in the brain damages in zebrafish, provide a developmental basis for evaluating the neurotoxicity of subchronic exposure, and raise the caution of aquatic application of TiO₂ NPs. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 163–175, 2016.     

Evaluation of cytogenotoxicity and oxidative stress parameters in male Swiss mice co-exposed to titanium dioxide and zinc oxide nanoparticles

A number of studies have investigated the adverse toxic effects of titanium dioxide (TiO₂) nanoparticles (NPs) or zinc oxide (ZnO) NPs. Information on the potential genotoxic effects of the interactions of TiO₂ NPs and ZnO NPs in vivo is lacking. Therefore, this study was designed to investigate the cytogenotoxicity of TiO₂ NPs or ZnO NPs alone or their mixtures using the bone marrow micronucleus assay, and mechanism of damage through the evaluation of oxidative stress parameters in the liver and kidney tissues of Swiss mice. Intraperitoneal administration of doses between 9.38 and 150.00 mg/kg of TiO₂ NPs or ZnO NPs or TiO₂ NPs + ZnO NPs was performed for 5 and 10 days, respectively. TiO₂ NPs alone induced a significant (P < 0.05) increase in micronucleated (Mn) polychromatic erythrocytes (PCEs) at the applied doses compared with the negative controls, with a significant difference between 5 and 10 days for TiO₂ NPs alone and TiO₂ NPs + ZnO NPs. Concurrently, TiO₂ NPs alone for 5 days and TiO₂ NPs and TiO₂ NPs + ZnO NPs for 10 days significantly (P < 0.05) decreased the percentage PCE: normochromatic erythrocyte (NCE) indicating cytotoxicity; with a significant difference between the two periods. Significant (P < 0.001) changes in the activities of superoxide dismutase (SOD) and catalase (CAT), and levels of reduced glutathione (GSH) and malondialdehyde (MDA) were observed in the liver and kidney of mice exposed to TiO₂ NPs or ZnO NPs alone or their mixtures. These results suggest that TiO₂ NPs alone was genotoxic; TiO₂ NPs and TiO₂ NPs + ZnO NPs were noticeably cytotoxic while ZnO NPs was not cytogenotoxic. The individual NPs or their mixtures induced oxidative stress.     

In vitro screening of metal oxide nanoparticles for effects on neural function using cortical networks on microelectrode arrays

Nanoparticles (NPs) may translocate to the brain following inhalation or oral exposures, yet higher throughput methods to screen NPs for potential neurotoxicity are lacking. The present study examined effects of 5 CeO₂ (5– 1288?nm), and 4 TiO₂ (6–142?nm) NPs and microparticles (MP) on network function in primary cultures of rat cortex on 12 well microelectrode array (MEA) plates. Particles were without cytotoxicity at concentrations ≤50?µg/ml. After recording 1?h of baseline activity prior to particle (3–50?µg/ml) exposure, changes in the total number of spikes (TS) and # of active electrodes (#AEs) were assessed 1, 24, and 48?h later. Following the 48?h recording, the response to a challenge with the GABA_A antagonist bicuculline (BIC; 25?µM) was assessed. In all, particles effects were subtle, but 69?nm CeO₂ and 25?nm TiO₂ NPs caused concentration-related decreases in TS following 1?h exposure. At 48?h, 5 and 69?nm CeO₂ and 25 and 31?nm TiO₂ decreased #AE, while the two MPs increased #AEs. Following BIC, only 31?nm TiO₂ produced concentration-related decreases in #AEs, while 1288?nm CeO₂ caused concentration-related increases in both TS and #AE. The results indicate that some metal oxide particles cause subtle concentration-related changes in spontaneous and/or GABA_A receptor-mediated neuronal activity in vitro at times when cytotoxicity is absent, and that MEAs can be used to screen and prioritize nanoparticles for neurotoxicity hazard.     

Evaluation of cytotoxic,genotoxic and inflammatory response in human alveolar and bronchial epithelial cells exposed to titanium dioxide nanoparticles

The toxicity of titanium dioxide nanoparticles (TiO₂‐NPs), used in several applications, seems to be influenced by their specific physicochemical characteristics. Cyto‐genotoxic and inflammatory effects induced by a mixture of 79% anatase/21% rutile TiO₂‐NPs were investigated in human alveolar (A549) and bronchial (BEAS‐2B) cells exposed to 1–40 µg ml^–1 30 min, 2 and 24 h to assess potential pulmonary toxicity. The specific physicochemical properties such as crystallinity, NP size and shape, agglomerate size, surface charge and specific surface area (SSA) were analysed. Cytotoxic effects were studied by evaluating cell viability using the WST1 assay and membrane damage using LDH analysis. Direct/oxidative DNA damage was assessed by the Fpg‐comet assay and the inflammatory potential was evaluated as interleukin (IL)‐6, IL‐8 and tumour necrosis factor (TNF)‐α release by enzyme‐linked immunosorbant assay (ELISA). In A549 cells no significant viability reduction and moderate membrane damage, only at the highest concentration, were detected, whereas BEAS‐2B cells showed a significant viability reduction and early membrane damage starting from 10 µg ml^–1. Direct/oxidative DNA damage at 40 µg ml^–1 and increased IL‐6 release at 5 µg ml^–1 were found only in A549 cells after 2 h. The secretion of pro‐inflammatory cytokine IL‐6, involved in the early acute inflammatory response, and oxidative DNA damage indicate the promotion of early and transient oxidative‐inflammatory effects of tested TiO₂‐NPs on human alveolar cells. The findings show a higher susceptibility of normal bronchial cells to cytotoxic effects and higher responsiveness of transformed alveolar cells to genotoxic, oxidative and early inflammatory effects induced by tested TiO₂‐NPs. This different cell behaviour after TiO₂‐NPs exposure suggests the use of both cell lines and multiple end‐points to elucidate NP toxicity on the respiratory system. Copyright © 2014 John Wiley & Sons, Ltd.     

RNA sequencing analysis shows that titanium dioxide nanoparticles induce endoplasmic reticulum stress,which has a central role in mediating plasma glucose in mice

Titanium dioxide nanoparticles (TiO₂ NPs) constitute the top five NPs in use today. In this study, oral administration of 50, 100, and 200?mg/kg body weight (b.w.) TiO₂ NPs increases plasma glucose in mice, whereas 10 and 20?mg/kg b.w. TiO₂ NPs did not. RNA sequencing (RNA-seq) technology was used to investigate genome-wide effects of TiO₂ NPs. Clustering analysis of the RNA-seq data showed the most significantly enriched gene ontology terms and KEGG pathways related to the endoplasmic reticulum (ER) and ER stress. Molecular biology verification showed that 50?mg/kg b.w. and higher doses TiO₂ NPs activated a xenobiotic biodegradation response and increased expression of cytochrome P450 family genes in mouse livers, thus inducing ER stress in mice. ER stress-activated MAPK and NF-κB pathways and induced an inflammation response, resulting in phosphorylation of the insulin receptor substrate 1 and, consequently, insulin resistance. This was the main mechanism by which TiO₂ NPs increased plasma glucose in mice. Meanwhile, ER stress disturbed the monooxygenase system, and thus generated reactive oxygen species (ROS). Relief of ER stress with 4-phenylbutyric acid inhibited all the above effects of TiO₂ NPs, including the generation of ROS. Therefore, TiO₂ NP-induced ER stress was a decisive factor with a central role in plasma glucose disturbance in mice.     

Bioaccumulation of ionic titanium and titanium dioxide nanoparticles in zebrafish eleutheroembryos

Abstract

The production of titanium dioxide nanoparticles (TiO₂ NPs) for commercial applications has greatly increased over the last years and consequently the potential risk for human health. There is a growing awareness of the need to understand the behavior and influence these nanoparticles exert on the environment. Bioaccumulation serves as a good integrator to assess chemical exposure in aquatic systems and is dependent on factors, such as the exposure routes, diet and the aqueous medium. We analyzed the experimental bioaccumulation capability of ionic titanium and TiO₂ NPs by zebrafish (Danio rerio) eleutheroembryos through bioconcentration factors (BCFs), after 48 or 72?h of exposure. The stability of both chemical forms in an aquatic medium was fully characterized for further bioaccumulation studies. Several stabilizing agents (humic acids, soluble starch, polyethylene glycol, Na₄P₂O₇ and Na₂HPO₄) for anatase and rutile, the two allotrophs of TiO₂ NPs, were evaluated to check the evolution of the aggregation process. Around 60% of TiO₂ NPs remained disaggregated under simulated environmental conditions with the addition of 50?mg?L^?1 of humic acids. However, the presence of eleutheroembryos in the exposure medium increased TiO₂ NPs aggregation in the experimental tests. The BCFs values obtained in all cases were <100, which classifies ionic titanium and TiO₂ NPs as non-bioaccumulative substances, under the REACH regulations.     

In vitro evaluation of the toxicity and underlying molecular mechanisms of Janus Fe3O4‐TiO2 nanoparticles in human liver cells

Recent studies show that Janus Fe₃O₄‐TiO₂ nanoparticles (NPs) have potential applications as a multifunctional agent of magnetic resonance imaging (MRI) and photodynamic therapy (PDT) for the diagnosis and therapy of cancer. However, little work has been done on their biological effects. To evaluate the toxicity and underlying molecular mechanisms of Janus Fe₃O₄‐TiO₂ nanoparticles, an in vitro study using a human liver cell line HL‐7702 cells was conducted. For comparison, the Janus Fe₃O₄‐TiO₂ NPs parent material TiO₂ NPs was also evaluated. Results showed that both Fe₃O₄‐TiO₂ NPs and TiO₂ NPs decreased cell viability and ATP levels when applied in treatment, but increased malonaldehyde (MDA) and reactive oxygen species (ROS) generation. Mitochondria JC‐1 staining assay showed that mitochondrial membrane permeability injury occurred in both NPs treated cells. Cell viability analysis showed that TiO₂ NPs induced slightly higher cytotoxicity than Fe₃O₄‐TiO₂ NPs in HL7702 cells. Western blotting indicated that both TiO₂ NPs and Fe₃O₄‐TiO₂ NPs could induce apoptosis, inflammation, and carcinogenesis related signal protein alterations. Comparatively, Fe₃O₄‐TiO₂ NPs induced higher signal protein expressions than TiO₂ NPs under a high treatment dose. However, under a low dose (6.25 μg/cm²), neither NPs had any significant toxicity on HL7702 cells. In addition, our results suggest both Fe₃O₄‐TiO₂ NPs and TiO₂ NPs could induce oxidative stress and have a potential carcinogenetic effect in vitro. Further studies are needed to elaborate the detailed mechanisms of toxicity induced by a high dose of Fe₃O₄‐TiO₂ NPs.     

Biological effect of food additive titanium dioxide nanoparticles on intestine: an in vitro study

Titanium dioxide nanoparticles (TiO₂ NPs) are widely found in food‐related consumer products. Understanding the effect of TiO₂ NPs on the intestinal barrier and absorption is essential and vital for the safety assessment of orally administrated TiO₂ NPs. In this study, the cytotoxicity and translocation of two native TiO₂ NPs, and these two TiO₂ NPs pretreated with the digestion simulation fluid or bovine serum albumin were investigated in undifferentiated Caco‐2 cells, differentiated Caco‐2 cells and Caco‐2 monolayer. TiO₂ NPs with a concentration less than 200 µg ml^–1 did not induce any toxicity in differentiated cells and Caco‐2 monolayer after 24 h exposure. However, TiO₂ NPs pretreated with digestion simulation fluids at 200 µg ml^–1 inhibited the growth of undifferentiated Caco‐2 cells. Undifferentiated Caco‐2 cells swallowed native TiO₂ NPs easily, but not pretreated NPs, implying the protein coating on NPs impeded the cellular uptake. Compared with undifferentiated cells, differentiated ones possessed much lower uptake ability of these TiO₂ NPs. Similarly, the traverse of TiO₂ NPs through the Caco‐2 monolayer was also negligible. Therefore, we infer the possibility of TiO₂ NPs traversing through the intestine of animal or human after oral intake is quite low. This study provides valuable information for the risk assessment of TiO₂ NPs in food. Copyright © 2015 John Wiley & Sons, Ltd.     

Amplification of arsenic genotoxicity by TiO2 nanoparticles in mammalian cells: new insights from physicochemical interactions and mitochondria

Titanium dioxide nanoparticles (TiO₂ NPs) have shown great adsorption capacity for arsenic (As); however, the potential impact of TiO₂ NPs on the behavior and toxic responses of As remains largely unexplored. In the present study, we focused on the physicochemical interaction between TiO₂ NPs and As(III) to clarify the underlying mechanisms involved in their synergistic genotoxic effect on mammalian cells. Our data showed that As(III) mainly interacted with TiO₂ NPs by competitively occupying the sites of hydroxyl groups on the surface of TiO₂ NP aggregates, resulting in more aggregation of TiO₂ NPs. Although TiO₂ NPs at concentrations used here had no cytotoxic or genotoxic effects on cells, they efficiently increased the genotoxicity of As(III) in human-hamster hybrid (A_L) cells. The synergistic genotoxicity of TiO₂ NPs and As(III) was partially inhibited by various endocytosis pathway inhibitors while it was completely blocked by an As(III)-specific chelator. Using a mitochondrial membrane potential fluorescence probe, a reactive oxygen species (ROS) probe together with mitochondrial DNA-depleted ρ⁰ A_L cells, we discovered that mitochondria were essential for mediating the synergistic DNA-damaging effects of TiO₂ NPs and As(III). These data provide novel mechanistic proof that TiO₂ NPs enhanced the genotoxicity of As(III) via physicochemical interactions, which were mediated by mitochondria-dependent ROS.     

Evaluation of distribution,redox parameters,and genotoxicity in Wistar rats co-exposed to silver and titanium dioxide nanoparticles

ABSTRACT

The increasing production of silver nanoparticles (AgNPs) and titanium dioxide nanoparticles (TiO₂NPs) has resulted in their elevated concentrations in the environment. This study was, therefore, aimed at determining the distribution, redox parameters, and genotoxic effects in male Wistar rats that were treated with either AgNP or TiO₂NP individually, as well as under a co-exposure scenario. Animals were exposed via oral gavage to either sodium citrate buffer (vehicle), 0.5 mg/kg/day TiO₂NP, 0.5 mg/kg/day AgNP or a mixture of TiO₂NPs and AgNPs. Exposure lasted 45 days after which rats were sacrificed, and tissue biodistribution of Ag and Ti measured. The blood concentration of glutathione (GSH) and activities of glutathione peroxidase (GPx) and catalase (CAT) were determined while the genotoxicity was analyzed using the comet assay in peripheral blood and liver cells. The tissue concentrations of Ag followed the order; blood > liver > kidneys while for Ti the order was kidneys > liver > blood. There was no significant change in the measured redox parameters in animals that were exposed to TiO₂NPs. However, there was a significant increase in GSH levels accompanied by a reduction in the GPx activity in AgNP-treated and co-exposed groups. The individual or co-exposure to TiO₂NP and AgNP did not markedly induce genotoxicity in blood or liver cells. Data showed that TiO₂NP did not produce significant oxidative stress or genotoxicity in rats at the dose used in this study while the same dose level of AgNPs resulted in oxidative stress, but no noticeable adverse genotoxic effects.     

Genotoxic evaluation of titanium dioxide nanoparticles in vivo and in vitro

With the extensive application of titanium dioxide (TiO₂) nanoparticles (NPs) in food industry, there is a rising debate concerning the possible risk associated with exposure to TiO₂ NPs. The purpose of this study is to evaluate the genotoxicity of TiO₂ NPs using in vivo and in vitro test systems. In vivo study, the adult male Sprague-Dawley rats were exposed to anatase TiO₂ NPs (75 ± 15 nm) through intragastric administration at 0, 10, 50 and 200 mg/kg body weight every day for 30 days. The γ-H₂AX assay showed TiO₂ NPs could induce DNA double strand breaks in bone marrow cells after oral administration. However, the micronucleus test revealed that the oral-exposed TiO₂ NPs did not cause damage to chromosomes or mitotic apparatus observably in rat bone marrow cells. In vitro study, Chinese hamster lung fibroblasts (V79 cells) were exposed to TiO₂ NPs at the dose of 0, 5, 10, 20, 50 and 100 μg/mL. Significant decreases in cell viability were detected in all the treated groups after 24 h and 48 h exposure. Significant DNA damage was only observed at the concentration of 100 μg/mL after 24 h treatment using the comet assay. The obvious gene mutation was observed at the concentration of 20 and 100 μg/mL after 2 h treatment using hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene mutation assay. This study presented a comprehensive genotoxic evaluation of TiO₂ NPs, and TiO₂ NPs were shown to be genotoxic both in vivo and in vitro tests. The gene mutation and DNA strand breaks seem to be more sensitive genetic endpoints for the detection of TiO₂ NPs induced genotoxic effects.