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.     

Analysis of titanium dioxide and zinc oxide nanoparticles in cosmetics

There have been rapid increases in consumer products containing nanomaterials, raising concerns over the impact of nanoparticles (NPs) to humankind and the environment, but little information has been published about mineral filters in commercial sunscreens. It is urgent to develop methods to characterize the nanomaterials in products. Titanium dioxide (TiO₂) and zinc oxide (ZnO) NPs in unmodified commercial sunscreens were characterized by laser scanning confocal microscopy, atomic force microscopy, X-ray diffraction (XRD), and transmission electron microscopy (TEM). The results showed that laser scanning confocal microscopy evaluated primary particle aggregates and dispersions but could not size NPs because of the diffraction limited resolution of optical microscopy (200 nm). Atomic force microscopy measurements required a pretreatment of the sunscreens or further calibration in phase analysis, but could not provide their elemental composition of commercial sunscreens. While XRD gave particle size and crystal information without a pretreatment of sunscreen, TEM analysis required dilution and dispersion of the commercial sunscreens before imaging. When coupled with energy-dispersive X-ray spectroscopy, TEM afforded particle size information and compositional analysis. XRD characterization of six commercial sunscreens labeled as nanoparticles revealed that three samples contained TiO₂ NPs, among which two listed ZnO and TiO₂, and displayed average particle sizes of 15 nm, 21 nm, and 78 nm. However, no nanosized ZnO particles were found in any of the samples by XRD. In general, TEM can resolve nanomaterials that exhibit one or more dimensions between 1 nm and 100 nm, allowing the identification of ZnO and TiO₂ NPs in all six sunscreens and ZnO/TiO₂ mixtures in two of the samples. Overall, the combination of XRD and TEM was suitable for analyzing ZnO and TiO₂ NPs in commercial sunscreens.     

Phytosynthesis and modification of metal and metal oxide nanoparticles/nanocomposites for antibacterial and anticancer activities: Recent advances

Overcoming multidrug resistance mechanisms is an intricate issue to fight against pathogenic bacterial and cancer cells wherein smart application of metal nanoparticles (MNPs: Ag, Au, Cu, Pt, and Pd NPs), metal oxide NPs (MONPs: CuO, Cu₂O, ZnO, TiO₂ and Fe₃O₄ NPs), and metal nanocomposites (MNCs) can be an alluring option compared to the conventional drugs. However, the production and processing of these nanomaterials in relatively safer forms is critical to secure competent therapeutic results. Phytosynthesis and modification using various plant sources are garnering attention in view of the abundance of resources, eco-friendly nature, biodegradability, and biocompatibility in comparison to the conventional chemical and physical methods. Herein, this review discourses recent progress and the future potentials of these greener methods.     

No evidence of the genotoxic potential of gold,silver, zinc oxide and titanium dioxide nanoparticles in the SOS chromotest

Gold nanoparticles (Au NPs), silver nanoparticles (Ag NPs), zinc oxide nanoparticles (ZnO NPs) and titanium dioxide nanoparticles (TiO₂ NPs) are widely used in cosmetic products such as preservatives, colorants and sunscreens. This study investigated the genotoxicity of Au NPs, Ag NPs, ZnO NPs and TiO₂ NPs using the SOS chromotest with Escherichia coli PQ37. The maximum exposure concentrations for each nanoparticle were 3.23 mg l^–1 for Au NPs, 32.3 mg l^–1 for Ag NPs and 100 mg l^–1 for ZnO NPs and TiO₂ NPs. Additionally, in order to compare the genotoxicity of nanoparticles and corresponding dissolved ions, the ions were assessed in the same way as nanoparticles. The genotoxicity of the titanium ion was not assessed because of the extremely low solubility of TiO₂ NPs. Au NPs, Ag NPs, ZnO NPs, TiO₂ NPs and ions of Au, Ag and Zn, in a range of tested concentrations, exerted no effects in the SOS chromotest, evidenced by maximum IF (IFmax) values of below 1.5 for all chemicals. Owing to the results, nanosized Au NPs, Ag NPs, ZnO NPs, TiO₂ NPs and ions of Au, Ag and Zn are classified as non‐genotoxic on the basis of the SOS chromotest used in this study. To the best of our knowledge, this is the first study to evaluate the genotoxicity of Au NPs, Ag NPs, ZnO NPs and TiO₂ NPs using the SOS chromotest. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

纳米二氧化钛神经毒性及其机制的研究进展

纳米二氧化钛（TiO2 NPs）作为一种新型纳米材料,已成为多个领域的研究热点。随着在日常生活中接触TiO2 NPs愈发容易,TiO2 NPs的毒性也逐渐受到关注。TiO2 NPs能够通过血脑屏障进入脑内并蓄积,引起脑组织、神经细胞的损伤,影响情感与认知,降低学习记忆能力。本文主要对近年来TiO2 NPs引起的体内、外神经毒性,以及其毒性机制如氧化应激、炎症反应、凋亡等的研究现状进行综述,旨在丰富TiO2 NPs的毒性研究数据库,为其日常安全使用提供科学依据。     

Mechanistic characterization of titanium dioxide nanoparticle-induced toxicity using electron spin resonance

Titanium dioxide nanoparticles (TiO₂ NPs) are one of the most widely used nanomaterials that have been manufactured worldwide and applied in different commercial realms. The well-recognized ability of TiO₂ to promote the formation of reactive oxygen species (ROS) has been extensively studied as one of the important mechanisms underlying TiO₂ NPs toxicity. As the “gold standard” method to quantify and identify ROS, electron spin resonance (ESR) spectroscopy has been employed in many studies aimed at evaluating TiO₂ NPs safety. This review aims to provide a thorough discussion of current studies using ESR as the primary method to unravel the mechanism of TiO₂ NPs toxicity. ESR spin label oximetry and immune-spin trapping techniques are also briefly introduced, because the combination of spin trapping/labeling techniques offers a promising tool for studying the oxidative damage caused by TiO₂ NPs.     

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.     

Rutile TiO₂ particles exert size and surface coating dependent retention and lesions on the murine brain

The rising commercial use and large-scale production of engineered nanoparticles (NPs) may lead to unintended exposure to humans. The central nervous system (CNS) is a potential susceptible target of the inhaled NPs, but so far the amount of studies on this aspect is limited. Here, we focus on the potential neurological lesion in the brain induced by the intranasally instilled titanium dioxide (TiO₂) particles in rutile phase and of various sizes and surface coatings. Female mice were intranasally instilled with four different types of TiO₂ particles (i.e. two types of hydrophobic particles in micro- and nano-sized without coating and two types of water-soluble hydrophilic nano-sized particles with silica surface coating) every other day for 30 days. Inductively coupled plasma mass spectrometry (ICP-MS) were used to determine the titanium contents in the sub-brain regions. Then, the pathological examination of brain tissues and measurements of the monoamine neurotransmitter levels in the sub-brain regions were performed. We found significant up-regulation of Ti contents in the cerebral cortex and striatum after intranasal instillation of hydrophilic TiO₂ NPs. Moreover, TiO₂ NPs exposure, in particular the hydrophilic NPs, caused obvious morphological changes of neurons in the cerebral cortex and significant disturbance of the monoamine neurotransmitter levels in the sub-brain regions studied. Thus, our results indicate that the surface modification of the NPs plays an important role on their effects on the brain. In addition, the difference in neurotoxicity of the two types of hydrophilic NPs may be induced by the shape differences of the materials. The present results suggest that physicochemical properties like size, shape and surface modification of the nanomaterials should be considered when evaluating their neurological effects.     

Limit-test toxicity screening of selected inorganic nanoparticles to the earthworm Eisenia fetida

The toxicity of a range of inorganic (Ag, Cu, Ni, Al₂O₃, SiO₂, TiO₂ and ZrO₂) nanoparticles (NP) and their corresponding metal salt or bulk metal oxide were screened for toxicity toward the earthworm Eisenia fetida using the limit-test design (1000 mg/kg). This study provides the first ecotoxicological life history trait data on earthworms for each these NPs, as well as for AgNO₃, Al₂O₃, SiO₂, TiO₂ and ZrO₂. Significant effects were observed on survival for AgNO₃ (2.5% of controls), CuCl₂ (17.5% of controls) and NiCl₂ (32.5% of controls) and on reproduction (AgNO₃, CuCl₂, NiCl₂, Ag-NP, Cu-NP, TiO₂-NP); with total reproductive failure in both silver treatments. Ag-NP, Cu-NP and TiO₂-NP were the only NPs that caused toxic effects to E. fetida. The toxicity could not be singularly related to particle size or zeta potential or to the inherent element constituting the NPs (e.g. Ag).     

Immunotoxic effects of thymus in mice following exposure to nanoparticulate TiO2

Titanium dioxide nanoparticles (TiO₂ NPs) have been extensively used in industry, medicine, and daily life, and have shown potential toxic effects for animals or humans. We noted that the effects of TiO₂ NPs on the immune system and its mechanism of action in animals or humans have not been elucidated. Thus, mice were exposed to the TiO₂ NPs (0, 1.25, 2.5, or 5 mg kg^?1 body weight) for 9 consecutive months. Exposure to TiO₂ NPs was accumulated in the thymus, leading to a decrease in body weight and increases in the weight of the thymus or thymus indices. In the blood, exposure to TiO₂ NPs significantly decreased white blood cell, red blood cell, reticulocyte, haemoglobin, and mean corpuscular haemoglobin concentration; and increased mean corpuscular volume, mean corpuscular haemoglobin, platelets, and mean platelet volume. The reductions of lymphocyte subsets, including CD3+, CD4+, CD8+, B cell, and natural killer cell, were observed in the TiO₂ NP‐treated mouse thymus. Appearance of starry‐sky aspect of the cortex that is given by the body of macrophages, bleeding, severe hemolysis or congestion, fatty degeneration, and cell apoptosis or necrosis were observed in the thymus following TiO₂ NPs exposure. Importantly, TiO₂ NPs increased expression of nucleic factor‐κB(NF‐κB), IκB kinase1/2, interleukin‐1β, interleukin ?4, regulated upon activation normal T‐cell expressed and secreted, cyclooxygenase 2, neutrophil gelatinase‐associated lipocalin, purinergic receptors‐7, interferon‐inducible protein 10, hypoxia inducible factor 1‐α, p‐c‐Jun N‐terminal kinase, p‐p38, and p‐extracellular signal‐regulated kinase 1/2 protein, respectively; whereas suppressed expression of IκB, peroxisome proliferater‐activated receptor‐γ, trefoil factor 1, peroxisome proliferator activated receptor gamma coactivator‐1α, and prostaglandin E2 proteins in the thymus, respectively. Taken together, these results suggest that TiO₂ NPs exerts toxic effects on lymphoid organs and T cell and innate immune cell homeostasis in mice and that these immunotoxic potential effects may result from the activation of NF‐κB‐mediated mitogen‐activated protein kinases (MAPKs) pathway.     

Reprint of: Cytotoxicity,cell uptake and microscopic analysis of titanium dioxide and silver nanoparticles in vitro

Commercially manufactured nanomaterials are used massively for modification of products of everyday use, including products intended for children. Therefore their potential risks have to be ultimately studied. Aside from toxicity of nanomaterials with known specific parameters, the end-consumer is potentially endangered by materials with unknown specification. Commercially available products are not usually accompanied by parameter/specification sheet providing the consumer with sufficient chemico-physical parameters allowing the evaluation of possible toxic effects.The aim of this work was to evaluate the declared parameters of commercially available TiO₂ and Ag NPs employing chemico-physical methods and consequently in vitro cytotoxicity and genotoxicity tests performed on non-cancer cell lines. Based on the results of our complex study we can conclude that the data provided by the producers are not in good agreement with the performed measurements. Furthermore, all tested NPs penetrated into the SVK14 cells and all NPs had significant effect on the kinetics of ROS production in all cell lines (note: the ROS production has not been established as the major mechanism of cell damage elicited by Ag NPs). The study revealed greater cytotoxic potential of Ag NPs in comparison with TiO₂ NPs and all of the studied NPs caused significant DNA damage.     

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.     

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.     

Role of the crystalline form of titanium dioxide nanoparticles: Rutile,and not anatase,induces toxic effects in Balb/3T3 mouse fibroblasts

The wide use of titanium dioxide nanoparticles (TiO₂ NPs) in industrial applications requires the investigation of their effects on human health. In this context, we investigated the effects of nanosized and bulk titania in two different crystalline forms (anatase and rutile) in vitro. By colony forming efficiency assay, a dose-dependent reduction of the clonogenic activity of Balb/3T3 mouse fibroblasts was detected in the presence of rutile, but not in the case of anatase NPs. Similarly, the cell transformation assay and the micronucleus test showed that rutile TiO₂ NPs were able to induce type-III foci formation in Balb/3T3 cells and appeared to be slightly genotoxic, whereas anatase TiO₂ NPs did not induce any significant neoplastic or genotoxic effect. Additionally, we investigated the interaction of TiO₂ NPs with Balb/3T3 cells and quantified the in vitro uptake of titania using mass spectrometry. Results showed that the internalization was independent of the crystalline form of TiO₂ NPs but size-dependent, as nano-titania were taken up more than their respective bulk materials.In conclusion, we demonstrated that the cytotoxic, neoplastic and genotoxic effects triggered in Balb/3T3 cells by TiO₂ NPs depend on the crystalline form of the nanomaterial, whereas the internalization is regulated by the particle size.     

Assessment of cellular toxicity of TiO2 nanoparticles for cardiac tissue engineering applications

Abstract

Because of the increased use of titanium dioxide (TiO₂) nanoparticles (NPs) in tissue engineering (TE), and in new constructs for cardiac TE, their effect was studied on three relevant cell types: Adult rat ventricular cardiomyocytes, human embryonic stem cell-derived cardiomyocytes (hESC-CM) and fibroblasts. For adult rat myocytes, 10 μg/mL TiO₂ NPs showed no significant effect on myocyte survival over 24 h or acute myocyte contractility. Increasing the concentration to 100 μg/mL was seen to reduce contraction amplitude (p < 0.05). For hESC-CM, 10 μg/mL TiO₂ reduced the beating rate significantly by 24 h. No arrhythmias or cessation of beating were observed in either cell type. Culturing fibroblasts in 5–150 μg/mL TiO₂ significantly reduced cell proliferation at day 4 and increased cell death. We conclude that there may be modest but potentially adverse effects of TiO₂ NPs if used in fast degrading polymers for myocardial tissue engineering (MTE) applications.     

Distinct toxic interactions of TiO2 nanoparticles with four coexisting organochlorine contaminants on algae

Engineered nanoparticles are increasingly discharged into the environment. After discharge, these nanoparticles can interact with co-existing organic contaminants, resulting in a phenomena referred to as ‘joint toxicity’. This study evaluated joint toxicities of TiO₂ nanoparticles (TiO₂NPs) with four different (atrazine, hexachlorobenzene, pentachlorobenzene, and 3,3′,4,4′-tetrachlorobiphenyl) organochlorine contaminants (OCs) toward algae (Chlorella pyrenoidosa). The potential mechanisms underlying the joint toxicity were discussed, including TiO₂NPs–OC interactions, effects of TiO₂NPs and OCs on biophysicochemical properties of algae and effects of TiO₂NPs and OCs on each other’s bioaccumulation in algae. The results indicate that coexposure led to a synergistic effect on the joint toxicity for TiO₂NPs–atrazine, antagonistic effect for TiO₂NPs–hexachlorobenzene and TiO₂NPs–3,3',4,4'-tetrachlorobiphenyl, and an additive effect for TiO₂NPs–pentachlorobenzene. There was nearly no adsorption of OCs by TiO₂NPs, and the physicochemical properties of TiO₂NPs were largely unaltered by the presence of OCs. However, both OCs and NPs affected the biophysicochemical properties of algal cells and thereby influenced the cell surface binding and/or internalization. TiO₂NPs significantly increased the bioaccumulation of each OC. However, with the exception of atrazine, the bioaccumulation of TiO₂NPs decreased when used with each OC. The distinct joint toxicity outcomes were a result of the balance between the increased toxicities of OCs (increased bioaccumulations) and the altered toxicity of TiO₂NPs (bioaccumulation can either increase or decrease). These results can significantly improve our understanding of the potential environmental risks associated with NPs.     

Combined effect of titanium dioxide nanoparticles and glucose on the cardiovascular system in young rats after oral administration

Titanium dioxide nanoparticles (TiO₂ NPs) have already been used as food additive in various products and are usually consumed with a considerable amount of sugar. Oral consumption of TiO₂ NPs poses concerning health risks; however, research on the combined effect of ingested TiO₂ NPs and glucose is limited. We examined young Sprague‐Dawley rats administrated TiO₂ NPs orally at doses of 0, 2, 10 and 50 mg/kg body weight per day with and without 1.8 g/kg body weight glucose for 30 and 90 days. Heart rate, systolic and diastolic blood pressure, blood biochemical parameters and histopathology of cardiac tissues was assessed to quantify cardiovascular damage. The results showed that oral exposure to TiO₂ NPs and high doses of glucose both could induce cardiovascular injuries. The toxic effects were dose‐, time‐ and gender‐dependent. The interaction effects between oral‐exposed TiO₂ NPs and glucose existed and revealed to be antagonism in most of the biological parameters. However, toxic effects of the high‐dose glucose seemed to be more severe than TiO₂ NPs and the interaction of TiO₂ NPs with glucose. These results suggest that it may be more important to control the sugar intake than TiO₂ NPs for protecting the health of TiO₂ NP consumers.     

A 3D co-culture of three human cell lines to model the inflamed intestinal mucosa for safety testing of nanomaterials

Oral exposure to nanomaterials is a current concern, asking for innovative biological test systems to assess their safety, especially also in conditions of inflammatory disorders. Aim of this study was to develop a 3D intestinal model, consisting of Caco-2 cells and two human immune cell lines, suitable to assess nanomaterial toxicity, in either healthy or diseased conditions. Human macrophages (THP-1) and human dendritic cells (MUTZ-3) were embedded in a collagen scaffold and seeded on the apical side of transwell inserts. Caco-2 cells were seeded on top of this layer, forming a 3D model of the intestinal mucosa. Toxicity of engineered nanoparticles (NM101 TiO₂, NM300 Ag, Au) was evaluated in non-inflamed and inflamed co-cultures, and also compared to non-inflamed Caco-2 monocultures. Inflammation was elicited by IL-1β, and interactions with engineered NPs were addressed by different endpoints. The 3D co-culture showed well preserved ultrastructure and significant barrier properties. Ag NPs were found to be more toxic than TiO₂ or Au NPs. But once inflamed with IL-1β, the co-cultures released higher amounts of IL-8 compared to Caco-2 monocultures. However, the cytotoxicity of Ag NPs was higher in Caco-2 monocultures than in 3D co-cultures. The naturally higher IL-8 production in the co-cultures was enhanced even further by the Ag NPs. This study shows that it is possible to mimic inflamed conditions in a 3D co-culture model of the intestinal mucosa. The fact that it is based on three easily available human cell lines makes this model valuable to study the safety of nanomaterials in the context of inflammation.     

Combined effect of titanium dioxide nanoparticles and glucose on the blood glucose homeostasis in young rats after oral administration

Titanium dioxide nanoparticles (TiO₂ NPs) were usually consumed with a high content of sugar, and children were identified as having the highest exposures due to sweet food preferences. Research on the combined effect of ingested TiO₂ NPs and glucose has great significance, particularly in young people. We examined young Sprague-Dawley rats administered TiO₂ NPs (0, 2, 10 and 50 mg/kg) orally with and without glucose (1.8 g/kg) for 90 days. Blood glucose homeostasis was assessed by monitoring blood glucose and detecting glycoproteins. Glucose tolerance was also evaluated by the oral glucose tolerance test. The levels of blood glucose-related hormones such as insulin, C-peptide and glucagon were measured. We found that subchronic co-exposure of TiO₂ NPs and glucose caused slight imbalance of blood glucose homeostasis in vivo. Mild and temporary hypoglycemia, impaired glucose tolerance and changes of glucose-regulating hormones were shown in the exposure groups. The combined effect of TiO₂ NPs and glucose was more apparent than that of TiO₂ NPs alone, which may be due to the effects of excess glucose and the interactions between TiO₂ NPs and glucose. The antagonistic effect of TiO₂ NPs with glucose did exist in the level of glycosylated hemoglobin in female rats. Gender differences were apparent in these effects induced by TiO₂ NPs and glucose. Female rats seemed to be more susceptible for blood glucose disorders. Co-exposure of TiO₂ NPs and excessive glucose could induce gender-dependent imbalance of blood glucose homeostasis in rats. It may be the reason that these consumers face greater health risks glycosylated hemoglobin.