Hazard assessment of nanoplastics is driven by their surface-functionalization. Effects in human-derived primary endothelial cells.

During plastic waste degradation into micro/nanoplastics (MNPLs) their physicochemical characteristics including surface properties (charge, functionalization, biocorona, etc.) can change, potentially affecting their biological effects. This paper focuses on the surface functionalization of MNPLs to determine if it has a direct impact on the toxicokinetic and toxicodynamic interactions in human umbilical vein endothelial cells (HUVECs), at different exposure times. Pristine polystyrene nanoplastics (PS-NPLs), as well as their carboxylated (PS-C-NPLs) and aminated (PS-A-NPLs) forms, all around 50 nm, were used in a wide battery of toxicological assays. These assays encompassed evaluations on cell viability, cell internalization, induction of intracellular reactive oxygen species (iROS), and genotoxicity. The experiments were conducted at a concentration of 100 µg/mL, chosen to ensure a high internalization rate across all treatments while maintaining a sub-toxic concentration. Our results show that all PS-NPLs are internalized by HUVECs, but the internalization dynamic depends on the particle's functionalization. PS-NPLs and PS-C-NPLs internalization modify the morphology of the cell increasing its inner complexity/granularity. Regarding cell toxicity, only PS-A-NPLs reduced cell viability. Intracellular ROS was induced by the three different PS-NPLs but at different time points. Genotoxic damage was induced by the three PS-NPLs at short exposures (2 h), but not for PS-C-NPLs at 24 h. Overall, this study suggests that the toxicological effects of PSNPLs on HUVEC cells are surface-dependent, highlighting the relevance of using human-derived primary cells as a target.

40S Ribosomal Protein S6 Kinase Integrates Daylength Perception and Growth Regulation in Arabidopsis thaliana.

Plant growth occurs via the interconnection of cell growth and proliferation in each organ following specific developmental and environmental cues. Therefore, different photoperiods result in distinct growth patterns due to the integration of light and circadian perception with specific Carbon (C) partitioning strategies. In addition, the TARGET OF RAPAMYCIN (TOR) kinase pathway is an ancestral signaling pathway that integrates nutrient information with translational control and growth regulation. Recent findings in Arabidopsis (Arabidopsis thaliana) have shown a mutual connection between the TOR pathway and the circadian clock. However, the mechanistical network underlying this interaction is mostly unknown. Here, we show that the conserved TOR target, the 40S ribosomal protein S6 kinase (S6 K) is under circadian and photoperiod regulation both at the transcriptional and post-translational level. Total S6 K (S6K1 and S6K2) and TOR-dependent phosphorylated-S6 K protein levels were higher during the light period and decreased at dusk especially under short day conditions. Using chemical and genetic approaches we found that the diel pattern of S6 K accumulation results from 26S proteasome-dependent degradation and is altered in mutants lacking the circadian F-box protein ZEITLUPE (ZTL), further strengthening our hypothesis that S6 K could incorporate metabolic signals via TOR, which are also under circadian regulation. Moreover, under short days when C/energy levels are limiting, changes in S6K1 protein levels affected starch, sucrose and glucose accumulation and consequently impacted root and rosette growth responses. In summary, we propose that S6K1 constitutes a missing molecular link where day-length perception, nutrient availability and TOR pathway activity converge to coordinate growth responses with environmental conditions.

The release of polylactic acid nanoplastics (PLA-NPLs) from commercial teabags. Obtention, characterization, and hazard effects of true-to-life PLA-NPLs.

This study investigates MNPLs release from commercially available teabags and their effects on both undifferentiated monocultures of Caco-2 and HT29 and in the in vitro model of the intestinal Caco-2/HT29 barrier. Teabags were subjected to mechanical and thermodynamic forces simulating the preparation of a cup of tea. The obtained dispersions were characterized using TEM, SEM, DLS, LDV, NTA, and FTIR. Results confirmed that particles were in the nano-range, constituted by polylactic acid (PLA-NPLs), and about one million of PLA-NPLs per teabag were quantified. PLA-NPLs internalization, cytotoxicity, intracellular reactive oxygen species induction, as well as structural and functional changes in the barrier were assessed. Results show that PLA-NPLs present high uptake rates, especially in mucus-secretor cells, and bio-persisted in the tissue after 72 h of exposure. Although no significant cytotoxicity was observed after the exposure to 100 µg/mL PLA-NPLs during 48 h, a slight barrier disruption could be detected at short-time periods. The present work reveals new insights into the safety of polymer-based teabags, the behavior of true-to-life MNPLs in the human body, as well as new questions on how repeated and prolonged exposures could affect the structure and function of the human intestinal epithelium.

The potential effects of in vitro digestion on the physicochemical and biological characteristics of polystyrene nanoplastics.

The presence of plastic waste in our environment has continued growing and become an important environmental concern. Because of its degradation into micro- and nanoplastics (MNPLs), MNPLs are becoming environmental pollutants of special environmental/health concern. Since ingestion is one of the main exposure routes to MNPLs, the potential effects of digestion on the physicochemical/biological characteristics of polystyrene nanoplastics (PSNPLs) were determined. The results indicated a high tendency of digested PSNPLs to agglomerate and a differential presence of proteins on their surface. Interestingly, digested PSNPLs showed greater cell uptake than undigested PSNPLs in all three tested cell lines (TK6, Raji-B, and THP-1). Despite these differences in cell uptake, no differences in toxicity were observed except for high and assumed unrealistic exposures. When oxidative stress and genotoxicity induction were determined, the low effects observed after exposure to undigested PDNPLs were not observed in the digested ones. This indicated that the greater ability of digested PSNPLs to internalize was not accompanied by a greater hazard. This type of analysis should be performed with other MNPLs of varying sizes and chemical compositions.

Food-Grade Metal Oxide Nanoparticles Exposure Alters Intestinal Microbial Populations, Brush Border Membrane Functionality and Morphology, In Vivo (Gallus gallus).

Among food additive metal oxide nanoparticles (NP), titanium dioxide (TiO2) and silicon dioxide (SiO2) are commonly used as food coloring or anti-caking agents, while zinc oxide (ZnO) and iron oxide (Fe2O3) are added as antimicrobials and coloring agents, respectively, and can be used as micronutrient supplements. To elucidate potential perturbations associated with NP consumption on gastrointestinal health and development, this in vivo study utilized the Gallus gallus (broiler chicken) intraamniotic administration to assess the effects of physiologically relevant concentrations of food-grade metal oxide NP on brush border membrane (BBM) functionality, intestinal morphology and intestinal microbial populations in vivo. Six groups with 1 mL injection of the following treatments were utilized: non-injected, 18 MΩ DI H2O; 1.4 × 10-6 mg TiO2 NP/mL, 2.0 × 10-5 mg SiO2 NP/mL, 9.7 × 10-6 mg ZnO NP/mL, and 3.8 × 10-4 mg Fe2O3 NP/mL (n = 10 per group). Upon hatch, blood, cecum, and duodenum were collected to assess mineral (iron and zinc) metabolism, BBM functional, and pro-inflammatory-related protein gene expression, BBM morphometric analysis, and the relative abundance of intestinal microflora. Food additive NP altered mineral transporter, BBM functionality, and pro-inflammatory cytokine gene expression, affected intestinal BBM development and led to compositional shifts in intestinal bacterial populations. Our results suggest that food-grade TiO2 and SiO2 NP have the potential to negatively affect intestinal functionality; food-grade ZnO NP exposure effects were associated with supporting intestinal development or compensatory mechanisms due to intestinal damage, and food-grade Fe2O3 NP was found to be a possible option for iron fortification, though with potential alterations in intestinal functionality and health.

Lack of mutagenicity of TiO2 nanoparticles in vitro despite cellular and nuclear uptake.

The potential genotoxicity of titanium dioxide (TiO2) nanoparticles (NPs) is a conflictive topic because both positive and negative findings have been reported. To add clarity, we have carried out a study with two cell lines (V79-4 and A549) to evaluate the effects of TiO2 NPs (NM-101), with a diameter ranging from 15 to 60 nm, at concentrations 1-75 µg/cm2. Using two different dispersion procedures, cell uptake was determined by Transmission Electron Microscopy (TEM). Mutagenicity was evaluated using the Hprt gene mutation test, while genotoxicity was determined with the comet assay, detecting both DNA breaks and oxidized DNA bases (with formamidopyrimidine glycosylase - Fpg). Cell internalization, as determined by TEM, shows TiO2 NM-101 in cytoplasmic vesicles, as well as close to and inside the nucleus. Such internalization did not depend on the state of agglomeration, nor the dispersion used. In spite of such internalization, no cytotoxicity was detected in V79-4 cells (relative growth activity and plating efficiency assays) or in A549 cells (AlamarBlue assay) after exposure lasting for 24 h. However, a significant decrease in the relative growth activity was detected at longer exposure times (48 and 72 h) and at the highest concentration 75 µg/cm2. When the modified enzyme-linked alkaline comet assay was performed on A549 cells, although no significant induction of DNA damage was detected, a positive concentration-effects relationship was observed (Spearman's correlation = 0.9, p 0.0001). Furthermore, no significant increase of DNA oxidized purine bases was observed. When the frequency of Hprt gene mutants was determined in V79-4 cells, no increase was observed in the exposed cells, relative to the unexposed cultures. Our general conclusion is that, under our experimental conditions, TiO2 NM-101 exposure does not exert mutagenic effects despite the evidence of NP uptake by V79-4 cells.

Analysis of the albumin-bilirubin score as an indicator of improved liver function among hepatitis C virus patients with sustained viral response after direct-acting antiviral therapy.

Background and Aim: To investigate the performance of the albumin-bilirubin (ALBI) score as an indicator of improved hepatic function using a cohort of hepatitis C virus (HCV) patients with sustained viral response (SVR) after direct-acting antiviral therapy (DAA). Methods: HCV patients who achieved SVR after DAAs between 2015 and 2016 were followed for at least 24 months. Changes in ALBI were evaluated in the entire cohort and according to liver function and liver stiffness status at baseline. Results: Four hundred ninety-seven patients were enrolled. Exactly 96.92% were in Child-Pugh (CTP) class A, and 42% had grade 2 fibrosis. Median ALBI was -3.02, while 87.7 and 11.3% of patients were in ALBI grades 1 and 2, respectively. ALBI improved significantly over time, particularly in patients who had a worse ALBI at baseline. Exactly 77% of patients initially in ALBI grade 1 and 93.9% of those in ALBI grades 2-3 improved their ALBI score in different amounts. Improved ALBI was observed irrespective of CTP score at baseline. Median ALBI at baseline and after 24 months were -3.03 and -3.27 for CTP 5, 2.02 and -2.88 for CTP 6, and -1.59 and -2.84 for CTP >6. Similarly, a significant improvement in ALBI was observed within each stage of fibrosis at baseline. Conclusion: ALBI was a good indicator of improved hepatic function in HCV patients with SVR after DAA therapy, able to identify changes even in those patients who started DAA therapy with well-preserved function and mild fibrosis. This simple, objective, and noninvasive test should be evaluated in other clinical scenarios where liver function is relevant.

The mechanistic effects of human digestion on magnesium oxide nanoparticles: implications for probiotics Lacticaseibacillus rhamnosus GG and Bifidobacterium bifidum VPI 1124.

The effects of nanoparticles (NPs) on the human gut microbiota are of high interest due to the link between the gut homeostasis and overall human health. The human intake of metal oxide NPs has increased due to its use in the food industry as food additives. Specifically, magnesium oxide nanoparticles (MgO-NPs) have been described as antimicrobial and antibiofilm. Therefore, in this work we investigated the effects of the food additive MgO-NPs, on the probiotic and commensal Gram-positive Lactobacillus rhamnosus GG and Bifidobacterium bifidum VPI 1124. The physicochemical characterization showed that food additive MgO is formed by nanoparticles (MgO-NPs) and after a simulated digestion, MgO-NPs partially dissociate into Mg2+. Moreover, nanoparticulate structures containing magnesium were found embedded in organic material. Exposures to MgO-NPs for 4 and 24 hours increased the bacterial viability of both L. rhamnosus and B. bifidum when in biofilms but not when as planktonic cells. High doses of MgO-NPs significantly stimulated the biofilm development of L. rhamnosus, but not B. bifidum. It is likely that the effects are primarily due to the presence of ionic Mg2+. Evidence from the NPs characterization indicate that interactions bacteria/NPs are unfavorable as both structures are negatively charged, which would create repulsive forces.

TiO2 Nanoparticles and Commensal Bacteria Alter Mucus Layer Thickness and Composition in a Gastrointestinal Tract Model.

Nanoparticles (NPs) are used in food packaging and processing and have become an integral part of many commonly ingested products. There are few studies that have focused on the interaction between ingested NPs, gut function, the mucus layer, and the gut microbiota. In this work, an in vitro model of gastrointestinal (GI) tract is used to determine whether, and how, the mucus layer is affected by the presence of Gram-positive, commensal Lactobacillus rhamnosus; Gram-negative, opportunistic Escherichia coli; and/or exposure to physiologically relevant doses of pristine or digested TiO2 NPs. Caco-2/HT29-MTX-E12 cell monolayers are exposed to physiological concentrations of bacteria (expressing fluorescent proteins) and/or TiO2 nanoparticles for a period of 4 h. To determine mucus thickness and composition, cell monolayers are stained with alcian blue, periodic acid schiff, or an Alexa Fluor 488 conjugate of wheat germ agglutinin. It is found that the presence of both bacteria and nanoparticles alter the thickness and composition of the mucus layer. Changes in the distribution or pattern of mucins can be indicative of pathological conditions, and this model provides a platform for understanding how bacteria and/or NPs may interact with and alter the mucus layer.

Effects of Titanium Dioxide Nanoparticles on the Hprt Gene Mutations in V79 Hamster Cells.

The genotoxicity of anatase/rutile TiO2 nanoparticles (TiO2 NPs, NM105 at 3, 15 and 75 µg/cm2) was assessed with the mammalian in-vitro Hypoxanthine guanine phosphoribosyl transferase (Hprt) gene mutation test in Chinese hamster lung (V79) fibroblasts after 24 h exposure. Two dispersion procedures giving different size distribution and dispersion stability were used to investigate whether the effects of TiO2 NPs depend on the state of agglomeration. TiO2 NPs were fully characterised in the previous European FP7 projects NanoTEST and NanoREG2. Uptake of TiO2 NPs was measured by transmission electron microscopy (TEM). TiO2 NPs were found in cytoplasmic vesicles, as well as close to the nucleus. The internalisation of TiO2 NPs did not depend on the state of agglomeration and dispersion used. The cytotoxicity of TiO2 NPs was measured by determining both the relative growth activity (RGA) and the plating efficiency (PE). There were no substantial effects of exposure time (24, 48 and 72 h), although a tendency to lower RGA at longer exposure was observed. No significant difference in PE values and no increases in the Hprt gene mutant frequency were found in exposed relative to unexposed cultures in spite of evidence of uptake of NPs by cells.

The Role of Metal Oxide Nanoparticles, Escherichia coli, and Lactobacillus rhamnosus on Small Intestinal Enzyme Activity.

Engineered nanomaterials (ENMs) have become common in the food industry, which motivates the need to evaluate ENM effects on human health. Gastrointestinal (GI) in vitro models (e.g. Caco-2, Caco-2/HT29-MTX) have been used in nanotoxicology research. However, the human gut environment is composed of both human cells and the gut microbiota. The goal of this study is to increase the complexity of the Caco-2/HT29-MTX in vitro model by co-culturing human cells with the Gram-positive, commensal Lactobacillus rhamnosus or the Gram-negative, opportunistic Escherichia coli; with the hypothesis that the presence of bacteria would ameliorate the effects of exposure to metal oxide nanoparticles (NPs) such as iron oxide (Fe2O3), silicone dioxide (SiO2), titanium dioxide (TiO2), or zinc oxide (ZnO). To understand this relationship, Caco-2/HT29-MTX cell barriers were acutely co-exposed (4 hours) to bacteria and/or NPs (pristine or in vitro digested). The activity of the brush border membrane (BBM) enzymes intestinal alkaline phosphatase (IAP), aminopeptidase-N (APN), sucrase isomaltase (SI) and the basolateral membrane enzyme (BLM) Na+/K+ ATPase were assessed. Findings show that (i) the human digestion process alters the physicochemical properties of NPs, (ii) large agglomerates of NPs remain entrapped on the apical side of the intestinal barrier, which (iii) affects the activity of BBM enzymes. Interestingly, some NPs effects were attenuated in the presence of either bacterial strains. Confocal microscopy detected bacteria-NPs interactions, which may impede the NP-intestinal cell contact. These results highlight the importance of improving in vitro models to closely mimic the complexities of the human body.

Micronuclei Detection by Flow Cytometry as a High-Throughput Approach for the Genotoxicity Testing of Nanomaterials.

Thousands of nanomaterials (NMs)-containing products are currently under development or incorporated in the consumer market, despite our very limited understanding of their genotoxic potential. Taking into account that the toxicity and genotoxicity of NMs strongly depend on their physicochemical characteristics, many variables must be considered in the safety evaluation of each given NM. In this scenario, the challenge is to establish high-throughput methodologies able to generate rapid and robust genotoxicity data that can be used to critically assess and/or predict the biological effects associated with those NMs being under development or already present in the market. In this study, we have evaluated the advantages of using a flow cytometry-based approach testing micronucleus (MNs) induction (FCMN assay). In the frame of the EU NANoREG project, we have tested six different NMs-namely NM100 and NM101 (TiO2NPs), NM110 (ZnONPs), NM212 (CeO2NPs), NM300K (AgNPs) and NM401 (multi-walled carbon nanotubes (MWCNTs)). The obtained results confirm the ability of AgNPs and MWCNTs to induce MN in the human bronchial epithelial BEAS-2B cell line, whereas the other tested NMs retrieved non-significant increases in the MN frequency. Based on the alignment of the results with the data reported in the literature and the performance of the FCMN assay, we strongly recommend this assay as a reference method to systematically evaluate the potential genotoxicity of NMs.

The Comet Assay as a Tool to Detect the Genotoxic Potential of Nanomaterials.

The interesting physicochemical characteristics of nanomaterials (NMs) has brought about their increasing use and, consequently, their increasing presence in the environment. As emergent contaminants, there is an urgent need for new data about their potential side-effects on human health. Among their potential effects, the potential for DNA damage is of paramount relevance. Thus, in the context of the EU project NANoREG, the establishment of common robust protocols for detecting genotoxicity of NMs became an important aim. One of the developed protocols refers to the use of the comet assay, as a tool to detect the induction of DNA strand breaks. In this study, eight different NMs-TiO2NP (2), SiO2NP (2), ZnONP, CeO2NP, AgNP, and multi-walled carbon nanotubes (MWCNT)-were tested using two different human lung epithelial cell lines (A549 and BEAS-2B). The comet assay was carried out with and without the use of the formamidopyrimidine glycosylase (FPG) enzyme to detect the induction of oxidatively damaged DNA bases. As a high throughput approach, we have used GelBond films (GBF) instead of glass slides, allowing the fitting of 48 microgels on the same GBF. The results confirmed the suitability of the comet assay as a powerful tool to detect the genotoxic potential of NMs. Specifically, our results indicate that most of the selected nanomaterials showed mild to significant genotoxic effects, at least in the A549 cell line, reflecting the relevance of the cell line used to determine the genotoxic ability of a defined NM.

Biodistribution of Liposome-Encapsulated Bacteriophages and Their Transcytosis During Oral Phage Therapy.

This study sheds light on the biodistribution of orally administered, liposome-encapsulated bacteriophages, and their transcytosis through intestinal cell layers. Fluorochrome-labeled bacteriophages were used together with a non-invasive imaging methodology in the in vivo visualization of bacteriophages in the stomach and intestinal tract of mice. In those studies, phage encapsulation resulted in a significant increase of the labeled phages in the mouse stomach, even 6 h after their oral administration, and without a decrease in their concentration. By contrast, the visualization of encapsulated and non-encapsulated phages in the intestine were similar. Our in vivo observations were corroborated by culture methods and ex vivo experiments, which also showed that the percentage of encapsulated phages in the stomach remained constant (50%) compared to the amount of initially administered product. However, the use of conventional microbiological methods, which employ bile salts to break down liposomes, prevented the detection of encapsulated phages in the intestine. The ex vivo data showed a higher concentration of non-encapsulated than encapsulated phages in liver, kidney, and even muscle up to 6 h post-administration. Encapsulated bacteriophages were able to reach the liver, spleen, and muscle, with values of 38% ± 6.3%, 68% ± 8.6%, and 47% ± 7.4%, respectively, which persisted over the course of the experiment. Confocal laser scanning microscopy of an in vitro co-culture of human Caco-2/HT29/Raji-B cells revealed that Vybrant-Dil-stained liposomes containing labeled bacteriophages were preferably embedded in cell membranes. No transcytosis of encapsulated phages was detected in this in vitro model, whereas SYBR-gold-labeled non-encapsulated bacteriophages were able to cross the membrane. Our work demonstrates the prolonged persistence of liposome-encapsulated phages in the stomach and their adherence to the intestinal membrane. These observations could explain the greater long-term efficacy of phage therapy using liposome-encapsulated phages.

Assessing the effectiveness of green synthetized silver nanoparticles with Cryptocarya alba extracts for remotion of the organic pollutant methylene blue dye.

In the present work, silver nanoparticles (AgNPs) synthetized with Cryptocarya alba (Peumo) leaf extract were studied. The fabrication method was fast, low cost, and eco-friendly, and the final properties of AgNPs were determined by experimental parameters, such as AgNO3 and Peumo extract concentrations used. Setting suitable experimental conditions, crystalline AgNPs with apparent spherical forms and average diameter around 3.5 nm were obtained. In addition, the capability of synthesized Peumo-AgNPs to remove methylene blue dye (MB) in aqueous solution as well as their catalytic effectiveness was also investigated. The results showed that green synthesized AgNPs can remove fast and effectively the MB dye from aqueous medium by itself, but better results were found acting like catalyst by using sodium borohydride (NaBH4) in the reaction. In addition, this green nanomaterial can be recycling several times maintaining initial properties for removal of MB. Thus, AgNPs synthetized with Peumo leaf extracts could be an excellent catalyst candidate for degradation of blue methylene dye in chemical industries.

Assessing the relevance of exposure time in differentiated Caco-2/HT29 cocultures. Effects of silver nanoparticles.

In vitro models of the intestinal barrier are being increasingly used to evaluate nanoparticles (NPs) exposure risk. Nevertheless, most of these studies have focused on short-term exposures lasting no more than 24 h of duration, which could underestimate the toxic effects of a given compound under a more realistic setting. Since the assessment of longer exposure time-points is crucial to evaluate the risk of cumulative exposure to NPs, we have analyzed the effects of AgNPs at different exposure time-points between 6 h and 4 days on the barrier model system constituted by Caco-2/HT29 cells. Our results indicate that i) the system is stable during this time frame; ii) AgNPs affect the barrier's integrity only at the highest concentration tested (100 µg/mL), and only after 96 h of exposure; iii) cellular uptake of AgNPs showed a time-dependent and concentration-dependent increase; iv) translocation through the barrier was only observed at the highest concentration and only after 96 h of exposure; v) the expression of genes involved in the barrier's structure differs depending on the exposure time analyzed. All these results reinforce our proposal of expanding exposure times beyond 24 h when performing assays for hazard assessment of NPs using in vitro models of the intestinal barrier.

Genotoxicity of Copper and Nickel Nanoparticles in Somatic Cells of Drosophila melanogaster.

Copper and nickel nanoparticles (Cu-NPs and Ni-NPs, respectively) are used in a variety of industrial applications, such as semiconductors, catalysts, sensors, and antimicrobial agents. Although studies on its potential genotoxicity already exist, few of them report in vivo data. In the present study we have used the wing-spot assay in Drosophila melanogaster to determine the genotoxic activity of Cu-NPs and Ni-NPs, and these data have been compared with those obtained with their microparticle forms (MPs). Additionally, a complete physical characterization of NPs using transmission electronic microscopy (TEM), dynamic light scattering (DLS), and laser Doppler velocimetry (LDV) techniques was also performed. Results obtained with Cu-NPs and Cu-MPs indicate that both failed to induce an increase in the frequency of mutant spots formation in the wings of the adults, suggesting a lack of genotoxicity in somatic cells of D. melanogaster. However, when Ni-NPs and Ni-MPs were evaluated, a significant increase of small single spots and total mutant spots was observed only for Ni-NPs (P<0.05) at the highest dose assessed. Thus, the genotoxicity of Ni-NPs seem to be related to their nanoscale size, because no genotoxic effects have been reported with their microparticles and ions. This study is the first assessing the in vivo genotoxic potential of Cu-NPs and Ni-NPs in the Drosophila model.

Effects of differently shaped TiO2NPs (nanospheres, nanorods and nanowires) on the in vitro model (Caco-2/HT29) of the intestinal barrier.

BACKGROUND: The biological effects of nanoparticles depend on several characteristics such as size and shape that must be taken into account in any type of assessment. The increased use of titanium dioxide nanoparticles (TiO2NPs) for industrial applications, and specifically as a food additive, demands a deep assessment of their potential risk for humans, including their abilities to cross biological barriers. METHODS: We have investigated the interaction of three differently shaped TiO2NPs (nanospheres, nanorods and nanowires) in an in vitro model of the intestinal barrier, where the coculture of Caco-2/HT29 cells confers inherent intestinal epithelium characteristics to the model (i.e. mucus secretion, brush border, tight junctions, etc.). RESULTS: Adverse effects in the intestinal epithelium were detected by studying the barrier's integrity (TEER), permeability (LY) and changes in the gene expression of selected specific markers. Using Laser Scanning Confocal Microscopy, we detected a different behaviour in the bio-adhesion and biodistribution of each of the TiO2NPs. Moreover, we were able to specifically localize each type of TiO2NPs inside the cells. Interestingly, general DNA damage, but not oxidative DNA damage effects, were detected by using the FPG version of the comet assay. CONCLUSIONS: Results indicate different interactions and cellular responses related to differently shaped TiO2NPs, nanowires showing the most harmful effects.

Titanium dioxide nanoparticles translocate through differentiated Caco-2 cell monolayers, without disrupting the barrier functionality or inducing genotoxic damage.

The widespread use of titanium dioxide nanoparticles (TiO2 NPs) in commercial food products makes intestinal cells a suitable target. Accordingly, we have used the human colon adenocarcinoma Caco-2 cells to detect their potential harmful effects. Caco-2 cells can differentiate in to enterocytic-like cells, forming consistent cell monolayers and are used as a model of the intestinal barrier. Using both undifferentiated and differentiated Caco-2 cells, we have explored a set of biomarkers, aiming to evaluate undesirable effects associated to TiO2 NP exposure. Results indicate non-toxic effects in exposures ranging 1-200 µg ml-1 . Significant differences were observed in cell uptake, with a higher amount of incorporated TiO2 NPs in undifferentiated cells, as visualized using confocal microscopy. In well-established monolayers, translocation was detected using both confocal microscopy and transmission electron microscopy with energy-dispersive X-ray spectroscopy. In spite of the observed uptake and translocation, TiO2 NP exposures did not modify the integrity of the monolayer, as measured using the transepithelial electrical resistance and Lucifer yellow methods. The potential genotoxic effects in differentiated cells were evaluated in the comet assay, with and without formamidopyrimidine DNA glycosylase enzyme to detect oxidatively the damaged DNA bases. Although some changes were detected at the lower dose (10 µg ml-1 ), no effects were observed at higher doses.

Assessing the effects of silver nanoparticles on monolayers of differentiated Caco-2 cells, as a model of intestinal barrier.

Since ingestion is one of the main routes of entry of nanoparticles (NPs) in our organism, simple and fast in vitro models of the intestinal barrier can be helpful to evaluate NPs risk. The human colon adenocarcinoma Caco-2 cell line has been extensively used due to its ability to differentiate, forming a well-structured cell monolayer. In this study, we have used these differentiated cells as a model of intestinal barrier to evaluate a wide set of effects caused by exposure to silver nanoparticles (AgNPs) with an average size of 7.74 nm. Different parameters such as toxicity, monolayer integrity and permeability (assessed by changes in cells' morphology and gene expression pattern), internalization (uptake), translocation, and induction of DNA damage (DNA breaks and oxidative DNA damage) were evaluated. No significant effects were observed on the monolayer's integrity/permeability after exposure to silver nanoparticles, although cellular uptake was demonstrated by using confocal microscopy. Despite the observed uptake, no translocation of AgNPs to the basolateral chamber was demonstrated with any of the different experimental approaches used. The genotoxic effects evaluated using the comet assay indicate that, although AgNPs were not able to induce direct DNA breaks, its exposure induced a significant increase in the oxidative DNA damage levels, at non-toxic concentrations.