Particulate iron oxide food colorants (E 172) during artificial digestion and their uptake and impact on intestinal cells.

Iron oxide of various structures is frequently used as food colorant (E 172). The spectrum of colors ranges from yellow over orange, red, and brown to black, depending on the chemical structure of the material. E 172 is mostly sold as solid powder. Recent studies have demonstrated the presence of nanoscaled particles in E 172 samples, often to a very high extent. This makes it necessary to investigate the fate of these particles after oral uptake. In this study, 7 differently structured commercially available E 172 food colorants (2 x Yellow FeO(OH), 2 x Red Fe2O3, 1 x Orange Fe2O3 + FeO(OH) and 2 x Black Fe3O4) were investigated for particle dissolution, ion release, cellular uptake, crossing of the intestinal barrier and toxicological impact on intestinal cells. Dissolution was analyzed in water, cell culture medium and artificial digestion fluids. Small-angle X-ray scattering (SAXS) was employed for determination of the specific surface area of the colorants in the digestion fluids. Cellular uptake, transport and toxicological effects were studied using human differentiated Caco-2 cells as an in vitro model of the intestinal barrier. For all materials, a strong interaction with the intestinal cells was observed, albeit there was only a limited dissolution, and no toxic in vitro effects on human cells were recorded.

Microplastics and nanoplastics: Size, surface and dispersant - What causes the effect?

There is increasing evidence that humans are exposed to microplastic particles through contaminated food. Although suitable analytical methods are still lacking, it is likely that these contaminations also contain a nanoplastics fraction. It is known from nanotoxicology that particles may acquire altered toxicological properties with decreasing particle sizes. Particles can also have different surface modalities and functionalizations. Moreover, nano- and microplastics as materials with probably a relatively low toxicity are often applied at high concentrations in in vitro tests, and therefore the solvating agent, namely the dispersant in which the particles are supplied may have a major impact on the outcome. This might be misinterpreted as particle effect. Therefore, it is crucial to determine what causes the effect - size, surface or dispersant? In this study this question was investigated by applying established in vitro models for the intestinal barrier (differentiated Caco-2 monoculture and mucus- and M-cell co-culture) and hepatocytes (differentiated HepaRG cells), mimicking the oral route of particle uptake. A complex set of nine different polystyrene micro- and nanoparticles was used to elucidate the effect of particle size, surface modification and dispersant. Uptake and transport as well as biochemical endpoints were measured, complemented by particle characterization. The results show that indeed some dispersants can cause a more pronounced cytotoxic effect than the particles themselves. Surface modification and particle size show a clear influence on the uptake and cytotoxicity of nano- and microplastic particles.

Counterions determine uptake and effects of aluminum in human intestinal and liver cells.

Aluminum (Al) is highly abundant in the biosphere and can occur in different physico-chemical states. It is present in human food and undergoes transitions between dissolved and particulate species during the passage of the gastrointestinal tract. Moreover, in a complex matrix such as food different inorganic and organic counterions can affect the chemical behavior of Al following oral uptake. In this work, the effects of different counterions, namely chloride, citrate, sulfate, lactate and acetylacetonate, on Al uptake and toxicity in the human intestine are studied. The respective Al salts showed different dissolution behavior in biological media and formed nanoscaled particles correlating in reverse with the amount of their dissolved fraction. The passage through the intestinal barrier was studied using a Caco-2 Transwell® system, showing counterion-dependent variance in cellular uptake and transport. In addition, Al toxicity was investigated using Al species (Al3+, metallic Al0 and oxidic γAl2O3 nanoparticles) and counterions individually or in mixtures on Caco-2 and HepG2 cells. The strongest toxicity was observed using a combination of Al species, depending on solubility, and the lipophilic counterion acetylacetonate. Notably, only the combination of both led to toxicity, while both substances individually did not show toxic effects. A toxification of previously non-toxic Al-species by the presence of acetylacetonate is shown here for the first time. The dependency on the concentration of free Al ions was demonstrated using sodium hydrogen phosphate, which was able to counteract the toxic effects by complexing free Al ions. These findings, using Al salts as an example for a common food contaminant, underline the importance of a consideration of the chemical properties of human nutrition, especially dissolution and hydrophobicity, which can significantly influence the cellular uptake and effects of xenobiotic substances.

Intestinal and hepatic effects of iron oxide nanoparticles.

Iron oxide nanoparticles gain increasing attention due to their broad industrial use. However, safety concerns exist since their effects on human cells are still under investigation. The presence of iron oxide nanoparticles in the food pigment E172 has been shown recently. Here, we studied four iron oxide nanoparticles, one food pigment E172 and the ionic control FeSO4 regarding dissolution in biological media, uptake and transport, and cellular effects in vitro in human intestinal Caco-2 and HepaRG hepatocarcinoma cells. The iron oxide nanoparticles passed the gastrointestinal passage without dissolution and reached the intestine in the form of particles. Minor uptake was seen into Caco-2 cells but almost no transport to the basolateral site was detected for any of the tested particles. HepaRG cells showed higher particle uptake. Caco-2 cells showed no alterations in reactive oxygen species production, apoptosis, or mitochondrial membrane potential, whereas two particles induced apoptosis in HepaRG cells, and one altered mitochondrial membrane potential at non-cytotoxic concentrations. No correlation between physicochemical particle characteristics and cellular effects was observed, thus emphasizing the need for case-by-case assessment of iron oxide nanoparticles.

Uptake and cellular effects of PE, PP, PET and PVC microplastic particles.

Humans are exposed to small plastic particles through contaminated food. Such contaminations usually comprise different particulate plastic materials differing in size, shape and surface. Up to now, data on intestinal uptake and adverse effects resulting from plastic particles other than polystyrene are scarce. In order to fill these knowledge gaps, this study aims to elucidate the gastrointestinal uptake and effects of microplastic particles of the materials polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET) and polyvinyl chloride (PVC) using human in vitro systems. The human intestinal epithelial cell line Caco-2 was used to study particle uptake in vitro, including an inverse culture system for buoyant particle species like PE and PP. Cytotoxicity was investigated using the human cell lines Caco-2, HepG2 and HepaRG in order to detect a possible impact on the first organs which come into contact with ingested particles: the intestine and the liver. The results of the study demonstrate that especially 1-4 µm PE microparticles were transported to a small but significant extent through the intestinal epithelium in vitro, to a substantially higher amount than PS particles of the same size. The present results suggest that intestinal exposure to plastic microparticles is material- and size-dependent. Only excessively high concentrations far beyond realistic dietary exposure of consumers induce cytotoxic effects.

NanoPASS: an easy-to-use user interface for nanoparticle dosimetry with the 3DSDD model.

Nanoparticles exhibit a specific diffusion and sedimentation behavior under cell culture conditions as used in nantoxicological in vitro testing. How a particular particle suspension behaves depends on the particular physicochemical characteristics of the particles and the cell culture system. Only a fraction of the nanoparticles applied to a cell culture will thus reach the cells within a given time frame. Therefore, dosimetric calculations are essential not only to determine the exact fraction of nanoparticles that has come into contact with the cells, but also to ensure experimental comparability and correct interpretation of results, respectively. Yet, the use of published dosimetry models is limited. Not the least because the correct application of these in silico tools usually requires bioinformatics knowledge, which often is perceived a hurdle. Moreover, not all models are freely available and accessible. In order to overcome this obstacle, we have now developed an easy-to-use interface for our recently published 3DSDD dosimetry model, called NanoPASS (NanoParticle Administration Sedimentation Simulator). The interface is freely available to all researchers. It will facilitate the use of in silico dosimetry in nanotoxicology and thus improve interpretation and comparability of in vitro results in the field.

Impact of iron oxide nanoparticles on xenobiotic metabolism in HepaRG cells.

Iron oxide nanoparticles are used in various industrial fields, as a tool in biomedicine as well as in food colorants, and can therefore reach human metabolism via oral uptake or injection. However, their effects on the human body, especially the liver as one of the first target organs is still under elucidation. Here, we studied the influence of different representative iron oxide materials on xenobiotic metabolism of HepaRG cells. These included four iron oxide nanoparticles, one commercially available yellow food pigment (E172), and non-particulate ionic control FeSO4. The nanoparticles had different chemical and crystalline structures and differed in size and shape and were used at a concentration of 50 µg Fe/mL. We found that various CYP enzymes were downregulated by some but not all iron oxide nanoparticles, with the Fe3O4-particle, both γ-Fe2O3-particles, and FeSO4 exhibiting the strongest effects, the yellow food pigment E172 showing a minor effect and an α-Fe2O3 nanoparticle leading to almost no inhibition of phase I machinery. The downregulation was seen at the mRNA, protein expression, and activity levels. Thereby, no dependency on the size or chemical structure was found. This underlines the difficulty of the grouping of nanomaterials regarding their physiological impact, suggesting that every iron oxide nanoparticle species needs to be evaluated in a case-by-case approach.

The presence of iron oxide nanoparticles in the food pigment E172.

Iron oxides used as food colorants are listed in the European Union with the number E172. However, there are no specifications concerning the fraction of nanoparticles in these pigments. Here, seven E172 products were thoroughly characterized. Samples of all colors were analyzed with a broad spectrum of methods to assess their physico-chemical properties. Small-Angle X-ray Scattering (SAXS), Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM), zeta-potential, Inductively Coupled Plasma-Mass Spectrometry (ICP-MS), X-ray diffraction (XRD), Brunauer-Emmett-Teller analysis (BET), Asymmetric Flow Field-Flow Fractionation (AF4) and in vitro cell viability measurements were used. Nanoparticles were detected in all E172 samples by TEM or SAXS measurements. Quantitative results from both methods were comparable. Five pigments were evaluated by TEM, of which four had a size median below 100 nm, while SAXS showed a size median below 100 nm for six evaluated pigments. Therefore, consumers may be exposed to iron oxide nanoparticles through the consumption of food pigments.

Identification of a transcriptomic signature of food-relevant genotoxins in human HepaRG hepatocarcinoma cells.

The conventional approach for testing the genotoxic potential of chemicals in vitro includes a battery of bacterial and mammalian mutagenicity tests. Toxicogenomics analyses may provide information about DNA-damaging properties of test compounds but are not routinely used for identification of a genotoxic potential. In this study, metabolically active human HepaRG hepatocarcinoma cells were exposed to five food-relevant genotoxic carcinogens. Transcriptomic responses were analyzed using RNA sequencing technology and validated by real-time polymerase chain reaction. Biostatistical approaches revealed a characteristic transcript signature of 37 differentially expressed genes, which were commonly regulated by the test chemicals. Specificity of the transcript signature was confirmed by using non-genotoxic carcinogens as comparators. Pathway analyses showed that the obtained transcript signature was closely related to DNA damage response and p53 activation. In conclusion, we have established a characteristic transcript marker pattern to monitor genotoxicity in human HepaRG cells, and to distinguish genotoxic from non-genotoxic carcinogens. Our analyses underline that a common response related to DNA damages response, cell cycle alterations and cell death is initiated in HepaRG cells upon exposure to genotoxic compounds and allows for the identification of a common transcriptomic signature for genotoxic stress.

Impact of artificial digestion on the sizes and shapes of microplastic particles.

Current analyses show a widespread occurrence of microplastic particles in food products and raise the question of potential risks to human health. Plastic particles are widely considered to be inert due to their low chemical reactivity and therefore supposed to pose, if at all only minor hazards. However, variable physicochemical conditions during the passage of the gastrointestinal tract gain strong importance, as they may affect particle characteristics. This study aims to analyze the impact of the gastrointestinal passage on the physicochemical particle characteristics of the five most produced and thus environmentally relevant plastic materials polyethylene, polypropylene, polyvinyl chloride, polyethylene terephthalate and polystyrene. Scanning electron microscopy (SEM) and subsequent image analysis were employed to characterize microplastic particles. Our results demonstrate a high resistance of all plastic particles to the artificial digestive juices. The present results underline that the main stages of the human gastrointestinal tract do not decompose the particles. This allows a direct correlation between the physicochemical particle characteristics before and after digestion. Special attention must be paid to the adsorption of organic compounds like proteins, mucins and lipids on plastic particles since it could lead to misinterpretations of particle sizes and shapes.

Transcriptomic effect marker patterns of genotoxins - a comparative study with literature data.

Microarray approaches are frequently used experimental tools which have proven their value for example in the characterization of the molecular mode of action of toxicologically relevant compounds. In a regulatory context, omics techniques are still not routinely used, amongst others due to lacking standardization in experimental setup and data processing, and also due to issues with the definition of adversity. In order to exemplarily determine whether consensus transcript biomarker signatures for a certain toxicological endpoint can be derived from published microarray datasets, we here compared transcriptome data from human HepaRG hepatocarcinoma cells treated with different genotoxins, based on re-analyzed datasets extracted from the literature. Comparison of the resulting data show that even with similarly-acting compounds in the same cell line, considerable variation was observed with respect to the numbers and identities of differentially expressed genes. Greater concordance was observed when considering the whole data sets and biological functions associated with the genes affected. The present results highlight difficulties and possibilities in inter-experiment comparisons of omics data and underpin the need for future efforts towards improved standardization to facilitate the use of omics data in risk assessment. Existing omics datasets may nonetheless prove valuable in establishing biological context information essential for the development of adverse outcome pathways.

An inverse cell culture model for floating plastic particles.

Plastic waste has become a major environmental problem. An increasing number of studies investigate microplastic particles with regard to their uptake and effects in cell culture systems. Individual plastic materials vary in their molecular structure, composition, size distribution, material density, and may also differ with respect to their toxicological effects. Plastic particles with lower densities than the cell culture medium, for example polyethylene (PE), pose a particular problem for in vitro assays as they float up during the incubation and thus do not contact the cells located on the bottom of the culture dish. We thus developed a practical and easy-to-use in vitro inverse cell culture model for investigating cellular effects of floating plastic particles. Cytotoxicity tests with floating PE particles were performed to demonstrate the utility of the inverted cell model. PE particles incubated in overhead culture were cytotoxic to HepG2 cells, while under the same cultivation conditions, except for inversion, no cytotoxicity occurred. These positive results demonstrate that inverted cell culture was required to detect the effects of PE particles and underlines the necessity to adapt cell culture conditions to the physicochemical properties of particles in order to obtain a more accurate estimate of the effects of floating particles on cells.

Uptake and effects of orally ingested polystyrene microplastic particles in vitro and in vivo.

Evidence exists that humans are exposed to plastic microparticles via diet. Data on intestinal particle uptake and health-related effects resulting from microplastic exposure are scarce. Aim of the study was to analyze the uptake and effects of microplastic particles in human in vitro systems and in rodents in vivo. The gastrointestinal uptake of microplastics was studied in vitro using the human intestinal epithelial cell line Caco-2 and thereof-derived co-cultures mimicking intestinal M-cells and goblet cells. Different sizes of spherical fluorescent polystyrene (PS) particles (1, 4 and 10 µm) were used to study particle uptake and transport. A 28-days in vivo feeding study was conducted to analyze transport at the intestinal epithelium and oxidative stress response as a potential consequence of microplastic exposure. Male reporter gene mice were treated three times per week by oral gavage with a mixture of 1 µm (4.55 × 107 particles), 4 µm (4.55 × 107 particles) and 10 µm (1.49 × 106 particles) microplastics at a volume of 10 mL/kg/bw. Effects of particles on macrophage polarization were investigated using the human cell line THP-1 to detect a possible impact on intestinal immune cells. Altogether, the results of the study demonstrate the cellular uptake of a minor fraction of particles. In vivo data show the absence of histologically detectable lesions and inflammatory responses. The particles did not interfere with the differentiation and activation of the human macrophage model. The present results suggest that oral exposure to PS microplastic particles under the chosen experimental conditions does not pose relevant acute health risks to mammals.

Aluminum in liver cells - the element species matters.

Aluminum (Al) can be ingested from food and released from packaging and can reach key organs involved in human metabolism, including the liver via systemic distribution. Recent studies discuss the occurrence of chemically distinct Al-species and their interconversion by contact with biological fluids. These Al species can vary with regard to their intestinal uptake, systemic transport, and therefore could have species-specific effects on different organs and tissues. This work aims to assess the in vitro hepatotoxic hazard potential of three different relevant Al species: soluble AlCl3 and two nanoparticulate Al species were applied, representing for the first time an investigation of metallic nanoparticles besides to mineral bound γ-Al2O3 on hepatic cell lines. To investigate the uptake and toxicological properties of the Al species, we used two different human hepatic cell lines: HepG2 and differentiated HepaRG cells. Cellular uptake was determined by different methods including light microscopy, transmission electron microscopy, side-scatter analysis, and elemental analysis. Oxidative stress, mitochondrial dysfunction, cell death mechanisms, and DNA damage were monitored as cellular parameters. While cellular uptake into hepatic cell lines occurred predominantly in the particle form, only ionic AlCl3 caused cellular effects. Since it is known, that Al species can convert one into another, and mechanisms including 'trojan-horse'-like uptake can lead to an Al accumulation in the cells. This could result in the slow release of Al ions, for which reason further hazard cannot be excluded. Therefore, individual investigation of the different Al species is necessary to assess the toxicological potential of Al particles.

Uptake and molecular impact of aluminum-containing nanomaterials on human intestinal caco-2 cells.

Aluminum (Al) is one of the most common elements in the earth crust and increasingly used in food, consumer products and packaging. Its hazard potential for humans is still not completely understood. Besides the metallic form, Al also exists as mineral, including the insoluble oxide, and in soluble ionic forms. Representatives of these three species, namely a metallic and an oxidic species of Al-containing nanoparticles and soluble aluminum chloride, were applied to human intestinal cell lines as models for the intestinal barrier. We characterized physicochemical particle parameters, protein corona composition, ion release and cellular uptake. Different in vitro assays were performed to determine potential effects and molecular modes of action related to the individual chemical species. For a deeper insight into signaling processes, microarray transcriptome analyses followed by bioinformatic data analysis were employed. The particulate Al species showed different solubility in biological media. Metallic Al nanoparticles released more ions than Al2O3 nanoparticles, while AlCl3 showed a mixture of dissolved and agglomerated particulate entities in biological media. The protein corona composition differed between both nanoparticle species. Cellular uptake, investigated in transwell experiments, occurred predominantly in particulate form, whereas ionic Al was not taken up by intestinal cell lines. Transcellular transport was not observed. None of the Al species showed cytotoxic effects up to 200 µg Al/mL. The transcriptome analysis indicated mainly effects on oxidative stress pathways, xenobiotic metabolism and metal homeostasis. We have shown for the first time that intestinal cellular uptake of Al occurs preferably in the particle form, while toxicological effects appear to be ion-related.

Regulation of Drug Metabolism by the Interplay of Inflammatory Signaling, Steatosis, and Xeno-Sensing Receptors in HepaRG Cells.

Nonalcoholic fatty liver disease (NAFLD), which is characterized by triglyceride deposition in hepatocytes resulting from imbalanced lipid homeostasis, is of increasing concern in Western countries, along with progression to nonalcoholic steatohepatitis (NASH), liver fibrosis, and cirrhosis. Previous studies suggest a complex, mutual influence of hepatic fat accumulation, NASH-related inflammatory mediators, and drug-sensing receptors regulating xenobiotic metabolism. Here, we investigated the suitability of human HepaRG hepatocarcinoma cells as a model for NAFLD and NASH. Cells were incubated for up to 14 days with an oleate/palmitate mixture (125 µM each) and/or with 10 ng/ml of the inflammatory mediator interleukin-6 (IL-6). Effects of these conditions on the regulation of drug metabolism were studied using xenobiotic agonists of the aryl hydrocarbon receptor (AHR), pregnane X receptor (PXR), constitutive androstane receptor (CAR), nuclear factor (erythroid-derived 2)-like 2, and peroxisome proliferator-activated receptor α (PPARα). Results underpin the suitability of HepaRG cells for NAFLD- and NASH-related research and constitute a broad-based analysis of the impact of hepatic fatty acid accumulation and inflammation on drug metabolism and its inducibility by xenobiotics. IL-6 exerted pronounced negative regulatory effects on basal as well as on PXR-, CAR-, and PPARα-, but not AHR-dependent induction of drug-metabolizing enzymes. This inhibition was related to diminished transactivation potential of the respective receptors rather than to reduced transcription of nuclear receptor-encoding mRNAs. The most striking effects of IL-6 and/or fatty acid treatment were observed in HepaRG cells after 14 days of treatment, making these cultures appear a suitable model for studying the relationship of fatty acid accumulation, inflammation, and xenobiotic-induced drug metabolism.

Comparative proteomic analysis of silver nanoparticle effects in human liver and intestinal cells.

Consumers are orally exposed to nanoparticulate or soluble species of the non-essential element silver due to its use in food contact materials or as a food additive. Potential toxicity of silver nanoparticles has gained special scientific attention. A fraction of ingested ionic or particulate silver is taken up in the intestine and transported to the liver, where it may induce oxidative stress and elicit subsequent adverse responses. Here, we present a comprehensive analysis of global proteomic changes induced in human Hep G2 hepatocarcinoma cells by different concentrations of AgPURE silver nanoparticles or by corresponding concentrations of ionic silver. Bioinformatic analysis of proteomic data confirms and substantiates previous findings on silver-induced alterations related to redox stress, mitochondrial dysfunction, intermediary metabolism, inflammatory responses, posttranslational protein modification and other cellular parameters. Similarities between the effects exerted by the two silver species are in line with the assumption that silver ions released from nanoparticles substantially contribute to their toxicity. Moreover, a comparative bioinformatic evaluation of proteomic effects in hepatic and intestinal cells exerted either by silver nanoparticles or bionic silver is presented. Our results show that, despite remarkable differences at the level of affected proteins in the different cell lines, highly similar biological consequences, corresponding to previous in vivo findings, can be deduced by applying appropriate bioinformatic data mining.

Impact of an Artificial Digestion Procedure on Aluminum-Containing Nanomaterials.

Aluminum has gathered toxicological attention based on relevant human exposure and its suspected hazardous potential. Nanoparticles from food supplements or food contact materials may reach the human gastrointestinal tract. Here, we monitored the physicochemical fate of aluminum-containing nanoparticles and aluminum ions when passaging an in vitro model of the human gastrointestinal tract. Small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), ion beam microscopy (IBM), secondary ion beam mass spectrometry (TOF-SIMS), and inductively coupled plasma mass spectrometry (ICP-MS) in the single-particle mode were employed to characterize two aluminum-containing nanomaterials with different particle core materials (Al0, γAl2O3) and soluble AlCl3. Particle size and shape remained unchanged in saliva, whereas strong agglomeration of both aluminum nanoparticle species was observed at low pH in gastric fluid together with an increased ion release. The levels of free aluminum ions decreased in intestinal fluid and the particles deagglomerated, thus liberating primary particles again. Dissolution of nanoparticles was limited and substantial changes of their shape and size were not detected. The amounts of particle-associated phosphorus, chlorine, potassium, and calcium increased in intestinal fluid, as compared to nanoparticles in standard dispersion. Interestingly, nanoparticles were found in the intestinal fluid after addition of ionic aluminum. We provide a comprehensive characterization of the fate of aluminum nanoparticles in simulated gastrointestinal fluids, demonstrating that orally ingested nanoparticles probably reach the intestinal epithelium. The balance between dissolution and de novo complex formation should be considered when evaluating nanotoxicological experiments.

Cytotoxicity of peptide-coated silver nanoparticles on the human intestinal cell line Caco-2.

Silver nanoparticles are used in a wide range of consumer products such as clothing, cosmetics, household goods, articles of daily use and pesticides. Moreover, the use of a nanoscaled silver hydrosol has been requested in the European Union for even nutritional purposes. However, despite the wide applications of silver nanoparticles, there is a lack of information concerning their impact on human health. In order to investigate the effects of silver nanoparticles on human intestinal cells, we used the Caco-2 cell line and peptide-coated silver nanoparticles with defined colloidal, structural and interfacial properties. The particles display core diameter of 20 and 40 nm and were coated with the small peptide L-cysteine L-lysine L-lysine. Cell viability and proliferation were measured using Promegas CellTiter-Blue® Cell Viability assay, DAPI staining and impedance measurements. Apoptosis was determined by Annexin-V/7AAD staining and FACS analysis, membrane damage with Promegas LDH assay and reactive oxygen species by dichlorofluorescein assay. Exposure of proliferating Caco-2 cells to silver nanoparticle induced decreasing adherence capacity and cytotoxicity, whereby the formation of reactive oxygen species could be the mode of action. The effects were dependent on particle size (20, 40 nm), doses (5-100 µg/mL) and time of incubation (4-48 h). Apoptosis or membrane damage was not detected.