A multi-omics approach to elucidate okadaic acid-induced changes in human HepaRG hepatocarcinoma cells.

Okadaic acid (OA), a prevalent marine biotoxin found in shellfish, is known for causing acute gastrointestinal symptoms. Despite its potential to reach the bloodstream and the liver, the hepatic effects of OA are not well understood, highlighting a significant research gap. This study aims to comprehensively elucidate the impact of OA on the liver by examining the transcriptome, proteome, and phosphoproteome alterations in human HepaRG liver cells exposed to non-cytotoxic OA concentrations. We employed an integrative multi-omics approach, encompassing RNA sequencing, shotgun proteomics, phosphoproteomics, and targeted DigiWest analysis. This enabled a detailed exploration of gene and protein expression changes, alongside phosphorylation patterns under OA treatment. The study reveals concentration- and time-dependent deregulation in gene and protein expression, with a significant down-regulation of xenobiotic and lipid metabolism pathways. Up-regulated pathways include actin crosslink formation and a deregulation of apoptotic pathways. Notably, our results revealed that OA, as a potent phosphatase inhibitor, induces alterations in actin filament organization. Phosphoproteomics data highlighted the importance of phosphorylation in enzyme activity regulation, particularly affecting proteins involved in the regulation of the cytoskeleton. OA's inhibition of PP2A further leads to various downstream effects, including alterations in protein translation and energy metabolism. This research expands the understanding of OA's systemic impact, emphasizing its role in modulating the phosphorylation landscape, which influences crucial cellular processes. The results underscore OA's multifaceted effects on the liver, particularly through PP2A inhibition, impacting xenobiotic metabolism, cytoskeletal dynamics, and energy homeostasis. These insights enhance our comprehension of OA's biological significance and potential health risks.

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.

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.

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.

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.

In vitro nanoparticle dosimetry for adherent growing cell monolayers covering bottom and lateral walls.

BACKGROUND: Even though a continuously high number of in vitro studies on nanoparticles are being published, the issue of correct dose matter is often not sufficiently taken into account. Due to their size, the diffusion of nanoparticles is slower, as compared to soluble chemicals, and they sediment slowly. Therefore, the administered dose of particles in in vitro experiments is not necessarily the same (effective) dose that comes into contact with the cellular system. This can lead to misinterpretations of experimental toxic effects and disturbs the meaningfulness of in vitro studies. In silico calculations of the effective nanoparticle dose can help circumventing this problem. RESULTS: This study addresses more complex in vitro models like the human intestinal cell line Caco-2 or the human liver cell line HepaRG, which need to be differentiated over a few weeks to reach their full complexity. During the differentiation time the cells grow up the wall of the cell culture dishes and therefore a three-dimensional-based in silico model of the nanoparticle dose was developed to calculate the administered dose received by different cell populations at the bottom and the walls of the culture dish. Moreover, the model can perform calculations based on the hydrodynamic diameter which is measured by light scattering methods, or based on the diffusion coefficient measured by nanoparticle tracking analysis (NTA). This 3DSDD (3D-sedimentation-diffusion-dosimetry) model was experimentally verified against existing dosimetry models and was applied to differentiated Caco-2 cells incubated with silver nanoparticles. CONCLUSIONS: The 3DSDD accounts for the 3D distribution of cells in in vitro cell culture dishes and is therefore suitable for differentiated cells. To encourage the use of dosimetry calculating software, our model can be downloaded from the supporting information.

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.

Disturbance of firefly luciferase-based bioassays by different aluminum species.

Luciferase-dependent assays, important for biochemical analyses of cytotoxicity and reporter genes, may be perturbed by compounds interfering with the luciferase reaction. We analyzed the impact of different aluminum (Al) species on a luciferase-based assay for determination of cellular adenosine triphosphate. Al(0) nanoparticles (Al(0)-NPs) but not Al2O3-NPs decreased luminescence, correlated to high absorbance of Al(0)-NPs. By contrast, Al ions increased the luminescent signal. Data demonstrate that luciferase-dependent assays can be reciprocally disturbed by Al-NPs or Al ions in a specific manner, depending on the particular Al species. Careful interpretation of data from such experiments is essential in order to obtain conclusive results.

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.

Influence of artificial digestion on characteristics and intestinal cellular effects of micro-, submicro- and nanoplastics.

The production of plastics is rising since they have been invented. Micro, submicro- and nanoplastics are produced intentionally or generated by environmental processes, and constitute ubiquitous contaminants which are ingested orally by consumers. Reported health concerns include intestinal translocation, inflammatory response, oxidative stress and cytotoxicity. Every digestive milieu in the gastrointestinal tract does have an influence on the properties of particles and can cause changes in their effect on biological systems. In this study, we subjected plastic particles of different materials (polylactic acid, polymethylmethacrylate, melamine formaldehyde) and sizes (micro- to nano-range) to a complex artificial digestion model consisting of three intestinal fluid simulants (saliva, gastric and intestinal juice). We monitored the impact of the digestion process on the particles by performing Dynamic Light Scattering, Scanning Electron Microscopy and Asymmetric Flow Field-Flow Fractionation. An in vitro model of the intestinal epithelial barrier was used to monitor cellular effects and translocation behavior of (un)digested particles. In conclusion, artificial digestion decreased cellular interaction and slightly increased transport of all particles across the intestinal barrier. The interaction with organic matter resulted in clear differences in the agglomeration behavior. Moreover, we provide evidence for polymer-, size- and surface-dependent cellular effects of the test particles.

Model peptides uncover the role of the ß-secretase transmembrane sequence in metal ion mediated oligomerization.

The ß-secretase or ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the enzyme responsible for the formation of amyloid-ß peptides, which have a major role in Alzheimer pathogenesis. BACE1 has a transmembrane sequence (TMS), which makes it unique among related proteases. We noticed that the BACE1 TMS contains an uncommon sulfur-rich motif. The sequence MxxxCxxxMxxxCxMxC spans the entire TMS, resembles metal ion binding motifs, and is highly conserved among homologues. We used a synthetic 31-mer model peptide comprising the TMS to study metal ion binding and oligomerization. Applying diverse biochemical and biophysical techniques, we detected dimer and trimer formation of the TMS peptide with copper ions. Replacement of the central Cys466 by Ala essentially abolished these effects. We show that the peptide undergoes a redox reaction with copper ions resulting in a disulfide bridge involving Cys466. Further, we find peptide trimerization that depends on the presence of monovalent copper ions and the sulfhydryl group of Cys466. We identified Cys466 as a key residue for metal ion chelation and to be the core of an oligomerization motif of the BACE1-TMS peptide. Our results demonstrate a novel metal ion controlled oligomerization of the BACE1 TMS, which could have an enormous therapeutic importance against Alzheimer disease.

Complex intestinal and hepatic in vitro barrier models reveal information on uptake and impact of micro-, submicro- and nanoplastics.

Plastic particles are found almost ubiquitously in the environment and can get ingested orally by humans. We have used food-relevant microplastics (2 µm polylactic acid), submicroplastics (250 nm polylactic acid and 366 nm melamine formaldehyde resin) and nanoplastics (25 nm polymethylmethacrylate) to study material- and size-dependent uptake and transport across the human intestinal barrier and liver. Therefore, different Transwell™-based in vitro (co-)culture models were used: Differentiated Caco-2 cells mimicking the intestinal enterocyte monolayer, an M-cell model complementing the Caco-2 monoculture with antigen uptake-specialized cells, a mucus model complementing the barrier with an intestinal mucus layer, and an intestinal-liver co-culture combining differentiated Caco-2 cells with differentiated HepaRG cells. Using these complex barrier models, uptake and transport of particles were analyzed based on the fluorescence of the particles using confocal microscopy and a fluorescence-based quantification method. Additionally, the results were verified by Time-of-Flight - Secondary Ion Mass Spectrometry (ToF-SIMS) analysis. Furthermore, an effect screening at the mRNA level was done to investigate oxidative stress response, inflammation and changes to xenobiotic metabolism in intestinal and hepatic cells after exposure to plastic particles. Oxidative stress and inflammation were additionally analyzed using a flow-cytometric assay for reactive oxygen species and cytokine measurements. The results reveal a noteworthy uptake into and transport of microplastic and submicroplastic particles across the intestinal epithelium. Particularly, we show a pronounced uptake of particles into liver cells after crossing of the intestinal epithelium, using the intestinal-liver co-culture. The particles evoke some alterations in xenobiotic metabolism, but did not cause increased oxidative stress or inflammatory response on protein level. Taken together, these complex barrier models can be applied on micro-, submicro- and nanoplastics and reveal information in particle uptake, transport and cellular impact.

Proteomic analysis of hepatic effects of okadaic acid in HepaRG human liver cells.

The marine biotoxin okadaic acid (OA) is produced by dinoflagellates and enters the human food chain by accumulating in the fatty tissue of filter-feeding shellfish. Consumption of highly contaminated shellfish can lead to diarrheic shellfish poisoning. However, apart from the acute effects in the intestine, OA can also provoke toxic effects in the liver, as it is able to pass the intestinal barrier into the blood stream. However, molecular details of OA-induced hepatotoxicity are still insufficiently characterized, and especially at the proteomic level data are scarce. In this study, we used human HepaRG liver cells and exposed them to non-cytotoxic OA concentrations for 24 hours. Global changes in protein expression were analyzed using 2-dimensional gel electrophoresis in combination with mass-spectrometric protein identification. The results constitute the first proteomic analysis of OA effects in human liver cells and indicate, amongst others, that OA affects the energy homeostasis, induces oxidative stress, and induces cytoskeletal changes.

Okadaic Acid Activates JAK/STAT Signaling to Affect Xenobiotic Metabolism in HepaRG Cells.

Okadaic acid (OA) is a marine biotoxin that is produced by algae and accumulates in filter-feeding shellfish, through which it enters the human food chain, leading to diarrheic shellfish poisoning (DSP) after ingestion. Furthermore, additional effects of OA have been observed, such as cytotoxicity. Additionally, a strong downregulation of the expression of xenobiotic-metabolizing enzymes in the liver can be observed. The underlying mechanisms of this, however, remain to be examined. In this study, we investigated a possible underlying mechanism of the downregulation of cytochrome P450 (CYP) enzymes and the nuclear receptors pregnane X receptor (PXR) and retinoid-X-receptor alpha (RXRα) by OA through NF-κB and subsequent JAK/STAT activation in human HepaRG hepatocarcinoma cells. Our data suggest an activation of NF-κB signaling and subsequent expression and release of interleukins, which then activate JAK-dependent signaling and thus STAT3. Moreover, using the NF-κB inhibitors JSH-23 and Methysticin and the JAK inhibitors Decernotinib and Tofacitinib, we were also able to demonstrate a connection between OA-induced NF-κB and JAK signaling and the downregulation of CYP enzymes. Overall, we provide clear evidence that the effect of OA on the expression of CYP enzymes in HepaRG cells is regulated through NF-κB and subsequent JAK signaling.

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.

Okadaic acid influences xenobiotic metabolism in HepaRG cells.

Okadaic acid (OA) is an algae-produced lipophilic marine biotoxin that accumulates in the fatty tissue of filter-feeding shellfish. Ingestion of contaminated shellfish leads to the diarrheic shellfish poisoning syndrome. Furthermore, several other effects of OA like genotoxicity, liver toxicity and tumor-promoting properties have been observed, probably linked to the phosphatase-inhibiting properties of the toxin. It has been shown that at high doses OA can disrupt the physical barrier of the intestinal epithelium. As the intestine and the liver do not only constitute a physical, but also a metabolic barrier against xenobiotic exposure, we here investigated the impact of OA on the expression of cytochrome P450 (CYP) enzymes and transporter proteins in human HepaRG cells liver cells in vitro at non-cytotoxic concentrations. The interplay of OA with known CYP inducers was also studied. Data show that the expression of various xenobiotic-metabolizing CYPs was downregulated after exposure to OA. Moreover, OA was able to counteract the activation of CYPs by their inducers. A number of transporters were also mainly downregulated. Overall, we demonstrate that OA has a significant effect on xenobiotic metabolism barrier in liver cells, highlighting the possibility for interactions of OA exposure with the metabolism of drugs and xenobiotics.

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.

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.

Risk assessment and toxicological research on micro- and nanoplastics after oral exposure via food products.

Plastics are used ubiquitously and have become part of our everyday life. The global production of plastics is rising, which in consequence is leading to increasing amounts of plastics being released into the environment. Recently, the issue of human exposure to micro- and nanoplastic particles and potentially resulting toxicological consequences has been broached, triggered by the discovery of microplastics in foodstuff. In addition to dietary exposure via contaminated food and beverages, other exposure paths such as via air and cosmetics, have to be considered. Currently there is no legislation for microplastics and nanoplastics as contaminants in food. Substantial data gaps with respect to exposure as well as toxicity of such particles impede the risk assessment. Within this EU-FORA fellowship project, a comprehensive data mining approach was followed, focusing on up-to-date knowledge on the occurrence and possible toxic effects associated with micro- and nanoplastics after oral exposure, especially via food products and beverages, in order to provide a basis for risk assessment and to identify important research gaps. The fellowship project was further complemented by practical work aimed at the determination of in vitro toxicity of micro-sized polylactic acid particles.