Efficient strategies for the human health risk assessment of micro- and nanoplastics (MNPs) are urgently needed to address the complexity and diversity of these materials. Here, we propose a risk assessment framework for MNPs following the oral route of exposure. The framework is based on a flexible and modular approach, drawing on modern concepts in risk assessment, such as Integrated Approaches to Testing and Assessment (IATAs) or Adverse Outcome Pathways (AOPs), and substantially relies on New Approach Methodologies (NAMs). The framework is structured into three different main steps: i) basic physicochemical characterization of testing materials, ii) intestinal crossing, iii) hazard assessment testing. Each step of the framework is supported by scientifically sound methods, enabling a mechanistic and hypothesis-driven risk assessment of MNPs.

The topic of micro- and nanoplastics received significant attention in recent decades due to increasing environmental exposure, strong public perception, and emerging health concerns. While knowledge regarding detection and material characteristics has improved, the understanding of impact on cells remained unclear. As biological effects are initially caused by molecular interactions, consequently direct interactions with biomolecules, such as enzymes, are of particular relevance. In this occasion, effects may vary depending on the plastic type and particle properties. The specific aim of this study was to characterize the direct molecular interactions by means of selected model proteins and a variety of different nanoplastic particles. Therefore, the aim of the study was to exemplarily characterize α-amylase's (as a model enzyme) interactions with different nanoplastics and the resulting effects on enzyme structure and function, as well as cellular responses. The properties of the α-amylase-nanoplastic mixtures were analyzed using dynamic light scattering (DLS), Fourier-transform infrared spectroscopy (FTIR), fluorescence spectroscopy, and Phadebas amylase activity test. Additionally, Caco-2 cells were used as a model system for the human intestinal barrier and exposed to these complexes to evaluate cellular uptake through flow cytometry, microscopy, and viability testing. All applied nanoplastics interacted with α-amylase, forming complexes with adsorption affinities that depended on the particle type (PP ≫ PE > PET ≫ PLA). FTIR and fluorescence analyses showed particle-specific structural changes. Despite these differences in structural response, concentration-dependent enzyme inhibition was measurable, depending on the particle type. Uptake studies on Caco-2 cells indicated no internalization or cytotoxicity. These findings suggest that nanoplastics influence the enzyme structure and function based on their chemical properties, offering new insights into direct enzyme-nanoplastics interactions and their potential impacts on enzymes and cells.

Copper is an essential trace element, yet concerns regarding its toxic potential have gained increasing attention, particularly with regard to nanoparticulate forms of copper oxides. Both CuO and Cu2O are currently authorised in the EU for use in agriculture, biocides, and animal nutrition, but regulatory clarity regarding their nanoforms remains limited. This comprehensive review—combining narrative, bibliometric, and systematic approaches—addressed three key questions: (i) whether CuO and Cu2O nanoparticles are sufficiently considered within EU regulations to prevent unintentional oral exposure via the food and feed chain, (ii) whether these nanoparticles can cross the intestinal barrier as intact particles and accumulate in tissues, and (iii) whether they elicit distinct biological responses, including regulated cell death pathways such as cuproptosis. With respect to question (i), a detailed analysis of EU legal frameworks revealed a heterogeneous regulatory landscape and a lack of binding provisions that would preclude unintended presence of copper oxide nanoforms in regulated materials. Although some nano-specific data requirements exist, empirical information on nanoparticulate fractions in commercial products is still lacking. With respect to questions (ii) and (iii), our systematic literature analysis supports consistent intracellular uptake of CuO and Cu2O nanoparticles, yet direct evidence for transport of intact particles across the intestinal barrier is scarce. Mechanistic evidence supports cuproptosis as a key pathway for CuO nanoparticle toxicity, while the extent to which these effects differ quantitatively from ionic copper remains unclear due to insufficient data using appropriate experimental controls. Notably, a pronounced data gap was identified for Cu2O nanoparticles across all evaluated domains. Finally, to organise and integrate the mechanistic evidence presented, we developed an adverse outcome pathway (AOP), registered as AOP 590 in the AOP Wiki, describing how increased intracellular copper can induce cuproptosis via disruption of energy metabolism. Collectively, these findings highlight the need for coordinated analytical, mechanistic, and regulatory research to support evidence-based risk assessment of CuO and Cu2O nanoparticles in the food and feed context.

Nanotechnology is a powerful technological basis for the design of materials for various areas of application. It is becoming increasingly important in our complex world and a growing number of applications are proving its high-performance capacity on a regular basis. Today, its unique ability to define materials and structures at the nanometer scale opens up new opportunities to tackle some of the most pressing challenges. In this review, we discuss key examples of nanotechnology's contributions to microplastics, packaging and textiles, all three of which have proven to be omnipresent and particularly transformative. We present current approaches to characterizing and addressing the challenge of microplastics, explain concepts of sustainable packaging solutions and demonstrate the applicability of improved textile materials. In all three areas, key examples will be given of how nanotechnology as an enabling technology can pave the way to a more sustainable and technologically advanced future.

Nanoplastic particles and their additives are increasingly present in the food chain, interacting with biomacromolecules with not yet known consequences. A protein corona forms around the particles in these usually complex matrices, primarily with a first contact at surface-active proteins. However, systematic studies on the interactions between the particles and proteins -especially regarding protein affinity and structural changes due to surface properties like polarity - are limited. It is also unclear whether the protein corona can "mask" the particles, mimic protein properties, and induce cytotoxic effects when internalized by mammalian cells. This study aimed at investigating the physicochemical properties of model particle-protein complexes, the structural changes of adsorbed proteins, and their effects on Caco-2 cells. Whey protein β-lactoglobulin (β-Lg) was used as a well-characterized model protein and studied in a mixture with nanoparticles of varying polarity, specifically silica, polylactic acid (PLA), and polyethylene terephthalate (PET). The physicochemical analyses included measurements of the hydrodynamic diameter and the zeta potential, while the protein conformational changes were analyzed using Fourier-transform-infrared spectroscopy (FTIR) and intrinsic fluorescence. Cellular uptake in Caco-2 cells was assessed through flow cytometry, cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay, and cellular impedance was analyzed with xCELLigence® technology. The results indicated that β-Lg had the highest affinity for hydrophilic silica particles, forming silica-β-Lg complexes and large aggregates through electrostatic interactions. The affinity decreased for PLA and was lowest for hydrophobic PET, which formed smaller complexes. Adsorption onto silica caused partial unfolding and refolding of β-Lg. The silica-β-Lg complexes were internalized by Caco-2 cells, impairing cell proliferation. In contrast, PLA- and PET-protein complexes were not internalized, though PLA complexes slightly reduced cell viability. This study enhances our understanding of protein adsorption on nanoparticles and its potential biological effects.

BACKGROUND:Microplastics, i.e., plastic particles ranging from 1 μm to 5 mm in size, are ubiquitous in the environment. They are to be distinguished from nanoplastics, which are defined as particles less than 1 μm or less than 100 nm in size (depending on the study). Microplastics have increasingly become a topic of discussion in the media. In this article we present the state of scientific and medical knowledge about microplastics and how they are perceived by the public. METHODS:This narrative review is based on pertinent publications retrieved by a search in PubMed and Scopus, supplemented by the findings of a random-quota online survey among the Germanspeaking population (N = 1135). RESULTS:Microplastic particles mainly enter the body by being inhaled or swallowed. According to current knowledge, most are excreted without being resorbed, while a small fraction of them could reach the tissues or the bloodstream depending on their size and become systemically bioavailable. Approximately 0.3% of particles measuring 1-10 μm in size are resorbed in the intestinal tract. Microplastics have been found in organs and tissues (e.g., placenta, atherosclerotic plaques), but no causal relation between their uptake and any health effects has yet been proven. Given the limited available evidence on microplastics, some segments of the general population are concerned about their potential effects on human health. In our random-quota survey, 84% of respondents considered the statement that microplastics in the body can worsen pre-existing medical conditions to be true. CONCLUSION:Current evidence does not permit any definitive conclusion about the effects of microplastics on health. The information presented here may help physicians counsel their patients on this matter.

Abstract Based on their ubiquitous distribution in various ecosystems, plastic particles of different origin and sizes enter the (human) food chain, are ingested and reach the gastrointestinal tract, where they interact with the intestinal (epithelial) tissue. To a certain extent, they may enter or cross the intestinal epithelium, getting into contact with enteric neurons. Therefore, a potential risk for gastrointestinal health has been suggested. In the current study, we aimed to investigate the effect of polystyrene particles of nano- (100 nm) and micro-size (1.0 μm), firstly, on intestinal epithelial electrophysiological processes using the Ussing chamber technique and, secondly, on enteric neuronal properties using neuroimaging on primary cultured enteric neurons. The results indicate a size-dependent impairment of epithelial integrity and electrogenic glucose transport upon particle application. Additionally, both sizes of particles induced a change in neuronal response parameters to nicotinergic stimulation after a 24 h incubation. Taken together, these results emphasise the potential of nano- and micro polystyrene particles to interact and affect intestinal epithelial as well as enteric neuronal functionality and stress the need for more detailed investigations of the effect of plastic particles present in the (human) diet on intestinal physiology and involvement of the enteric nervous system.

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.

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.

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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.

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 (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.

The intestinal barrier is a complex interface of the human body, possessing the largest contact surface to nutrients and antigens and containing a major part of the immune system. It has to deal with continuous exposure to a broad mixture of essential, harmful, or useless substances and particles. In the context of plastic pollution and the ubiquitous occurrence of micro- and nanoplastics, oral exposure to such particles is of particular interest. Standard intestinal in vitro models, however, are unable to mimic the role of the immune system in the particle-exposed intestine. To allow for a closer look on the effect of particles on the intestinal immune system, we here developed a co-culture model to enable investigation of the epithelial brush border monolayer in a healthy and inflamed state. The model is based on well-established Caco-2 intestinal epithelial cells cultured in a Transwell™ system. Intraepithelial immune cells were mimicked by THP-1-derived M0-macrophages and MUTZ-3-derived dendritic cells. To fulfill the requirements needed for the investigation of particles, the co-culture system was developed without an additional matrix layer. Cell–cell contacts were established between interstitial and immune cells, and the Caco-2 standard cell culture medium was used, which is well-characterized for its role in defining the identity of particle dispersions. The model was characterized using confocal microscopy, membrane integrity measurements, and cytokine release assays from inflamed and healthy cells. Finally, the new co-culture model was used for investigation on polylactic acid, melamine formaldehyde resin, and polymethylmethacrylate plastic micro- and nanoparticles.

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.