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.

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.

Micro- and nanoplastics - current state of knowledge with the focus on oral uptake and toxicity.

The production and use of plastics has constantly increased over the last 30 years. Over one third of the plastics is used in disposables, which are discarded within three years of their production. Despite efforts towards recycling, a substantial volume of debris has accumulated in the environment and is slowly degraded to micro- and nanoplastics by weathering and aging. It has recently been discovered that these small particles can enter the food chain, as for example demonstrated by the detection of microplastic particles in honey, beer, salt, sea food and recently in mineral water. Human exposure has further been documented by the detection of plastic microparticles in human feces. Potential toxic consequences of oral exposure to small plastic particles are discussed. Due to lacking data concerning exposure, biodistribution and related effects, the risk assessment of micro- and nanoplastics is still not possible. This review focuses on the oral uptake of plastic and polymer micro- and nanoparticles. Oral exposure, particle fate, changes of particle properties during ingestion and gastrointestinal digestion, and uptake and transport at the intestinal epithelium are reviewed in detail. Moreover, the interaction with intestinal and liver cells and possibly resulting toxicity are highlighted.

Isolation methods for particle protein corona complexes from protein-rich matrices.

Background: Nanoparticles become rapidly encased by a protein layer when they are in contact with biological fluids. This protein shell is called a corona. The composition of the corona has a strong influence on the surface properties of the nanoparticles. It can affect their cellular interactions, uptake and signaling properties. For this reason, protein coronae are investigated frequently as an important part of particle characterization. Main body of the abstract: The protein corona can be analyzed by different methods, which have their individual advantages and challenges. The separation techniques to isolate corona-bound particles from the surrounding matrices include centrifugation, magnetism and chromatographic methods. Different organic matrices, such as blood, blood serum, plasma or different complex protein mixtures, are used and the approaches vary in parameters such as time, concentration and temperature. Depending on the investigated particle type, the choice of separation method can be crucial for the subsequent results. In addition, it is important to include suitable controls to avoid misinterpretation and false-positive or false-negative results, thus allowing the achievement of a valuable protein corona analysis result. Conclusion: Protein corona studies are an important part of particle characterization in biological matrices. This review gives a comparative overview about separation techniques, experimental parameters and challenges which occur during the investigation of the protein coronae of different particle types.

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.

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.

A tripartite mode of action approach for investigating the impact of aneugens on tubulin polymerization.

Chemical-induced disruption of the cellular microtubule network is one key mechanism of aneugenicity. Since recent data indicate that genotoxic effects of aneugens show nonlinear dose-response relationships, margins of safety can be derived with the ultimate goal to perform a risk assessment for the support of drug development. Furthermore, microtubule-interacting compounds are widely used for cancer treatment. While there is a need to support the risk assessment of tubulin-interacting chemicals using reliable mechanistic assays, no standard assays exist to date in regulatory genotoxicity testing for the distinction of aneugenic mechanisms. Recently reported methods exclusively rely on either biochemical, morphological, or cytometric endpoints. Since data requirements for the diverse fields of application of those assays differ strongly, the use of multiple assays for a correct classification of aneugens is ideal. We here report a tripartite mode of action approach comprising a cell-free biochemical polymerization assay and the cell-based methods cellular imaging and flow cytometry. The biochemical assay measures tubulin polymerization over time whereas the two cell-based assays quantify tubulin polymer mass. We herein show that the flow cytometric method yielded IC50 values for tubulin destabilizers and EC50 values for tubulin stabilizers as well as cell cycle information. In contrast, cellular imaging complemented these findings with characteristic morphological patterns. Biochemical analysis yielded kinetic information on tubulin polymerization. This multiplex approach is able to create holistic effect profiles which can be individually customized to the research question with regard to quality, quantity, usability, and economy. Environ. Mol. Mutagen. 59:188-201, 2018. © 2017 Wiley Periodicals, Inc.