Birgit Niemann

Analytically monitored digestion of silver nanoparticles and their toxicity on human intestinal cells

Abstract Orally ingested nanoparticles may overcome the gastrointestinal barrier, reach the circulatory system, be distributed in the organism and cause adverse health effects. However, ingested nanoparticles have to pass through different physicochemical environments, which may alter their properties before they reach the intestinal cells. In this study, silver nanoparticles are characterised physicochemically during the course of artificial digestion to simulate the biochemical processes occurring during digestion. Their cytotoxicity on intestinal cells was investigated using the Caco-2 cell model. Using field-flow fractionation combined with dynamic light scattering and small-angle X-ray scattering, the authors found that particles only partially aggregate as a result of the digestive process. Cell viabilities were determined by means of CellTiter-Blue® assay, 4′,6-diamidino-2-phenylindole-staining and real-time impedance. These measurements reveal small differences between digested and undigested particles (1–100 µg/ml or 1–69 particles/cell). The findings suggest that silver nanoparticles may indeed overcome the gastrointestinal juices in their particulate form without forming large quantities of aggregates. Consequently, the authors presume that the particles can reach the intestinal epithelial cells after ingestion with only a slight reduction in their cytotoxic potential. The study indicates that it is important to determine the impact of body fluids on the nanoparticles of interest to provide a reliable interpretation of their nano-specific cytotoxicity testing in vivo and in vitro.

Impact of food components during in vitro digestion of silver nanoparticles on cellular uptake and cytotoxicity in intestinal cells

Abstract Because of the rising application of nanoparticles in food and food-related products, we investigated the influence of the digestion process on the toxicity and cellular uptake of silver nanoparticles for intestinal cells. The main food components – carbohydrates, proteins and fatty acids – were implemented in an in vitro digestion process to simulate realistic conditions. Digested and undigested silver nanoparticle suspensions were used for uptake studies in the well-established Caco-2 model. Small-angle X-ray scattering was used to estimate particle core size, size distribution and stability in cell culture medium. Particles proved to be stable and showed radii from 3.6 to 16.0 nm. Undigested particles and particles digested in the presence of food components were comparably taken up by Caco-2 cells, whereas the uptake of particles digested without food components was decreased by 60%. Overall, these findings suggest that in vivo ingested poly (acrylic acid)-coated silver nanoparticles may reach the intestine in a nanoscaled form even if enclosed in a food matrix. While appropriate for studies on the uptake into intestinal cells, the Caco-2 model might be less suited for translocation studies. Moreover, we show that nanoparticle digestion protocols lacking food components may lead to misinterpretation of uptake studies and inconclusive results.

Simulation of prospective phytosterol intake in Germany by novel functional foods

A blood cholesterol-lowering margarine containing plant sterolesters was the first functional food placed on the European food market pursuant to the regulation (EC) 258/97. In the following years nine further applicants submitted the request to add plant sterol compounds to dairy products, cheeses, bakery products, sausages, plant oils and other products. The European Scientific Committee on Food (SCF) declared a precautionary intake limit of 3 g plant sterols per d by multiple dietary sources. Using the consumption data of the German National Food Consumption Study, carried out from 1985 to 1988 with 23 209 participants, we hypothetically added 0.3-2 g plant sterols to usual daily servings of ten different food products, selected from the novel food applications. We calculated the prospective plant sterol intake regarding each kind of enriched food and by stepwise accumulation of different functional foods in three enrichment scenarios. Within our enrichment context we find a phytosterol intake satiation, if multiple plant sterol-enriched foods are eaten. An enrichment amount of 2 g plant sterols per proposed food serving size results in an intake maximum of 13 g/d.

Molecular mechanism of silver nanoparticles in human intestinal cells

Abstract Silver nanoparticles are used in consumer products like food contact materials, drinking water technologies and supplements, due to their antimicrobial properties. This leads to an oral uptake and exposure of intestinal cells. In contrast to other studies we found no apoptosis induction by surfactant-coated silver nanoparticles in the intestinal cell model Caco-2 in a previous study, although the particles induced oxidative stress, morphological changes and cell death. Therefore, this study aimed to analyze the molecular mechanism of silver nanoparticles in Caco-2 cells. We used global gene expression profiling in differentiated Caco-2 cells, supported by verification of the microarray data by quantitative real-time RT-PCR and microscopic analysis, impedance measurements and assays for apoptosis and oxidative stress. Our results revealed that surfactant-coated silver nanoparticles probably affect the cells by outside-in signaling. They induce oxidative stress and have an influence on canonical pathways related to FAK, ILK, ERK, MAPK, integrins and adherence and tight junctions, thereby inducing transcription factors like AP1, NFkB and NRF2, which mediate cellular reactions in response to oxidative stress and metal ions and induce changes in the cytoskeleton and cell–cell and cell–matrix contacts. The present data confirm the absence of apoptotic cell death. Non-apoptotic, necrotic cell death, especially in the intestine, can cause inflammation and influence the mucosal immune response.

Processing nanoparticles with A4F-SAXS for toxicological studies: Iron oxide in cell-based assays.

Nanoparticles are not typically ready-to-use for in vitro cell culture assays. Prior to their use in assays, powder samples containing nanoparticles must be dispersed, de-agglomerated, fractionated by size, and characterized with respect to size and size distribution. For this purpose we report exemplarily on polyphosphate-stabilized iron oxide nanoparticles in aqueous suspension. Fractionation and online particle size analysis was performed in a time-saving procedure lasting 50 min by combining asymmetrical flow field-flow fractionation (A4F) and small-angle X-ray scattering (SAXS). Narrowly distributed nanoparticle fractions with radii of gyration (R(g)) from 7 to 21 nm were obtained from polydisperse samples. The A4F-SAXS combination is introduced for the preparation of well-characterized sample fractions originating from a highly polydisperse system as typically found in engineered nanoparticles. A4F-SAXS processed particles are ready-to-use for toxicological studies. The results of preliminary tests of the effects of fractionated iron oxide nanoparticles with a R(g) of 15 nm on a human colon model cell line are reported.

It takes more than a coating to get nanoparticles through the intestinal barrier in vitro

Graphical abstract Figure. No caption available. ABSTRACT Size and shape are crucial parameters which have impact on the potential of nanoparticles to penetrate cell membranes and epithelial barriers. Current research in nanotoxicology additionally focuses on particle coating. To distinguish between core‐ and coating‐related effects in nanoparticle uptake and translocation, two nanoparticles equal in size, coating and charge but different in core material were investigated. Silver and iron oxide nanoparticles coated with poly (acrylic acid) were chosen and extensively characterized by small‐angle x‐ray scattering, nanoparticle tracing analysis and transmission electron microscopy (TEM). Uptake and transport were studied in the intestinal Caco‐2 model in a Transwell system with subsequent elemental analysis. TEM and ion beam microscopy were conducted for particle visualization. Although equal in size, charge and coating, the behavior of the two particles in Caco‐2 cells was different: while the internalized amount was comparable, only iron oxide nanoparticles additionally passed the epithelium. Our findings suggest that the coating material influenced only the uptake of the nanoparticles whereas the translocation was determined by the core material. Knowledge about the different roles of the particle coating and core materials in crossing biological barriers will facilitate toxicological risk assessment of nanoparticles and contribute to the optimization of pharmacokinetic properties of nano‐scaled pharmaceuticals.

Trans Fatty Acids Affect Cellular Viability of Human Intestinal Caco-2 Cells and Activate Peroxisome Proliferator-Activated Receptors

Trans fatty acids (TFA) are hypothesized to have an impact not only on coronary heart diseases but also on the development of colon cancer. To analyze if TFA exhibit cellular and molecular effects which could be involved in colon tumor progression, cells of the human colorectal adenocarcinoma-derived cell line Caco-2 were treated with various TFA isomers differing in the number and position of trans double bonds. The TFA tested in this study did not increase cellular proliferation but displayed growth-inhibitory effects at concentrations higher than 500 μM. In case of the TFA isomer C18:3 t9, t11, t13, an IC50 value of 23 μM was estimated for cytotoxicity indicating a high cytotoxic potential of this compound. In addition to the cytotoxicity studies, the TFA isomers were tested for their ability to activate peroxisome proliferator-activated receptors (PPAR) by taking advantage of a PPAR-dependent reporter gene assay. In contrast to PPARγ that was not activated by the TFA isomers tested in this study, the substances were shown to moderately activate PPARα, and strong activation was observed for PPARδ. The putative impact of TFA on colon cancer development with respect to PPARδ activation is being discussed.

Höchstmengen für Vitamine und Mineralstoffe in Nahrungsergänzungsmitteln

ZusammenfassungIn Deutschland greift etwa ein Drittel der Erwachsenen regelmäßig zu Nahrungsergänzungsmitteln (NEM). Neben Vitaminen und Mineralstoffen enthalten die Produkte teilweise auch sonstige Stoffe mit physiologischer Wirkung wie Aminosäuren, Fettsäuren, Pflanzenextrakte oder Mikroorganismen. Die Werbung verspricht positive Effekte für Gesundheit, Wohlbefinden und verbesserte Leistungsfähigkeit. Fragen zur Produktsicherheit werden selten thematisiert. Schon im Jahr 2002 wurde in der Europäischen Union (EU) eine Richtlinie (2002/46/EG) zur Angleichung der Rechtsvorschriften der Mitgliedstaaten über NEM erlassen, die auch die Festlegung von Höchstmengen vorsieht. In Deutschland und anderen europäischen Ländern wurden seither verschiedene Modelle für die Höchstmengenableitung für Vitamine und Mineralstoffe entwickelt und diskutiert. Bis heute wurden jedoch auf EU-Ebene keine verbindlichen Höchstmengen festgelegt. Länder wie Dänemark und Frankreich haben daher mittlerweile nationale Regelungen getroffen. Vor diesem Hintergrund hat das Bundesinstitut für Risikobewertung (BfR) auf Basis des derzeitigen wissenschaftlichen Kenntnisstandes Vorschläge für Vitamin- und Mineralstoffhöchstmengen in NEM erarbeitet. Produkte, die diese Empfehlungen einhalten und entsprechend den Herstelleranweisungen eingenommen werden, sind für Personen ab 15 Jahren sicher.AbstractAbout one third of the adults in Germany are using food supplements regularly. Besides vitamins and minerals, these products also contain other substances such as amino acids, fatty acids, plant extracts or microorganisms. Advertising promises positive effects for health, well-being and improved performance. Product safety issues are rarely discussed. Already in the year 2002, a directive (2002/46/EC) on the approximation of the laws of the EU Member States concerning food supplements was adopted, which also provides settings of maximum levels. Since then, different models for the quantitative derivation of maximum levels for vitamins and minerals have been developed and discussed in Germany and other European countries. However, to date no binding maximum levels for vitamins and minerals in food supplements have been set at EU level. Meanwhile, EU member states such as Denmark and France have adopted national regulations. Against this background, the Federal Institute for Risk Assessment (BfR) has developed proposals for maximum levels for vitamins and minerals in food supplements. Vitamin and mineral supplements which comply with these recommendations and are taken according to the manufacturer’s instructions are safe for persons from 15 years of age.

Erratum zu: Höchstmengen für Vitamine und Mineralstoffe in Nahrungsergänzungsmitteln

In Tabelle 1, erste Zeile, letzte Spalte (Bemerkungen zu Vitamin D) wurde angegeben, dass laut Gemeinsamer Expertenkommission von BVL/BfArM Vitamin D-haltige Präparate bis zu einer Tagesdosis von 20 µg noch als Nahrungsergänzungsmittel (NEM) eingestuft werden können, während Präparate mit höheren Dosierungen als Arzneimittel anzusehen sind (BVL/BfArM 2016).

Folic acid modulates cancer-associated micro RNAs and inflammatory mediators in neoplastic and non-neoplastic colonic cells in a different way

SCOPE Scientific evidence suggests that folic acid (FA) supplementation protects the healthy colonic mucosa from neoplastic transformation but may promote the progression of precancerous lesions. The underlying molecular mechanisms are not fully understood. Therefore, we explored, if high physiological FA doses provoke changes in (i) promoter-specific DNA methylation (ii) expression of cancer-associated micro RNAs (miRNAs) and (iii) inflammatory mediators in human neoplastic and non-neoplastic colonic cell lines. METHODS AND RESULTS The malignant and the non-malignant colonic cell lines HT29 and HCEC were adapted to different near-physiological FA concentrations. Using DNA methylation and pathway specific PCR arrays, high-physiological FA concentrations revealed no relevant impact on promoter methylation but a number of differences between the cell lines in the expression of miRNAs and inflammatory mediators. In the HCEC cell line pro-inflammatory genes were repressed and the miRNA expression remained nearly unaffected. In contrast, in the HT29 cell line tumour-suppressive miRNAs were predominantly down-regulated and the expression of genes involved in chemotaxis and immunity were modulated. CONCLUSION The different effects of high-physiological FA concentrations in malignant and non-malignant colonic cell lines regarding cancer-associated miRNAs and inflammatory mediators may contribute to the different effects of FA supplementation on colonic carcinogenesis.