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Organisationseinheit der BAM
Increased use of nanomaterials in everyday products leads to their environmental release and therefore, the information need on their fate and behaviour. Nanomaterials have to be suspended with high repeatability and comparability for studies on environmental effects. They also have to be well characterised with a focus on the state of agglomeration and particle size distribution. Dynamic light-scattering (DLS) is a common technique used for these measurements. If suspensions are prepared in different laboratories, then concern has risen about the comparability of the measured results, especially when different DLS instruments are used. Therefore, for quality assurance, a round-robin test was conducted to assess the comparability of different DLS instruments and a dispersion protocol in ten independent laboratories. Polystyrene and TiO2 were chosen as test (nano)materials. For the comparability of the DLS instruments, the average sizes of the PSL and a stabilised TiO2 suspension were measured. The measured average hydrodynamic diameter shows an overall good inter-laboratory comparability. For the PSL suspension, an average hydrodynamic diameter of 201 ± 13 nm and for the TiO2 suspension an average diameter of 224 ± 24 nm were detected. For the TiO2 suspension that was prepared at each laboratory following an established suspension preparation protocol, an average hydrodynamic diameter of 211 ± 11 nm was detected. The measured average particle size (mode) increased up to 284 nm with a high standard deviation of 119 nm if the preparation protocol could not established and different procedures or different equipment were employed. This study shows that no significant differences between the employed DLS instrument types were determined. It was also shown that comparable measurements and suspension preparation could be achieved if well-defined suspension preparation protocols and comparable equipment can be used.
Silver nanoparticles (SNPs) are among the most commercialized nanoparticles because of their antibacterial effects. Besides being employed, e.g. as a coating material for sterile surfaces in household articles and appliances, the particles are also used in a broad range of medical applications. Their antibacterial properties make SNPs especially useful for wound disinfection or as a coating material for prostheses and surgical instruments. Because of their optical characteristics, the particles are of increasing interest in biodetection as well. Despite the widespread use of SNPs, there is little knowledge of their toxicity. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) and laser post-ionization secondary neutral mass spectrometry (Laser-SNMS) were used to investigate the effects of SNPs on human macrophages derived from THP-1 cells in vitro. For this purpose, macrophages were exposed to SNPs. The SNP concentration ranges were chosen with regard to functional impairments of the macrophages. To optimize the analysis of the macrophages, a special silicon wafer sandwich preparation technique was employed; ToF-SIMS was employed to characterize fragments originating from macrophage cell membranes. With the use of this optimized sample preparation method, the SNP-exposed macrophages were analyzed with ToF-SIMS and with Laser-SNMS. With Laser-SNMS, the three-dimensional distribution of SNPs in cells could be readily detected with very high efficiency, sensitivity, and submicron lateral resolution. We found an accumulation of SNPs directly beneath the cell membrane in a nanoparticular state as well as agglomerations of SNPs inside the cells.
Silver nanoparticles (SNP) are among the most commercialized nanoparticles. Here, we show that peptide-coated SNP cause functional impairment of human macrophages. A dose-dependent inhibition of phagocytosis is observed after nanoparticle treatment, and pretreatment of cells with N-acetyl cysteine (NAC) can counteract the phagocytosis disturbances caused by SNP.
Using the surface-sensitive mode of time-of-flight secondary ion mass spectrometry, in combination with multivariate statistical methods, we studied the composition of cell membranes in human macrophages upon exposure to SNP with and without NAC preconditioning. This method revealed characteristic changes in the lipid pattern of the cellular membrane outer leaflet in those cells challenged by SNP. Statistical analyses resulted in 19 characteristic ions, which can be used to distinguish between NAC pretreated and untreated macrophages. The present study discusses the assignments of surface cell membrane phospholipids for the identified ions and the resulting changes in the phospholipid pattern of treated cells. We conclude that the adverse effects in human macrophages caused by SNP can be partially reversed through NAC administration. Some alterations, however, remained.
Silver nanoparticles (SNP) are among the most commercialized nanoparticles worldwide. They can be found in many diverse products, mostly because of their antibacterial properties. Despite its widespread use only little data on possible adverse health effects exist. It is difficult to compare biological data from different studies due to the great variety in sizes, coatings or shapes of the particles. Here, we applied a novel synthesis approach to obtain SNP, which are covalently stabilized by a small peptide. This enables a tight control of both size and shape. We applied these SNP in two different sizes of 20 or 40 nm (Ag20Pep and Ag40Pep) and analyzed responses of THP-1-derived human macrophages. Similar gold nanoparticles with the same coating (Au20Pep) were used for comparison and found to be non-toxic. We assessed the cytotoxicity of particles and confirmed their cellular uptake via transmission electron microscopy and confocal Raman microscopy. Importantly a majority of the SNP could be detected as individual particles spread throughout the cells. Furthermore we studied several types of oxidative stress related responses such as induction of heme oxygenase I or formation of protein carbonyls. In summary, our data demonstrate that even low doses of SNP exerted adverse effects in human macrophages.
Silver nanoparticles (SNP) are the subject of worldwide commercialization because of their antimicrobial effects. Yet only little data on their mode of action exist. Further, only few techniques allow for visualization and quantification of unlabeled nanoparticles inside cells. To study SNP of different sizes and coatings within human macrophages, we introduce a novel laser postionization secondary neutral mass spectrometry (Laser-SNMS) approach and prove this method superior to the widely applied confocal Raman and transmission electron microscopy. With time-of-flight secondary ion mass spectrometry (TOF-SIMS) we further demonstrate characteristic fingerprints in the lipid pattern of the cellular membrane indicative of oxidative stress and membrane fluidity changes. Increases of protein carbonyl and heme oxygenase-1 levels in treated cells confirm the presence of oxidative stress biochemically. Intriguingly, affected phagocytosis reveals as highly sensitive end point of SNP-mediated adversity in macrophages. The cellular responses monitored are hierarchically linked, but follow individual kinetics and are partially reversible.
Nicht zuletzt bedingt durch den Pferdefleischskandal im Jahr 2013 wurden in Deutschland die Aktivitäten bei der Methodenstandardisierung im Bereich der Tierarten-Differenzierung in den vergangenen Jahren intensiviert. Wenn auch positive Befunde bei dem Nachweis von Bestandteilen aus Pferd in Lebensmitteln wie Lasagne derzeit so gut wie nicht mehr anzutreffen sind, so hat die Tierartendifferenzierung insgesamt beim Nachweis von Verfälschungen in Lebensmitteln einen hohen Stellenwert.
Diese Arbeit fasst daher den aktuellen Stand der Analytik in Deutschland mit Schwerpunkt bei der Standardisierung zusammen. Sie wurde erstellt durch die Arbeitsgruppe „Biochemische und molekularbiologische Analytik“ der Lebensmittelchemischen Gesellschaft mit Unterstützung von Experten der Arbeitsgruppe „Molekularbiologische Methoden zur Pflanzen- und Tierartendifferenzierung“ (§ 64 LFGB) sowie der ALTS-Arbeitsgruppe „Immunologie und Molekularbiologie“ (jeweils D).
In the field of animal species differentiation, work on standardizing methods has been intensified in Germany in recent years, not least due to the horsemeat scandal in 2013. Even though there are now hardly ever any positive findings anymore in examinations to detect horse adulterations in foods such as lasagne, animal species differentiation altogether ranks high in detecting adulteration of foods. This article, therefore, summarises the current status of analytical techniques used in Germany with standardization at German level. It has been established by the working group "Biochemical and Molecular Biological Analytics" of the Lebensmittelchemische Gesellschaft (Food Chemistry Society within the German Chemical Society) with support of experts in the working group “Molecular biology techniques for differentiating plant and animal species" (§64 of the German Food and Feed Code - LFGB) and the "Immunology and molecular biology" task force of the food hygiene and food of animal origin working group (ALTS), both from Germany.
Work on standardising methods in the field of animal species differentiation has been intensified in Germany in recent years, not least due to the horsemeat scandal in 2013. Even though there are now hardly ever any positive findings any more in examinations to detect horse adulterations in foods such as lasagne, animal species differentiation altogether ranks high in detecting adulteration of foods. This article therefore summarises the current status of analytical techniques used in Germany with standardisation at German level. It has been established by the working group “Biochemical and Molecular Biological Analytics” of the Lebensmittelchemische Gesellschaft (Food Chemistry Society within the German Chemical Society) with support of experts in the working group “Molecular biology techniques for differentiating plant and animal species” (§ 64 of the German Food and Feed Code – LFGB) and the “Immunology and molecular biology” task force of the food hygiene and Food of animal origin working group (ALTS), both from Germany.
Development and market introduction of new nanomaterials trigger the need for an adequate risk assessment of such products alongside suitable risk communication measures. Current application of classical and new nanomaterials is analyzed in context of regulatory requirements and standardization for chemicals, food and consumer products. The challenges of nanomaterial characterization as the main bottleneck of risk assessment and regulation are presented. In some areas, e.g., quantification of nanomaterials within complex matrices, the establishment and adaptation of analytical techniques such as laser ablation inductively coupled plasma mass spectrometry and others are potentially suited to meet the requirements. As an example, we here provide an approach for the reliable characterization of human exposure to nanomaterials resulting from food packaging. Furthermore, results of nanomaterial toxicity and ecotoxicity testing are discussed, with concluding key criteria such as solubility and fiber rigidity as important parameters to be considered in material development and regulation. Although an analysis of the public opinion has revealed a distinguished rating depending on the particular field of application, a rather positive perception of nanotechnology could be ascertained for the German public in general. An improvement of material characterization in both toxicological testing as well as end-product control was concluded as being the main obstacle to ensure not only safe use of materials, but also wide acceptance of this and any novel technology in the general public.
Ergot alkaloids are mycotoxins formed by fungi of the Claviceps genus, which are some of the most common contaminants of food and feed worldwide. These toxins are a structurally heterogeneous group of compounds, sharing an ergoline backbone. Six structures and their corresponding stereoisomers are typically quantified by either HPLC-FLD or HPLC-MS/MS and the values subsequently summed up to determine the total ergot alkaloid content. For the development of a screening method targeting all ergot alkaloids simultaneously, the alkaloids need to be transferred to one homogeneous structure: a lysergic acid derivative. In this study, two promising cleaving methods—acidic esterification and hydrazinolysis—are compared, using dihydroergocristine as a model compound. While the acidic esterification proved to be unsuitable, due to long reaction times and oxidation sensitivity, hydrazinolysis reached a quantitative yield in 40-60 min. Parallel workup of several samples is possible. An increasing effect on the reaction rate by the addition of ammonium iodide was demonstrated. Application of hydrazinolysis to a major ergot alkaloid mix solution showed that all ergopeptines were cleaved, but ergometrine/-inine was barely affected. Still, hydrazinolysis is a suitable tool for the development of a sum parameter screening method for ergot alkaloids in food and feed.