Versailles project on advanced materials and standards (VAMAS) interlaboratory study on measuring the number concentration of colloidal gold nanoparticles.

We describe the outcome of a large international interlaboratory study of the measurement of particle number concentration of colloidal nanoparticles, project 10 of the technical working area 34, "Nanoparticle Populations" of the Versailles Project on Advanced Materials and Standards (VAMAS). A total of 50 laboratories delivered results for the number concentration of 30 nm gold colloidal nanoparticles measured using particle tracking analysis (PTA), single particle inductively coupled plasma mass spectrometry (spICP-MS), ultraviolet-visible (UV-Vis) light spectroscopy, centrifugal liquid sedimentation (CLS) and small angle X-ray scattering (SAXS). The study provides quantitative data to evaluate the repeatability of these methods and their reproducibility in the measurement of number concentration of model nanoparticle systems following a common measurement protocol. We find that the population-averaging methods of SAXS, CLS and UV-Vis have high measurement repeatability and reproducibility, with between-labs variability of 2.6%, 11% and 1.4% respectively. However, results may be significantly biased for reasons including inaccurate material properties whose values are used to compute the number concentration. Particle-counting method results are less reproducibile than population-averaging methods, with measured between-labs variability of 68% and 46% for PTA and spICP-MS respectively. This study provides the stakeholder community with important comparative data to underpin measurement reproducibility and method validation for number concentration of nanoparticles.

Soluble silver ions from silver nanoparticles induce a polarised secretion of interleukin-8 in differentiated Caco-2 cells.

Because of their antimicrobial properties, silver nanoparticles are increasingly incorporated in food-related and hygiene products, which thereby could lead to their ingestion. Although their cytotoxicity mediated by oxidative stress has been largely studied, their effects on inflammation remain controversial. Moreover, the involvement of silver ions (originating from Ag0 oxidation) in their mode of action is still unclear. In this context, the present study aims at assessing the impact of silver nanoparticles on the secretion of the pro-inflammatory chemokine interleukin-8 by Caco-2 cells forming an in vitro model of the intestinal mucosal barrier. Silver nanoparticles induced a vectorized secretion of interleukin-8 towards the apical compartment, which is found in the medium 21 h after the incubation. This secretion seems mediated by Nrf2 signalling pathway that orchestrates cellular defense against oxidative stress. The soluble silver fraction of silver nanoparticles suspensions led to a similar amount of secreted interleukin-8 than silver nanoparticles, suggesting an involvement of silver ions in this interleukin-8 secretion.

In vitro exposure of a 3D-tetraculture representative for the alveolar barrier at the air-liquid interface to silver particles and nanowires.

BACKGROUND: The present study aimed to evaluate the potential differences in the biological effects of two types of spherical silver particles of 20 and 200 nm (Ag20 and Ag200), and of PVP-coated silver nanowires (AgNWs) with a diameter of 50 nm and length up to 50 µm, using a complex 3D model representative for the alveolar barrier cultured at air-liquid interface (ALI). The alveolar model was exposed to 0.05, 0.5 and 5 µg/cm2 of test compounds at ALI using a state-of-the-art exposure system (Vitrocell™Cloud System). Endpoints related to the oxidative stress induction, anti-oxidant defence mechanisms, pro-inflammatory responses and cellular death were selected to evaluate the biocompatibility of silver particles and nanowires (AgNMs) and to further ascribe particular biological effects to the different morphologic properties between the three types of AgNMs evaluated. RESULTS: Significant cytotoxic effect was observed for all three types of AgNMs at the highest tested doses. The increased mRNA levels of the pro-apoptotic gene CASP7 suggests that apoptosis may occur after exposure to AgNWs. All three types of AgNMs increased the mRNA level of the anti-oxidant enzyme HMOX-1 and of the metal-binding anti-oxidant metallothioneins (MTs), with AgNWs being the most potent inducer. Even though all types of AgNMs induced the nuclear translocation of NF-kB, only AgNWs increased the mRNA level of pro-inflammatory mediators. The pro-inflammatory response elicited by AgNWs was further confirmed by the increased secretion of the 10 evaluated interleukins. CONCLUSION: In the current study, we demonstrated that the direct exposure of a complex tetra-culture alveolar model to different types of AgNMs at ALI induces shape- and size-specific biological responses. From the three AgNMs tested, AgNWs were the most potent in inducing biological alterations. Starting from 50 ng/cm2, a dose representative for an acute exposure in a high exposure occupational setting, AgNWs induced prominent changes indicative for a pro-inflammatory response. Even though the acute responses towards a dose representative for a full-lifetime exposure were also evaluated, chronic exposure scenarios at low dose are still unquestionably needed to reveal the human health impact of AgNMs during realistic conditions.

Do the pristine physico-chemical properties of silver and gold nanoparticles influence uptake and molecular effects on Gammarus fossarum (Crustacea Amphipoda)?

The specific and unique properties of silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs), make them of high interest for different scientific and industrial applications. Their increasing use will inevitably lead to their release in the environment and aquatic ecosystems where they may represent a threat to aquatic organisms. Being a widespread and important component of the aquatic macroinvertebrate assemblage, amphipods and more specifically Gammarus fossarum will certainly be exposed to AgNPs and AuNPs. For these reasons, G. fossarum was selected as model organism for this study. The aim of the present work was the evaluation of the influence of both size (20, 40 and 80 nm) and surface coating (citrate CIT, polyethylene glycol PEG) on the acute toxicity of AgNPs and AuNPs on G. fossarum. We investigated the effects of AgNPs and AuNPs on the uptake by G. fossarum, NP tissue distribution and the expression of stress related genes by the use of ICP-MS, NanoSIMS50, Cytoviva®, and Rt-qPCR, respectively. Ag and Au bioaccumulation revealed a significant surface-coating dependence, with CIT-AgNPs and CIT-AuNPs showing the higher bio-accumulation potential in G. fossarum as compared to PEG-NPs. Opposite to that, no size-dependent effects on the bioaccumulation potential was observed. SIMS imaging and CytoViva® revealed an influence of the type of metal on the tissue distribution after uptake, with AgNPs detected in the cuticle and the gills of G. fossarum, while AuNPs were detected in the gut area. Furthermore, AgNPs were found to up-regulate CuZnSOD gene expression while AuNPs led to its down-regulation. Modulation of SOD may indicate generation of reactive species of oxygen and a possible activation of antioxidant defence in order to prevent and defend the organism from oxidative stress. However, further investigations are still needed to better define the mechanisms underlying the observed AgNPs and AuNPs effects.

Inhibition of multixenobiotic resistance transporters (MXR) by silver nanoparticles and ions in vitro and in Daphnia magna.

The P-glycoprotein (P-gp, ABCB1) and multidrug resistance associated protein 1 (MRP1), important members of the ABC (ATP-binding cassette) transporters, protect cells and organisms via efflux of xenobiotics and are responsible for the phenomenon of multidrug or multixenobiotic resistance (MXR). In this study we first evaluated, in vitro, the interaction of silver nanoparticles (Ag NPs, 20, 23 and 27nm), Ag 200nm particles and Ag ions (AgNO3) with MXR efflux transporters using MDCKII and the P-gp over-expressing MDCKII-MDR1 cells and calcein-AM as a substrate of the transporters. Next the in vivo modulation of MXR activity was studied in Daphnia magna juveniles with the model P-gp and MRP1 inhibitors verapamil-HCl and MK571, respectively. The common environmental contaminants perfluorooctane sulfonate and bisphenol A, previously observed to interfere with the P-gp in vitro, also inhibited the efflux of calcein in vivo. Small-sized Ag NPs (with biomolecules present on the surface) and AgNO3 inhibited the MXR activity in daphnids and MDCKII-MDR1 cells, but abcb1 gene expression remained unchanged. Both Ag NPs and dissolved ions contributed to the effects. This study provides evidence of the interference of Ag NPs and AgNO3 with the MXR activity both in vitro and in D. magna, and should be taken into account when Ag NP toxicity is assessed.

Effects of silver nanoparticles and ions on a co-culture model for the gastrointestinal epithelium.

BACKGROUND: The increased incorporation of silver nanoparticles (Ag NPs) into consumer products makes the characterization of potential risk for humans and other organisms essential. The oral route is an important uptake route for NPs, therefore the study of the gastrointestinal tract in respect to NP uptake and toxicity is very timely. The aim of the present study was to evaluate the effects of Ag NPs and ions on a Caco-2/TC7:HT29-MTX intestinal co-culture model with mucus secretion, which constitutes an important protective barrier to exogenous agents in vivo and may strongly influence particle uptake. METHODS: The presence of the mucus layer was confirmed with staining techniques (alcian blue and toluidine blue). Mono and co-cultures of Caco-2/TC7 and HT29-MTX cells were exposed to Ag NPs (Ag 20 and 200 nm) and AgNO3 and viability (alamar blue), ROS induction (DCFH-DA assay) and IL-8 release (ELISA) were measured. The particle agglomeration in the media was evaluated with DLS and the ion release with ultrafiltration and ICP-MS. The effects of the Ag NPs and AgNO3 on cells in co-culture were studied at a proteome level with two-dimensional difference in gel electrophoresis (2D-DIGE) followed by Matrix Assisted Laser Desorption Ionization - Time Of Flight/ Time Of Flight (MALDI-TOF/TOF) mass spectrometry (MS). Intracellular localization was assessed with NanoSIMS and TEM. RESULTS: The presence of mucus layer led to protection against ROS and decrease in IL-8 release. Both Ag 20 and 200 nm NPs were taken up by the cells and Ag NPs 20 nm were mainly localized in organelles with high sulfur content. A dose- and size-dependent increase in IL-8 release was observed with a lack of cytotoxicity and oxidative stress. Sixty one differentially abundant proteins were identified involved in cytoskeleton arrangement and cell cycle, oxidative stress, apoptosis, metabolism/detoxification and stress. CONCLUSIONS: The presence of mucus layer had an impact on modulating the induced toxicity of NPs. NP-specific effects were observed for uptake, pro-inflammatory response and changes at the proteome level. The low level of overlap between differentially abundant proteins observed in both Ag NPs and AgNO3 treated co-culture suggests size-dependent responses that cannot only be attributed to soluble Ag.

In Vitro Bioaccessibility and Bioavailability of Iron from Potatoes with Varying Vitamin C, Carotenoid, and Phenolic Concentrations.

The bioaccessibility and bioavailability of iron from 12 Andean potato clones were estimated using an in vitro gastrointestinal digestion procedure and the Caco-2 cell line as a model of human intestine, with ferritin formation as a marker of iron absorption. We first showed that 63.7% (for the genotype CIP_311422.016) to 79.0% (for the genotype CIP_311575.003) of the iron is released from the potato tuber matrix during in vitro gastrointestinal digestion and is therefore available at the intestinal level. On average, 32 and 24.5% of the hydrophilic bioactive components, vitamin C and chlorogenic acid, respectively, were also bioaccessible from boiled tubers. Intestinal absorption of intrinsic iron from potato tubers could not be detected using our in vitro Caco-2 cell model. When an extrinsic source of iron (20 µM FeCl3 and 1 mM ascorbic acid) was added to the digestion mixture, iron absorption varied from 1.8 to 8% for the genotypes CIP_311422.016 and CIP_311624.021, respectively, as compared to the reference control. Principal component analysis revealed negative relationships between bioavailable iron values and phenolic concentrations, whereas vitamin C concentrations were positively associated with the ferritin values. Further controlled intervention trials would be needed to conclusively assess the bioavailability of intrinsic iron from potato tubers.

Determination of oral uptake and biodistribution of platinum and chromium by the garden snail (Helix aspersa) employing nano-secondary ion mass-spectrometry.

Environmental heavy metal contamination is a case of concern for both animal and human health. Studying the fate of metals in plant or animal tissues may provide information on pollution. In the present study, we investigated the possibility to follow the biological fate of chromium and platinum uptake in common garden snails (Helix aspersa), typically accumulating high concentrations of metals from their environment. Chromium and platinum were administered orally to snails in 5 groups (n=25/group): control, food contaminated by ca. 2.5 µg g(-1) and 19 µg g(-1) chromium and 2.5 µg g(-1) and 25 µg g(-1) platinum, for 8 weeks. Following exposure, surviving snails were sacrificed, shell and remaining tissue investigated by ICP-MS, and shell, midgut gland and mantle by nano-secondary ion mass-spectrometry (Nano-SIMS). (12)C(14)N-normalized platinum and (40)Ca-normalized chromium measurements indicated highest enrichments in cellular vesicles of the midgut gland, and lower concentrations in mantle and shell, with significantly higher platinum and chromium concentrations in the 2 exposure groups vs. control (P<0.05), with somewhat differing distribution patterns for chromium and platinum. Comparable results were obtained by ICP-MS, with both chromium and platinum fed snails showing drastically elevated concentrations of metals in shell (up to 78 and 122 µg g(-1) dw platinum and chromium, respectively) and in other tissues (up to 200 and 1125 µg g(-1) dw platinum and chromium, respectively). Nano-SIMS allowed for semi-quantitative comparison of metal fate in snail tissues, making this an interesting technique for future studies in the area of environmental pollution.

Iron uptake and homeostasis related genes in potato cultivated in vitro under iron deficiency and overload.

Potato is one of the most important staple food in the world because it is a good source of vitamin C, vitamin B6 but also an interesting source of minerals including mainly potassium, but also magnesium, phosphorus, manganese, zinc and iron to a lesser extent. The lack of iron constitutes the main form of micronutrient deficiency in the world, namely iron deficiency anemia, which strongly affects pregnant women and children from developing countries. Iron biofortification of major staple food such as potato is thus a crucial issue for populations from these countries. To better understand mechanisms leading to iron accumulation in potato, we followed in an in vitro culture experiment, by qPCR, in the cultivar Désirée, the influence of media iron content on the expression of genes related to iron uptake, transport and homeostasis. As expected, plantlets grown in a low iron medium (1 mg L(-1) FeNaEDTA) displayed a decreased iron content, a strong induction of iron deficiency-related genes and a decreased expression of ferritins. Inversely, plantlets grown in a high iron medium (120 mg L(-1) FeNaEDTA) strongly accumulated iron in roots; however, no significant change in the expression of our set of genes was observed compared to control (40 mg L(-1) FeNaEDTA).

Chemical contaminants in fish species from rivers in the North of Luxembourg: Potential impact on the Eurasian otter (Lutra lutra).

Contamination levels of PCBs, and of the heavy metals cadmium (Cd), lead (Pb) and mercury (Hg) were analyzed in four fish species from seven rivers in the North of Luxembourg. During August and September 2007, 85 samples of fish were collected belonging to four species: the stone loach (Barbatula barbatula, n=12 pools), the chub (Squalius cephalus, n=36), the barbel (Barbus barbus, n=23) and eel (Anguilla anguilla, n=14). The concentration of seven indicator PCBs ( summation operator(7)PCBs) reached a mean of 39ngg(-1) and varied between 4.0 and 346.2ngg(-1) (wet wt) depending on the site and species. Fish collected at Wallendorf on the Our River and sites on the Wiltz and the Clerve rivers showed the highest concentrations for PCBs. In comparison with 1994, PCB levels in fish decreased strongly during the last decade in these rivers. Lead was detected at low levels (0-181.4ngg(-1) wet wt). Mercury concentrations ranged between 10.3 and 534.5ngg(-1) (wet wt) exceeding maximum tolerable levels for human consumption of 500ngg(-1) in two fish out of 85. Chubs and eels from the Sûre River were the most contaminated by mercury. Cadmium levels varied between 4.0 and 103.9ngg(-1) (wet wt). In addition to mercury in fish, cadmium was the most problematic pollutant on the Our, the Wiltz, the Clerve and the Troine Rivers, because values found in 20% of fish exceeded the threshold of about 10-50ngg(-1) (wet wt) recommended for human health. The total PCB level predicted to accumulate in livers from otter potentially feeding on these fish based on a previously published mathematical model is 37.7microgg(-1) (lipid wt), which is between a proposed "safe level" and a "critical level" for otters. Rivers in the North of Luxembourg are thus to some extent polluted, and the establishment of otter populations could be affected by current levels of contamination.

Combining proteomics and metabolite analyses to unravel cadmium stress-response in poplar leaves.

A proteomic analysis of poplar leaves exposed to cadmium, combined with biochemical analysis of pigments and carbohydrates revealed changes in primary carbon metabolism. Proteomic results suggested that photosynthesis was slightly affected. Together with a growth inhibition, photoassimilates were less needed for developmental processes and could be stored in the form of hexoses or complex sugars, acting also as osmoprotectants. Simultaneously, mitochondrial respiration was upregulated, providing energy needs of cadmium-exposed plants.