Improving quality in nanoparticle-induced cytotoxicity testing by a tiered inter-laboratory comparison study

The quality and relevance of nanosafety studies constitute major challenges to ensure their key role as a supporting tool in sustainable innovation, and subsequent competitive economic advantage. However, the number of apparently contradictory and inconclusive research results has increased in the past few years, indicating the need to introduce harmonized protocols and good practices in the nanosafety research community. Therefore, we aimed to evaluate if best-practice training and inter-laboratory comparison (ILC) of performance of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay for the cytotoxicity assessment of nanomaterials among 15 European laboratories can improve quality in nanosafety testing. We used two well-described model nanoparticles, 40-nm carboxylated polystyrene (PS-COOH) and 50-nm amino-modified polystyrene (PS-NH2). We followed a tiered approach using well-developed standard operating procedures (SOPs) and sharing the same cells, serum and nanoparticles. We started with determination of the cell growth rate (tier 1), followed by a method transfer phase, in which all laboratories performed the first ILC on the MTS assay (tier 2). Based on the outcome of tier 2 and a survey of laboratory practices, specific training was organized, and the MTS assay SOP was refined. This led to largely improved intra- and inter-laboratory reproducibility in tier 3. In addition, we confirmed that PS-COOH and PS-NH2 are suitable negative and positive control nanoparticles, respectively, to evaluate impact of nanomaterials on cell viability using the MTS assay. Overall, we have demonstrated that the tiered process followed here, with the use of SOPs and representative control nanomaterials, is necessary and makes it possible to achieve good inter-laboratory reproducibility, and therefore high-quality nanotoxicological data.Web of Science108art. no. 143

The impact of oxidative stress and potential antioxidant therapy on function and survival of cultured pancreatic β-islet cells

Dietary antioxidant curcumin derived from turmeric has been suggested to decrease the risk of many chronic diseases. Much of the existing data for curcumin stem from experiments performed at supra-physiological concentrations (μM-mM) that are impossible to attain through oral ingestion. It was therefore hypothesized that curcumin at low plasma achievable concentration, though itself not acting as a direct antioxidant might up-regulate the intracellular antioxidants and thus helping combat oxidative stress and protect β-islet cells. The results indicated that Curcumin, DMC and BDMC were able to scavenge hydroxyl radicals, but showed little scavenging ability against superoxide and nitric oxide radicals. Nanomolar concentrations of curcuminoids easily prevented the deleterious effects of H2O2 in pancreatic β-islet RINm5F cells. Non of the curcuminoids showed a detrimental effect on insulin secretion, but the model did not allow assessment of any potential positive effect on insulin secretion. The findings confirmed that nanomolar concentrations of curcumin offered protection in pancreatic β-islet cells against H2O2-indicated damage by modulating the proportion of oxidised GSH (GSSG): reduced GSH in the favour of GSH and the increasing the activity of SOD. This increase in GSH and SOD levels was, at least in part, on account of an increase in GR, SOD-1 and SOD-2 gene expression. The intracellular mechanism driving this modulation of antioxidant gene was, by virtue of blocking the H2O2  induced NF-κB activation.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Development of an in vitro co-culture model to mimic the human intestine in healthy and diseased state.

The intestine forms the largest interface between the environment and the human organism. Its integrity and functioning are crucial for the uptake of nutrients while preventing access of harmful antigens. Inflammatory conditions can significantly change the normal functioning of the intestine. In vitro models that adequately reproduce both healthy and inflamed intestinal tissue could provide a useful tool for studying the mechanisms of intestinal inflammation and investigating new therapeutic drugs. We established a co-culture of Caco-2 and PMA-differentiated THP-1 cells that mimics the intestine in healthy and controlled inflamed states. In homoeostatic conditions without stimulation, Caco-2 and THP-1 cells were co-cultured for 48 h without affecting the barrier integrity and with no increase in the release of cytokines, nitric oxide or lactate dehydrogenase. To simulate the inflamed intestine, the Caco-2 barrier was primed with IFN-γ and THP-1 cells were pre-stimulated with LPS and IFN-γ. In these conditions a significant but temporary reduction in barrier integrity was measured, and large concentrations of pro-inflammatory cytokines and cytotoxicity markers detected. With its ability to feature numerous hallmarks of intestinal inflammation the presented co-culture model of epithelial cells and macrophages offers a unique possibility to study exposure effects in relation to the health status of the intestine.JRC.F.1-Health in Societ

Ongoing inflammation enhances the toxicity of engineered nanomaterials: Application of an in vitro co-culture model of the healthy and inflamed intestine

Chronic inflammatory conditions can negatively impact intestinal barrier function and affect the epithelium's interaction with nano-sized materials. We demonstrate the application of a Caco-2/THP-1 co-culture mimicking the intestine in healthy (i.e. stable) or inflamed state in nanotoxicological research. The co-cultures were exposed to non-toxic concentrations of silver nanoparticles (AgNPs) or silver nitrate (AgNO3) for 24 h. The barrier integrity and cytokine release as well as necrotic and apoptotic cell death were investigated. AgNPs and AgNO3 most strongly affected the inflamed co-culture. Higher concentrations of AgNPs induced a significant increase in barrier integrity in the inflamed but not the stable co-culture. Necrotic and apoptotic cell death was detected in both conditions but were significantly more pronounced in the inflamed condition. The exposure to AgNO3 affected barrier integrity in all experimental set-ups, but caused nuclear condensation only in the Caco-2 monoculture and the inflamed co-culture. AgNPs reduced the release of monocyte chemoattractant protein-1 in the stable model. Clear differences were observed in the effects of AgNPs and AgNO3 in relation to the model's health status. The results suggest an increased vulnerability of the inflamed epithelial barrier towards AgNPs underlining the importance to consider the intestinal health status in the safety assessment of nanomaterials.JRC.F.2-Consumer Products Safet

The role of Kupffer cells in the hepatic response to silver nanoparticles

Engineered nanoparticles are increasingly used in medical applications and day-to-day consumer products, leading to concerns about the potential environmental and human health impacts. Silver nanoparticles are particularly prevalent because of their use as anti-bacterial agents in many commonly available products. Nanoparticles (NPs) are believed to accumulate, often preferentially, in the liver. This study therefore investigates the effect of a silver NP (20 nm) on the liver, and in particular, the role of Kupffer cells (KCs; resident liver macrophages) in the overall inflammatory response in the organ. Cytokine expression in the normal liver was measured in terms of IL2, IL4, TNF-α, IFN-γ and IL10 released from the organ with significant up-regulation of TNF-α and IL10 being observed. For livers in which the KC population was specifically targeted and destroyed this cytokine increase was significantly decreased in comparison to the normal tissue. IL10 was secreted at approximately three times the concentration of TNF-α in all the test cases. The high levels of IL10 released from the normal tissue in comparison to the KC depleted livers suggest that the cytokine may help to protect against a pro-inflammatory response to these Ag NPs. This may indicate a potentially important role for KCs in the anti-inflammatory response and suggests that tolerance to the Ag NPs is favoured over a fully activated immune response. In addition, albumin production was measured as an indicator of hepatic function. It was noted that the liver function was unaffected by the Ag NPs