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Given the multiplicity of nanoparticles (NPs), there is a requirement to develop screening strategies to evaluate their toxicity. Within the EU-funded FP7 NanoTEST project, a panel of medically relevant NPs has been used to develop alternative testing strategies of NPs used in medical diagnostics. As conventional toxicity tests cannot necessarily be directly applied to NPs in the same manner as for soluble chemicals and drugs, we determined the extent of interference of NPs with each assay process and components. In this study, we fully characterized the panel of NP suspensions used in this project (poly(lactic-co-glycolic acid)-polyethylene oxide [PLGA-PEO], TiO2, SiO2, and uncoated and oleic-acid coated Fe3O4) and showed that many NP characteristics (composition, size, coatings, and agglomeration) interfere with a range of in vitro cytotoxicity assays (WST-1, MTT, lactate dehydrogenase, neutral red, propidium iodide, (3)H-thymidine incorporation, and cell counting), pro-inflammatory response evaluation (ELISA for GM-CSF, IL-6, and IL-8), and oxidative stress detection (monoBromoBimane, dichlorofluorescein, and NO assays). Interferences were assay specific as well as NP specific. We propose how to integrate and avoid interference with testing systems as a first step of a screening strategy for biomedical NPs.

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Diesel exhaust particles (DEP) induce a proinflammatory response in human bronchial epithelial cells (16HBE) characterized by the release of proinflammatory cytokines after activation of transduction pathways involving MAPK and the transcription factor NF-kappaB. Because cellular effects induced by DEP are prevented by antioxidants, they could be mediated by reactive oxygen species (ROS). Using fluorescent probes, we detected ROS production in bronchial and nasal epithelial cells exposed to native DEP, organic extracts of DEP (OE-DEP), or several polyaromatic hydrocarbons. Carbon black particles mimicking the inorganic part of DEP did not increase ROS production. DEP and OE-DEP also induced the expression of genes for phase I [cytochrome P-450 1A1 (CYP1A1)] and phase II [NADPH quinone oxidoreductase-1 (NQO-1)] xenobiotic metabolization enzymes, suggesting that DEP-adsorbed organic compounds become bioavailable, activate transcription, and are metabolized since the CYP1A1 enzymatic activity is increased. Because NQO-1 gene induction is reduced by antioxidants, it could be related to the ROS generated by DEP, most likely through the activation of the stress-sensitive Nrf2 transcription factor. Indeed, DEP induced the translocation of Nrf2 to the nucleus and increased protein nuclear binding to the antioxidant responsive element. In conclusion, we show that DEP-organic compounds generate an oxidative stress, activate the Nrf2 transcription factor, and increase the expression of genes for phase I and II metabolization enzymes.

The involvement of diesel exhaust particles (DEPs) in respiratory diseases was evaluated by studying their effects on two in vitro models of human airway epithelial cells. The cytotoxicity of DEPs, their phagocytosis, and the resulting immune response were investigated in a human bronchial epithelial cell line (16HBE14o−) as well as in human nasal epithelial cells in primary culture. DEP exposure induced a time- and dose-dependent membrane damage. Transmission electron microscopy showed that DEPs underwent endocytosis by epithelial cells and translocated through the epithelial cell sheet. Flow cytometric measurements allowed establishment of the time and dose dependency of this phagocytosis and its nonspecificity with different particles (DEPs, carbon black, and latex particles). DEPs also induced a time-dependent increase in interleukin-8, granulocyte-macrophage colony-stimulating factor, and interleukin-1β release. This inflammatory response occurred later than phagocytosis, and its extent seems to depend on the content of adsorbed organic compounds because carbon black had no effect on cytokine release. Furthermore, exhaust gas posttreatments, which diminished the adsorbed organic compounds, reduced the DEP-induced increase in granulocyte-macrophage colony-stimulating factor release. These results suggest that DEPs could 1) be phagocytosed by airway epithelial cells and 2) induce a specific inflammatory response.

BackgroundIncreasing environmental and occupational exposures to nanoparticles (NPs) warrant deeper insight into the toxicological mechanisms induced by these materials. The present study was designed to characterize the cell death induced by carbon black (CB) and titanium dioxide (TiO2) NPs in bronchial epithelial cells (16HBE14o- cell line and primary cells) and to investigate the implicated molecular pathways.ResultsDetailed time course studies revealed that both CB (13 nm) and TiO2(15 nm) NP exposed cells exhibit typical morphological (decreased cell size, membrane blebbing, peripheral chromatin condensation, apoptotic body formation) and biochemical (caspase activation and DNA fragmentation) features of apoptotic cell death. A decrease in mitochondrial membrane potential, activation of Bax and release of cytochrome c from mitochondria were only observed in case of CB NPs whereas lipid peroxidation, lysosomal membrane destabilization and cathepsin B release were observed during the apoptotic process induced by TiO2 NPs. Furthermore, ROS production was observed after exposure to CB and TiO2 but hydrogen peroxide (H2O2) production was only involved in apoptosis induction by CB NPs.ConclusionsBoth CB and TiO2 NPs induce apoptotic cell death in bronchial epithelial cells. CB NPs induce apoptosis by a ROS dependent mitochondrial pathway whereas TiO2 NPs induce cell death through lysosomal membrane destabilization and lipid peroxidation. Although the final outcome is similar (apoptosis), the molecular pathways activated by NPs differ depending upon the chemical nature of the NPs.

Increasing evidence linking nanoparticles (NPs) with different cellular outcomes necessitate an urgent need for the better understanding of cellular signalling pathways triggered by NPs. Oxidative stress has largely been reported to be implicated in NP-induced toxicity. It could activate a wide variety of cellular events such as cell cycle arrest, apoptosis, inflammation and induction of antioxidant enzymes. These responses occur after the activation of different cellular pathways. In this context, three groups of MAP kinase cascades [ERK (extracellular signal-regulated kinases), p38 mitogen-activated protein kinase and JNK (c-Jun N-terminal kinases)] as well as redox-sensitive transcription factors such as NFκB and Nrf-2 were specially investigated. The ability of NPs to interact with these signalling pathways could partially explain their cytotoxicity. The induction of apoptosis is also closely related to the modulation of signalling pathways induced by NPs. Newly emerged scientific areas of research are the studies on interactions between NPs and biological molecules in body fluids, cellular microenvironment, intracellular components or secreted cellular proteins such as cytokines, growth factors and enzymes and use of engineered NPs to target various signal transduction pathways in cancer therapy. Recently published data present the ability of NPs to interact with membrane receptors leading to a possible aggregation of these receptors. These interactions could lead to a sustained modulation of specific signalling in the target cells or paracrine and even "by-stander" effects of the neighbouring cells or tissues. However, oxidative stress is not sufficient to explain specific mechanisms which could be induced by NPs, and these new findings emphasize the need to revise the paradigm of oxidative stress to explain the effects of NPs.

BackgroundThe uptake of nanoparticles (NPs) by cells remains to be better characterized in order to understand the mechanisms of potential NP toxicity as well as for a reliable risk assessment. Real NP uptake is still difficult to evaluate because of the adsorption of NPs on the cellular surface.ResultsHere we used two approaches to distinguish adsorbed fluorescently labeled NPs from the internalized ones. The extracellular fluorescence was either quenched by Trypan Blue or the uptake was analyzed using imaging flow cytometry. We used this novel technique to define the inside of the cell to accurately study the uptake of fluorescently labeled (SiO2) and even non fluorescent but light diffracting NPs (TiO2). Time course, dose-dependence as well as the influence of surface charges on the uptake were shown in the pulmonary epithelial cell line NCI-H292. By setting up an integrative approach combining these flow cytometric analyses with confocal microscopy we deciphered the endocytic pathway involved in SiO2 NP uptake. Functional studies using energy depletion, pharmacological inhibitors, siRNA-clathrin heavy chain induced gene silencing and colocalization of NPs with proteins specific for different endocytic vesicles allowed us to determine macropinocytosis as the internalization pathway for SiO2 NPs in NCI-H292 cells.ConclusionThe integrative approach we propose here using the innovative imaging flow cytometry combined with confocal microscopy could be used to identify the physico-chemical characteristics of NPs involved in their uptake in view to redesign safe NPs.