Modulation of Heat Shock Protein Expression in Alveolar Adenocarcinoma Cells through Gold Nanoparticles and Cisplatin Treatment.

Heat-shock proteins (HSPs) are stress-responsive molecules belonging to the family of evolutionary molecular chaperones known to be crucial in many cancer types, including human alveolar adenocarcinoma cells (A549). These proteins are highly overexpressed in cancers to support their ability to accommodate imbalances in cell signalling, DNA alterations, proteins, and energy metabolism associated with oncogenesis. The current study evaluated the effects of gold nanoparticles (AuNPs) combined with cisplatin (CDDP) on molecular chaperone HSPs in A549 cells. It was found that AuNPs:CDDP decreased the percentage of cell viability (38.5%) measured using the modified lactated dehydrogenase (mLDH) and 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays. AuNPs:CDDP exposure caused a significant (p < 0.05) increase in reactive oxygen species (ROS) generation by 1.81-fold, apoptosis induction, and a decrease in the mitochondrial membrane potential (MMP) compared to AuNPs or CDDP alone. Similarly, exposure to the AuNPs:CDDP combination had pronounced cytotoxic effects on the expression of HSPs and PI3K/AKT/mTOR, as well as apoptosis-related proteins. The results demonstrate that the combination of AuNPs with CDDP might enhance the anticancer efficacy of CDDP.

Functionalized Gold Nanoparticles Suppress the Proliferation of Human Lung Alveolar Adenocarcinoma Cells by Deubiquitinating Enzymes Inhibition.

Functionalized gold nanoparticles (AuNPs) are widely used in therapeutic applications, but little is known regarding the impact of their surface functionalization in the process of toxicity against cancer cells. This study investigates the anticancer effects of 5 nm spherical AuNPs functionalized with tannate, citrate, and PVP on deubiquitinating enzymes (DUBs) in human lung alveolar adenocarcinoma (A549) cells. Our findings show that functionalized AuNPs reduce the cell viability in a concentration- and time-dependent manner as measured by modified lactate dehydrogenase (mLDH) and 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays. An increased generation of intracellular reactive oxygen species (ROS) and depletion of glutathione (GSH/GSSG) ratio was observed with the highest AuNP concentration of 10 µg/mL. The expression of DUBs such as ubiquitin specific proteases (USP7, USP8, and USP10) was slightly inhibited when treated with concentrations above 2.5 µg/mL. Moreover, functionalized AuNPs showed an inhibitory effect on protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) and wingless-related integration site (Wnt) signaling proteins, and this could further trigger mitochondrial related-apoptosis by the upregulation of caspase-3, caspase-9, and PARP in A549 cells. Furthermore, our study shows a mechanistic understanding of how functionalized AuNPs inhibit the DUBs, consequently suppressing cell proliferation, and can be modulated as an approach toward anticancer therapy. The study also warrants the need for future work to investigate the effect of functionalized AuNPs on DUB on other cancer cell lines both in vitro and in vivo.

Gold Nanoparticles Induced Size Dependent Cytotoxicity on Human Alveolar Adenocarcinoma Cells by Inhibiting the Ubiquitin Proteasome System.

Gold nanoparticles (AuNPs) are widely used in biomedicine due to their remarkable therapeutic applications. However, little is known about their cytotoxic effects on the ubiquitin proteasome system (UPS). Herein, the cytotoxicity of different sizes of AuNPs (5, 10, and 80 nm) on the UPS was investigated with a particular focus on deubiquitinating enzymes (DUBs) such as ubiquitin-specific proteases (USP) and ubiquitin carboxyl-terminal hydrolases (UCHL-1) in human alveolar epithelial adenocarcinoma (A549). It was found that all sizes of AuNPs reduced the percentage of viable A549 cells and increased lactate dehydrogenase (LDH) release, measured using the MTT and LDH assays, respectively. Furthermore, the 5 nm AuNPs were found to exhibit greater cytotoxicity than the 10 and 80 nm AuNPs. In addition, apoptosis and necrosis were activated through reactive oxygen species (ROS) generation due to AuNPs exposure. The internalisation of AuNPs in A549 cells increased with increasing particle size (80 > 10 > 5 nm). Interestingly, the expression of USP7, USP8, USP10, and UCHL-1 was significantly (p < 0.001) downregulated upon treatment with 5-30 µg/mL of all the AuNPs sizes compared to control cells. Moreover, the inhibition of these proteins triggered mitochondrial-related apoptosis through the upregulation of poly (ADP-ribose) polymerase (PARP), caspase-3, and caspase-9. Collectively, these results indicate that AuNPs suppress the proliferation of A549 cells and can potentially be used as novel inhibitors of the proteasome.

Scanning transmission X-ray microscopy study of subcellular granules in human platelets at the carbon K- and calcium L2,3-edges.

Platelets and their subcellular components (e.g., dense granules) are essential components in hemostasis. Understanding their chemical heterogeneities at the sub-micrometer scale, particularly their activation during hemostasis and production of platelet-derived extracellular vesicles, may provide important insights into their mechanisms; however, this has rarely been investigated, mainly owing to the lack of appropriate chemical characterization tools at nanometer scale. Here, the use of scanning transmission X-ray microscopy (STXM) combined with X-ray absorption near edge structure (XANES) to characterize human platelets and their subcellular components at the carbon K-edge and calcium L2,3-edge, is reported. STXM images can identify not only the spatial distribution of subcellular components in human platelets, such as dense granules (DGs) with sizes of ~200 nm, but also their granule-to-granule chemical heterogeneities on the sub-micrometer scale, based on their XANES spectra. The calcium distribution map as well as the principal component analysis of the STXM image stacks clearly identified the numbers and locations of the calcium-rich DGs within human platelets. Deconvolution of the carbon K-edge XANES spectra, extracted from various locations in the platelets, showed that amide carbonyl and carboxylic acid functional groups were mainly found in the cytoplasm, while ketone-phenol-nitrile-imine, aliphatic, and carbonate functional groups were dominant in the platelet DGs. These observations suggest that platelet DGs are most likely composed of calcium polyphosphate associated with adenosine triphosphate (ATP) and adenosine diphosphate (ADP), with significant granule-to-granule variations in their compositions, while the cytoplasm regions of platelets contain significant amounts of proteins.

An Untargeted Thermogravimetric Analysis-Fourier Transform Infrared-Gas Chromatography-Mass Spectrometry Approach for Plastic Polymer Identification.

Reliable chemical identification of specific polymers in environmental samples represents a major challenge in plastic research, especially with the wide range of commercial polymers available, along with variable additive mixtures. Thermogravimetric analysis-Fourier transform infrared-gas chromatography-mass spectrometry (TGA-FTIR-GC-MS) offers a unique characterization platform that provides both physical and chemical properties of the analyzed polymers. This study presents a library of 11 polymers generated using virgin plastics and post-consumer products. TGA inflection points and mass of remaining residues following pyrolysis, in some cases, proved to be indicative of the polymer type. FTIR analysis of the evolved gas was able to differentiate between all but polypropylene (PP) and polyethylene (PE). Finally, GC-MS was able to differentiate between the unique chemical fingerprints of all but one polymer in the library. This library was then used to characterize real environmental samples of mesoplastics collected from beaches in the U.K. and South Africa. Unambiguous identification of the polymer types was achieved, with PE being the most frequently detected polymer and with South African samples indicating variations that potentially resulted from aging and weathering.

Predicting Cytotoxicity of Metal Oxide Nanoparticles using Isalos Analytics Platform.

A literature curated dataset containing 24 distinct metal oxide (MexOy) nanoparticles (NPs), including 15 physicochemical, structural and assay-related descriptors, was enriched with 62 atomistic computational descriptors and exploited to produce a robust and validated in silico model for prediction of NP cytotoxicity. The model can be used to predict the cytotoxicity (cell viability) of MexOy NPs based on the colorimetric lactate dehydrogenase (LDH) assay and the luminometric adenosine triphosphate (ATP) assay, both of which quantify irreversible cell membrane damage. Out of the 77 total descriptors used, 7 were identified as being significant for induction of cytotoxicity by MexOy NPs. These were NP core size, hydrodynamic size, assay type, exposure dose, the energy of the MexOy conduction band (EC), the coordination number of the metal atoms on the NP surface (Avg. C.N. Me atoms surface) and the average force vector surface normal component of all metal atoms (v⟂ Me atoms surface). The significance and effect of these descriptors is discussed to demonstrate their direct correlation with cytotoxicity. The produced model has been made publicly available by the Horizon 2020 (H2020) NanoSolveIT project and will be added to the project's Integrated Approach to Testing and Assessment (IATA).

Elucidating the mechanism of the surface functionalization dependent neurotoxicity of graphene family nanomaterials.

Graphene family nanomaterials (GFNs) have shown great potential for biological and environmental applications; however, their future use has been debated due to their reported potential neurotoxicity. Moreover, the effects of surface functionalization on their biological end points are largely unknown. Here, we compared the effects of reduced graphene oxide (RGO), and carboxylated (G-COOH), hydroxylated (G-OH) and aminated (G-NH2) graphene nanosheets on human neuroblastoma cells (SK-N-SH). All GFNs inhibited cellular growth at concentrations of 0.1-10 mg L-1 after 24 h exposure. The toxicity was attenuated over longer exposure times, with the exception of G-NH2. Although the overall acute toxicity followed the order: G-OH ≈ G-COOH > RGO > G-NH2, G-NH2 induced more persistent toxicity and more metabolic disturbance compared to the other GFNs, with lipid and carbohydrate metabolism being the most affected. The potential for physical disruption of the lipid membrane and oxidative damage induced by GFNs varied with different functionalization, which accounts for the observed differences in neurotoxicity. This study provides significant insights into the neurological effects of GFNs, and suggests that G-NH2 is not as safe as reported in many previous studies. The neurological effect of GFNs over longer term exposure should be considered in future studies.

Graphene Oxide-Induced pH Alteration, Iron Overload, and Subsequent Oxidative Damage in Rice (Oryza sativa L.): A New Mechanism of Nanomaterial Phytotoxicity.

The mechanism of graphene-based nanomaterial (GBM)-induced phytotoxicity and its association with the GBM physicochemical properties are not yet fully understood. The present study compared the effects of graphene oxide (GO) and reduced GO (rGO) on rice seedling growth under hydroponic conditions for 3 weeks. GO at 100 and 250 mg/L reduced shoot biomass (by 25 and 34%, respectively) and shoot elongation (by 17 and 43%, respectively) and caused oxidative damage, while rGO exhibited no overt effect except for the enhancement of the antioxidant enzyme activities, suggesting that the surface oxygen content is a critical factor affecting the biological impacts of GBMs. GO treatments (100 and 250 mg/L) enhanced the iron (Fe) translocation and caused excessive Fe accumulation in shoots (2.2 and 3.6 times higher than control), which was found to be the main reason for the oxidative damage in shoots. GO-induced acidification of the nutrient solution was the main driver for the Fe overload in plants. In addition to the antioxidant regulators, the plants triggered other pathways to defend against the Fe toxicity via downregulation of the Fe transport associated metabolites (mainly coumarins and flavonoids). Plant root exudates facilitated the reduction of toxic GO to nontoxic rGO, acting as another route for plant adaption to GO-induced phytotoxicity. This study provides new insights into the mechanism of the phytotoxicity of GBMs. It also provides implications for the agricultural application of GBM that the impacts of GBMs on the uptake of multiple nutrients in plants should be assessed simultaneously and reduced forms of GBMs are preferential to avoid toxicity.

A high throughput imaging database of toxicological effects of nanomaterials tested on HepaRG cells.

The large amount of existing nanomaterials demands rapid and reliable methods for testing their potential toxicological effect on human health, preferably by means of relevant in vitro techniques in order to reduce testing on animals. Combining high throughput workflows with automated high content imaging techniques allows deriving much more information from cell-based assays than the typical readouts (i.e. one measurement per well) with optical plate-readers. We present here a dataset including data based on a maximum of 14 different read outs (including viable cell count, cell membrane permeability, apoptotic cell death, mitochondrial membrane potential and steatosis) of the human hepatoma HepaRG cell line treated with a large set of nanomaterials, coatings and supernatants at different concentrations. The database, given its size, can be utilized in the development of in silico hazard assessment and prediction tools or can be combined with toxicity results from other in vitro test systems.

Cytotoxicity and cellular mechanisms of toxicity of CuO NPs in mussel cells in vitro and comparative sensitivity with human cells.

There is a need to assess human and ecosystem health effects of copper oxide nanoparticles (CuO NPs), extensively used in many industrial products. Here, we aimed to determine the cytotoxicity and cellular mechanisms involved in the toxicity of CuO NPs in mussel cells (hemocytes and gill cells) in parallel with exposures to ionic Cu and bulk CuO, and to compare the sensitivity of mussel primary cells with a well-established human cell line (pulmonary TT1 cells). At similar doses, CuO NPs promoted dose-dependent cytotoxicity and increased reactive oxygen species (ROS) production in mussel and human cells. In mussel cells, ionic Cu was more toxic than CuO NPs and the latter more than bulk CuO. Ionic Cu and CuO NPs increased catalase and acid phosphatase activities in both mussel cells and decreased gill cells Na-K-ATPase activity. All Cu forms produced DNA damage in hemocytes, whereas in gill cells only ionic Cu and CuO NPs were genotoxic. Induction of the MXR transport activity was found in gill cells exposed to all forms of Cu and in hemocytes exposed to ionic Cu and CuO NPs. Phagocytosis increased only in hemocytes exposed to CuO NPs, indicating a nanoparticle-specific immunostimulatory effect. In conclusion, toxicity of CuO NPs is driven by ROS in human and mussel cells. Mussel cells respond to CuO NP exposure by triggering an array of defensive mechanisms.

Microscopy-based high-throughput assays enable multi-parametric analysis to assess adverse effects of nanomaterials in various cell lines.

Manufactured nanomaterials (MNMs) selected from a library of over 120 different MNMs with varied compositions, sizes, and surface coatings were tested by four different laboratories for toxicity by high-throughput/-content (HT/C) techniques. The selected particles comprise 14 MNMs composed of CeO2, Ag, TiO2, ZnO and SiO2 with different coatings and surface characteristics at varying concentrations. The MNMs were tested in different mammalian cell lines at concentrations between 0.5 and 250 µg/mL to link physical-chemical properties to multiple adverse effects. The cell lines are derived from relevant organs such as liver, lung, colon and the immune system. Endpoints such as viable cell count, cell membrane permeability, apoptotic cell death, mitochondrial membrane potential, lysosomal acidification and steatosis have been studied. Soluble MNMs, Ag and ZnO, were toxic in all cell types. TiO2 and SiO2 MNMs also triggered toxicity in some, but not all, cell types and the cell type-specific effects were influenced by the specific coating and surface modification. CeO2 MNMs were nearly ineffective in our test systems. Differentiated liver cells appear to be most sensitive to MNMs, Whereas most of the investigated MNMs showed no acute toxicity, it became clear that some show adverse effects dependent on the assay and cell line. Hence, it is advised that future nanosafety studies utilise a multi-parametric approach such as HT/C screening to avoid missing signs of toxicity. Furthermore, some of the cell type-specific effects should be followed up in more detail and might also provide an incentive to address potential adverse effects in vivo in the relevant organ.

Mechanisms of Iron Uptake from Ferric Phosphate Nanoparticles in Human Intestinal Caco-2 Cells.

Food fortification programs to reduce iron deficiency anemia require bioavailable forms of iron that do not cause adverse organoleptic effects. Rodent studies show that nano-sized ferric phosphate (NP-FePO4) is as bioavailable as ferrous sulfate, but there is controversy over the mechanism of absorption. We undertook in vitro studies to examine this using a Caco-2 cell model and simulated gastrointestinal (GI) digestion. Supernatant iron concentrations increased inversely with pH, and iron uptake into Caco-2 cells was 2-3 fold higher when NP-FePO4 was digested at pH 1 compared to pH 2. The size and distribution of NP-FePO4 particles during GI digestion was examined using transmission electron microscopy. The d50 of the particle distribution was 413 nm. Using disc centrifugal sedimentation, a high degree of agglomeration in NP-FePO4 following simulated GI digestion was observed, with only 20% of the particles ≤1000 nm. In Caco-2 cells, divalent metal transporter-1 (DMT1) and endocytosis inhibitors demonstrated that NP-FePO4 was mainly absorbed via DMT1. Small particles may be absorbed by clathrin-mediated endocytosis and micropinocytosis. These findings should be considered when assessing the potential of iron nanoparticles for food fortification.

Role of the crystalline form of titanium dioxide nanoparticles: Rutile, and not anatase, induces toxic effects in Balb/3T3 mouse fibroblasts.

The wide use of titanium dioxide nanoparticles (TiO2 NPs) in industrial applications requires the investigation of their effects on human health. In this context, we investigated the effects of nanosized and bulk titania in two different crystalline forms (anatase and rutile) in vitro. By colony forming efficiency assay, a dose-dependent reduction of the clonogenic activity of Balb/3T3 mouse fibroblasts was detected in the presence of rutile, but not in the case of anatase NPs. Similarly, the cell transformation assay and the micronucleus test showed that rutile TiO2 NPs were able to induce type-III foci formation in Balb/3T3 cells and appeared to be slightly genotoxic, whereas anatase TiO2 NPs did not induce any significant neoplastic or genotoxic effect. Additionally, we investigated the interaction of TiO2 NPs with Balb/3T3 cells and quantified the in vitro uptake of titania using mass spectrometry. Results showed that the internalization was independent of the crystalline form of TiO2 NPs but size-dependent, as nano-titania were taken up more than their respective bulk materials. In conclusion, we demonstrated that the cytotoxic, neoplastic and genotoxic effects triggered in Balb/3T3 cells by TiO2 NPs depend on the crystalline form of the nanomaterial, whereas the internalization is regulated by the particle size.

Cadmium sulfide quantum dots induce oxidative stress and behavioral impairments in the marine clam Scrobicularia plana.

Cadmium sulfide (CdS) quantum dots have a number of current applications in electronics and solar cells and significant future potential in medicine. The aim of the present study was to examine the toxic effects of CdS quantum dots on the marine clam Scrobicularia plana exposed for 14 d to these nanomaterials (10 µg Cd L(-1) ) in natural seawater and to compare them with soluble Cd. Measurement of labile Cd released from CdS quantum dots showed that 52% of CdS quantum dots remained in the nanoparticulate form. Clams accumulated the same levels of Cd regardless of the form in which it was delivered (soluble Cd vs CdS quantum dots). However, significant changes in biochemical responses were observed in clams exposed to CdS quantum dots compared with soluble Cd. Increased activities of catalase and glutathione-S-transferase were significantly higher in clams exposed in seawater to Cd as the nanoparticulate versus the soluble form, suggesting a specific nano effect. The behavior of S. plana in sediment showed impairments of foot movements only in the case of exposure to CdS quantum dots. The results show that oxidative stress and behavior biomarkers are sensitive predictors of CdS quantum dots toxicity in S. plana. Such responses, appearing well before changes might occur at the population level, demonstrate the usefulness of this model species and type of biomarker in the assessment of nanoparticle contamination in estuarine ecosystems.

Accumulation dynamics and acute toxicity of silver nanoparticles to Daphnia magna and Lumbriculus variegatus: implications for metal modeling approaches.

Frameworks commonly used in trace metal ecotoxicology (e.g., biotic ligand model (BLM) and tissue residue approach (TRA)) are based on the established link between uptake, accumulation and toxicity, but similar relationships remain unverified for metal-containing nanoparticles (NPs). The present study aimed to (i) characterize the bioaccumulation dynamics of PVP-, PEG-, and citrate-AgNPs, in comparison to dissolved Ag, in Daphnia magna and Lumbriculus variegatus; and (ii) investigate whether parameters of bioavailability and accumulation predict acute toxicity. In both species, uptake rate constants for AgNPs were ∼ 2-10 times less than for dissolved Ag and showed significant rank order concordance with acute toxicity. Ag elimination by L. variegatus fitted a 1-compartment loss model, whereas elimination in D. magna was biphasic. The latter showed consistency with studies that reported daphnids ingesting NPs, whereas L. variegatus biodynamic parameters indicated that uptake and efflux were primarily determined by the bioavailability of dissolved Ag released by the AgNPs. Thus, principles of BLM and TRA frameworks are confounded by the feeding behavior of D. magna where the ingestion of AgNPs perturbs the relationship between tissue concentrations and acute toxicity, but such approaches are applicable when accumulation and acute toxicity are linked to dissolved concentrations. The uptake rate constant, as a parameter of bioavailability inclusive of all available pathways, could be a successful predictor of acute toxicity.

Nano-titanium dioxide bioreactivity with human alveolar type-I-like epithelial cells: Investigating crystalline phase as a critical determinant.

There can be significant variability between bioreactivity studies of nanomaterials that are apparently the same, possibly reflecting differences in the models used and differing sources of experimental material. In this study, we have generated two crystal forms of titanium dioxide nanoparticles (nano-TiO2), pure anatase and pure rutile to address the hypothesis that the bioreactivity of these nanoparticles with human alveolar epithelium will depend on their crystal phase. We used a human alveolar type-I-like epithelial cell model (TT1; generated in-house from primary human alveolar epithelial type II cells); these cells cover 95% of the alveolar epithelial surface area and are an important target cell for inhaled nanomaterials. Using literature as a guide, we hypothesised that pure anatase nano-TiO2 would display greater bioreactivity with TT1 cells in comparison to pure rutile nano-TiO2. However, we found the profile and pattern of inflammatory mediator release was similar between these two nano-TiO2 formats, although pure rutile treatment caused a small, but consistently greater, response for IL-6, IL-8 and MCP-1. Interestingly, the temporal induction of oxidative stress (increased reactive oxygen species levels and depleted glutathione) varied markedly between the different nano-TiO2 formats. We have shown that a combination of using nanomaterials synthesised specifically for toxicological study and the use of a highly relevant, reproducible human lung cell model, offers a useful approach to delineating the physicochemical properties of nanomaterials that may be important in their cellular reactivity.

Aneuploidogenic effects and DNA oxidation induced in vitro by differently sized gold nanoparticles.

Gold nanoparticles (Au NPs) are used in many fields, including biomedical applications; however, no conclusive information on their potential cytotoxicity and genotoxicity mechanisms is available. For this reason, experiments in human primary lymphocytes and murine macrophages (Raw264.7) were performed exposing cells to spherical citrate-capped Au NPs with two different nominal diameters (5 nm and 15 nm). The proliferative activity, mitotic, apoptotic, and necrotic markers, as well as chromosomal damage were assessed by the cytokinesis-block micronucleus cytome assay. Fluorescence in situ hybridization with human and murine pancentromeric probes was applied to distinguish between clastogenic and aneuploidogenic effects. Our results indicate that 5 nm and 15 nm Au NPs are able to inhibit cell proliferation by apoptosis and to induce chromosomal damage, in particular chromosome mis-segregation. DNA strand breaks were detected by comet assay, and the modified protocol using endonuclease-III and formamidopyrimidine-DNA glycosylase restriction enzymes showed that pyrimidines and purines were oxidatively damaged by Au NPs. Moreover, we show a size-independent correlation between the cytotoxicity of Au NPs and their tested mass concentration or absolute number, and genotoxic effects which were more severe for Au NP 15 nm compared to Au NP 5 nm. Results indicate that apoptosis, aneuploidy, and DNA oxidation play a pivotal role in the cytotoxicity and genotoxicity exerted by Au NPs in our cell models.

Biochemical and behavioural responses of the marine polychaete Hediste diversicolor to cadmium sulfide quantum dots (CdS QDs): waterborne and dietary exposure.

Cadmium sulfide (CdS) quantum dots are widely used in medical imaging. The aim of this study was to examine toxicity effects of CdS engineered nanoparticles (CdS NPs) compared to soluble Cd, on marine ragworms (Hediste diversicolor) exposed for 14 d to these contaminants (10 µg Cd L(-1)) in seawater or via their food (contaminated worm tissue). In our experimental media, Dynamic Light Scattering studies showed that the majority of CdS remained in the nanoscale (1-10 nm) with the exception of few aggregates (100-300 nm). Labile Cd fractions released from CdS NPs were estimated by diffusive gradient in thin films, showing that about 50% of CdS NPs remained in nanoparticulate form. Ragworms accumulated Cd in both soluble Cd and CdS NPs in waterborne exposures only. Greater significant changes of biochemical responses were observed in worms exposed to CdS NPs in seawater compared to contaminated food. Catalase and glutathione-S-transferase activities were the most sensitive biochemical biomarkers responding to both Cd treatments for waterborne exposure. Inductions of CAT were higher in diet-exposed worms to Cd as NPs vs soluble form suggesting a specific "nano" effect. Caspase activities increased in worms exposed to soluble Cd and Cd NPs for the two routes of exposure compared to controls. Defences, may be insufficient to prevent reactive oxygen species generation and the associated apoptosis. Behaviour of invertebrates inside sediment showed impairments of body movements in worms exposed to CdS NPs. This study points out oxidative processes as the main consequences of exposure to Cd based NPs in worms.

Multi-walled carbon nanotube length as a critical determinant of bioreactivity with primary human pulmonary alveolar cells.

Multiwalled carbon nanotube (MWCNT) length is suggested to critically determine their pulmonary toxicity. This stems from in vitro and in vivo rodent studies and in vitro human studies using cell lines (typically cancerous). There is little data using primary human lung cells. We addressed this knowledge gap, using highly relevant, primary human alveolar cell models exposed to precisely synthesized and thoroughly characterized MWCNTs. In this work, transformed human alveolar type-I-like epithelial cells (TT1), primary human alveolar type-II epithelial cells (ATII) and alveolar macrophages (AM) were treated with increasing concentrations of MWCNTs before measuring cytotoxicity, inflammatory mediator release and MAP kinase signalling. Strikingly, we observed that short MWCNTs (~0.6 µm in length) induced significantly greater responses from the epithelial cells, whilst AM were particularly susceptible to long MWCNTs (~20 µm). These differences in the pattern of mediator release were associated with alternative profiles of JNK, p38 and ERK1/2 MAP kinase signal transduction within each cell type. This study, using highly relevant target human alveolar cells and well defined and characterized MWCNTs, shows marked cellular responses to the MWCNTs that vary according to the target cell type, as well as the aspect ratio of the MWCNT.

Elucidation of toxicity pathways in lung epithelial cells induced by silicon dioxide nanoparticles.

A study into the effects of amorphous nano-SiO2 particles on A549 lung epithelial cells was undertaken using proteomics to understand the interactions that occur and the biological consequences of exposure of lung to nanoparticles. Suitable conditions for treatment, where A549 cells remained viable for the exposure period, were established by following changes in cell morphology, flow cytometry, and MTT reduction. Label-free proteomics was used to estimate the relative level of proteins from their component tryptic peptides detected by mass spectrometry. It was found that A549 cells tolerated treatment with 100 µg/ml nano-SiO2 in the presence of 1.25% serum for at least 4 h. After this time detrimental changes in cell morphology, flow cytometry, and MTT reduction were evident. Proteomics performed after 4 h indicated changes in the expression of 47 proteins. Most of the proteins affected fell into four functional groups, indicating that the most prominent cellular changes were those that affected apoptosis regulation (e.g. UCP2 and calpain-12), structural reorganisation and regulation of actin cytoskeleton (e.g. PHACTR1), the unfolded protein response (e.g. HSP 90), and proteins involved in protein synthesis (e.g. ribosomal proteins). Treatment with just 10 µg/ml nano-SiO2 particles in serum-free medium resulted in a rapid deterioration of the cells and in medium containing 10% serum the cells were resistant to up to 1000 µg/ml nano-SiO2 particles, suggesting interaction of serum components with the nanoparticles. A variety of serum proteins were found which bound to nano-SiO2 particles, the most prominent of which were albumin, apolipoprotein A-I, hemoglobin, vitronectin and fibronectin. The use of a proteomics platform, with appropriately designed experimental conditions, enabled the early biological perturbations induced by nano-SiO2 in a model target cell system to be identified. The approach facilitates the design of more focused test systems for use in tiered evaluations of nanomaterials.