Assessment of the physicochemical properties of chrysotile-containing brake debris pertaining to toxicity.

Grinding and drilling of chrysotile asbestos-containing brake pads during the 20th century led to release of chrysotile, resulting in varying levels of workplace exposures of mechanics. Despite exposures, excess risk of mesothelioma remains in doubt. Objectives: The toxicity of particulates is primarily derived through a combination of physicochemical properties and dose and as such this study aimed to determine properties of asbestos-containing brake debris (BD) which may influence pathogenicity and potential of mesothelioma. Materials and Methods: Chrysotile-containing brake pads were ground - to reflect occupational activities, aerosolized, and size-fractionated to isolate respirable fractions. Analysis of morphology, biodurability, surface charge, and interactions with macrophages were undertaken. Results: The respirable fraction of BD contained ∼15-17% free chrysotile fibers thereby constituting a small but relevant potential long fiber dose. Acellular biodurability studies showed rapid dissolution and fragmentation of chrysotile fibers that was consistent for pure chrysotile control and BD samples. Conclusions: The long, free, respirable chrysotile fibers were present in BD, yet were of low bio-durability; incubation in artificial lysosomal fluid led to destruction of free fibers.

Assessing the bioactivity of crystalline silica in heated high-temperature insulation wools.

High-Temperature Insulation Wools (HTIW), such as alumino silicate wools (Refractory Ceramic Fibers) and Alkaline Earth Silicate wools, are used in high-temperature industries for thermal insulation. These materials have an amorphous glass-like structure. In some applications, exposure to high temperatures causes devitrification resulting in the formation of crystalline species including crystalline silica. The formation of this potentially carcinogenic material raises safety concerns regarding after-use handling and disposal. This study aims to determine whether cristobalite formed in HTIW is bioactive in vitro. Mouse macrophage (J774A.1) and human alveolar epithelial (A549) cell lines were exposed to pristine HTIW of different compositions, and corresponding heat-treated samples. Cell death, cytokine release, and reactive oxygen species (ROS) formation were assessed in both cell types. Cell responses to aluminum lactate-coated fibers were assessed to determine if responses were caused by crystalline silica. DQ12 α-quartz was used as positive control, and TiO2 as negative control. HTIW did not induce cell death or intracellular ROS, and their ability to induce pro-inflammatory mediator release was low. In contrast, DQ12 induced cytotoxicity, a strong pro-inflammatory response and ROS generation. The modest pro-inflammatory mediator responses of HTIW did not always coincide with the formation of cristobalite in heated fibers; therefore, we cannot confirm that devitrification of HTIW results in bioactive cristobalite in vitro. In conclusion, the biological responses to HTIW observed were not attributable to a single physicochemical characteristic; instead, a combination of physicochemical characteristics (cristobalite content, fiber chemistry, dimensions and material solubility) appear to contribute to induction of cellular responses.

Pan-European inter-laboratory studies on a panel of in vitro cytotoxicity and pro-inflammation assays for nanoparticles.

The rapid development of nanotechnologies and increased production and use of nanomaterials raise concerns about their potential toxic effects for human health and environment. To evaluate the biological effects of nanomaterials, a set of reliable and reproducible methods and development of standard operating procedures (SOPs) is required. In the framework of the European FP7 NanoValid project, three different cell viability assays (MTS, ATP content, and caspase-3/7 activity) with different readouts (absorbance, luminescence and fluorescence) and two immune assays (ELISA of pro-inflammatory cytokines IL1-ß and TNF-α) were evaluated by inter-laboratory comparison. The aim was to determine the suitability and reliability of these assays for nanosafety assessment. Studies on silver and copper oxide nanoparticles (NPs) were performed, and SOPs for particle handling, cell culture, and in vitro assays were established or adapted. These SOPs give precise descriptions of assay procedures, cell culture/seeding conditions, NPs/positive control preparation and dilutions, experimental well plate preparation, and evaluation of NPs interference. The following conclusions can be highlighted from the pan-European inter-laboratory studies: Testing of NPs interference with the toxicity assays should always be conducted. Interference tests should be designed as close as possible to the cell exposure conditions. ATP and MTS assays gave consistent toxicity results with low inter-laboratory variability using Ag and CuO NPs and different cell lines and therefore, could be recommended for further validation and standardization. High inter-laboratory variability was observed for Caspase 3/7 assay and ELISA for IL1-ß and TNF-α measurements.

Copper oxide nanoparticle toxicity profiling using untargeted metabolomics.

BACKGROUND: The rapidly increasing number of engineered nanoparticles (NPs), and products containing NPs, raises concerns for human exposure and safety. With this increasing, and ever changing, catalogue of NPs it is becoming more difficult to adequately assess the toxic potential of new materials in a timely fashion. It is therefore important to develop methods which can provide high-throughput screening of biological responses. The use of omics technologies, including metabolomics, can play a vital role in this process by providing relatively fast, comprehensive, and cost-effective assessment of cellular responses. These techniques thus provide the opportunity to identify specific toxicity pathways and to generate hypotheses on how to reduce or abolish toxicity. RESULTS: We have used untargeted metabolome analysis to determine differentially expressed metabolites in human lung epithelial cells (A549) exposed to copper oxide nanoparticles (CuO NPs). Toxicity hypotheses were then generated based on the affected pathways, and critically tested using more conventional biochemical and cellular assays. CuO NPs induced regulation of metabolites involved in oxidative stress, hypertonic stress, and apoptosis. The involvement of oxidative stress was clarified more easily than apoptosis, which involved control experiments to confirm specific metabolites that could be used as standard markers for apoptosis; based on this we tentatively propose methylnicotinamide as a generic metabolic marker for apoptosis. CONCLUSIONS: Our findings are well aligned with the current literature on CuO NP toxicity. We thus believe that untargeted metabolomics profiling is a suitable tool for NP toxicity screening and hypothesis generation.

Enhanced Deposition by Electrostatic Field-Assistance Aggravating Diesel Exhaust Aerosol Toxicity for Human Lung Cells.

Air pollution is associated with increased risk of cardiovascular and pulmonary diseases, but conventional air quality monitoring gives no information about biological consequences. Exposing human lung cells at the air-liquid interface (ALI) to ambient aerosol could help identify acute biological responses. This study investigated electrode-assisted deposition of diesel exhaust aerosol (DEA) on human lung epithelial cells (A549) in a prototype exposure chamber. A549 cells were exposed to DEA at the ALI and under submerged conditions in different electrostatic fields (EFs) and were assessed for cell viability, membrane integrity, and IL-8 secretion. Qualitative differences of the DEA and its deposition under different EFs were characterized using scanning mobility particle sizer (SMPS) measurements, transmission electron microscopy (TEM), and electron energy loss spectroscopy (EELS). Upon exposure to DEA only, cell viability decreased and membrane impairment increased for cells at the ALI; submerged cells were unaffected. These responses were enhanced upon application of an EF, as was DEA deposition. No adverse effects were observed for filtered DEA or air only, confirming particle-induced responses. The prototype exposure chamber proved suitable for testing DEA-induced biological responses of cells at the ALI using electrode-assisted deposition and may be useful for analysis of other air pollutants.

Assessment of a panel of interleukin-8 reporter lung epithelial cell lines to monitor the pro-inflammatory response following zinc oxide nanoparticle exposure under different cell culture conditions.

BACKGROUND: Stably transfected lung epithelial reporter cell lines pose an advantageous alternative to replace complex experimental techniques to monitor the pro-inflammatory response following nanoparticle (NP) exposure. Previously, reporter cell lines have been used under submerged culture conditions, however, their potential usefulness in combination with air-liquid interface (ALI) exposures is currently unknown. Therefore, the aim of the present study was to compare a panel of interleukin-8 promoter (pIL8)-reporter cell lines (i.e. green or red fluorescent protein (GFP, RFP), and luciferase (Luc)), originating from A549 lung epithelial type II-like cells cells, following NPs exposure under both submerged and ALI conditions. METHODS: All cell lines were exposed to zinc oxide (ZnO) NPs at 0.6 and 6.2 µg/cm(2) for 3 and 16 hours under both submerged and ALI conditions. Following physicochemical characterization, the cytotoxic profile of the ZnO-NPs was determined for each exposure scenario. Expression of IL-8 from all cell types was analyzed at the promoter level and compared to the mRNA (qRT-PCR) and protein level (ELISA). RESULTS: In summary, each reporter cell line detected acute pro-inflammatory effects following ZnO exposure under each condition tested. The pIL8-Luc cell line was the most sensitive in terms of reporter signal strength and onset velocity following TNF-α treatment. Both pIL8-GFP and pIL8-RFP also showed a marked signal induction in response to TNF-α, although only after 16 hrs. In terms of ZnO-NP-induced cytotoxicity pIL8-RFP cells were the most affected, whilst the pIL8-Luc were found the least responsive. CONCLUSIONS: In conclusion, the use of fluorescence-based reporter cell lines can provide a useful tool in screening the pro-inflammatory response following NP exposure in both submerged and ALI cell cultures.

Chitosan functionalisation of gold nanoparticles encourages particle uptake and induces cytotoxicity and pro-inflammatory conditions in phagocytic cells, as well as enhancing particle interactions with serum components.

BACKGROUND: Gold nanoparticles (AuNPs) are a popular choice for use in medical and biomedical research applications. With suitable functionalisation AuNPs can be applied in drug delivery systems, or can aid in disease diagnosis. One such functionalisation is with chitosan, which enables efficient interaction and permeation of cellular membranes, providing an effective adjuvant. As both AuNPs and chitosan have been shown to have low toxicity and high biocompatibility their proposed use in nanomedicine, either individually or combined, is expanding. However, further toxicological and immunological assessments of AuNP-chitosan conjugates are still needed. Therefore, we have evaluated how AuNP functionalisation with chitosan can affect uptake, cytotoxicity, and immunological responses within mononuclear cells, and influence the interaction of AuNPs with biomolecules within a complex biofluid. The AuNPs used were negatively charged through citrate-coating, or presented either low or high positive charge through chitosan-functionalisation. Uptake by THP-1 cells was assessed via transmission electron microscopy and electron energy loss spectroscopy, pro-inflammatory responses by ELISA and qRT-PCR, and cell death and viability via lactate dehydrogenase release and mitochondrial activity, respectively. Interactions of AuNPs with protein components of a frequently used in vitro cell culture medium supplement, foetal calf serum, were investigated using mass spectrometry. RESULTS: Although cells internalised all AuNPs, uptake rates and specific routes of intracellular trafficking were dependent upon chitosan-functionalisation. Accordingly, an enhanced immune response was found to be chitosan-functionalisation-dependent, in the form of CCL2, IL-1ß, TNF-α and IL-6 secretion, and expression of IL-1ß and NLRP3 mRNA. A corresponding increase in cytotoxicity was found in response to chitosan-coated AuNPs. Furthermore, chitosan-functionalisation was shown to induce an increase in unique proteins associating with these highly charged AuNPs. CONCLUSIONS: It can be concluded that functionalisation of AuNPs with the perceived non-toxic biocompatible molecule chitosan at a high density can elicit functionalisation-dependent intracellular trafficking mechanisms and provoke strong pro-inflammatory conditions, and that a high affinity of these NP-conjugates for biomolecules may be implicit in these cellular responses.

A comparison of dermal toxicity models; assessing suitability for safe(r)-by-design decision-making and for screening nanomaterial hazards.

The objective of Safe-by-Design (SbD) is to support the development of safer products and production processes, and enable safe use throughout a materials' life cycle; an intervention at an early stage of innovation can greatly benefit industry by reducing costs associated with the development of products later found to elicit harmful effects. Early hazard screening can support this process, and is needed for all of the expected nanomaterial exposure routes, including inhalation, ingestion and dermal. In this study, we compare in vitro and ex vivo cell models that represent dermal exposures (including HaCaT cells, primary keratinocytes, and reconstructed human epidermis (RhE)), and when possible consider these in the context of regulatory accepted OECD TG for in vitro dermal irritation. Various benchmark nanomaterials were used to assess markers of cell stress in each cell model. In addition, we evaluated different dosing strategies that have been used when applying the OECD TG for dermal irritation in assessment of nanomaterials, and how inconsistencies in the approach used can have considerable impact of the conclusions made. Although we could not demonstrate alignment of all models used, there was an indication that the simpler in vitro cell model aligned more closely with RhE tissue than ex vivo primary keratinocytes, supporting the use of HaCaT cells for screening of dermal toxicity of nanomaterials and in early-stage SbD decision-making.

Multi-walled carbon nanotube induced frustrated phagocytosis, cytotoxicity and pro-inflammatory conditions in macrophages are length dependent and greater than that of asbestos.

The potential toxicity of carbon nanotubes (CNTs) has been compared to pathogenic fibres such as asbestos. It is important to test this hypothesis to ascertain safe methods for CNT production, handling and disposal. In this study aspects reported to contribute to CNT toxicity were assessed: length, aspect ratio, iron content and crystallinity; with responses compared to industrially produced MWCNTs and toxicologically relevant materials such as asbestos. The impacts of these particles on a range of macrophage models in vitro were assessed due to the key role of macrophages in particle clearance and particle/fibre-induced disease. Industrially produced and long MWCNTs were cytotoxic to cells, and were potent in inducing pro-inflammatory and pro-fibrotic immune responses. Short CNTs did not induce any cytotoxicity. Frustrated phagocytosis was most evident in response to long CNTs, as was respiratory burst and reduction in phagocytic ability. Short CNTs, metal content and crystallinity had less or no influence on these endpoints, suggesting that many responses were fibre-length dependent. This study demonstrates that CNTs are potentially pathogenic, as they were routinely found to induce detrimental responses in macrophages greater than those induced by asbestos at the same mass-based dose.

An investigation into the potential for different surface-coated quantum dots to cause oxidative stress and affect macrophage cell signalling in vitro.

The aim of this study was to investigate the ability of a series of different surface-coated quantum dots (QDs) to cause oxidative stress and affect cell signalling in J774.A1 macrophages. Organic QDs caused a significant (p < 0.001) decrease in glutathione (GSH) levels over 24 h, while COOH and NH(2) (PEG) QDs induced a significant decrease (p < 0.05) in GSH at 6 and 24 h only. J774.A1 cytosolic Ca(2+) concentration significantly increased (p < 0.01) 30 min after treatment with all QDs. Trolox was, however, able to prevent the COOH and NH(2) (PEG) QD-induced Ca(2+) signal, but not the organic QD induced effect. All QDs tested were observed to have a relatively low ability to stimulate increased expression of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha). In conclusion, QDs differ in their interactions with macrophages according to their specific surface properties.