Characterisation of inhalable aerosols from carbon fibres

Carbon fibres (CF) and CF-reinforced plastics (CFRPs) are innovative materials, which are increasingly produced, recycled and disposed, possibly releasing particles and fibres. The BMBF project “Carbon Fibre Cycle – CFC” has the aim to identify respirable particles, fibres and fibre fragments after thermal and mechanical treatment of CF/CFRP, to analyse them and to assess pulmonary toxicity. The physical properties of CF being similar to asbestos raise serious concerns about potentially harmful effects in the lung. Inhalable aerosols of mechanically or mechanical-thermally treated CF are provided and characterized, which are deposited at the air-liquid interface onto human lung cells in an exposure system, where toxicological investigations are carried out, i.e. directly on the apical surface of cell cultures, in order to simulate lung-like conditions. Lung epithelial cells and macrophages in mono and co-culture are used for toxicological evaluation of respirable CF fragments focussing on determination of cytotoxicity, gene expression analyses and determination of proinflammatory, profibrotic and genotoxic potential. Commercial short carbon fibres based on polyacrylnitrile (PAN) were investigated after mechanically or mechanical-thermal treatment. The aerosol from the exposure system was on the one hand sampled on filters which were analysed by different microscopy methods and on the other hand the deposited dose on the cell surfaces was measured. All images from digital and scanning electron microscopy were evaluated using the image analysis software FibreShape (IST AG, Switzerland) in combination with own data post processing. The fibres were analysed regarding length, diameter and in a further step the aerodynamic equivalent diameter was calculated. Fibre characterisation is discussed in the context with biological responses caused by inhalable CF. This project is financed by the Federal Ministry of Education and Research under project number FK03XP0195 which is greatly acknowledged

Regulation of the arachidonic acid mobilization in macrophages by combustion-derived particles

<p>Abstract</p> <p>Background</p> <p>Acute exposure to elevated levels of environmental particulate matter (PM) is associated with increasing morbidity and mortality rates. These adverse health effects, e.g. culminating in respiratory and cardiovascular diseases, have been demonstrated by a multitude of epidemiological studies. However, the underlying mechanisms relevant for toxicity are not completely understood. Especially the role of particle-induced reactive oxygen species (ROS), oxidative stress and inflammatory responses is of particular interest.</p> <p>In this <it>in vitro </it>study we examined the influence of particle-generated ROS on signalling pathways leading to activation of the arachidonic acid (AA) cascade. Incinerator fly ash particles (MAF02) were used as a model for real-life combustion-derived particulate matter. As macrophages, besides epithelial cells, are the major targets of particle actions in the lung murine RAW264.7 macrophages and primary human macrophages were investigated.</p> <p>Results</p> <p>The interaction of fly ash particles with macrophages induced both the generation of ROS and as part of the cellular inflammatory responses a dose- and time-dependent increase of free AA, prostaglandin E₂/thromboxane B₂(PGE₂/TXB₂), and 8-isoprostane, a non-enzymatically formed oxidation product of AA. Additionally, increased phosphorylation of the mitogen-activated protein kinases (MAPK) JNK1/2, p38 and ERK1/2 was observed, the latter of which was shown to be involved in MAF02-generated AA mobilization and phosphorylation of the cytosolic phospolipase A₂. Using specific inhibitors for the different phospolipase A₂isoforms the MAF02-induced AA liberation was shown to be dependent on the cytosolic phospholipase A₂, but not on the secretory and calcium-independent phospholipase A₂. The initiation of the AA pathway due to MAF02 particle exposure was demonstrated to depend on the formation of ROS since the presence of the antioxidant N-acetyl-cysteine (NAC) prevented the MAF02-mediated enhancement of free AA, the subsequent conversion to PGE₂/TXB₂via the induction of COX-2 and the ERK1/2 and JNK1/2 phosphorylation. Finally we showed that the particle-induced formation of ROS, liberation of AA and PGE₂/TXB₂together with the phosphorylation of ERK1/2 and JNK1/2 proteins was decreased after pre-treatment of macrophages with the metal chelator deferoxamine mesylate (DFO).</p> <p>Conclusions</p> <p>These results indicate that one of the primary mechanism initiating inflammatory processes by incinerator fly ash particles seems to be the metal-mediated generation of ROS, which triggers via the MAPK cascade the activation of AA signalling pathway.</p

Comparing α-Quartz-Induced Cytotoxicity and Interleukin-8 Release in Pulmonary Mono- and Co-Cultures Exposed under Submerged and Air-Liquid Interface Conditions

The occupational exposure to particles such as crystalline quartz and its impact on the respiratory tract have been studied extensively in recent years. For hazard assessment, the development of physiologically more relevant in-vitro models, i.e., air-liquid interface (ALI) cell cultures, has greatly progressed. Within this study, pulmonary culture models employing A549 and differentiated THP-1 cells as mono-and co-cultures were investigated. The different cultures were exposed to α-quartz particles (Min-U-Sil5) with doses ranging from 15 to 66 µg/cm(2) under submerged and ALI conditions and cytotoxicity as well as cytokine release were analyzed. No cytotoxicity was observed after ALI exposure. Contrarily, Min-U-Sil5 was cytotoxic at the highest dose in both submerged mono- and co-cultures. A concentration-dependent release of interleukin-8 was shown for both exposure types, which was overall stronger in co-cultures. Our findings showed considerable differences in the toxicological responses between ALI and submerged exposure and between mono- and co-cultures. A substantial influence of the presence or absence of serum in cell culture media was noted as well. Within this study, the submerged culture was revealed to be more sensitive. This shows the importance of considering different culture and exposure models and highlights the relevance of communication between different cell types for toxicological investigations

Gene Expression Profiling of Mono- and Co-Culture Models of the Respiratory Tract Exposed to Crystalline Quartz under Submerged and Air-Liquid Interface Conditions

In vitro lung cell models like air-liquid interface (ALI) and 3D cell cultures have advanced greatly in recent years, being especially valuable for testing advanced materials (e.g., nanomaterials, fibrous substances) when considering inhalative exposure. Within this study, we established submerged and ALI cell culture models utilizing A549 cells as mono-cultures and co-cultures with differentiated THP-1 (dTHP-1), as well as mono-cultures of dTHP-1. After ALI and submerged exposures towards α-quartz particles (Min-U-Sil5), with depositions ranging from 15 to 60 µg/cm(2), comparison was made with respect to their transcriptional cellular responses employing high-throughput RT-qPCR. A significant dose- and time-dependent induction of genes coding for inflammatory proteins, e.g., IL-1A, IL-1B, IL-6, IL-8, and CCL22, as well as genes associated with oxidative stress response such as SOD2, was observed, even more pronounced in co-cultures. Changes in the expression of similar genes were more pronounced under submerged conditions when compared to ALI exposure in the case of A549 mono-cultures. Hereby, the activation of the NF-κB signaling pathway and the NLRP3 inflammasome seem to play an important role. Regarding genotoxicity, neither DNA strand breaks in ALI cultivated cells nor a transcriptional response to DNA damage were observed. Altogether, the toxicological responses depended considerably on the cell culture model and exposure scenario, relevant to be considered to improve toxicological risk assessment

Agglomeration State of Titanium-Dioxide (TiO2) Nanomaterials Influences the Dose Deposition and Cytotoxic Responses in Human Bronchial Epithelial Cells at the Air-Liquid Interface

Extensive production and use of nanomaterials (NMs), such as titanium dioxide (TiO(2)), raises concern regarding their potential adverse effects to humans. While considerable efforts have been made to assess the safety of TiO(2) NMs using in vitro and in vivo studies, results obtained to date are unreliable, possibly due to the dynamic agglomeration behavior of TiO(2) NMs. Moreover, agglomerates are of prime importance in occupational exposure scenarios, but their toxicological relevance remains poorly understood. Therefore, the aim of this study was to investigate the potential pulmonary effects induced by TiO(2) agglomerates of different sizes at the air–liquid interface (ALI), which is more realistic in terms of inhalation exposure, and compare it to results previously obtained under submerged conditions. A nano-TiO(2) (17 nm) and a non-nano TiO(2) (117 nm) was selected for this study. Stable stock dispersions of small agglomerates and their respective larger counterparts of each TiO(2) particles were prepared, and human bronchial epithelial (HBE) cells were exposed to different doses of aerosolized TiO(2) agglomerates at the ALI. At the end of 4h exposure, cytotoxicity, glutathione depletion, and DNA damage were evaluated. Our results indicate that dose deposition and the toxic potential in HBE cells are influenced by agglomeration and exposure via the ALI induces different cellular responses than in submerged systems. We conclude that the agglomeration state is crucial in the assessment of pulmonary effects of NMs