Systems toxicology of complex wood combustion aerosol reveals gaseous carbonyl compounds as critical constituents.

Epidemiological studies identified air pollution as one of the prime causes for human morbidity and mortality, due to harmful effects mainly on the cardiovascular and respiratory systems. Damage to the lung leads to several severe diseases such as fibrosis, chronic obstructive pulmonary disease and cancer. Noxious environmental aerosols are comprised of a gas and particulate phase representing highly complex chemical mixtures composed of myriads of compounds. Although some critical pollutants, foremost particulate matter (PM), could be linked to adverse health effects, a comprehensive understanding of relevant biological mechanisms and detrimental aerosol constituents is still lacking. Here, we employed a systems toxicology approach focusing on wood combustion, an important source for air pollution, and demonstrate a key role of the gas phase, specifically carbonyls, in driving adverse effects. Transcriptional profiling and biochemical analysis of human lung cells exposed at the air-liquid-interface determined DNA damage and stress response, as well as perturbation of cellular metabolism, as major key events. Connectivity mapping revealed a high similarity of gene expression signatures induced by wood smoke and agents prompting DNA-protein crosslinks (DPCs). Indeed, various gaseous aldehydes were detected in wood smoke, which promote DPCs, initiate similar genomic responses and are responsible for DNA damage provoked by wood smoke. Hence, systems toxicology enables the discovery of critical constituents of complex mixtures i.e. aerosols and highlights the role of carbonyls on top of particulate matter as an important health hazard.

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 (TiO2), raises concern regarding their potential adverse effects to humans. While considerable efforts have been made to assess the safety of TiO2 NMs using in vitro and in vivo studies, results obtained to date are unreliable, possibly due to the dynamic agglomeration behavior of TiO2 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 TiO2 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-TiO2 (17 nm) and a non-nano TiO2 (117 nm) was selected for this study. Stable stock dispersions of small agglomerates and their respective larger counterparts of each TiO2 particles were prepared, and human bronchial epithelial (HBE) cells were exposed to different doses of aerosolized TiO2 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.

A novel TEM grid sampler for airborne particles to measure the cell culture surface dose.

The applied surface dose is a key parameter for the measurement of toxic effects of airborne particles by air liquid interface exposure of human lung cells. Besides online measurement of the deposited particle mass by quartz crystal microbalance frequently other dose metrics such as particle size distribution, surface and agglomeration state are required. These particle properties and their spatial distribution can be determined by digital processing of micrographs obtained by transmission electron microscopy (TEM). Here, we report the development and characterization of a novel holder for film coated TEM copper grids, which allows for sampling under identical geometric and ambient conditions as in a cell culture chamber. The sample holder avoids artefacts by reliable grounding of the grids and improves handling of the grids to prevent damage of the sensitive film. This sample holder is applied during exposure experiments with titanium dioxide nanoparticles. The measured dose of 0.2 µg/cm² corresponds well to the mass loading signal of the quartz crystal microbalance. Additionally, the spatial distribution of particles on the sampling surface shows a good homogeneity of deposition. This novel sampling method allows verifying other dosimetry methods and gives additional information about particle properties and homogeneity of the dose.

Air-Liquid Interface Exposure of Lung Epithelial Cells to Low Doses of Nanoparticles to Assess Pulmonary Adverse Effects.

Reliable and predictive in vitro assays for hazard assessments of manufactured nanomaterials (MNMs) are still limited. Specifically, exposure systems which more realistically recapitulate the physiological conditions in the lung are needed to predict pulmonary toxicity. To this end, air-liquid interface (ALI) systems have been developed in recent years which might be better suited than conventional submerged exposure assays. However, there is still a need for rigorous side-by-side comparisons of the results obtained with the two different exposure methods considering numerous parameters, such as different MNMs, cell culture models and read outs. In this study, human A549 lung epithelial cells and differentiated THP-1 macrophages were exposed under submerged conditions to two abundant types of MNMs i.e., ceria and titania nanoparticles (NPs). Membrane integrity, metabolic activity as well as pro-inflammatory responses were recorded. For comparison, A549 monocultures were also exposed at the ALI to the same MNMs. In the case of titania NPs, genotoxicity was also investigated. In general, cells were more sensitive at the ALI compared to under classical submerged conditions. Whereas ceria NPs triggered only moderate effects, titania NPs clearly initiated cytotoxicity, pro-inflammatory gene expression and genotoxicity. Interestingly, low doses of NPs deposited at the ALI were sufficient to drive adverse outcomes, as also documented in rodent experiments. Therefore, further development of ALI systems seems promising to refine, reduce or even replace acute pulmonary toxicity studies in animals.

Characterization of Nanoparticle Batch-To-Batch Variability.

A central challenge for the safe design of nanomaterials (NMs) is the inherent variability of NM properties, both as produced and as they interact with and evolve in, their surroundings. This has led to uncertainty in the literature regarding whether the biological and toxicological effects reported for NMs are related to specific NM properties themselves, or rather to the presence of impurities or physical effects such as agglomeration of particles. Thus, there is a strong need for systematic evaluation of the synthesis and processing parameters that lead to potential variability of different NM batches and the reproducible production of commonly utilized NMs. The work described here represents over three years of effort across 14 European laboratories to assess the reproducibility of nanoparticle properties produced by the same and modified synthesis routes for four of the OECD priority NMs (silica dioxide, zinc oxide, cerium dioxide and titanium dioxide) as well as amine-modified polystyrene NMs, which are frequently employed as positive controls for nanotoxicity studies. For 46 different batches of the selected NMs, all physicochemical descriptors as prioritized by the OECD have been fully characterized. The study represents the most complete assessment of NMs batch-to-batch variability performed to date and provides numerous important insights into the potential sources of variability of NMs and how these might be reduced.

Metabolic Profiling as Well as Stable Isotope Assisted Metabolic and Proteomic Analysis of RAW 264.7 Macrophages Exposed to Ship Engine Aerosol Emissions: Different Effects of Heavy Fuel Oil and Refined Diesel Fuel.

Exposure to air pollution resulting from fossil fuel combustion has been linked to multiple short-term and long term health effects. In a previous study, exposure of lung epithelial cells to engine exhaust from heavy fuel oil (HFO) and diesel fuel (DF), two of the main fuels used in marine engines, led to an increased regulation of several pathways associated with adverse cellular effects, including pro-inflammatory pathways. In addition, DF exhaust exposure was shown to have a wider response on multiple cellular regulatory levels compared to HFO emissions, suggesting a potentially higher toxicity of DF emissions over HFO. In order to further understand these effects, as well as to validate these findings in another cell line, we investigated macrophages under the same conditions as a more inflammation-relevant model. An air-liquid interface aerosol exposure system was used to provide a more biologically relevant exposure system compared to submerged experiments, with cells exposed to either the complete aerosol (particle and gas phase), or the gas phase only (with particles filtered out). Data from cytotoxicity assays were integrated with metabolomics and proteomics analyses, including stable isotope-assisted metabolomics, in order to uncover pathways affected by combustion aerosol exposure in macrophages. Through this approach, we determined differing phenotypic effects associated with the different components of aerosol. The particle phase of diluted combustion aerosols was found to induce increased cell death in macrophages, while the gas phase was found more to affect the metabolic profile. In particular, a higher cytotoxicity of DF aerosol emission was observed in relation to the HFO aerosol. Furthermore, macrophage exposure to the gas phase of HFO leads to an induction of a pro-inflammatory metabolic and proteomic phenotype. These results validate the effects found in lung epithelial cells, confirming the role of inflammation and cellular stress in the response to combustion aerosols.

Toxicity of wood smoke particles in human A549 lung epithelial cells: the role of PAHs, soot and zinc.

Indoor air pollution is associated with increased morbidity and mortality. Specifically, the health impact of emissions from domestic burning of biomass and coal is most relevant and is estimated to contribute to over 4 million premature deaths per year worldwide. Wood is the main fuel source for biomass combustion and the shift towards renewable energy sources will further increase emissions from wood combustion even in developed countries. However, little is known about the constituents of wood smoke and biological mechanisms that are responsible for adverse health effects. We exposed A549 lung epithelial cells to collected wood smoke particles and found an increase in cellular reactive oxygen species as well as a response to bioavailable polycyclic aromatic hydrocarbons. In contrast, cell vitality and regulation of the pro-inflammatory cytokine interleukin-8 were not affected. Using a candidate approach, we could recapitulate WSP toxicity by the combined actions of its constituents soot, metals and PAHs. The soot fraction and metals were found to be the most important factors for ROS formation, whereas the PAH response can be mimicked by the model PAH benzo[a]pyrene. Strikingly, PAHs adsorbed to WSPs were even more potent in activating target gene expression than B[a]P individually applied in suspension. As PAHs initiate multiple adverse outcome pathways and are prominent carcinogens, their role as key pollutants in wood smoke and its health effects warrants further investigation. The presented results suggest that each of the investigated constituents soot, metals and PAHs are major contributors to WSP toxicity. Mitigation strategies to prevent adverse health effects of wood combustion should therefore not only aim at reducing the emitted soot and PAHs but also the metal content, through the use of more efficient combustion appliances, and particle precipitation techniques, respectively.

Particulate matter from both heavy fuel oil and diesel fuel shipping emissions show strong biological effects on human lung cells at realistic and comparable in vitro exposure conditions.

BACKGROUND: Ship engine emissions are important with regard to lung and cardiovascular diseases especially in coastal regions worldwide. Known cellular responses to combustion particles include oxidative stress and inflammatory signalling. OBJECTIVES: To provide a molecular link between the chemical and physical characteristics of ship emission particles and the cellular responses they elicit and to identify potentially harmful fractions in shipping emission aerosols. METHODS: Through an air-liquid interface exposure system, we exposed human lung cells under realistic in vitro conditions to exhaust fumes from a ship engine running on either common heavy fuel oil (HFO) or cleaner-burning diesel fuel (DF). Advanced chemical analyses of the exhaust aerosols were combined with transcriptional, proteomic and metabolomic profiling including isotope labelling methods to characterise the lung cell responses. RESULTS: The HFO emissions contained high concentrations of toxic compounds such as metals and polycyclic aromatic hydrocarbon, and were higher in particle mass. These compounds were lower in DF emissions, which in turn had higher concentrations of elemental carbon ("soot"). Common cellular reactions included cellular stress responses and endocytosis. Reactions to HFO emissions were dominated by oxidative stress and inflammatory responses, whereas DF emissions induced generally a broader biological response than HFO emissions and affected essential cellular pathways such as energy metabolism, protein synthesis, and chromatin modification. CONCLUSIONS: Despite a lower content of known toxic compounds, combustion particles from the clean shipping fuel DF influenced several essential pathways of lung cell metabolism more strongly than particles from the unrefined fuel HFO. This might be attributable to a higher soot content in DF. Thus the role of diesel soot, which is a known carcinogen in acute air pollution-induced health effects should be further investigated. For the use of HFO and DF we recommend a reduction of carbonaceous soot in the ship emissions by implementation of filtration devices.

Silica nanoparticles are less toxic to human lung cells when deposited at the air-liquid interface compared to conventional submerged exposure.

BACKGROUND: Investigations on adverse biological effects of nanoparticles (NPs) in the lung by in vitro studies are usually performed under submerged conditions where NPs are suspended in cell culture media. However, the behaviour of nanoparticles such as agglomeration and sedimentation in such complex suspensions is difficult to control and hence the deposited cellular dose often remains unknown. Moreover, the cellular responses to NPs under submerged culture conditions might differ from those observed at physiological settings at the air-liquid interface. RESULTS: In order to avoid problems because of an altered behaviour of the nanoparticles in cell culture medium and to mimic a more realistic situation relevant for inhalation, human A549 lung epithelial cells were exposed to aerosols at the air-liquid interphase (ALI) by using the ALI deposition apparatus (ALIDA). The application of an electrostatic field allowed for particle deposition efficiencies that were higher by a factor of more than 20 compared to the unmodified VITROCELL deposition system. We studied two different amorphous silica nanoparticles (particles produced by flame synthesis and particles produced in suspension by the Stöber method). Aerosols with well-defined particle sizes and concentrations were generated by using a commercial electrospray generator or an atomizer. Only the electrospray method allowed for the generation of an aerosol containing monodisperse NPs. However, the deposited mass and surface dose of the particles was too low to induce cellular responses. Therefore, we generated the aerosol with an atomizer which supplied agglomerates and thus allowed a particle deposition with a three orders of magnitude higher mass and of surface doses on lung cells that induced significant biological effects. The deposited dose was estimated and independently validated by measurements using either transmission electron microscopy or, in case of labelled NPs, by fluorescence analyses. Surprisingly, cells exposed at the ALI were less sensitive to silica NPs as evidenced by reduced cytotoxicity and inflammatory responses. CONCLUSION: Amorphous silica NPs induced qualitatively similar cellular responses under submerged conditions and at the ALI. However, submerged exposure to NPs triggers stronger effects at much lower cellular doses. Hence, more studies are warranted to decipher whether cells at the ALI are in general less vulnerable to NPs or specific NPs show different activities dependent on the exposure method.

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

BACKGROUND: 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.In this in vitro 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. RESULTS: 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 A2 isoforms 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/TXB2 via 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/TXB2 together with the phosphorylation of ERK1/2 and JNK1/2 proteins was decreased after pre-treatment of macrophages with the metal chelator deferoxamine mesylate (DFO). CONCLUSIONS: 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.

The response of a co-culture lung model to fine and ultrafine particles of incinerator fly ash at the air-liquid interface.

Elevated concentrations of particulate matter in the environmental atmosphere constitute a potential risk to human health. In vitro cell-based assays are therefore necessary to evaluate the toxicological potential of inhaled particulate emissions. In this study, the exposure of a co-culture cell model at the air-liquid interface was used to evaluate the dose-dependent biological effects of a test aerosol. The CULTEX system was used to expose human cells to an environmentally-relevant aerosol, generated from fly ash collected in a commercial municipal waste incinerator and resuspended in filtered air. Human bronchial epithelial cells, BEAS-2B, co-cultured with differentiated THP-1 macrophages growing on Transwell inserts, were employed in the bioassay. Analyses of cell viability, interleukin-8 (IL-8) release, intracellular glutathione, and haeme oxygenase-1 enzyme expression were performed. Transportation of the cells and exposure to humidified filtered air or the test aerosol, at 100 ml/min for 1 to 6 hours, were well tolerated by the cells and had no effect on their viability. Levels of IL-8 release and haeme oxygenase-1 expression were elevated by exposure to fly ash aerosol as a function of time, but not by exposure to clean air. For IL-8 release, a dose-dependent effect was demonstrated with the assumption that the deposited mass of the particles correlated with exposure time. Exposure to the test aerosol did not affect the intracellular glutathione concentration. This in vitro approach simulates particle deposition in the human lung more realistically than does submerged exposure, and it preserves the inherent properties of the particles. It shows promise for use to detect particulate emissions which are potentially detrimental to human health.

Electron-beam treatment of aromatic hydrocarbons that can be air-stripped from contaminated groundwater. 2. Gas-phase studies.

The electron-beam (EB) degradation of volatile aromatics (benzene, toluene, ethylbenzene, xylenes: BTEX) in groundwater strip gas, which in the present work has been modeled by the introduction of the desired aromatic(s) to a stream of air or another gas, such as oxygen, is initiated essentially by the addition of *OH radicals to the aromatic ring, giving rise to hydroxycyclohexadienyl radicals, which form the corresponding peroxyl radicals upon addition of oxygen. As studied in some detail with benzene as a BTEX representative, various reactions of these lead to numerous oxidation products in a cascade of reactions, including the decomposition of products under the prevailing conditions of high turnover of the initial aromatic. Importantly, hydroxycyclohexadienylperoxyl radical formation is partly reversible, and the reactions of the hydroxycyclohexadienyl radicals, which thus have a significant presence in these systems, must therefore also be taken into consideration. In the gas phase, in contrast to the aqueous phase (see Part 1), the reactions of the hydroxycyclohexadienyl radicals lead to oligomeric products that appear to contribute, in addition to ionic clusters, to nucleation for the aerosols observed. Various nitrated products, among them nitrophenols, are observed when air is used for the stripping. However, these studies did not clear the pilot plant stage, since BTEX degradation using a bioreactor carried out in parallel was so successful that the EB technology was judged to be noncompetitive. As for the latter, expensive equipment consisting of a stripper, the EB machine, and an aerosol precipitator would be required. The condensed aerosols are biorefractory and would require further treatment for detoxification.