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 benzoapyrene. Strikingly, PAHs adsorbed to WSPs were even more potent in activating target gene expression than BaP 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.
The introduction of engineered nanostructured materials into a rapidly increasing number of industrial and consumer products will result in enhanced exposure to engineered nanoparticles. Workplace ...exposure has been identified as the most likely source of uncontrolled inhalation of engineered aerosolized nanoparticles, but release of engineered nanoparticles may occur at any stage of the lifecycle of (consumer) products. The dynamic development of nanomaterials with possibly unknown toxicological effects poses a challenge for the assessment of nanoparticle induced toxicity and safety.
In this consensus document from a workshop on in-vitro cell systems for nanoparticle toxicity testing
1
1
Workshop on ‘In-Vitro Exposure Studies for Toxicity Testing of Engineered Nanoparticles’ sponsored by the Association for Aerosol Research (GAeF), 5–6 September 2009, Karlsruhe, Germany.
an overview is given of the main issues concerning exposure to airborne nanoparticles, lung physiology, biological mechanisms of (adverse) action, in-vitro cell exposure systems, realistic tissue doses, risk assessment and social aspects of nanotechnology. The workshop participants recognized the large potential of in-vitro cell exposure systems for reliable, high-throughput screening of nanoparticle toxicity. For the investigation of lung toxicity, a strong preference was expressed for air–liquid interface (ALI) cell exposure systems (rather than submerged cell exposure systems) as they more closely resemble in-vivo conditions in the lungs and they allow for unaltered and dosimetrically accurate delivery of aerosolized nanoparticles to the cells. An important aspect, which is frequently overlooked, is the comparison of typically used in-vitro dose levels with realistic in-vivo nanoparticle doses in the lung. If we consider average ambient urban exposure and occupational exposure at 5
mg/m
3 (maximum level allowed by Occupational Safety and Health Administration (OSHA)) as the boundaries of human exposure, the corresponding upper-limit range of nanoparticle flux delivered to the lung tissue is 3×10
−5–5×10
-3
μg/h/cm
2 of lung tissue and 2–300
particles/h/(epithelial) cell. This range can be easily matched and even exceeded by almost all currently available cell exposure systems.
The consensus statement includes a set of recommendations for conducting in-vitro cell exposure studies with pulmonary cell systems and identifies urgent needs for future development. As these issues are crucial for the introduction of safe nanomaterials into the marketplace and the living environment, they deserve more attention and more interaction between biologists and aerosol scientists. The members of the workshop believe that further advances in in-vitro cell exposure studies would be greatly facilitated by a more active role of the aerosol scientists. The technical know-how for developing and running ALI in-vitro exposure systems is available in the aerosol community and at the same time biologists/toxicologists are required for proper assessment of the biological impact of nanoparticles.
► Air–liquid interface (ALI) cell systems facilitate pulmonary nanotoxicity testing. ► Overview on aerosol and biology issues relevant for ALI cell exposure systems. ► Upper limits for realistic nanoparticle doses delivered to the lung tissue. ► Most ALI exposure systems match or exceed realistic nanoparticle tissue doses. ► Recommendations for conducting pulmonary in-vitro cell exposure studies.
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.
•OMICs analysis reveals a strong impact of the gas phase of wood smoke.•CMAP indicates DPC agents as drivers of OMICs response.•Carbonyls were identified as DPC agents and key pollutants triggering ...DNA damage and stress signalling.
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.
In this paper, trace metals (As, Be, Cd, Cr, Hg Ni, Pb, Se and V) emission from the atmospheric entrained flow gasifier REGA at Karlsruhe Institute of Technology, Germany is presented. Elemental ...composition and metals concentration in glycol (model fuel) and straw char samples were determined. In order to achieve well defined and reproducible operating conditions, gasification experiments were carried out with glycol and slurry (straw char mixed with glycol) as fuel. Trace metals emission and distribution into particle and gas phase in syngas from gasification were measured according to US EPA method 29. During glycol gasification, metals concentration in syngas ranged from 0.1 mug/Nm super(3) (V) to 4.6 mug/Nm super(3) (Cr). In slurry gasification, metals concentration in syngas ranged from 2.4 mug/Nm super(3) (Hg) to 53.2 mug/Nm super(3) (Ni). Trace metals tend to volatilize more in reducing gasification environment. Tendency of metals to speciate into particle phase increased in slurry gasification than glycol gasification due to increased in particle concentration and unburned carbon content in syngas. The increase of particle and carbon content in syngas possessed strong influence in distribution behavior of As, Be and Pb, however, such influence was less for other metals studied. Mercury in syngas was speciated predominantly into elemental form followed by oxidized form whereas share of particle bond mercury was the least.
In vitro toxicity testing of airborne particles usually takes place in multi-well plates, where the cells are exposed to a suspension of particles in cell culture medium. Due to the artefacts caused ...by particle collection and preparation of suspensions, the air–liquid interface (ALI) exposure is challenging this conventional exposure technique to become the method of choice. The ALI technique allows for direct sampling of an aerosol and exposure of cell cultures to airborne particles. At the same time, it reflects the physiological conditions in the lung to a greater extent. So far, the available ALI systems have mostly been laboratory set-ups of the single components. Here, we present a mobile and complete system providing all process technology required for cell exposure experiments at dynamic aerosol sources. The system is controlled by a human machine interface (HMI) with standard routines for experiments and internal testing to assure reproducibility. It also provides documentation of the exposure experiment regarding process parameters and measured doses. The performance of this system is evaluated using fluorescein-sodium dosimetry, which is also used to determine the factor of dose enhancement by optional electrostatic deposition. The application of the system is shown for two different technical aerosol sources: wood smoke particles emitted by a household log wood stove and emissions from a ship diesel engine. After exposure of lung cells, cytotoxicity and gene regulation on a genome-wide scale were analysed.
•We present a fully automated air–liquid-interface exposure system.•System characterisation shows a high reproducibility.•Wood combustion aerosol causes cytotoxicity in human lung epithelial cells.•Gene regulation caused by ship diesel emissions in human lung epithelial cells.
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.
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.
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.
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.
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.
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.
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