Abstract
The use of nanostructured silica (SiO2) particles is no longer restricted to biomedical and (bio-) technological fields but rather finding applications in products of the food industry. ...Thus, our studies on the toxicological relevance of SiO2 nanoparticles focused on cytotoxic effects, the modulation of the cellular redox status and the impact on DNA integrity in human colon carcinoma cells (HT29). The results indicate that these SiO2 nanoparticles stimulate the proliferation of HT29 cells, depending on the incubation time and the particle size. The cytotoxicity of the investigated SiO2 nanoparticles was found to depend on the concentration, size and on the FCS content of the culture medium. Furthermore, SiO2 seem to interfere with glutathione biosynthesis. The results indicate further that effects of SiO2 NPs are not mediated by oxidative stress but by interference with the MAPK/ERK1/2 as well as the Nrf2/ARE signalling pathways. Additionally, investigations regarding DNA integrity revealed no substantial (oxidative) DNA damage.
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.
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).
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.
Today nanosciences are experiencing massive investment worldwide although research on toxicological aspects of these nano-sized particles has just begun and to date, no clear guidelines exist to ...quantify the effects. In the present study, we focus on carbon nanotubes (CNTs), which represent one of the most widely investigated carbon nanoparticles. The present data indicate that CNTs are able to cross the cell membrane of rat macrophages (NR8383) and, therefore, might have an influence on cell physiology and function. NR8383 and human A549 lung cells were incubated with commercial single-walled (NT-1) and multi-walled (NT-2, NT-3) CNTs, carbon black and quartz as reference particles as well as an acid-treated single-walled CNT preparation (SWCNT a.t.) with reduced metal catalyst content. We did not observe any acute toxicity on cell viability (WST-1, PI-staining) upon incubation with all CNT products. None of the CNTs induced the inflammatory mediators NO, TNF-α and IL-8. A rising tendency of TNF-α release from LPS-primed cells due to CNT treatment could be observed. We detected however, a dose- and time-dependent increase of intracellular reactive oxygen species and a decrease of the mitochondrial membrane potential with the commercial CNTs in both cell types after particle treatment whereas incubation with the purified CNTs (SWCNT a.t.) had no effect. This leads us to the conclusion that metal traces associated with the commercial nanotubes are responsible for the biological effects.
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.
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 flyash 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 100ml/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.
Nanostructured silica particles are commonly used in biomedical and biotechnical fields, as well as, in cosmetics and food industry. Thus, their environmental and health impacts are of great interest ...and effects after oral uptake are only rarely investigated. In the present study, the toxicological effects of commercially available nano-scaled silica with a nominal primary diameter of 12 nm were investigated on the human gastric carcinoma cell line GXF251L. Besides the analysis of cytotoxic and proliferative effects and the comparison with effects of particles with a nominal primary diameter of 200 nm, emphasis was also given to their influence on the cellular epidermal growth factor receptor (EGFR) and mitogen-activated protein kinases (MAPK) signaling pathways-both of them deeply involved in the regulation of cellular processes like cell cycle progression, differentiation or proliferation. The investigated silica nanoparticles (NPs) were found to stimulate cell proliferation as measured by microscopy and the sulforhodamine B assay. In accordance, the nuclear level of the proliferation marker Ki-67 was enhanced in a concentration-dependent manner. At high particle concentrations also necrosis was induced. Finally, silica NPs affected the EGFR and MAPK pathways at various levels dependent on concentration and time. However, classical activation of the EGFR, to be reflected by enhanced levels of phosphorylation, could be excluded as major trigger of the proliferative stimulus. After 45 min of incubation the level of phosphorylated EGFR did not increase, whereas enhanced levels of total EGFR protein were observed. These results indicate interference with the complex homeostasis of the EGFR protein, whereby up to 24 h no impact on the transcription level was detected. In addition, downstream on the level of the MAP kinases ERK1/2 short term incubation appeared to affect total protein levels without clear increase in phosphorylation. Depending on the concentration range, enhanced levels of ERK1/2 phosphorylation were only observed after 24 h of incubation. Taken together, the present study demonstrates the potential of the tested silica particles to enhance the growth of gastric carcinoma cells. Although interference with the EGFR/MAPK cascade is observed, additional mechanisms are likely to be involved in the onset of the proliferative stimulus.
Engineered amorphous silica nanoparticles (nanosilica) are widely used in industry yet can induce adverse effects, which might be classified according to the oxidative stress model. However, the ...underlying mechanisms as well as the potential interactions of the three postulated different tiers of toxicity—i.e. oxidative-, pro-inflammatory- and cytotoxic-stress response—are poorly understood. As macrophages are primary targets of nanoparticles, we used several macrophage models, primarily murine RAW264.7 macrophages, and monitored pro-inflammatory and anti-oxidative reactions as well as cytotoxicity in response to nanosilica at max. 50 µg/mL. Special attention was given to the activation of mitogen-activated protein kinases (MAPKs) as potential regulators of the cellular stress response. Indeed, according to the oxidative stress model, also nanosilica elicits an, albeit modest, anti-oxidative response as well as pronounced pro-inflammatory reactions and cytotoxicity in macrophages. Interestingly however, these three tiers of toxicity seem to operate separately of each other for nanosilica. Specifically, impeding the anti-oxidative response by scavenging of reactive oxygen species does not prevent the pro-inflammatory and cytotoxic response. Furthermore, blocking the pro-inflammatory response by inhibition of MAPKs does not impair cell death. As hazard assessment has been guided by the prevailing assumption of a dose-dependent coupling of sequential tiers of toxicity, identification of critical physico-chemical parameters to assist the safe-by-design concept should be enabled by simply monitoring one of the toxicity read-outs. Our results indicate a more complex scenario in the case of nanosilica, which triggers independent pleiotropic effects possibly also related to different material properties and primary cellular targets.
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.
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