Impact of the FcgammaII-receptor on quartz uptake and inflammatory response by alveolar macrophages.

The inflammatory response following particle inhalation is described as a key event in the development of lung diseases, e.g., fibrosis and cancer. The essential role of alveolar macrophages (AM) in the pathogenicity of particles through their functions in lung clearance and mediation of inflammation is well known. However, the molecular mechanisms and direct consequences of particle uptake are still unclear. Inhibition of different classic phagocytosis receptors by flow cytometry shows a reduction of the dose-dependent quartz particle (DQ12) uptake in the rat AM cell line NR8383. Thereby the strongest inhibitory effect was observed by blocking the FcgammaII-receptor (FcgammaII-R). Fluorescence immunocytochemistry, demonstrating FcgammaII-R clustering at particle binding sites as well as transmission electron microscopy, visualizing zippering mechanism-like morphological changes, confirmed the role of the FcgammaII-R in DQ12 phagocytosis. FcgammaII-R participation in DQ12 uptake was further strengthened by the quartz-induced activation of the Src-kinase Lyn, the phospho-tyrosine kinases Syk (spleen tyrosine kinase) and PI3K (phosphatidylinositol 3-kinase), as shown by Western blotting. Activation of the small GTPases Rac1 and Cdc42, shown by immunoprecipitation, as well as inhibition of tyrosine kinases, GTPases, or Rac1 provided further support for the role of the FcgammaII-R. Consistent with the uptake results, FcgammaII-R activation with its specific ligand caused a similar generation of reactive oxygen species and TNF-alpha release as observed after treatment with DQ12. In conclusion, our results indicate a major role of FcgammaII-R and its downstream signaling cascade in the phagocytosis of quartz particles in AM as well as in the associated generation and release of inflammatory mediators.

Surface-dependent quartz uptake by macrophages: potential role in pulmonary inflammation and lung clearance.

Inhalation of quartz particles is associated with a variety of adverse lung effects. Since particle surface is considered to be crucial for particle pathogenicity, we investigated the influence of quartz surface properties on lung burden, inflammation (bronchoalveolar lavage cells), and cytotoxicity (protein, lactate dehydrogenase, beta-glucuronidase) 90 days after a single intratracheal instillation of 2 mg DQ12 into rats. The role of particle surface characteristics was investigated by comparative investigation of native versus surface-modified quartz, using polyvinylpyridine N-oxide (PVNO) or aluminum lactate (AL) coating. Uptake and subcellular localization of quartz samples as well as tumor necrosis factor (TNF)-alpha release were determined using NR8383 rat alveolar macrophages. Surface modification of quartz particles resulted in marked in vivo and in vitro changes. Compared to native quartz, modified quartz samples showed lower lung burden at 90 days, as well as decreased inflammatory and cytotoxic responses. Coating with polyvinylpyridine N-oxide (PVNO) appeared to be more effective than aluminium lactate (AL). PVNO-coating of quartz also resulted in an enhanced particle uptake by macrophages up to 24 h, whereas AL coating caused a transient reduction of quartz uptake at 2 h. At 24 h differences with the native quartz were absent. Subcellular localization of quartz particles was not affected by surface modifications. However, surface modification resulted in a reduced release of TNF-alpha. In conclusion, surface properties of quartz particles appear to be crucial for rate and extent of in vitro particle uptake in macrophages. Our in vivo findings also indicate that quartz surface properties may affect clearance kinetics. Particle surface-specific interactions between quartz and macrophages may therefore play a major role in the pulmonary pathogenicity of quartz.

Endocytosis, oxidative stress and IL-8 expression in human lung epithelial cells upon treatment with fine and ultrafine TiO2: role of the specific surface area and of surface methylation of the particles.

Inhaled ultrafine particles show considerably stronger pulmonary inflammatory effects when tested at equal mass dose with their fine counterparts. However, the responsible mechanisms are not yet fully understood. We investigated the role of particle size and surface chemistry in initiating pro-inflammatory effects in vitro in A549 human lung epithelial cells on treatment with different model TiO(2) particles. Two samples of TiO(2), i.e. fine (40-300 nm) and ultrafine (20-80 nm) were tested in their native forms as well as upon surface methylation, as was confirmed by Fourier transformed infrared spectroscopy. Radical generation during cell treatment was determined by electron paramagnetic resonance with 5,5-dimethyl-1-pyrroline-N-oxide or 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl. Interleukin-8 mRNA expression/release was determined by RT-PCR and ELISA, whereas particle uptake was evaluated by transmission electron microscopy. TiO(2) particles were rapidly taken up by the cells, generally as membrane bound aggregates and large intracellular aggregates in vesicles, vacuoles and lamellar bodies. Aggregate size tended to be smaller for the ultrafine samples and was also smaller for methylated fine TiO(2) when compared to non-methylated fine TiO(2). No particles were observed inside nuclei or any other vital organelle. Both ultrafine TiO(2) samples but not their fine counterparts elicited significantly stronger oxidant generation and IL-8 release, despite their aggregation state and irrespective of their methylation. The present data indicate that ultrafine TiO(2), even as aggregates/agglomerates, can trigger inflammatory responses that appear to be driven by their large surface area. Furthermore, our results indicate that these effects result from oxidants generated during particle-cell interactions through a yet to be elucidated mechanism(s).

Actin plays a crucial role in the phagocytosis and biological response to respirable quartz particles in macrophages.

The uptake of respirable quartz particles by alveolar macrophages (AM) is believed to cause an inflammatory response, which is discussed as a crucial step in quartz pathogenicity. However, little is known about the mechanism and the relevance of particle uptake. Therefore, the aim of this study was to analyze the role of the actin cytoskeleton in quartz particle uptake, reactive oxygen species generation (ROS) and tumour necrosis factor alpha (TNF-alpha) release. Primary rat alveolar and interstitial macrophages (IM) as well as a rat alveolar macrophage cell line (NR8383) were treated with quartz particles at various concentrations and time intervals. Particle uptake was studied using flow cytometry and light/fluorescence microscopy to analyze particle uptake and cytoskeleton recruitment. Intra- as well as extracellular ROS generation was analyzed by flow cytometry and electron spin resonance (ESR). Flow cytometric investigations demonstrated a dose- and time-dependent particle uptake. Primary AM showed a similar uptake indicating that the cell line provides a good model to investigate the mechanisms of particle uptake while primary IM had a lower uptake rate. Inhibition of actin polymerization using cytochalasin-D caused a significant reduction of particle uptake in NR8383 cells. The quartz induced dose-dependent increase of ROS generation and TNF-alpha release was also blocked by inhibition of actin polymerization. Our results demonstrate an active involvement of the cytoskeleton in uptake of quartz particles and suggest a role of the actin framework and/or the particle uptake in DQ12-induced ROS generation and cytokine release.

Inhibition of the mitochondrial respiratory chain function abrogates quartz induced DNA damage in lung epithelial cells.

Respirable quartz dust has been classified as a human carcinogen by the International Agency for Research on Cancer. The aim of our study was to investigate the mechanisms of DNA damage by DQ12 quartz in RLE-6TN rat lung epithelial type II cells (RLE). Transmission electron microscopy and flow-cytometry analysis showed a rapid particle uptake (30 min to 4 h) of quartz by the RLE cells, but particles were not found within the cell nuclei. This suggests that DNA strand breakage and induction of 8-hydroxydeoxyguanosine - as also observed in these cells during these treatment intervals - did not result from direct physical interactions between particles and DNA, or from short-lived particle surface-derived reactive oxygen species. DNA damage by quartz was significantly reduced in the presence of the mitochondrial inhibitors rotenone and antimycin-A. In the absence of quartz, these inhibitors did not affect DNA damage, but they reduced cellular oxygen consumption. No signs of apoptosis were observed by quartz. Flow-cytometry analysis indicated that the reduced DNA damage by rotenone was not due to a possible mitochondria-mediated reduction of particle uptake by the RLE cells. Further proof of concept for the role of mitochondria was shown by the failure of quartz to elicit DNA damage in mitochondria-depleted 143B (rho-0) osteosarcoma cells, at concentrations where it elicited DNA damage in the parental 143B cell line. In conclusion, our data show that respirable quartz particles can elicit oxidative DNA damage in vitro without entering the nuclei of type II cells, which are considered to be important target cells in quartz carcinogenesis. Furthermore, our observations indicate that such indirect DNA damage involves the mitochondrial electron transport chain function, by an as-yet-to-be elucidated mechanism.

Induction of CYP1A1 in rat lung cells following in vivo and in vitro exposure to quartz.

Respirable quartz has been classified as a human lung carcinogen, but the mechanism by which quartz exposure leads to lung cancer has not been clarified. Consistently higher risks of lung cancer are reported in smokers with quartz exposure and we therefore hypothesised that quartz exposure may alter the expression of enzyme systems involved in activation/detoxification of pre-carcinogens in cigarette smoke. More specifically we studied cytochrome P4501A1 (CYP1A1) expression using reverse transcriptase polymerase chain reaction and immunohistochemistry (IHC) upon in vitro and in vivo quartz exposure. In vitro incubation of rat lung epithelial cells with DQ12 quartz for 24 h showed a dose-dependent induction of CYP1A1-mRNA. On the other hand, CYP1A1 message was not increased in lung epithelial cells isolated from rats at 3, 28 or 90 days after intratracheal instillation of 2 mg DQ12. Following IHC for CYP1A1 protein in rat lung sections from later time-points (180 and 360 days), we observed an increase in the number of CYP1A1 positive cells. After in vivo quartz exposure, protein expression of the Aryl hydrocarbon receptor (AhR) was increased and nuclear translocation of AhR was observed at the same time-points. In conclusion, our findings demonstrate an effect of quartz exposure on chronic CYP1A1 expression in vivo, whereas the in vitro models show an immediate upregulation. We suggest that this upregulation of CYP1A1 may act as a co-carcinogenic pathway in quartz exposed workers by activation of pre-carcinogens such as those present in cigarette smoke.

The crucial role of particle surface reactivity in respirable quartz-induced reactive oxygen/nitrogen species formation and APE/Ref-1 induction in rat lung.

Persistent inflammation and associated excessive oxidative stress have been crucially implicated in quartz-induced pulmonary diseases, including fibrosis and cancer. We have investigated the significance of the particle surface reactivity of respirable quartz dust in relation to the in vivo generation of reactive oxygen and nitrogen species (ROS/RNS) and the associated induction of oxidative stress responses in the lung. Therefore, rats were intratracheally instilled with 2 mg quartz (DQ12) or quartz whose surface was modified by either polyvinylpyridine-N-oxide (PVNO) or aluminium lactate (AL). Seven days after instillation, the bronchoalveolar lavage fluid (BALF) was analysed for markers of inflammation (total/differential cell counts), levels of pulmonary oxidants (H2O2, nitrite), antioxidant status (trolox equivalent antioxidant capacity), as well as for markers of lung tissue damage, e.g. total protein, lactate dehydrogenase and alkaline phosphatase. Lung homogenates as well as sections were investigated regarding the induction of the oxidative DNA-lesion/oxidative stress marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) using HPLC/ECD analysis and immunohistochemistry, respectively. Homogenates and sections were also investigated for the expression of the bifunctional apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) by Western blotting and immunohistochemistry. Significantly increased levels of H2O2 and nitrite were observed in rats treated with non-coated quartz, when compared to rats that were treated with either saline or the surface-modified quartz preparations. In the BALF, there was a strong correlation between the number of macrophages and ROS, as well as total cells and RNS. Although enhanced oxidant generation in non-coated DQ12-treated rats was paralleled with an increased total antioxidant capacity in the BALF, these animals also showed significantly enhanced lung tissue damage. Remarkably however, elevated ROS levels were not associated with an increase in 8-OHdG, whereas the lung tissue expression of APE/Ref-1 protein was clearly up-regulated. The present data provide further in vivo evidence for the crucial role of particle surface properties in quartz dust-induced ROS/RNS generation by recruited inflammatory phagocytes. Our results also demonstrate that quartz dust can fail to show steady-state enhanced oxidative DNA damage in the respiratory tract, in conditions were it elicits a marked and persistent inflammation with associated generation of ROS/RNS, and indicate that this may relate to compensatory induction of APE/Ref-1 mediated base excision repair.

Variability in quartz exposure in the construction industry: implications for assessing exposure-response relations.

The aims of this study were to determine implications of inter- and intraindividual variation in exposure to respirable (quartz) dust and of heterogeneity in dust characteristics for epidemiologic research in construction workers. Full-shift personal measurements (n = 67) from 34 construction workers were collected. The between-worker and day-to-day variances of quartz and respirable dust exposure were estimated using mixed models. Heterogeneity in dust characteristics was evaluated by electron microscopic analysis and electron spin resonance. A grouping strategy based on job title resulted in a 2- and 3.5-fold reduction in expected attenuation of a hypothetical exposure-response relation for respirable dust and quartz exposure, respectively, compared to an individual based approach. Material worked on explained most of the between-worker variance in respirable dust and quartz exposure. However, for risk assessment in epidemiology, grouping workers based on the materials they work on is not practical. Microscopic characterization of dust samples showed large quantities of aluminum silicates and large quantities of smaller particles, resulting in a D(50) between 1 and 2 microm. For risk analysis, job title can be used to create exposure groups, although error is introduced by the heterogeneity of dust produced by different construction workers activities and by the nonuniformity of exposure groups. A grouping scheme based on materials worked on would be superior, for both exposure and risk assessment, but is not practical when assessing past exposure. In dust from construction sites, factors are present that are capable of influencing the toxicological potency.

Inflammatory time course after quartz instillation: role of tumor necrosis factor-alpha and particle surface.

Inflammation has been suggested as the key factor in the development of quartz-induced fibrosis and carcinogenesis, and particle surface properties are argued as an important characteristic responsible for these pathologic alterations. To evaluate the effect of surface modification on acute and subchronic inflammation, female Wistar rats were intratracheally instilled with 2 mg native quartz, or quartz coated either with polyvinyl-pyridine-N-oxide or with aluminium lactate. Various markers of lung toxicity, inflammation, and oxidative stress were found to be enhanced at 3, 7, 21, and 90 d after instillation of native quartz. Quartz-treated animals also showed enhanced immunostaining of nuclear factor-kappaB (NF-kappaB) in alveolar macrophages and lung epithelium, as well as reduced IkappaBalpha levels in whole lung homogenate. Both surface modifications were found to inhibit most of the effects as observed with native quartz. NF-kappaB activation was also observed in vitro in rat lung epithelial cells following treatment with lavage fluid from quartz-treated animals, as well as with conditioned medium of quartz-treated macrophages, and these effects appeared to be at least partly tumor necrosis factor-alpha-independent. In conclusion, the persistent subchronic inflammatory lung response after quartz exposure appears to be particle surface-driven and is associated with NF-kappaB activation in both alveolar macrophages and the lung epithelium.

Surface modification of quartz inhibits toxicity, particle uptake, and oxidative DNA damage in human lung epithelial cells.

Quartz (crystalline silica) is not consistently carcinogenic across different industries where similar quartz exposure occurs. In addition, there are reports that surface modification of quartz affects its cytotoxicity, inflammogenicity, and fibrogenicity. Taken together, these data suggest that the carcinogenicity of quartz is also related to particle surface characteristics, and so we determined the genotoxic effects of DQ12 quartz particles versus DQ12 whose surface was modified by treating with either aluminum lactate or polyvinylpyridine-N-oxide (PVNO). The different particle preparations were characterized for hydroxyl-radical generation using electron spin resonance (ESR). DNA damage was determined by immunocytochemical analysis of 8-hydroxydeoxyguanosine (8-OHdG) and the alkaline comet-assay using A549 human lung epithelial cells. Cytotoxicity was measured using the LDH- and MTT-assays, and particle uptake by the A549 cells was quantified by light microscopy, using digital light imaging evaluation of 800 nm sections. The ability of quartz to generate hydroxyl-radicals in the presence of hydrogen peroxide was markedly reduced upon surface modification with aluminum lactate or PVNO. DNA strand breakage and 8-OHdG formation, as produced by quartz at nontoxic concentrations, could be completely prevented by both coating materials. Particle uptake into A549 cells appeared to be significantly inhibited by the PVNO-coating, and to a lesser extent by the aluminum-lactate coating. Our data demonstrate that respirable quartz particles induce oxidative DNA damage in human lung epithelial cells and indicates that surface properties of the quartz as well as particle uptake by these target cells are important in the cytotoxic and the genotoxic effects of quartz in vitro.

DNA damage in lung epithelial cells isolated from rats exposed to quartz: role of surface reactivity and neutrophilic inflammation.

Respirable quartz has been classified as a human lung carcinogen (IARC, 1997). However, the mechanisms involved in quartz-induced carcinogenesis remain unclear. The aim of the present study was to investigate acute DNA damage in epithelial lung cells from rats exposed to quartz. Since surface reactivity is considered to play a crucial role in the toxicity of quartz, the effect of surface modifying agents polyvinylpyridine-N-oxide (PVNO) and aluminium lactate (AL) was evaluated. Therefore, rats were instilled with quartz (DQ12, 2 mg/rat) or quartz treated with PVNO or AL. After 3 days animals were killed and brochoalveolar lavage (BAL) was performed to evaluate inflammatory cell influx. BAL-fluid levels of lactate dehydrogenase (LDH), alkaline phosphatase (AP) and total protein were used as lung damage markers. Neutrophil activation was assessed by myeloperoxidase (MPO) measurement, and total antioxidant capacity of the BAL-fluid was determined using the TEAC (trolox equivalent antioxidant capacity) assay. Lung epithelial cells were isolated and DNA strand breakage was determined by single cell gel electrophoresis (comet assay). DNA damage was significantly increased in epithelial cells from rats instilled with DQ12, whereas no enhanced DNA strand breakage was observed when quartz was treated with PVNO or AL. Total protein, LDH and TEAC were increased in rats treated with native quartz, and this was inhibited by both coatings. A significant correlation between neutrophil numbers and MPO levels was observed, indicating neutrophil activation. Inhibition of DNA damage by both coatings was paralleled by a reduction of neutrophil influx as well as MPO activity. In this study we provide evidence that modification of the particle surface prevents DNA strand breakage in epithelial lung cells from quartz-exposed rats. Furthermore, the present data show the feasibility of our in vivo model to evaluate the role of inflammation, antioxidant status, and cytotoxicity in particle-induced DNA damage.

The surface area rather than the surface coating determines the acute inflammatory response after instillation of fine and ultrafine TiO2 in the rat.

Alterations in the hydrophobic status of particle surfaces have been suggested to modify the toxic properties of ultrafine TiO2. We investigated the acute inflammatory responses and cell damage after intratracheal instillation of surface modified (hydrophilic and hydrophobic) fine (180 nm) and ultrafine (20-30 nm) TiO2 particles 16 h at equivalent mass (1 or 6 mg) and surface doses (100, 500, 600 and 3000 cm2) in rats. Inflammatory response and most enzyme levels were significantly related to the administered surface dose. The hydrophobic surface of the TiO2 particles, achieved by methylation, induced a lower total cell number and influx of neutrophils (PMN) compared to rats instilled with the 1 mg of the untreated, fine or ultrafine TiO2 but the outcomes were not statistically significant. No differences were observed between fine/ultrafine and hydophilic/hydrophobic TiO2 at the high dose (6 mg) or surface dose over 600 cm2. The differences in BAL cellularity at the low dose were reflected in changes in the chemokine MIP-2, but no differences were seen in levels of macrophage cytokines. Considering the large influx of PMN little cell damage was seen when studying enzyme leakage in lavage fluid, although PMNs appeared to be activated as suggested by increased myeloperoxidase (MPO) activity in the lavage fluid. We conclude that the surface area rather than the hydrophobic surface determines the acute, pulmonary inflammation induced by both fine and ultrafine TiO2.

[Differentiation processes in the germinating Gemmula ofEphydatia fluviatilis]. / Differenzierungsvorgänge in der keimenden Gemmula vonEphydatia fluviatilis.

The dormant shelled gemmulae of the fresh water spongeEphydatia fluviatilis contain uniform, totipotent statocytes (thésocytes), which can differentiate either into archaeocytes (mono- and binucleated) or into histoblasts. The histoblasts accumulate at the villus near the micropyle. The other cell types orientate in a three-dimensional pattern at the micropyle, according to a developing gradient.After the primary pinacoderm is formed, the sponge primordium is released through the open micropyle. The primordium develops into a new sponge, into which archaeocytes, histoblasts and scleroblasts migrate.