Erwin Karg

Instillation of six different ultrafine carbon particles indicates a surface area threshold dose for acute lung inflammation in mice.

Increased levels of particulate air pollution are associated with increased respiratory and cardiovascular mortality and morbidity. Some epidemiologic and toxicologic research suggests ultrafine particles (UFPs) (< 100 nm) to be more harmful per unit mass than larger particles. Our study was aimed at a quantitative comparison of acute adverse effects of different types of carbonaceous UFPs at a dose range that causes a moderate inflammatory response in lungs. We used six different particle types (primary particle size 10–50 nm, specific surface area 30–800 m2/g, and organic content 1–20%): PrintexG, Printex90, flame soot particles with different organic content (SootL, SootH), spark-generated ultrafine carbon particles (ufCP), and the reference diesel exhaust particles (DEP) SRM1650a. Mice were instilled with 5, 20, and 50 μg of each particle type, and bronchoalveolar lavage was analyzed 24 hr after instillation for inflammatory cells and the level of proinflammatory cytokines. At respective mass-doses, particle-caused detrimental effects ranked in the following order: ufCP > SootL ≥ SootH > Printex90 > PrintexG > DEP. Relating the inflammatory effects to the particle characteristics—organic content, primary particle size, or specific surface area—demonstrates the most obvious dose response for particle surface area. Our study suggests that the surface area measurement developed by Brunauer, Emmett, and Teller is a valuable reference unit for the assessment of causative health effects for carbonaceous UFPs. Additionally, we demonstrated the existence of a threshold for the particle surface area at an instilled dose of approximately 20 cm2, below which no acute proinflammatory responses could be detected in mice.

Distribution Pattern of Inhaled Ultrafine Gold Particles in the Rat Lung

The role of alveolar macrophages in the fate of ultrafine particles in the lung was investigated. Male Wistar-Kyoto rats were exposed to ultrafine gold particles, generated by a spark generator, for 6 h at a concentration of 88 μg/m3 (4 × 106/cm3, 16 nm modal mobility diameter). Up to 7 days, the animals were serially sacrificed, and lavaged cells and lung tissues were examined by transmission electron microscopy. The gold concentration/content in the lung, lavage fluid, and blood was estimated by inductively coupled plasma–mass spectrometry. Gold particles used were spherical and electron dense with diameters of 5–8 nm. The particles were individual or slightly agglomerated. By inductively coupled plasma–mass spectrometry analysis of the lung, 1945 ± 57 ng (mean ± SD) and 1512 ± 184 ng of gold were detected on day 0 and on day 7, respectively, indicating that a large portion of the deposited gold particles was retained in the lung tissue. In the lavage fluid, 573 ± 67 ng and 96 ± 29 ng were found on day 0 and day 7, respectively, which means that 29% and 6% of the retained gold particles were lavageable on these days. A low but significant increase of gold (0.03 to 0.06% of lung concentration) was found in the blood. Small vesicles containing gold particles were found in the cytoplasm of alveolar macrophages. In the alveolar septum, the gold particles were enclosed in vesicles observed in the cytoplasm of alveolar type I epithelial cells. These results indicate that inhaled ultrafine gold particles in alveolar macrophages and type I epithelial cells are processed by endocytotic pathways, though the uptake of the gold particles by alveolar macrophages is limited. To a low degree, systemic particle translocation took place.

A dose-controlled system for air-liquid interface cell exposure and application to zinc oxide nanoparticles

BackgroundEngineered nanoparticles are becoming increasingly ubiquitous and their toxicological effects on human health, as well as on the ecosystem, have become a concern. Since initial contact with nanoparticles occurs at the epithelium in the lungs (or skin, or eyes), in vitro cell studies with nanoparticles require dose-controlled systems for delivery of nanoparticles to epithelial cells cultured at the air-liquid interface.ResultsA novel air-liquid interface cell exposure system (ALICE) for nanoparticles in liquids is presented and validated. The ALICE generates a dense cloud of droplets with a vibrating membrane nebulizer and utilizes combined cloud settling and single particle sedimentation for fast (~10 min; entire exposure), repeatable (<12%), low-stress and efficient delivery of nanoparticles, or dissolved substances, to cells cultured at the air-liquid interface. Validation with various types of nanoparticles (Au, ZnO and carbon black nanoparticles) and solutes (such as NaCl) showed that the ALICE provided spatially uniform deposition (<1.6% variability) and had no adverse effect on the viability of a widely used alveolar human epithelial-like cell line (A549). The cell deposited dose can be controlled with a quartz crystal microbalance (QCM) over a dynamic range of at least 0.02-200 μg/cm2. The cell-specific deposition efficiency is currently limited to 0.072 (7.2% for two commercially available 6-er transwell plates), but a deposition efficiency of up to 0.57 (57%) is possible for better cell coverage of the exposure chamber.Dose-response measurements with ZnO nanoparticles (0.3-8.5 μg/cm2) showed significant differences in mRNA expression of pro-inflammatory (IL-8) and oxidative stress (HO-1) markers when comparing submerged and air-liquid interface exposures. Both exposure methods showed no cellular response below 1 μg/cm2 ZnO, which indicates that ZnO nanoparticles are not toxic at occupationally allowed exposure levels.ConclusionThe ALICE is a useful tool for dose-controlled nanoparticle (or solute) exposure of cells at the air-liquid interface. Significant differences between cellular response after ZnO nanoparticle exposure under submerged and air-liquid interface conditions suggest that pharmaceutical and toxicological studies with inhaled (nano-)particles should be performed under the more realistic air-liquid interface, rather than submerged cell conditions.

Deducing in vivo toxicity of combustion-derived nanoparticles from a cell-free oxidative potency assay and metabolic activation of organic compounds.

Background The inhalation of combustion-derived nanoparticles (CDNPs) is believed to cause an oxidative stress response, which in turn may lead to pulmonary or even systemic inflammation. Objective and Methods In this study we assessed whether the in vivo inflammatory response—which is generally referred to as particle toxicity—of mice to CDNPs can be predicted in vitro by a cell-free ascorbate test for the surface reactivity or, more precisely, oxidative potency (OxPot) of particles. Results For six types of CDNPs with widely varying particle diameter (10–50 nm), organic content (OC; 1–20%), and specific Brunauer, Emmett, and Teller (BET) surface area (43–800 m2/g), OxPot correlated strongly with the in vivo inflammatory response (pulmonary polymorphonuclear neutrophil influx 24 hr after intratracheal particle instillation). However, for CDNPs with high organic content, OxPot could not explain the observed inflammatory response, possibly due to shielding of the OxPot of the carbon core of CDNPs by an organic coating. On the other hand, a pathway-specific gene expression screen indicated that, for particles rich in polycyclic aromatic hydrocarbon (PAHs), cytochrome P450 1A1 (CYP1A1) enzyme-mediated biotransformation of bio-available organics may generate oxidative stress and thus enhance the in vivo inflammatory response. Conclusion The compensatory nature of both effects (shielding of carbon core and biotransformation of PAHs) results in a good correlation between inflammatory response and BET surface area for all CDNPs. Hence, the in vivo inflammatory response can either be predicted by BET surface area or by a simple quantitative model, based on in vitro OxPot and Cyp1a1 induction.

Fate and toxic effects of inhaled ultrafine cadmium oxide particles in the rat lung.

Female Fischer 344 rats were exposed to ultrafine cadmium oxide particles, generated by spark discharging, for 6 h at a concentration of 70 μg Cd/m3 (1× 106/cm3) (40 nm modal diameter). Lung morphology and quantification of Cd content/concentration by inductively coupled plasma (ICP)–mass spectrometry were performed on days 0, 1, 4, and 7 after exposure. Cd content in the lung on day 0 was 0.53± 0.12 μg/lung, corresponding to 19% of the estimated total inhaled cumulative dose, and the amount remained constant throughout the study. In the liver no significant increase of Cd content was found up to 4 days. A slight but statistically significant increase was observed in the liver on day 7. We found neither exposure-related morphological changes of lungs nor inflammatory responses in lavaged cells. Another group of rats were exposed to a higher concentration of ultrafine CdO particles (550 μg Cd/m3 for 6 h, 51 nm modal diameter). The rats were sacrificed immediately and 1 day after exposure. The lavage study performed on day 0 showed an increase in the percentage of neutrophils. Multifocal alveolar inflammation was seen histologically on day 0 and day 1. Although the Cd content in the lung was comparable between day 0 and day 1 (3.9 μg/lung), significant elevation of Cd levels in the liver and kidneys was observed on both days. Two of 4 rats examined on day 0 showed elevation of blood cadmium, indicating systemic translocation of a fraction of deposited Cd from the lung in this group. These results and comparison with reported data using fine CdO particles indicate that inhalation of ultrafine CdO particles results in efficient deposition in the rat lung. With regard to the deposition dose, adverse health effects of ultrafine CdO and fine CdO appear to be comparable. Apparent systemic translocation of Cd took place only in animals exposed to a high concentration that induced lung injury.

Inflammatory and Oxidative Stress Responses of an Alveolar Epithelial Cell Line to Airborne Zinc Oxide Nanoparticles at the Air-Liquid Interface: A Comparison with Conventional, Submerged Cell-Culture Conditions

The biological effects of inhalable nanoparticles have been widely studied in vitro with pulmonary cells cultured under submerged and air-liquid interface (ALI) conditions. Submerged exposures are experimentally simpler, but ALI exposures are physiologically more realistic and hence potentially biologically more meaningful. In this study, we investigated the cellular response of human alveolar epithelial-like cells (A549) to airborne agglomerates of zinc oxide (ZnO) nanoparticles at the ALI, compared it to the response under submerged culture conditions, and provided a quantitative comparison with the literature data on different types of particles and cells. For ZnO nanoparticle doses of 0.7 and 2.5 μg ZnO/cm2 (or 0.09 and 0.33 cm2 ZnO/cm2), cell viability was not mitigated and no significant effects on the transcript levels of oxidative stress markers (HMOX1, SOD-2 and GCS) were observed. However, the transcript levels of proinflammatory markers (IL-8, IL-6, and GM-CSF) were induced to higher levels under ALI conditions. This is consistent with the literature data and it suggests that in vitro toxicity screening of nanoparticles with ALI cell culture systems may produce less false negative results than screening with submerged cell cultures. However, the database is currently too scarce to draw a definite conclusion on this issue.

Platelet adhesion and fibrinogen deposition in murine microvessels upon inhalation of nanosized carbon particles

Summary.  Background: The translocation of nanoparticles in the lung toward effector organs via the circulation is considered an important direct pathway for systemic effects of nanoparticles after inhalation. Recently, we have reported that a moderate dose of systemically administered nanosized carbon black particles exerted thrombogenic effects in hepatic microvessels of healthy mice. Objectives: This study addresses the questions of whether similar thrombogenic effects are also evoked upon inhalation of nanosized carbon particles (NCP) and whether NCP‐induced hepatic platelet accumulation is associated with pulmonary or systemic inflammation. Methods: Two and 8 h after a 24‐h exposure to either filtered air or to NCP, intravital fluorescence microscopy of the hepatic microcirculation was performed in C57Bl/6 mice. Parameters of pulmonary or systemic inflammatory response were determined in bronchoalveolar lavage and blood/plasma samples. Results: Inhalative exposure to NCP caused platelet accumulation in the hepatic microvasculature, whereas leukocyte recruitment and sinusoidal perfusion did not differ from controls. Fibrinogen deposition was detected by immunohistochemistry in both hepatic and cardiac microvessels from NCP‐exposed mice. In contrast, inhalation of NCP affected neither the plasma levels of proinflammatory cytokines nor blood cell counts. Moreover, the bronchoalveolar lavage data indicate that no significant inflammatory response occurred in the lung. Conclusions: Thus, exposure to NCP exerts thrombogenic effects in the microcirculation of healthy mice independent of the route of administration (i.e. inhalation or systemic intra‐arterial administration). The NCP‐induced thrombogenic effects are not liver specific, are associated with neither a local nor a systemic inflammatory response, and seem to be independent of pulmonary inflammation.

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.

Effects of ultrafine particles-induced oxidative stress on Clara cells in allergic lung inflammation

BackgroundClara cell protein (CC16), the main secretory product of bronchiolar Clara cells, plays an important protective role in the respiratory tract against oxidative stress and inflammation. The purpose of the study was to investigate the role of elemental carbon ultrafine particles (EC-UFP)-induced oxidative stress on Clara cells and CC16 in a mouse model of allergic lung inflammation.MethodsOvalbumin (OVA)-sensitized mice were exposed to EC-UFP (507 μg/m3 for 24 h) or filtered air immediately prior to allergen challenge and systemically treated with N-acetylcysteine (NAC) or vehicle prior and during EC-UFP inhalation. CC16 was measured up to one week after allergen challenge in bronchoalveolar lavage fluid (BALF) and in serum. The relative expression of CC16 and TNF-α mRNA were measured in lung homogenates. A morphometrical analysis of mucus hypersecretion and electron microscopy served to investigate goblet cell metaplasia and Clara cell morphological alterations.ResultsIn non sensitized mice EC-UFP inhalation caused alterations in CC16 concentration, both at protein and mRNA level, and induced Clara cell hyperplasia. In sensitized mice, inhalation of EC-UFP prior to OVA challenge caused most significant alterations of BALF and serum CC16 concentration, BALF total protein and TNF-α relative expression compared to relevant controls; their Clara cells displayed the strongest morphological alterations and strongest goblet cell metaplasia occurred in the small airways. NAC strongly reduced both functional and morphological alterations of Clara cells.ConclusionOur findings demonstrate that oxidative stress plays an important role in EC-UFP-induced augmentation of functional and morphological alterations of Clara cells in allergic lung inflammation.

Model for the Deposition of Aerosol Particles in the Respiratory Tract of the Rat. I. Nonhygroscopic Particle Deposition

Rats are used to test the toxicological and pharmacological effects of aerosol particles on the organism. For estimates of the delivered aerosol dose, lung deposition models provide a valuable tool. Here a previously developed deposition model for nonhygroscopic and hygroscopic aerosol particles in the lungs of man (Ferron et al., J. Aerosol Sci. 1988, 19:611) is adapted to the rat by implementing a lung structure for the rat combined with empirical equations for particle deposition due to impaction/sedimentation in the extrathoracic region and in bifurcations. To account for the effect of body weight (BW) on the physiological parameters (lung size, respiration frequency) we present BW-scaling laws with an estimated accuracy of about 16%. The present model shows good agreement with the measured total deposition (per breath) and other models from the literature to within the variability of the experimental data (20% absolute). Our calculations show that the variability of the experimental data is consistent with the combined effects from realistic variations in particle properties (mainly density) and physiological parameters (mainly activity level). For the alveolar region, which is of particular significance for pharmacological and health studies, we show that although the activity level may change the deposited dose by up to a factor of 2.2 for particles between 0.05 and 2.0 microm in diameter, the alveolar dose is almost independent (to within 10%) of activity level for particles between 0.5 and 1 microm, which makes this size range advantageous for pharmacological and toxicological experiments. The present model allows estimates of the total and regional particle dose deposited in the lungs of rats, which are consistent with experimental data. The advantage of the present model is that hygroscopic growth can be included in the calculations.