Showing papers in "Particle and Fibre Toxicology in 2007"

Kupffer cells are central in the removal of nanoparticles from the organism

Abstract: The study aims at revealing the fate of nanoparticles administered intravenously and intraperitoneally to adult female mice, some of which were pregnant. Gold nanoparticles were chosen as a model because these particles have been found to be chemically inert and at the same time are easily traced by autometallography (AMG) at both ultrastructural and light microscopic levels. Gold nanoparticles were injected intravenously (IV) or intraperitoneally (IP) and traced after 1, 4 or 24 hours. For IV injections 2 and 40 nm particles were used; for IP injections 40 nm particles only. The injected nanoparticles were found in macrophages only, and at moderate exposure primarily in the Kupffer cells in the liver. IV injections resulted in a rapid accumulation/clustering of nanoparticles in these liver macrophages, while the uptake in spleen macrophages was moderate. IP injections were followed by a delayed uptake in the liver and included a moderate uptake in macrophages located in mesenteric lymph nodes, spleen and small intestine. Ultrastructurally, the AMG silver enhanced nanocrystals were found in lysosome-like organelles of the Kupffer cells and other macrophages wherever located. Accumulations of gold nanoparticles were not found in any other organs analysed, i.e. kidneys, brain, lungs, adrenals, ovaries, placenta, and fetal liver, and the control animals were all void of AMG staining. Our results suggest that: (1) inert gold nanoparticles do not penetrate cell membranes by non-endocytotic mechanisms, but are rather taken up by endocytosis; (2) gold nanoparticles, independent of size, are taken up primarily by Kupffer cells in the liver and secondarily by macrophages in other places; (3) gold nanoparticles do not seem to penetrate the placenta barrier; (4) the blood-brain barrier seems to protect the central nervous system from gold nanoparticles; (5) 2 nanometer gold particles seem to be removed not only by endocytosis by macrophages, and we hypothesize that part of these tiny nanoparticles are released into the urine as a result of simple filtration in the renal glomeruli.

Ultrafine particles and platelet activation in patients with coronary heart disease – results from a prospective panel study

Abstract: Epidemiological studies on health effects of air pollution have consistently shown adverse cardiovascular effects. Toxicological studies have provided evidence for thrombogenic effects of particles. A prospective panel study in a susceptible population was conducted in Erfurt, Germany, to study the effects of daily changes in ambient particles on various blood cells and soluble CD40ligand (sCD40L, also known as CD154), a marker for platelet activation that can cause increased coagulation and inflammation. Blood cells and plasma sCD40L levels were repeatedly measured in 57 male patients with coronary heart disease (CHD) during winter 2000/2001. Fixed effects linear regression models were applied, adjusting for trend, weekday and meteorological parameters. Hourly data on ultrafine particles (UFP, number concentration of particles from 0.01 to 0.1 μm), mass concentration of particles less than 10 and 2.5 μm in diameter (PM10, PM2.5), accumulation mode particle counts (AP, 0.1–1.0 μm), elemental and organic carbon, gaseous pollutants and meteorological data were collected at central monitoring sites. An immediate increase in plasma sCD40L was found in association with UFP and AP (% change from geometric mean: 7.1; CI: [0.1, 14.5] and 6.9; CI: [0.5, 13.8], respectively). Platelet counts decreased in association with UFP showing an immediate, a three days delayed (lag 3) and a 5-day average response (% change from the mean: -1.8; CI: [-3.4,-0.2]; -2.4; CI: [-4.5,-0.3] and -2.2; CI: [-4.0,-0.3] respectively). The increased plasma sCD40L levels support the hypothesis that higher levels of ambient air pollution lead to an inflammatory response in patients with CHD thus providing a possible explanation for the observed association between air pollution and cardiovascular morbidity and mortality in susceptible parts of the population.

Cytokine responses of human lung cells (BEAS-2B) treated with micron-sized and nanoparticles of metal oxides compared to soil dusts

Abstract: The induction of cytokines by airway cells in vitro has been widely used to assess the effects of ambient and occupational particles. This study measured cytotoxicity and the release of the proinflammatory cytokines IL-6 and IL-8 by human bronchial epithelial cells treated with manufactured nano- and micron-sized particles of Al2O3, CeO2, Fe2O3, NiO, SiO2, and TiO2, with soil-derived particles from fugitive dust sources, and with the positive controls LPS, TNF-α, and VOSO4. The nano-sized particles were not consistently more potent than an equal mass of micron-sized particles of the same nominal composition for the induction of IL-6 and IL-8 secretion in the in vitro models used in this study. The manufactured pure oxides were much less potent than natural PM2.5 particles derived from soil dust, and the cells were highly responsive to the positive controls. The nano-sized particles in the media caused artifacts in the measurement of IL-6 by ELISA due to adsorption of the cytokine on the high-surface-area particles. The potency for inducing IL-6 secretion by BEAS-2B cells did not correlate with the generation of reactive oxygen species in cell-free media. Direct comparisons of manufactured metal oxide nanoparticles and previously studied types of particles and surrogate proinflammatory agonists showed that the metal oxide particles have low potency to induce IL-6 secretion in BEAS-2B cells. Particle artifacts from non-biological effects need to be considered in experiments of this type, and the limitations inherent in cell culture studies must be considered when interpreting in vitro results. This study suggests that manufactured metal oxide nanoparticles are not highly toxic to lung cells compared to environmental particles.

Relationship between redox activity and chemical speciation of size-fractionated particulate matter

Abstract: Although the mechanisms of airborne particulate matter (PM) related health effects remain incompletely understood, one emerging hypothesis is that these adverse effects derive from oxidative stress, initiated by the formation of reactive oxygen species (ROS) within affected cells. Typically, ROS are formed in cells through the reduction of oxygen by biological reducing agents, with the catalytic assistance of electron transfer enzymes and redox active chemical species such as redox active organic chemicals and metals. The purpose of this study was to relate the electron transfer ability, or redox activity, of the PM samples to their content in polycyclic aromatic hydrocarbons and various inorganic species. The redox activity of the samples has been shown to correlate with the induction of the stress protein, hemeoxygenase-1. Size-fractionated (i.e. < 0.15; < 2.5 and 2.5 – 10 μm in diameter) ambient PM samples were collected from four different locations in the period from June 2003 to July 2005, and were chemically analyzed for elemental and organic carbon, ions, elements and trace metals and polycyclic aromatic hydrocarbons. The redox activity of the samples was evaluated by means of the dithiothreitol activity assay and was related to their chemical speciation by means of correlation analysis. Our analysis indicated a higher redox activity on a per PM mass basis for ultrafine (< 0.15 μm) particles compared to those of larger sizes. The PM redox activity was highly correlated with the organic carbon (OC) content of PM as well as the mass fractions of species such as polycyclic aromatic hydrocarbons (PAH), and selected metals. The results of this work demonstrate the utility of the dithiothreitol assay for quantitatively assessing the redox potential of airborne particulate matter from a wide range of sources. Studies to characterize the redox activity of PM from various sources throughout the Los Angeles basin are currently underway.

Translocation of particles and inflammatory responses after exposure to fine particles and nanoparticles in an epithelial airway model.

Abstract: Experimental studies provide evidence that inhaled nanoparticles may translocate over the airspace epithelium and cause increased cellular inflammation. Little is known, however, about the dependence of particle size or material on translocation characteristics, inflammatory response and intracellular localization. Using a triple cell co-culture model of the human airway wall composed of epithelial cells, macrophages and dendritic cells we quantified the entering of fine (1 μm) and nano-sized (0.078 μm) polystyrene particles by laser scanning microscopy. The number distribution of particles within the cell types was significantly different between fine and nano-sized particles suggesting different translocation characteristics. Analysis of the intracellular localization of gold (0.025 μm) and titanium dioxide (0.02–0.03 μm) nanoparticles by energy filtering transmission electron microscopy showed differences in intracellular localization depending on particle composition. Titanium dioxide nanoparticles were detected as single particles without membranes as well as in membrane-bound agglomerations. Gold nanoparticles were found inside the cells as free particles only. The potential of the different particle types (different sizes and different materials) to induce a cellular response was determined by measurements of the tumour necrosis factor-α in the supernatants. We measured a 2–3 fold increase of tumour necrosis factor-α in the supernatants after applying 1 μm polystyrene particles, gold nanoparticles, but not with polystyrene and titanium dioxide nanoparticles. Quantitative laser scanning microscopy provided evidence that the translocation and entering characteristics of particles are size-dependent. Energy filtering transmission electron microscopy showed that the intracellular localization of nanoparticles depends on the particle material. Both particle size and material affect the cellular responses to particle exposure as measured by the generation of tumour necrosis factor-α.

Mechanisms of action of inhaled fibers, particles and nanoparticles in lung and cardiovascular diseases.

Abstract: Background A symposium on the mechanisms of action of inhaled airborne particulate matter (PM), pathogenic particles and fibers such as silica and asbestos, and nanomaterials, defined as synthetic particles or fibers less than 100 nm in diameter, was held on October 27 and 28, 2005, at the Environmental Protection Agency (EPA) Conference Center in Research Triangle Park, North Carolina. The meeting was the eighth in a series of transatlantic conferences first held in Penarth, Wales, at the Medical Research Council Pneumoconiosis Unit (1979), that have fostered long-standing collaborations between researchers in the fields of mineralogy, cell and molecular biology, pathology, toxicology, and environmental/occupational health.

Visualization and quantitative analysis of nanoparticles in the respiratory tract by transmission electron microscopy

Abstract: Nanotechnology in its widest sense seeks to exploit the special biophysical and chemical properties of materials at the nanoscale. While the potential technological, diagnostic or therapeutic applications are promising there is a growing body of evidence that the special technological features of nanoparticulate material are associated with biological effects formerly not attributed to the same materials at a larger particle scale. Therefore, studies that address the potential hazards of nanoparticles on biological systems including human health are required. Due to its large surface area the lung is one of the major sites of interaction with inhaled nanoparticles. One of the great challenges of studying particle-lung interactions is the microscopic visualization of nanoparticles within tissues or single cells both in vivo and in vitro. Once a certain type of nanoparticle can be identified unambiguously using microscopic methods it is desirable to quantify the particle distribution within a cell, an organ or the whole organism. Transmission electron microscopy provides an ideal tool to perform qualitative and quantitative analyses of particle-related structural changes of the respiratory tract, to reveal the localization of nanoparticles within tissues and cells and to investigate the 3D nature of nanoparticle-lung interactions. This article provides information on the applicability, advantages and disadvantages of electron microscopic preparation techniques and several advanced transmission electron microscopic methods including conventional, immuno and energy-filtered electron microscopy as well as electron tomography for the visualization of both model nanoparticles (e.g. polystyrene) and technologically relevant nanoparticles (e.g. titanium dioxide). Furthermore, we highlight possibilities to combine light and electron microscopic techniques in a correlative approach. Finally, we demonstrate a formal quantitative, i.e. stereological approach to analyze the distributions of nanoparticles in tissues and cells. This comprehensive article aims to provide a basis for scientists in nanoparticle research to integrate electron microscopic analyses into their study design and to select the appropriate microscopic strategy.

A comparison of dispersing media for various engineered carbon nanoparticles

Abstract: With the increased manufacture and use of carbon nanoparticles (CNP) there has been increasing concern about the potential toxicity of fugitive CNP in the workplace and ambient environment. To address this matter a number of investigators have conducted in vitro and in vivo toxicity assessments. However, a variety of different approaches for suspension of these particles (culture media, Tween 80, dimethyl sulfoxide, phosphate-buffered saline, fetal calf serum, and others), and different sources of materials have generated potentially conflicting outcomes. The quality of the dispersion of nanoparticles is very dependent on the medium used to suspend them, and this then will most likely affect the biological outcomes. In this work, the distributions of different CNP (sources and types) have been characterized in various media. Furthermore, the outcome of instilling the different agglomerates, or size distributions, was examined in mouse lungs after one and seven days. Our results demonstrated that CNP suspended in serum produced particle suspensions with the fewest large agglomerates, and the most uniform distribution in mouse lungs. In addition, no apparent clearance of instilled CNP took place from lungs even after seven days. This work demonstrates that CNP agglomerates are present in all dispersing vehicles to some degree. The vehicle that contains some protein, lipid or protein/lipid component disperses the CNP best, producing fewer large CNP agglomerates. In contrast, vehicles absent of lipid and protein produce the largest CNP agglomerates. The source of the CNP is also a factor in the degree of particle agglomeration within the same vehicle.

Initial in vitro screening approach to investigate the potential health and environmental hazards of Envirox™ – a nanoparticulate cerium oxide diesel fuel additive

Abstract: Nanotechnology is the new industrial revolution of the 21st Century as the various processes lead to radical improvements in medicine, manufacturing, energy production, land remediation, information technology and many other everyday products and applications. With this revolution however, there are undoubted concerns for health, safety and the environment which arise from the unique nature of materials and processes at the nanometre scale. The in vitro assays used in the screening strategy are all validated, internationally accepted protocols and provide a useful indication of potential toxicity of a chemical as a result of effects on various toxicological endpoints such as local site of contact (dermal) irritation, general cytotoxicity and mutagenicity. The initial in vitro screening strategy described in this paper to investigate the potential health implications, if any, which may arise following exposure to one specific application of nanoparticulate cerium oxide used as a diesel fuel borne catalyst, reflects a precautionary approach and the results will inform judgement on how best to proceed to ensure safe use.

Re-evaluation of pulmonary titanium dioxide nanoparticle distribution using the "relative deposition index": Evidence for clearance through microvasculature.

Abstract: Background: Translocation of nanoparticles (NP) from the pulmonary airways into other pulmonary compartments or the systemic circulation is controversially discussed in the literature. In a previous study it was shown that titanium dioxide (TiO 2 ) NP were "distributed in four lung compartments (air-filled spaces, epithelium/endothelium, connective tissue, capillary lumen) in correlation with compartment size". It was concluded that particles can move freely between these tissue compartments. To analyze whether the distribution of TiO2 NP in the lungs is really random or shows a preferential targeting we applied a newly developed method for comparing NP distributions. Methods: Rat lungs exposed to an aerosol containing TiO2 NP were prepared for light and electron microscopy at 1 h and at 24 h after exposure. Numbers of TiO2 NP associated with each compartment were counted using energy filtering transmission electron microscopy. Compartment size was estimated by unbiased stereology from systematically sampled light micrographs. Numbers of particles were related to compartment size using a relative deposition index and chi-squared analysis. Results: Nanoparticle distribution within the four compartments was not random at 1 h or at 24 h after exposure. At 1 h the connective tissue was the preferential target of the particles. At 24 h the NP were preferentially located in the capillary lumen. Conclusion: We conclude that TiO2 NP do not move freely between pulmonary tissue compartments, although they can pass from one compartment to another with relative ease. The residence time of NP in each tissue compartment of the respiratory system depends on the compartment and the time after exposure. It is suggested that a small fraction of TiO2 NP are rapidly transported from the airway lumen to the connective tissue and subsequently released into the systemic circulation.

Physicochemical characteristics and bronchial epithelial cell cytotoxicity of Folpan 80 WG® and Myco 500®, two commercial forms of folpet

Abstract: Pesticides, in particular folpet, have been found in rural and urban air in France in the past few years. Folpet is a contact fungicide and has been widely used for the past 50 years in vineyards in France. Slightly water-soluble and mostly present as particles in the environment, it has been measured at average concentration of 40.1 μg/m3 during its spraying, 0.16–1.2 μg/m3 in rural air and around 0.01 μg/m3 in urban air, potentially exposing both the workers and the general population. However, no study on its penetration by inhalation and on its respiratory toxicity has been published. The objective of this study was to determine the physicochemical characteristics of folpet particles (morphology, granulometry, stability) in its commercial forms under their typical application conditions. Moreover, the cytotoxic effect of these particles and the generation of reactive oxygen species were assessed in vitro on respiratory cells. Granulometry of two commercial forms of folpet (Folpan 80WG® and Myco 500®) under their typical application conditions showed that the majority of the particles (>75%) had a size under 5 μm, and therefore could be inhaled by humans. These particles were relatively stable over time: more than 75% of folpet remained in the particle suspension after 30 days under the typical application conditions. The inhibitory concentration (IC50) on human bronchial epithelial cells (16HBE14o-) was found to be between 2.89 and 5.11 μg/cm2 for folpet commercial products after 24 h of exposure. Folpet degradation products and vehicles of Folpan 80 WG® did not show any cytotoxicity at tested concentrations. At non-cytotoxic and subtoxic concentrations, Folpan 80 WG® was found to increase DCFH-DA fluorescence. These results show that the particles of commercial forms of folpet are relatively stable over time. Particles could be easily inhaled by humans, could reach the conducting airways and are cytotoxic to respiratory cells in vitro. Folpet particles may mediate its toxicity directly or indirectly through ROS-mediated alterations. These data constitute the first step towards the risk assessment of folpet particles by inhalation for human health. This work confirms the need for further studies on the effect of environmental pesticides on the respiratory system.

Differences in estimates of size distribution of beryllium powder materials using phase contrast microscopy, scanning electron microscopy, and liquid suspension counter techniques.

Abstract: Accurate characterization of the physicochemical properties of aerosols generated for inhalation toxicology studies is essential for obtaining meaningful results. Great emphasis must also be placed on characterizing particle properties of materials as administered in inhalation studies. Thus, research is needed to identify a suite of techniques capable of characterizing the multiple particle properties (i.e., size, mass, surface area, number) of a material that may influence toxicity. The purpose of this study was to characterize the morphology and investigate the size distribution of a model toxicant, beryllium. Beryllium metal, oxides, and alloy particles were aerodynamically size-separated using an aerosol cyclone, imaged dry using scanning electron microscopy (SEM), then characterized using phase contrast microscopy (PCM), a liquid suspension particle counter (LPC), and computer-controlled SEM (CCSEM). Beryllium metal powder was compact with smaller sub-micrometer size particles attached to the surface of larger particles, whereas the beryllium oxides and alloy particles were clusters of primary particles. As expected, the geometric mean (GM) diameter of metal powder determined using PCM decreased with aerodynamic size, but when suspended in liquid for LPC or CCSEM analysis, the GM diameter decreased by a factor of two (p < 0.001). This observation suggested that the smaller submicrometer size particles attached to the surface of larger particles and/or particle agglomerates detach in liquid, thereby shifting the particle size distribution downward. The GM diameters of the oxide materials were similar regardless of sizing technique, but observed differences were generally significant (p < 0.001). For oxides, aerodynamic cluster size will dictate deposition in the lung, but primary particle size may influence biological activity. The GM diameter of alloy particles determined using PCM became smaller with decreasing aerodynamic size fraction; however, when suspended in liquid for CCSEM and LPC analyses, GM particle size decreased by a factor of two (p < 0.001) suggesting that alloy particles detach in liquid. Detachment of particles in liquid could have significance for the expected versus actual size (and number) distribution of aerosol delivered to an exposure subject. Thus, a suite of complimentary analytical techniques may be necessary for estimating size distribution. Consideration should be given to thoroughly understanding the influence of any liquid vehicle which may alter the expected aerosol size distribution.