Quantitative energy dispersive X-ray analysis of submicrometric particles using a scanning electron microscope.

The quantitative scanning electron microscope-energy dispersive X-ray (SEM-EDX) analysis of a horneblende and two augite prismatic samples reduced to submicrometric particles was performed, and error due to the particle effects ("absent mass" and the "reduced absorption" effect) was minimized. Correction factors as a function of fragment size were obtained for O, Na, Mg, Si, Ca, and Fe. In addition, the influence of chemical composition of the samples used as standards (the matrix effect) on correction factors was evaluated. The results indicate that the absent mass effect is dominant for all elements except for the light elements O and Na, for which the reduced absorption effect is dominant. No significant matrix effect has been observed. By using corrected SEM-EDX data, the error on quantification of the element concentration has been estimated to be 3% relative for light elements and below 2% relative for heavy elements (notably, about 1% relative for Fe).

Exposure to fine and ultrafine particles from secondhand smoke in public places before and after the smoking ban, Italy 2005.

BACKGROUND: A smoking ban in all indoor public places was enforced in Italy on 10 January 2005. METHODS: We compared indoor air quality before and after the smoking ban by monitoring the indoor concentrations of fine (<2.5 microm diameter, PM2.5) and ultrafine particulate matter (<0.1 microm diameter, UFP). PM2.5 and ultrafine particles were measured in 40 public places (14 bars, six fast food restaurants, eight restaurants, six game rooms, six pubs) in Rome, before and after the introduction of the law banning smoking (after 3 and 12 months). Measurements were taken using real time particle monitors (DustTRAK Mod. 8520 TSI; Ultra-fine Particles Counter-TRAK Model 8525 TSI). The PM2.5 data were scaled using a correction equation derived from a comparison with the reference method (gravimetric measurement). The study was completed by measuring urinary cotinine, and pre-law and post-law enforcement among non-smoking employees at these establishments RESULTS: In the post-law period, PM2.5 decreased significantly from a mean concentration of 119.3 microg/m3 to 38.2 microg/m3 after 3 months (p<0.005), and then to 43.3 microg/m3 a year later (p<0.01). The UFP concentrations also decreased significantly from 76,956 particles/cm3 to 38,079 particles/cm3 (p<0.0001) and then to 51,692 particles/cm3 (p<0.01). Similarly, the concentration of urinary cotinine among non-smoking workers decreased from 17.8 ng/ml to 5.5 ng/ml (p<0.0001) and then to 3.7 ng/ml (p<0.0001). CONCLUSION: The application of the smoking ban led to a considerable reduction in the exposure to indoor fine and ultrafine particles in hospitality venues, confirmed by a contemporaneous reduction of urinary cotinine.

Inflammatory mediators induced by coarse (PM2.5-10) and fine (PM2.5) urban air particles in RAW 264.7 cells.

Increased incidence of mortality and morbidity due to cardiopulmonary complications has been found to associate with elevated levels of urban air particles with an aerodynamic diameter <10 micron, PM10 and <2.5 micron, PM2.5. Respirable particles reach the lower respiratory tract where they are phagocytized by alveolar macrophages. Depending on particle composition, exposed macrophages may produce inflammatory mediators. A cascade impactor sampler was used to collect size-fractionated urban air particles. Particulate matter from the city of Rome (Italy) were collected onto stainless steel plates, and recovered using alcohol. The murine monocytic/macrophagic RAW 264.7 cell line was used to compare the ability of PM2.5-10, PM2.5 and carbon black to cause cell injury, such as arachidonic acid (AA) release, tumour necrosis factor alpha (TNF alpha) and interleukin (IL)-6 production. All test particles have been used at the same concentrations 30 and 120 microg/ml. Treatment with PM2.5-10 and PM2.5 induced significant AA release after 5 h of exposure at both concentrations, while carbon black was effective only at the higher concentration. After 5 h of incubation, PM2.5-10 and PM2.5 at 120 microg/ml induced 10 times the amount of TNF alpha than carbon black particles. The urban air particles-stimulated TNF alpha production decreased after 24 h of incubation while carbon black-stimulated TNF alpha was not. IL-6 production was induced by PM2.5 and by PM2.5-10 but not by carbon black. Carbon black was consistently less effective than the urban particles, suggesting that, the contaminants adsorbed on the particles are responsible for the release of inflammatory mediators.

Effects of the new-identified amphibole fluoro-edenite in lung epithelial cells.

An epidemiological survey on mortality for malignant pleural neoplasm in Italy evidenced a number of patients in Biancavilla, a village located in a volcanic area of eastern Sicily, none of which had been significantly exposed to asbestos during their professional lives. Environmental studies suggested the involvement of the material derived from stone quarries in the disease onset. A detailed crystal-chemical analysis of amphiboles contained in this material allowed the discovery and the identification of a new fiber that was named fluoro-edenite. In order to define the mode of action of fluoro-edenite at a subcellular level, we have conducted a study by using A549 cells, a tumor-cell line from a human lung carcinoma with properties of alveolar epithelial cells. The results obtained showed a remarkable tropism of A549 cells toward fluoro-edenite fibers. In fact, these epithelial cells contacted the fibers via the extension of membrane ruffles and filopodia that allowed the capture and most probably the internalization of material into the cytoplasm. Moreover, fluoro-edenite interfered with epithelial cell physiology, by reducing the proliferation rate without perturbing the cell cycle and increasing the release of the proinflammatory cytokine IL-6, one of the main mediators of asbestos-induced pathophysiological response.

Physico-chemical characterisation of the inhalable particulate matter (PM10) in an urban area: an analysis of the seasonal trend.

Between October 1998 and February 2000, 11 particulate samplings were conducted in an urban area of Rome to evaluate the seasonal trend of PM10 characteristics: seven samplings were made at ground-level and four 30 m above ground level. The samples were analysed by scanning electron microscopy equipped with an EDS X-ray attachment and by transmission electron microscopy and an electron energy-loss spectrometer. The airborne particulate matter was characterised from a physico-chemical point of view to provide information on the particle composition and on the compounds carried on their surface. The data sets, consisting of the atomic concentrations of the constituent chemical elements of the fine (PM3.3) and of the coarse (PM10-3.3) particulate, were subjected to cluster analysis to determine the principal components of PM10. In the particulate matter, the statistical analysis methods allowed us to identify seven groups (clusters) of particles: C-rich particles; carbonates; silica; silicates; sulfates; Fe-rich particles; and metals. Carbonaceous and silicate particles with a surface coating containing S were observed. This sulfur-enriched coating turned out to be a PM3.3 feature strongly dependent upon season.

Pulmonary fibre burden in sheep living in the Biancavilla area (Sicily): preliminary results.

In a national survey on mortality from malignant pleural neoplasms in Italy, aimed at detecting geographic clusters of cases of the disease, the town of Biancavilla, located in a volcanic area of Eastern Sicily, showed high risk of pleural mesothelioma in the absence of occupational asbestos exposure. An environmental survey suggested the stone quarries located in 'Monte Calvario', south-east of the town of Biancavilla, as a possible source of asbestiform fibre exposure. A subsequent crystal-chemistry investigation of the 'Monte Calvario' amphiboles identified the mineral asbestiform fibres as 'fluoro-edenite', a new end-member of the edenite ==> fluoro-edenite series. A collaborative epidemiological and environmental study was initiated to investigate the characteristics of the outbreak of malignant mesothelioma and test the hypothesis of a causal association with exposure to naturally occurring fibres. To investigate if a sheep population could be used to monitor the environmental diffusion of the fibres, we examined lung specimens from 27 culled sheep, at least 3 years old and living near Monte Calvario to check for the presence of fluoro-edenite fibres, using scanning electron microscopy (SEM) analysis and X-ray microanalysis. Fourteen mineral species have been isolated in the mineral particulate matter taken from pulmonary parenchyma, and fluoro-edenite was detected in eight sheep. These preliminary data suggest a possible bio-indicative role of sheep as sentinel animals in the evaluation of environmental fibre diffusion, which merits further investigation.

Pleural mesothelioma cases in Biancavilla are related to a new fluoro-edenite fibrous amphibole.

A cluster of deaths from pleural mesothelioma was previously reported for Biancavilla, Italy, a city in eastern Sicily. An environmental survey suggested that the stone quarries located southeast of the city might be a source of asbestos exposure. The materials extracted from the quarries, used widely in the local building industry, contain large quantities of a fibrous amphibole that was initially referred to as an anomalous intermediate phase of sodium- and fluorine-rich tremolite-actinolite. A subsequent crystal chemistry investigation identified the mineral as fluoro-edenite, a new end-member of the edenite --> fluoro-edenite series. The material is very similar in morphology and composition to the minerals of the tremolite-actinolite series. To the authors' knowledge, fluoro-edenite becomes the 3rd mineral fiber (along with erionite and winchite), not yet classified as asbestos, with a demonstrable mesotheliomatogenous action in humans.

[Inhalable airborne particulate pollution in sites characterized by dissimilar environmental conditions]. / Inquinamento da polveri e da particolato fino in siti con differenti caratteristiche ambientali.

The aim of the present study is to characterise from a physico-chemical point of view the airborne particulate matter (PM10) sampled in different sites dissimilar with regard to the weather conditions, the residential density and the industrial activities. The particles were collected by an eight-stage cascade impactor, Andersen particle fractionating sampler. The analysis of the particle samples was performed by a scanning electron microscopy (SEM) equipped with a thin-window system for X-ray microanalysis by energy dispersion spectrometry. The hierarchical cluster analysis (HCA) on the analytical data revealed the presence of eight different particulate types (particle clusters) in the sampling sites: C-rich particles (cluster 1); Ca and Mg carbonates (cluster 2); Ca sulphates (cluster 3); silica particles (cluster 4); silicates (cluster 5); Fe-rich particles (cluster 6); metal compounds (cluster 7); carbonaceous particles with heavy metals (cluster 8). Data obtained in this study demonstrate a significant correlation between the PM10 composition and the anthropic activities present the sampling site.

Differences in the biological activity of two PM3.3 components: carbonaceous and silica particles.

In this study we compared the biological reactivity of PM3.3 with those of carbon black (CB) and respirable silica particles, monitored by in vitro hemolytic potential and morphological alterations, in order to evaluate the correlations between the different physico-chemical characteristics of the three types of particulate and their biological effects. Carbon black and silica particles were used as reference environmental particles in order to limit the number of the urban PM variables, which is a mixture highly heterogeneous. Our data suggest that the urban PM3.3 have a similar surface reactivity as CB. In fact, when the percent of hemolysis were plotted against particle surface per volume units, the PM3.3 activity did not differ significantly from that of CB. This observation is in agreement with the SEM morphological evaluations of treated erythrocytes because the more abundant alteration in PM3.3-treated cells was the stomatocytic transformation (main feature of CB-treated red blood cells), followed by echinocytic transformation (observed in silica-treated cells).

[Pleural mesothelioma and environmental exposure to mineral fibres: the case of a rural area in the Basilicata region, Italy]. / Mesotelioma pleurico ed esposizione ambientale a fibre minerali: il caso di un'area rurale in Basilicata.

The main aspects of the sites characterized by environmental exposure to mineral asbestiform fibres are described. Several adverse health effects including high incidence of pleural mesothelioma are reported. The average concentration of airborne fibres is generally low but it rises significantly in association with mechanical disturb of materials with fibres. Multiple sources of exposure have been identified, fibres can be found in the soil and in many materials locally used, mainly in buildings. Three mesothelioma cases were observed in a small rural area of the Basilicata region (Italy). Two of them had a possible occupational exposure to asbestos, the third had a proved environmental exposure to tremolite. This fibre, found in the area, is the same observed in two of the three biological samples analysed.

pH-sensitive hydrogels of dextran: synthesis, characterization and in vivo studies.

pH-Sensitive hydrogels of dextran were synthesized by photochemical cross-linking reaction of methacrylate dextran (DEX-MA) at different derivatization degree, functionalized with acidic residues through reaction with phthalic anhydride. The hydrogels were characterized by FT-IR spectra, swelling measurements, experiments of chemical and enzymatic hydrolyses. The swelling data agreed with the formation of networks having pH-sensitive behaviour. This property was confirmed by the morphological examination performed by scanning electron microscopy on samples maintained in media at different pH. (S)-4-Isobutyl-2-phenylpropionic acid (ibuprofen) was loaded into the polymeric matrices. The analysis of the release profiles of the drug from the three networks showed that all the matrices were able to retain ibuprofen during the transit through the stomach, releasing it in a sustained way in the intestinal tract at a rate strictly dependent on the derivatization degree in methacrylic groups. In vivo studies verified the biocompatibility of the materials. Moreover, when the matrix loaded with ibuprofen was administered to rats, it was able to protect them from the ulcerogenic effects of the drug.

Winter urban air particles from Rome (Italy): effects on the monocytic-macrophagic RAW 264.7 cell line.

Epidemiological data show an association between exposure to elevated levels of particulate matter (PM), in particular the fine fraction (<2.5 microm in diameter), and an increase in cardiovascular mortality and respiratory symptoms. The aim of this study was to compare the in vitro toxicity of coarse and fine particulate matter collected with a cascade impactor during winter in an urban area of Rome in relation to their physicochemical characterization (size distribution and chemical composition) as assessed by analytical electron microscopy (SEM/EDX). The X-ray microanalysis data of single particles of coarse and fine matter were analyzed by hierarchical cluster analysis to determine the principal component of the two granulometric fractions. The main chemical difference between the two fractions was the greater abundance of carbonaceous particles in the fine fraction. We compared the ability of coarse and fine fractions, carbon black (CB), and residual oil fly ash (ROFA) to induce arachidonic acid release and tumor necrosis factor-alpha (TNF-alpha) production in the monocytic-macrophagic RAW 264.7 cell line at concentrations of 30 and 120 microg/mL. Our results showed that CB and ROFA were consistently less effective than both fractions of urban particles at inducing an inflammatory reaction in RAW 264.7 cells. Both PM fractions dose-dependently increased TNF-alpha production in RAW 264.7 cells after 5 and 24h of incubation, and only the TNF-alpha production induced by coarse particles at 30 microg/mL decreased significantly (P<0.01) after 24h of treatment. In our in vitro model the winter fine fraction was more reactive than the winter coarse fraction, in contrast to a previously examined summer sample. In the summer sample, coarse particles produced higher levels of inflammatory mediators than fine particles and the CB was consistently less effective than the urban particles. The different behaviors between summer and winter urban fractions may be due to their different physicochemical characteristics; in fact, the comparison of the two samples' characterization by SEM/EDX and X-ray photoelectron spectroscopy (XPS) analysis showed that in winter the carbonaceous particles are more abundant than in summer and that winter particles carry a greater quantity of organic compounds. We suggest that the higher concentration of organic compounds on fine carbonaceous particles may partially explain the higher activation of RAW 264.7 cells by fine particles.

Fine environmental particulate engenders alterations in human lung epithelial A549 cells.

Particulate matter (PM), a component of urban air pollution that derives primarily from the combustion of fossil fuels, is responsible for a number of health effects in humans. Recent studies have demonstrated that the fine particles (PM(2.5)) present in high numbers in PM samples can be more harmful than larger particles, since they are more efficiently retained in the peripheral lung. In the present study, we have investigated the biological effects of PM(2.5) on human lung epithelial cell line A549. Morphological analysis performed by immunofluorescence and electron microscopy showed that fine particles interact with the cell surface, where they induce evident alterations and, subsequently, are internalized in the cytoplasm. Cytoskeletal components, in particular microfilaments and microtubules, cause modifications upon challenge with PM(2.5). Of interest, an early cell response to the fine particulate is an increase of reactive oxygen species content, which can account for the observed cytoskeletal changes and the production of proinflammatory cytokines in A549 cells. In particular, exposure to PM(2.5) promoted a dose- and time-dependent release of TNF-alpha and IL-6 in the cell medium.

Environmental fine particulate matter (PM 2.5) activates the RAW 264.7 macrophage cell line even at very low concentrations as revealed by 1H NMR.

Because of the association between inhalation of airborne particulate matter (PM) and human respiratory and cardiovascular disease, it is necessary to understand the tissue damage induced by these particles. One of the cell types principally involved in the body's reaction to PM are macrophages, which remove particles in the airway passages and the lungs through phagocytosis. In fact, when macrophages are exposed to a toxic agent such as PM, they undergo a series of changes (including variations in morphology, an increase in glycolysis, and consequent lactate production and the release of cytokines such as interleukin-6 and tumor necrosis factor-alpha) necessary to transform them from "resting" to "activated" macrophages. Because (1)H NMR is extremely useful in monitoring, noninvasively, macrophage metabolism and because this technique has never been utilized to examine macrophage activation after exposure to PM, it was the purpose of the present study to investigate the effects of PM exposure on the RAW 264.7 stabilized macrophage cell line using (1)H NMR spectroscopy. PM with a diameter <2.5 microm (PM 2.5) was utilized because a closer association to mortality and adverse respiratory health effects has been found with this fraction than with particles of a larger size. Measurements were conducted on whole cells at both 500 and 700 MHz as well as on perchloric acid extracts at 700 MHz. Significant variations in numerous metabolites were seen at very low concentrations of PM 2.5. Many of these changes point to activation of RAW 264.7 macrophages even at doses of PM 2.5 much lower than those commonly employed in cell studies. These results are particularly significant since the same concentrations of PM did not induce changes in morphology and release of cytokines in these cells. Therefore, (1)H NMR spectroscopy is an extremely sensitive probe in observing subtle variations in macrophages after exposure to PM 2.5.