Eleonora Longhin

Winter fine particulate matter from Milan induces morphological and functional alterations in human pulmonary epithelial cells (A549).

Samples of PM(2.5) were gravimetrically collected during the winter 2005/2006 in the urban area of Milan (North Italy). Samples were chemically characterized and the particles were detached from filters to determine their cytotoxic effects on the A549 cell line. Based on the potential toxicological relevance of its components, Milan winter PM(2.5) contained high concentrations of pro-oxidant transition metals and PAHs, while re-suspended particles showed a relatively high frequency of dimensional classes ranging from 40 nm to 300 nm. A549 cells exposed to particle suspensions showed a concentration-dependent decrease in viability, starting from 10 microg/cm(2). Phagocytosis of particles by A549 cells and particle aggregates were morphologically characterized and seemed to depend on both particle concentration and exposure time, with the majority of particles being engulfed in membrane-bound vacuoles after 24h of exposure. The ability of ultrafine particles to penetrate and spread throughout the cells was also verified. Cell membrane lysis and mitochondrial ultrastructural disruption appeared to be the main modifications induced by PM(2.5) on A549 cells. Concomitantly to the adverse effects observed in terms of cell mortality and ultrastructural lesions, a significant intracellular production of reactive oxygen species (ROS) was observed, suggesting that the cytotoxicity, exerted by the winter PM(2.5) in Milan, derived also from its oxidative potential, probably associated with particle-adsorbed metals and PAHs.

Gene expression profiling of A549 cells exposed to Milan PM2.5

BACKGROUND Particulate matter (PM) has been associated to adverse health effects in exposed population and DNA damage has been extensively reported in in vitro systems exposed to fine PM (PM2.5). The ability to induce gene expression profile modulation, production of reactive oxygen species (ROS) and strand breaks to DNA molecules has been investigated in A549 cells exposed to winter and summer Milan PM2.5. RESULTS A549 cells, exposed to 10 μg/cm(2) of both winter and summer PM2.5, showed increased cytotoxicity at 24h and a significant increase of ROS at 3h of treatment. Despite these similar effects winter PM induced a higher number of gene modulation in comparison with summer PM. Both PMs modulated genes related to the response to xenobiotic stimuli (CYP1A1, CYP1B1, TIPARP, ALDH1A3, AHRR) and to the cell-cell signalling (GREM1) pathways with winter PM2.5 inducing higher fold increases. Moreover the winter fraction modulated also JUN (cell-cell signalling), GDF15, SIPA1L2 (signal transduction), and HMOX1 (oxidative stress). Two genes, epiregulin (EREG) and FOS-like antigen1 (FOSL1), were significantly up-regulated by summer PM2.5. The results obtained with the microarray approach have been confirmed by qPCR and by the analysis of CYP1B1 expression. Comet assay evidenced that winter PM2.5 induced more DNA strand breaks than the summer one. CONCLUSION Winter PM2.5 is able to induce gene expression alteration, ROS production and DNA damage. These effects are likely to be related to the CYP enzyme activation in response to the polycyclic aromatic hydrocarbons (PAHs) adsorbed on particle surface.

Particle size, chemical composition, seasons of the year and urban, rural or remote site origins as determinants of biological effects of particulate matter on pulmonary cells

Particulate matter (PM), a complex mix of chemical compounds, results to be associated with various health effects. However there is still lack of information on the impact of its different components. PM2.5 and PM1 samples, collected during the different seasons at an urban, rural and remote site, were chemically characterized and the biological effects induced on A549 cells were assessed. A Partial Least Square Discriminant Analysis has been performed to relate PM chemical composition to the toxic effects observed. Results show that PM-induced biological effects changed with the seasons and sites, and such variations may be explained by chemical constituents of PM, derived both from primary and secondary sources. The first-time here reported biological responses induced by PM from a remote site at high altitude were associated with the high concentrations of metals and secondary species typical of the free tropospheric aerosol, influenced by long range transports and aging.

Release of IL-1 β triggered by milan Summer PM10: Molecular pathways involved in the cytokine release

Particulate matter (PM) exposure is related to pulmonary and cardiovascular diseases, with increased inflammatory status. The release of the proinflammatory interleukin- (IL-) 1β, is controlled by a dual pathway, the formation of inactive pro-IL-1β, through Toll-like receptors (TLRs) activation, and its cleavage by NLRP3 inflammasome. THP-1-derived macrophages were exposed for 6 h to 2.5 μg/cm2 of Milan PM10, and the potential to promote IL-1β release by binding TLRs and activating NLRP3 has been examined. Summer PM10, induced a marked IL-1β response in the absence of LPS priming (50-fold increase compared to unexposed cells), which was reduced by caspase-1 inhibition (91% of inhibition respect summer PM10-treated cells) and by TLR-2 and TLR-4 inhibitors (66% and 53% of inhibition, resp.). Furthermore, summer PM10 increased the number of early endosomes, and oxidative stress inhibition nearly abolished PM10-induced IL-1β response (90% of inhibition). These findings suggest that summer PM10 contains constituents both related to the activation of membrane TLRs and activation of the inflammasome NLPR3 and that TLRs activation is of pivotal importance for the magnitude of the response. ROS formation seems important for PM10-induced IL-1β response, but further investigations are needed to elucidate the molecular pathway by which this effect is mediated.

Milan PM1 Induces Adverse Effects on Mice Lungs and Cardiovascular System

Recent studies have suggested a link between inhaled particulate matter (PM) exposure and increased mortality and morbidity associated with cardiorespiratory diseases. Since the response to PM1 has not yet been deeply investigated, its impact on mice lungs and cardiovascular system is here examined. A repeated exposure to Milan PM1 was performed on BALB/c mice. The bronchoalveolar lavage fluid (BALf) and the lung parenchyma were screened for markers of inflammation (cell counts, tumor necrosis factor-α (TNF-α); macrophage inflammatory protein-2 (MIP-2); heme oxygenase-1 (HO-1); nuclear factor kappa-light-chain-enhancer of activated B cells p50 subunit (NFκB-p50); inducible nitric oxide synthetase (iNOS); endothelial-selectin (E-selectin)), cytotoxicity (lactate dehydrogenase (LDH); alkaline phosphatase (ALP); heat shock protein 70 (Hsp70); caspase-8-p18), and a putative pro-carcinogenic marker (cytochrome 1B1 (Cyp1B1)). Heart tissue was tested for HO-1, caspase-8-p18, NFκB-p50, iNOS, E-selectin, and myeloperoxidase (MPO); plasma was screened for markers of platelet activation and clot formation (soluble platelet-selectin (sP-selectin); fibrinogen; plasminogen activator inhibitor 1 (PAI-1)). PM1 triggers inflammation and cytotoxicity in lungs. A similar cytotoxic effect was observed on heart tissues, while plasma analyses suggest blood-endothelium interface activation. These data highlight the importance of lung inflammation in mediating adverse cardiovascular events following increase in ambient PM1 levels, providing evidences of a positive correlation between PM1 exposure and cardiovascular morbidity.

Synergistic inflammatory effect of PM10 with mycotoxin deoxynivalenol on human lung epithelial cells.

The presence of deoxynivalenol (DON), a mycotoxin produced by Fusarium species, has been reported worldwide in food and feedstuffs. Even though oral intake is the main route of exposure, DON inhalation is also of concern in workers and exposed population. Particulate matter (PM) is one of the most important causes of air quality detriment and it induces several adverse health effects. Therefore it is of primary importance to understand possible combined effects of DON and PM. The alveolar type II, A549, and the bronchial epithelial, BEAS-2B, cell lines were exposed for 24 h to different concentrations of DON (10-1000 ng/ml), PM10 (5 μg/cm(2), sampled in summer or winter season), and a combination of these pollutants. Cell death, interleukins release and cell cycle alteration were analysed; protein array technique was also applied to evaluate proteins activation related to MAP-kinases cascade. Our results demonstrate that low doses of PM and DON used alone have scarce toxic effects, while induce cytotoxicity and inflammation when used in combination. This observation outlines the importance of investigation on the combined effects of air pollutants for their possible outcomes on human health.

Adverse biological effects of Milan urban PM looking for suitable molecular markers of exposure

The results presented summarise the ones obtained in the coordinated research project Tosca, which extensively analysed the impact of Milan urban PM on human health. The molecular markers of exposure and effects of seasonally and size-fractionated PMs (summer and winter PM10, PM2.5) were investigated in in vitro (human lung cell lines) and in vivo (mice) systems. The results obtained by the analyses of cytotoxic, pro-inflammatory and genotoxic parameters demonstrate that the biological responses are strongly dependent upon the PM samples seasonal and dimensional variability, that ultimately reflect their chemical composition and source. In fact summer PM10, enriched in crustal elements and endotoxins, was the most cytotoxic and pro-inflammatory fraction, while fine winter PMs induced genotoxic effects and xenobiotic metabolizing enzymes (like CYP1B1) production, likely as a consequence of the higher content in combustion derived particles reach in PAHs and heavy toxic metals. These outcomes outline the need of a detailed knowledge of the PMs physico-chemical composition on a local scale, coupled with the biological hazard directly associated to PM exposure. Apparently this is the only way allowing scientists and police-makers to establish the proper relationships between the respirable PM quantity/quality and the health outcomes described by clinicians and epidemiologists.

Biological effects of Milan PM: the role of particles dimension and season of sampling

The biological effects induced in human pulmonary cells by atmospheric particles collected in Milan have been analysed according to their dimension and season of sampling. Summer and winter PM1, PM2.5 and PM10 were chemically characterized and used for exposure of pulmonary cultured cell lines. Cell viability, proinflammatory cytokine expression, cell cycle modifications and cytochrome P450 CYP1B1 expression were analysed. Chemical characterization showed that summer fractions had a higher endotoxin content and were enriched in metals while winter PMs in PAHs. In line with this aspect, summer PM10 induced stronger cytotoxic and inflammatory effects in comparison with the other fractions. Winter fine PMs provoked a cell cycle arrest in G2/M phases and remarkable cytochrome P450 CYP1B1 activation, suggesting a potential genotoxic effect.

Sphingoid esters from the molecular distillation of squid oil: A preliminary bioactivity determination.

A mixture of sphingoid esters was isolated (1.4% w/w) from the molecular distillation of crude squid visceral oil. A preliminary investigation on the bioactivity profile and toxic potential of this residue was carried out by in vitro experiments. No cytotoxicity and a moderate lipase inhibition activity were highlighted.

Transcriptional profiling of human bronchial epithelial cell BEAS-2B exposed to diesel and biomass ultrafine particles

BackgroundEmissions from diesel vehicles and biomass burning are the principal sources of primary ultrafine particles (UFP). The exposure to UFP has been associated to cardiovascular and pulmonary diseases, including lung cancer. Although many aspects of the toxicology of ambient particulate matter (PM) have been unraveled, the molecular mechanisms activated in human cells by the exposure to UFP are still poorly understood. Here, we present an RNA-seq time-course experiment (five time point after single dose exposure) used to investigate the differential and temporal changes induced in the gene expression of human bronchial epithelial cells (BEAS-2B) by the exposure to UFP generated from diesel and biomass combustion. A combination of different bioinformatics tools (EdgeR, next-maSigPro and reactome FI app-Cytoscape and prioritization strategies) facilitated the analyses the temporal transcriptional pattern, functional gene set enrichment and gene networks related to cellular response to UFP particles.ResultsThe bioinformatics analysis of transcriptional data reveals that the two different UFP induce, since the earliest time points, different transcriptional dynamics resulting in the activation of specific genes. The functional enrichment of differentially expressed genes indicates that the exposure to diesel UFP induces the activation of genes involved in TNFα signaling via NF-kB and inflammatory response, and hypoxia. Conversely, the exposure to ultrafine particles from biomass determines less distinct modifications of the gene expression profiles. Diesel UFP exposure induces the secretion of biomarkers associated to inflammation (CCXL2, EPGN, GREM1, IL1A, IL1B, IL6, IL24, EREG, VEGF) and transcription factors (as NFE2L2, MAFF, HES1, FOSL1, TGIF1) relevant for cardiovascular and lung disease. By means of network reconstruction, four genes (STAT3, HIF1a, NFKB1, KRAS) have emerged as major regulators of transcriptional response of bronchial epithelial cells exposed to diesel exhaust.ConclusionsOverall, this work highlights modifications of the transcriptional landscape in human bronchial cells exposed to UFP and sheds new lights on possible mechanisms by means of which UFP acts as a carcinogen and harmful factor for human health.