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Nanoparticle-induced genotoxicity can arise through different mechanisms, and generally, primary and secondary genotoxicity can be distinguished where the secondary is driven by an inflammatory response. It is, however, yet unclear how a secondary genotoxicity can be detected using in vitro methods. The aim of this study was to investigate inflammation and genotoxicity caused by agglomerated nickel (Ni) and nickel oxide (NiO) nanoparticles and, furthermore, to explore the possibility to test secondary (inflammation-driven) genotoxicity in vitro. As a benchmark particle to compare with, we used crystalline silica (quartz). A proteome profiler antibody array was used to screen for changes in release of 105 different cytokines and the results showed an increased secretion of various cytokines including vascular endothelial growth factor (VEGF) following exposure of macrophages (differentiated THP-1 cells). Both Ni and NiO caused DNA damage (comet assay) following exposure of human bronchial epithelial cells (HBEC) and interestingly conditioned media (CM) from exposed macrophages also resulted in DNA damage (2- and 3-fold increase for Ni and NiO, respectively). Similar results were also found when using a co-culture system of macrophages and epithelial cells. In conclusion, this study shows that it is possible to detect a secondary genotoxicity in lung epithelial cells by using in vitro methods based on conditioned media or co-cultures. Further investigation is needed in order to find out what factors that are causing this secondary genotoxicity and whether such effects are caused by numerous nanoparticles.

Exposure to urban particulate matter (PM) is correlated with increases in the emergence of health services due to adverse events and deaths and is mainly related to cardiorespiratory complications. The translocation of particles from the lung into circulation has been proposed as a factor that may trigger systemic effects. Monocytes may be exposed to PM, and if the monocytes are activated, then they are likely to adhere to endothelial cells in a distant organ due to the expression of receptors for adhesion molecules. In the present study, we evaluated the expression of receptors for adhesion molecules (sLex, PSGL-1, LFA-1, VLA-4 and αVβ3) in monocytes (U937 cells) exposed for 3 or 18 h to PM10 (0.001, 0.003, 0.010, 0.030, 0.300, 3 or 30 µg/mL). Exposed cells were co-cultured with human endothelial cells that were naive or previously exposed to the same particles. When U937 cells were exposed to PM10, similar levels of expression for early and late receptors for adhesion molecules were observed from 30 ng/mL as those induced by TNF-α. Cells exposed to particles at concentrations above 30 ng/mL were more adhesive to naive or exposed human endothelial cells. Taken together, our results suggest that it is plausible that activated monocytes may play a role in systemic effects induced by PM10 due to the size distribution of the particles and the concentrations required to trigger the expression of receptors for adhesion molecules in monocytes.

Endocrine disrupting chemicals (EDCs) from the environment are associated with reproductive abnormalities (i.e. decreased sperm concentration; increased endometriosis) and alterations of the cardiovascular system (i.e. increased blood pressure and risk of coronary disease). Some phthalates esters have been identified as EDCs, for which inhalation is considered as one of the routes of exposure. However, only little is known regarding inhalational exposure to EDCs via urban airborne particles. In the present study, we report the monthly concentration of 8 phthalate esters measured in PM10 and PM2.5 collected and recovered during 7 months in a highly populated area of Mexico City. Using the levels of PM10 and PM2.5 reported by the automatized network of environmental monitoring of Mexico City for the sampling site, we estimated exposure levels for people of different ages and gender. Two endocrine disrupting compounds, the phthalate esters DEHP and DnBP, were found on the particles in higher concentrations during the warmer months of the year. The highest concentration was reported for DEHP (229.7μg/g of particles) in PM2.5 collected in May 2013. After calculations of the DEHP concentration in the atmosphere, and using the respiratory flow rate, we determined males were potentially exposed to larger quantities of DEHP, reaching up to 18ng/8h in April 2013. Despite the concentrations of phthalates seem to be rather small, a comprehensive characterization of its presence is necessary in order to evaluate the overall exposure to these compounds, providing a clear view of exposure on children, adolescents and pregnant women.

Background Epidemiological evidence associates chronic exposure to particulate matter (PM) with respiratory damage and lung cancer. Inhaled PM may induce systemic effects including inflammation and metastasis. This study evaluated whether PM induces expression of adhesion molecules in lung cancer cells promoting interaction with monocytes. Methods The expression of early and late adhesion molecules and their receptors was evaluated in A549 (human lung adenocarcinoma) cells using a wide range of concentrations of PM2.5 and PM10. Then we evaluated cellular adhesion between A549 cells and U937 (human monocytes) cells after PM exposure. Results We found higher expression of both early and late adhesion molecules and their ligands in lung adenocarcinoma cells exposed to PM2.5 and PM10 particles present in the air pollution at Mexico City from 0.03 μg/cm2 with a statistically significant difference (p ≤ 0.05). PM10 had stronger effect than PM2.5. Both PM also stimulated cellular adhesion between tumor cells and monocytes. Conclusions This study reveals a comprehensive expression profile of adhesion molecules and their ligands upregulated by PM2.5 and PM10 in A549 cells. Additionally these particles induced cellular adhesion of lung cancer cells to monocytes. This highlights possible implications of PM in two cancer hallmarks i.e. inflammation and metastasis, underlying the high cancer mortality associated with air pollution.

Titanium dioxide nanoparticles (TiO2 NPs) are widely used for industrial and commercial applications. Once inside the body, they translocate into the bloodstream and reach different areas of the cardiovascular system including the heart, increasing the risk of developing cardiovascular diseases; consequently, the investigation of their interaction with cardiac cells is required. We previously showed that TiO2 NPs are internalized by H9c2 rat cardiomyoblasts, and here, we examined the molecular mechanisms underlying this process. TiO2 NPs internalization was evaluated by transmission electron microscopy, time-lapse microscopy, and flow cytometry. Changes in the actin cytoskeleton were studied by phalloidin staining. Endocytic uptake mechanisms for nanoparticles were probed with chemical inhibitors, whereas clathrin and dynamin expression was measured by Western blot. Cellular uptake of TiO2 NPs occurred early after 30 min exposure, and large aggregates were observed after 1 h. Actin cytoskeleton reorganization included cell elongation plus lower density and stability of actin fibers. Cytochalasin-D inhibited TiO2 NPs uptake, indicating actin-mediated internalization. Dynamin and clathrin levels increased early after TiO2 NPs exposure, and their inhibition reduced nanoparticle uptake. Therefore, TiO2 NPs internalization by H9c2 rat cardiomyoblasts involves actin cytoskeleton reorganization and clathrin/dynamin-mediated endocytosis.

Titanium dioxide nanoparticles (TiO2 NPs) are widely used in industry and daily life. TiO2 NPs can penetrate into the body, translocate from the lungs into the circulation and come into contact with cardiac cells. In this work, we evaluated the toxicity of TiO2 NPs on H9c2 rat cardiomyoblasts. Internalization of TiO2 NPs and their effect on cell proliferation, viability, oxidative stress and cell death were assessed, as well as cell cycle alterations. Cellular uptake of TiO2 NPs reduced metabolic activity and cell proliferation and increased oxidative stress by 19-fold measured as H2DCFDA oxidation. TiO2 NPs disrupted the plasmatic membrane integrity and decreased the mitochondrial membrane potential. These cytotoxic effects were related with changes in the distribution of cell cycle phases resulting in necrotic death and autophagy. These findings suggest that TiO2 NPs exposure represents a potential health risk, particularly in the development of cardiovascular diseases via oxidative stress and cell death.

Endocrine disrupting chemicals (EDCs) from the environment are associated with reproductive abnormalities (i.e. decreased sperm concentration; increased endometriosis) and alterations of the cardiovascular system (i.e. increased blood pressure and risk of coronary disease). Some phthalates esters have been identified as EDCs, for which inhalation is considered as one of the routes of exposure. However, only little is known regarding inhalational exposure to EDCs via urban airborne particles. In the present study, we report the monthly concentration of 8 phthalate esters measured in PM

Curcumin has protective effects against toxic agents and shows preventive properties for various diseases. Particulate material with an aerodynamic diameter of ≤10 μm (PM10) and titanium dioxide nanoparticles (TiO2-NPs) induce endothelial dysfunction and activation. We explored whether curcumin is able to attenuate different events related to endothelial activation. This includes adhesion, expression of adhesion molecules and oxidative stress induced by PM10 and TiO2-NPs. Human umbilical vein endothelial cells (HUVEC) were treated with 1, 10 and 100 μM curcumin for 1 h and then exposed to PM10 at 3 μg/cm2 or TiO2-NPs at 10 μg/cm2. Cell adhesion was evaluated by co-culture with U937 human myelomonocytic cells. Adhesion molecules expression was measured by flow cytometry after 3 or 24 h of exposure. Oxidative stress was determined by 2,7-dichlorodihydrofluorescein (H2DCF) oxidation. PM10 and TiO2-NPs induced the adhesion of U937 cells and the expression of E- and P-selectins, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and platelet-endothelial cell adhesion molecule-1 (PECAM-1). The expression of E- and P-selectins matched the adhesion of monocytes to HUVEC after 3 h. In HUVEC treated with 1 or 10 μM curcumin, the expression of adhesion molecules and monocytes adhesion was significantly diminished. Curcumin also partially reduced the H2DCF oxidation induced by PM10 and TiO2-NPs. Our results suggest an anti-inflammatory and antioxidant role by curcumin attenuating the activation caused on endothelial cells by exposure to particles. Therefore, curcumin could be useful in the treatment of diseases where an inflammatory process and endothelial activation are involved.

Nanoparticles can reach the blood and cause inflammation, suggesting that nanoparticles–endothelial cells interactions may be pathogenically relevant. We evaluated the effect of titanium dioxide nanoparticles (TiO2) on proliferation, death, and responses related with inflammatory processes such as monocytic adhesion and expression of adhesion molecules (E- and P-selectins, ICAM-1, VCAM-1, and PECAM-1) and with inflammatory molecules (tissue factor, angiotensin-II, VEGF, and oxidized LDL receptor-1) on human umbilical vein endothelial cells (HUVEC). We also evaluated the production of reactive oxygen species, nitric oxide production, and NF-κB pathway activation. Aggregates of TiO2 of 300 nm or smaller and individual nanoparticles internalized into HUVEC inhibited proliferation strongly and induced apoptotic and necrotic death starting at 5 μg/cm2. Besides, TiO2 induced activation of HUVEC through an increase in adhesion and in expression of adhesion molecules and other molecules involved with the inflamma...

Gene delivery has become an increasingly important strategy for treating a variety of human diseases, including infections, genetic disorders and tumours. To avoid the difficulties of using viral carriers, more and more non-viral gene delivery nanoparticles are developed. Among these new approaches polyethylene imine (PEI) is currently considered as one of the most effective polymer based method solution and considered as the gold standard. The toxicity of nanoparticles is a major concern when used for medical application. In this study we chose two nanoparticles for an in depth toxicological and ecotoxicological evaluation, one well characterized, PEI, and another novel polymer, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA). In the present study we have assessed the toxicity of these cation nanoparticles as such and of the polyplexes nanoparticles covered with DNA. As these nanoparticles are also frequently used in high volumes in various industries and as such may enter in the environment, we also made an initial assessment of ecotoxicological effects assessment. The following nanoparticles related aspects have been studied during the project: development and characterization, ecotoxicity, general toxicity and specific toxicity. To this end a battery of different tests was used. The conclusion of these tests is that toxicity is varying between different nanoparticles and between different DNA covering ratios. In general, in the different systems tested, the PEI polymer is more toxic than the PDMAEMA polymer. The same difference is seen for the polyplexes and the higher the charge ratio, the more toxic are the polyplexes. Our study also clearly shows the need for a broad spectrum of toxicity assays for a comprehensive risk assessment. Our study has performed such a comprehensive analysis of two biomedical nanoparticles.

Correlations between exposure to particle matter (PM) with an aerodynamic diameter

Exposure to particulate matter (PM) induces inflammatory cytokines. In the present study, we evaluated the secretion of IL-6 and IL-8 by an airway cell line exposed to PM with a mean aerodynamic size equal to or less than 10 or 2.5 microm (PM10 and PM2.5, respectively) collected in Mexico City, using a modified high-volume sampling method avoiding the use of solvents or introducing membrane components into the samples. PM was collected on cellulose-nitrate (CN) membranes modified for collection on high-volume samplers. Composition of the particles was evaluated by particle-induced X-ray emission (PIXE) and scanning electron microscopy. The particles (10-160 microg/cm2) were tested on Calu-3 cells. Control cultures were exposed to LPS (10 ng/mL to 100 microg/mL) or silica (10-160 microg/cm2). IL-6 and IL-8 secretions were evaluated by ELISA. An average of 10 mg of PM was recovered form each cellulose-nitrate filter. No evidence of contamination from the filter was found. Cells exposed to PM10 presented an increase in the secretion of IL-6 (up to 400%), while IL-8 decreased (from 40% to levels below the detection limit). A similar but weaker effect was observed with PM2.5. In conclusion, our modified sampling method provides a large amount of urban PM free of membrane contamination. The urban particles induce a decrease in IL-8 secretion that contrasts with the LPS and silica effects. These results suggest that the regulation of IL-8 expression is different for urban particles (complex mixture containing combustion-related particles, soil and biologic components) than for biogenic compounds or pure mineral particles.

Previous studies have used particle mass and size as metrics to link airborne particles with deleterious health effects. Recent evidence suggests that particle composition can play an important role in PM-toxicity; however, little is known about the specific participation of components (individually or acting in groups) present in such a complex mixture that accounts for toxicity. This work explores relationships among PM(10) components in order to identify their covariant structure and how they vary in three sites in Mexico City. Relationships between PM(10) with cell toxicity and geographical location were also explored. PM(10) was analyzed for elemental composition, organic and elemental carbon, endotoxins and the induction of inhibition of cell proliferation, IL-6, TNFalpha and p53. PM(10) variables were evaluated with principal component analysis and one-way ANOVA. The inhibition of cell proliferation, IL-6 and TNFalpha were evaluated with factorial ANOVA and p53 with the Welch test. The results indicate that there is heterogeneity in particle mass, composition and toxicity in samples collected at different sites. Multivariate analysis identified three major groups: (1) S/K/Ca/Ti/Mn/Fe/Zn/Pb; (2) Cl/Cr/Ni/Cu; and (3) endotoxins, organic and elemental carbon. Groups 1 and 3 showed significant differences among sites. Factorial ANOVA modeling indicated that cell proliferation was affected by PM concentration; TNFalpha and IL-6 by the interaction of concentration and site, and p53 was different by site. Radial plots suggest the existence of complex interactions between components, resulting in characteristic patterns of toxicity by site. We conclude that interactions of PM(10) components determine specific cellular outcomes.

Exposure to PM10 is associated with cardiovascular effects. We evaluated the effects of PM10 on E-Selectin expression and monocytic cell adhesion in human umbilical vein endothelial cells (HUVECs). HUVEC were exposed to PM10 (5-40 microg/cm2) for 6 h, following which surface E-Selectin expression was detected by fluorescence microscopy and flow cytometry. The effects of total particles, particles treated with polymixin-B to block the effects of endotoxin, and both soluble and insoluble fractions of particles, were assessed. Incubation with PM10 lead to a concentration-related increase of E-Selectin expression (>seven-fold increase at 40 microg/cm2). Particles pre-treated with polymixin-B inhibited E-Selectin expression to a level slightly higher than untreated particles. An increase in fluorescence was also observed with the insoluble fraction, while the soluble fraction had no significant effect. HUVEC exposed to PM10 were also evaluated for adhesivity of monocytic cells (U937). PM10 strongly increased the adhesion of U937 cells to HUVEC. In conclusion, PM10 induces endothelial cell activation, evidenced by enhanced E-Selectin expression. This activation is manifested functionally as an increase in monocytic cell adhesion. Insoluble components as well as endotoxins appear to be responsible for this activity.

Inhalation of urban particles (PM 10 ) has been linked to systemic effects such as alterations in the circulatory system. In previous studies, rats exposed to particles, presented monocytes adhering to the blood vessels distant from the lung (Nurkiewicz et al. Environ Health Perspect. 2006;114(3):412-419). The adhesion of monocytes to endothelial cells is mediated by adhesion molecules (i.e. selectins, ICAM-1, VCAM-1, PECAM-1) expressed in the endothelial cells and their receptors (i.e. PSGL-1, sLex, LFA-1, VLA-4 and aVb3), expressed in activated monocytes. In the present study we exposed in vitro human monocytes to PM 10 from Mexico City. The monocytes were exposed during 1, 2, 3, 18 and 24 h and the expression of PSGL-1, sLex, LFA-1, VLA-4 and aVb3 were measured by means of flow cytometry. The tested concentrations ranged from 0.001 μg/mL up to 30 μg/mL. At 3h, a significant increase (p < 0.05) of PSGL-1 and sLex expression was observed from 0.03 μg/mL (Fig 1). The concentrations of 0.001 to 0.01 μg/mL did not induce the expression of these molecules. After 18 h of exposure, the expression of LFA-1 and VLA-4 were induced at all concentrations from 0.03 μg/mL (p 10 to activate human monocytes. Thus, low concentrations of PM 10 inducing the expression of these molecules may be linked to systemic effects induced by inhaled particles.

Many nanoparticles (NPs) have toxic effects on multiple cell lines. This toxicity is assumed to be related to their accumulation within cells. However, the process of internalization of NPs has not yet been fully characterized. In this study, the cellular uptake, accumulation, and localization of titanium dioxide nanoparticles (TiO2 NPs) in rat (C6) and human (U373) glial cells were analyzed using time-lapse microscopy (TLM) and transmission electron microscopy (TEM). Cytochalasin D (Cyt-D) was used to evaluate whether the internalization process depends of actin reorganization. To determine whether the NP uptake is mediated by phagocytosis or macropinocytosis, nitroblue tetrazolium (NBT) reduction was measured and the 5-(N-ethyl-N-isopropyl)-amiloride was used. Expression of proteins involved with endocytosis and exocytosis such as caveolin-1 (Cav-1) and cysteine string proteins (CSPs) was also determined using flow cytometry. TiO2 NPs were taken up by both cell types, were bound to cellular membranes and were internalized at very short times after exposure (C6, 30 min; U373, 2h). During the uptake process, the formation of pseudopodia and intracellular vesicles was observed, indicating that this process was mediated by endocytosis. No specific localization of TiO2 NPs into particular organelles was found: in contrast, they were primarily localized into large vesicles in the cytoplasm. Internalization of TiO2 NPs was strongly inhibited by Cyt-D in both cells and by amiloride in U373 cells; besides, the observed endocytosis was not associated with NBT reduction in either cell type, indicating that macropinocytosis is the main process of internalization in U373 cells. In addition, increases in the expression of Cav-1 protein and CSPs were observed. In conclusion, glial cells are able to internalize TiO2 NPs by a constitutive endocytic mechanism which may be associated with their strong cytotoxic effect in these cells; therefore, TiO2 NPs internalization and their accumulation in brain cells could be dangerous to human health.

Coarse and fine particles were collected using an ambient particle concentrator (VACES system) in the north, center and south regions of Mexico City during May and November of 2003 with the aim of collecting enough particulate matter (PM) to examine their chemical and physical characteristics, biological content, and toxicity potential. The chemical, morphological and biological composition of PM was determined, together with the redox activity, induction of apoptosis and DNA damage. Carbonaceous species determined by thermal-optical transmittance (TOT) showed that the highest concentrations were found in PM2.5 from the north and in PM10 from the center. When analyzed by inductively coupling plasma (ICP), levels of metals were higher in the coarse fraction, mainly in the north. Morphological analysis by Scanning Electron Microscope & Energy Dispersive X-ray Spectrometer (SEM-EDX) is shown. Bacteria, fungi and endotoxin were present mostly in the coarse samples from the north. Fine PM had higher redox activity, than the coarse PM assessed by the dithiothreitol (DTT) assay. Early apoptotic cell death assessed by annexin V was observed in A549 cells exposed to PM from all regions, particularly with those collected in May. The fine fraction from the south induced higher apoptotic cell death compared to the coarse fraction, in contrast, the coarse fraction from the north induced significantly higher apoptosis than the fine fraction. All PM samples induced DNA damage assessed by the comet assay on THP-1 cells when exposed to a concentration of 10 μg/mL, the highest DNA damage was produced by both particle fractions collected in the north in May and November. In conclusion, PM from the north showed a higher metal and biological content, apoptotic cell death induction and more extensive DNA damage. Also, fine PM fractions from all sampled regions showed more redox activity than the coarse fraction. In summary, location, season and size of PM collection influenced their chemical, morphological and biological composition and thus their toxicity to cells.

Exposure to urban airborne particulate matter (PM) is associated with adverse health effects. We previously reported that the cytotoxic and proinflammatory effects of Mexico City PM10 (less than or equal to 10 micro m mean aerodynamic diameter) are determined by transition metals and endotoxins associated with these particles. However, PM2.5 (less than or equal to 2.5 micro m mean aerodynamic diameter) could be more important as a human health risk because this smaller PM has the potential to reach the distal lung after inhalation. In this study, we compared the cytotoxic and proinflammatory effects of Mexico City PM10 with those of PM2.5 using the murine monocytic J774A.1 cell line in vitro. PMs were collected from the northern zone or the southeastern zone of Mexico City. Elemental composition and bacterial endotoxin on PMs were measured. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) production by J774A.1 cells was measured in the presence or absence of recombinant endotoxin-neutralizing protein (rENP). Both northern and southeastern PMs contained endotoxin and a variety of transition metals. Southeastern PM10 contained the highest endotoxin levels, 2-fold higher than that in northern PM10. Northern and southeastern PM2.5 contained the lowest endotoxin levels. Accordingly, southeastern PM10 was the most potent in causing secretion of the proinflammatory cytokines TNF-alpha and IL-6. All PM2.5 and PM10 samples caused cytotoxicity, but northern PMs were the most toxic. Cytokine secretion induced by southeastern PM10 was reduced 50-75% by rENP. These results indicate major differences in PM10 and PM2.5. PM2.5 induces cytotoxicity in vitro through an endotoxin-independent mechanism that is likely mediated by transition metals. In contrast, PM10 with relatively high levels of endotoxin induces proinflammatory cytokine release via an endotoxin-dependent mechanism.

Exposure to urban airborne particles is associated with an increase in morbidity and mortality. There is little experimental evidence of the mechanisms involved and the role of particle composition. We assessed cytotoxicity (crystal violet assay), apoptosis [terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) or annexin V assay], DNA breakage (comet assay), and production of proinflammatory mediators [tumor necrosis factor Alpha (TNF-Alpha), interleukin 6 (IL-6), prostaglandin E2 (PGE2)] (enzyme-linked immunosorbent assay), and E-selectin (flow cytometry) in cell lines exposed to particulate matter < 10 microm in size (PM10) obtained from the northern, central, and southern zones of Mexico City. Particle concentrations ranged from 2.5 to 160 microg/cm(2). We used epithelial, endothelial, fibroblastic, and monocytic cells and assessed DNA damage in Balb-c cells, TNF-Alpha and IL-6 production in mouse monocytes, and PGE2 in rat lung fibroblasts. We determined the expression of E-selectin in human endothelial cells and evaluated the cytotoxic potential of the PM10 samples in all cell types. PM10 from all three zones of Mexico City caused cell death, DNA breakage, and apoptosis, with particles from the north and central zones being the most toxic. All of these PM10 samples induced secretion of proinflammatory molecules, and particles from the central zone were the most potent. Endothelial cells exposed to PM10 from the three zones expressed similar E-selectin levels. Mexico City PM10 induced biologic effects dependent on the zone of origin, which could be caused by differences in the mixture or size distribution within particle samples. Our data suggest that particle composition as well as particle size should be considered in assessing the adverse effects of airborne particulate pollution.

An aerobiological study was performed to evaluate the potential exposure of animals and workers to dust constituents generated during routine animal house work. Different rooms of air conditioned (A, control) and passively ventilated (B, non-air conditioned) animal facilities were sampled, in order to evaluate total airborne culturable fungi and bacteria, fungal spore concentrations and particle levels. Airborne room particles were analyzed gravimetrically and for endotoxin content. All parameters, except for culturable fungi, were higher in facility B and statistically significant, with respect to those from the control facility A. Median values for airborne particle concentration, endotoxin and fungal spores in facility B were: 115 µg m−3, 25 EU m−3, and 2173 spores m−3, respectively. Median values for facility A were: 66 µg m−3, 9 EU m−3, and 248 fungal spores m−3. Broncheoalveolar lavage from rats kept in the rat room of B, presented median concentrations of total cells and lactate dehydrogenase, higher than those found in the control facility (4.4 × 105vs. 1.1 × 105 and 2.7 UmL-1vs. 0.39 UmL−1, respectively). Values of total and biological particles of both facilities, as well as the time spent in different rooms, showed that worker exposure was higher during cage washing. It was especially high in the passively ventilated facility (airborne particles 686 µg m−3 3.5 h−1vs. 976 µg m−3 3.5 h−1, endotoxin 70 EU m−3 3.5 h−1vs. 108 EU m−3 3.5 h−1). The type of basidiospores and ascospores found, as well as the significant correlation between particle levels and endotoxin contents suggests that wood chip bedding disturbance during cage washing is an important source for airborne biological particles. The changes in broncheoalveolar lavage components found in rats from these facilities and previously reported changes in pro-inflammatory cellular responses found in workers, indicate that these relatively low levels of exposure are enough to induce a biological response. Studies considering the composition of mixed organic dusts, would be needed to reevaluate current occupational standards.

Objective: To investigate cellular immune responses in laboratory animal workers who are exposed to high levels of animal allergens but also to other biologically active substances containing lipopolysaccharides (LPS), i.e., endotoxins. Methods: A survey among 20 animal facility workers and 20 matched (gender, smoking) controls was conducted using exposure measurements (endotoxin, colony-forming units of bacteria and fungi) and a questionnaire on respiratory symptoms. Blood samples were taken to determine the ex vivo whole-blood release of tumor necrosis factor-alpha (TNF) and interleukin-8 (IL-8) as well as plasma levels of LPS-binding protein (LBP), bactericidal permeability-increasing protein (BPI), the 75-kDa soluble TNF receptor (sTNFR75), and total/specific IgE. Results: Although exposure to endotoxin was low (range 0.05–2.8 ng/m3), a significant (P < 0.05) increase in plasma BPI (4-fold) and srTNF75 (1.2-fold) was found, suggesting a response to inhalation of LPS. Also, the capacity of blood leukocytes to release TNF and IL-8 in response to ex vivo exposure to workplace dust was increased. Data were not confounded by specific allergies, levels of IgE, smoking, or respiratory symptoms. Conclusions: A profound effect on the cellular immune response was seen in animal workers with low endotoxin exposure and a high antigenic load. It remains to be determined which other biologically active substance(s) are involved in this effect.

Platelet-derived growth factor (PDGF) and its receptor system regulate mesenchymal cell proliferation. We recently reported that emission-source fly-ash particles and asbestos fibers induce the PDGF alpha-receptor through a macrophage-dependent pathway, and upregulation of this receptor greatly enhances the mitogenic response of lung myofibroblasts to PDGF (Lindroos and colleagues, Am. J. Respir. Cell Mol. Biol. 1997;16:283-292). In the present study we investigated the effect of particulate matter= 10 micrometers in size (PM10) from the southern, central, and northern regions of Mexico City on PDGF receptor induction and compared these urban, ambient particles with Mt. St. Helen's volcanic ash particles as a negative control. All Mexico City PM10 samples, but not volcanic ash, stimulated rat alveolar macrophages to secrete a soluble, upregulatory factor(s) for the PDGF alpha-receptor on early passage rat lung myofibroblasts. The macrophage-derived upregulatory activity was blocked by the interleukin (IL)-1 receptor antagonist. The ability of PM10 to stimulate IL-1beta release was blocked in part by a recombinant endotoxin neutralizing protein (rENP). Lipopolysaccharide/endotoxin (LPS) and vanadium, both constituents that were present within these PM10 samples, also stimulated macrophages to secrete factor(s) that upregulated PDGF-Ralpha on lung myofibroblasts. Direct exposure of myofibroblasts to PM10 also elicited upregulation of the PDGF alpha-receptor, and this effect was blocked by rENP and mimicked by LPS, but not vanadium. These findings suggest that PM10 particles induce expression of the PDGF receptor system through macrophage-dependent and -independent mechanisms involving endotoxin and metals.

Humans have been exposed for thousands of years to particulate matter (PM) from natural and anthropogenic sources. Since the first third of the twentieth century, health problems related to dust exposure in miners have been documented [1]. Early epidemiological data have shown evidence of the relation between inhalation of PM and several lung diseases, including lung fibrosis and lung cancer [2]. The Meuse valley fog of 1930 [3], the Donora smog incident of 1948 [4], and the London great smog event of 1952 [5] were the foundation to create a legislation regarding to air pollutants. In the US, the Clean Air Act was enacted in 1972. Before 1970, the main efforts in this field were aimed at measuring the PM suspended in the air and its relation to death increases associated to lung diseases. During the 1970s and 1980s, the measurements of environmental particles improved, and efforts to quantify particles with different aerodynamic sizes gave a view on how the particles could be related to different diseases. These methods helped to identify sources of different particles, leading to specific actions to control the emission of PM. During the 1990s and the first decade of this century, a great effort has been done to determine the cellular and molecular mechanisms related to the particle toxicity, and many studies have shown that particle size and composition play central roles in the biological effects. During the last 20 years, a great concern has grown regarding the use of nanoparticles (NPs) and its possible impact on workers and final users. To present, it is evident that inhaled particles may have local and systemic effects, and that the size, the composition, and the physicochemical characteristics of these airborne particles play a central role in their toxicity. Despite all the clinical, epidemiological, and toxicological evidence, we are far from understanding the toxicology of particles, in part by the combined effects and interactions of various substances mixed within the particles. In addition, the lack of evidence of a threshold value makes it difficult to set safe limit values. Therefore, a constant growth in the total number of publications related to urban PM and NP is easy to observe when a simple search is done on PUBMED. When the words “particles” and “air pollution” are searched, 101 publications are found from 1900 to 1970. The number rises to 149 from 1971 to 1980. During the 1980s, the first efforts were done to evaluate the effects of particles with different aerodynamical sizes but the number of publications remained in 150. Later on, during the 1990s, the evaluation of particles with different aerodynamic sizes (PM10, PM2.5, and ultrafine particles) made the number of publications to grow up to more than 600. The continuous evaluation of urban particles and the arising use of nanomaterials led to almost 2000 publications during the first decade of this century. If the rate of publications on these fields keeps the same rhythm as the first two years of the present decade, we will find about 2400 publications for the 2011–2020 decade (Figure 1). Figure 1 The previously-mentioned numbers gave a clear idea of why a special issue on particulate matter and nanoparticles toxicology is important for this field. In this special issue, we are publishing a selection of studies dealing with particle sampling and characterization, in vitro toxic effects characterization using traditional and novel models, in vivo effects of PM, and in vivo and in vitro effects of different types of NP. We also include three reviews, discussing the cellular effects of diesel particles, another one discussing the evidence relating the exposure to particles and other inhaled pollutants to the increased risk of Alzheimer and Parkinson's diseases. Finally, a review of the state of the art in the in vivo and in vitro toxicological characterization of particles was prepared for this issue by the guest editors, where we discuss the latest evidence of local and systemic effects induced by inhaled particles. Great efforts have been done during the last 50 years to understand the toxicology of particulate matter, and much information is available helping to understand the risks of exposure to different types of particles. Nevertheless, there is much to do in the field, and the efforts presented here will be of great value to push further the frontiers of our knowledge on the particulate matter toxicology field. Abderrahim Nemmar Ernesto Alfaro-Moreno Irma Rosas Per Schwarze Tim S. Nawrot

Epidemiological and clinical studies have linked exposure to particulate matter (PM) to adverse health effects, which may be registered as increased mortality and morbidity from various cardiopulmonary diseases. Despite the evidence relating PM to health effects, the physiological, cellular, and molecular mechanisms causing such effects are still not fully characterized. Two main approaches are used to elucidate the mechanisms of toxicity. One is the use ofin vivoexperimental models, where various effects of PM on respiratory, cardiovascular, and nervous systems can be evaluated. To more closely examine the molecular and cellular mechanisms behind the different physiological effects, the use of variousin vitromodels has proven to be valuable. In the present review, we discuss the current advances on the toxicology of particulate matter and nanoparticles based on these techniques.

Particulate matter (PM) and nanoparticles (NPs) induce activation and dysfunction of endothelial cells characterized by inhibition of proliferation, increase of adhesion and adhesion molecules expression, increase of ROS production, and death. DHEA has shown anti-inflammatory and antioxidant properties in HUVEC activated with proinflammatory agents. We evaluated if DHEA could protect against some inflammatory events produced by PM10and TiO2NPs in HUVEC. Adhesion was evaluated by a coculture with U937 cells, proliferation by crystal violet staining, and oxidative stress through DCFDA and Griess reagent. PM10and TiO2NPs induced adhesion and oxidative stress and inhibited proliferation of HUVEC; however, when particles were added in combination with DHEA, the effects previously observed were abolished independently from the tested concentrations and the time of addition of DHEA to the cultures. These results indicate that DHEA exerts significant anti-inflammatory and antioxidative effects on the damage induced by particles in HUVEC, suggesting that DHEA could be useful to counteract the harmful effects and inflammatory diseases induced by PM and NPs.

The effects of particulate matter (PM) on endothelial cells have been evaluated in vitro by exposing isolated endothelial cells to different types of PM. Although some of the findings from these experiments have been corroborated by in vivo studies, an in vitro model that assesses the interaction among different cell types is necessary to achieve more realistic assays. We developed an in vitro model that mimics the alveolar–capillary interface, and we challenged the model using TiO2 nanoparticles (TiO2-NPs). Human umbilical endothelial cells (HUVECs) were cultured on the basolateral side of a membrane and pneumocytes (A549) on the apical side. Confluent co-cultures were exposed on the apical side to 10 μg/cm2 of TiO2-NPs or 10 ng/mL of TNFα for 24 h. Unexposed cultures were used as negative controls. We evaluated monocyte adhesion to HUVECs, adhesion molecule expression, nitric oxide concentration and proinflammatory cytokine release. The TiO2-NPs added to the pneumocytes induced a 3- to 4-fold increase in monocyte adhesion to the HUVECs and significant increases in the expression of adhesion molecules (4-fold for P-selectin at 8 h, and about 8- and 10-fold for E-selectin, ICAM-1, VCAM-1 and PECAM-1 at 24 h). Nitric oxide production also increased significantly (2-fold). These results indicate that exposing pneumocytes to TiO2-NPs causes endothelial cell activation.

Nanoparticles can reach the blood and cause inflammation, suggesting that nanoparticles-endothelial cells interactions may be pathogenically relevant. We evaluated the effect of titanium dioxide nanoparticles (TiO₂) on proliferation, death, and responses related with inflammatory processes such as monocytic adhesion and expression of adhesion molecules (E- and P-selectins, ICAM-1, VCAM-1, and PECAM-1) and with inflammatory molecules (tissue factor, angiotensin-II, VEGF, and oxidized LDL receptor-1) on human umbilical vein endothelial cells (HUVEC). We also evaluated the production of reactive oxygen species, nitric oxide production, and NF-κB pathway activation. Aggregates of TiO₂ of 300 nm or smaller and individual nanoparticles internalized into HUVEC inhibited proliferation strongly and induced apoptotic and necrotic death starting at 5 μg/cm². Besides, TiO₂ induced activation of HUVEC through an increase in adhesion and in expression of adhesion molecules and other molecules involved with the inflammatory process. These effects were associated with oxidative stress and NF-κB pathway activation. In conclusion, TiO₂ induced HUVEC activation, inhibition of cell proliferation with increased cell death, and oxidative stress.

Chronic thromboembolic pulmonary hypertension (CTEPH) is characterised by proximal pulmonary vascular obstruction by thrombo-fibrotic material, the origin of which has not been elucidated. Enhanced inflammation could contribute to persistent obstruction by impairing pulmonary vascular cell function in CTEPH. We investigated C-reactive protein (CRP) effects on pulmonary vascular cell function in vitro. Primary cultures of proximal pulmonary endothelial cells (ECs) and smooth muscle cells (SMCs) from CTEPH and nonthromboembolic pulmonary hypertension (PH) patients were established. Recombinant CRP effects on mitogenic activity, adhesion capacity, endothelin-1 and von Willebrand factor (vWF) secretion and intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule-1 expression were investigated in ECs and/or SMCs. Expression of the CRP receptor, lectin-like oxidised low-density lipoprotein receptor (LOX)-1, was evaluated in proximal pulmonary arterial tissue and cells by Western blotting and immunofluorescence. CRP increased CTEPH-SMC proliferation by 250%. CRP increased adhesion capacity, endothelin-1 and vWF secretion by CTEPH-ECs by 37%, 129% and 694%, respectively. CRP-induced adhesion of CTEPH-ECs to monocytes was mediated by ICAM-1. CRP had no effect on cells from nonthromboembolic PH patients, probably because of overexpression of LOX-1 in CTEPH. Local expression of CRP was detected in ECs and SMCs within pulmonary arterial tissue. CRP may contribute to persistent obstruction of proximal pulmonary arteries in CTEPH by promoting vascular remodelling, endothelial dysfunction and in situ thrombosis.

Objective The aim of the present study was to evaluate differences in expression levels and localization status of PTEN, p53 and hDlg suppressor proteins in premalignant lesions and cervical cancer, and to analyze the possible correlation between them. Methods Expression levels (positivity/intensity) and localization (nuclear, membrane or cytoplasmic) of PTEN, hDlg and p53 were analyzed by immunohistochemistry in 43 cases with different stages of cervical intraepithelial neoplasia (CIN) and 105 invasive cervical carcinomas (ICC) (91 squamous carcinoma, 14 adenocarcinoma). Differences between proportions were evaluated. Results We found a decreased expression of PTEN in ICC that correlated with a loss of hDlg from the cell membrane. In contrast, no changes were found in p53 protein levels or localization in CIN and ICC. Conclusions These results suggest that the abnormal expression and localization of PTEN during cervical carcinogenesis may be a consequence of modifications in the expression patterns of hDlg.

Summary. Background: Epidemiological findings suggest an association between exposure to particulate matter (PM) and venous thrombo-embolism. Objectives: To investigate arterial vs. venous thrombosis, inflammation and coagulation in mice, (sub)acutely exposed to two types of PM. Methods: Various doses (25, 100 and 200 μg per animal) of urban particulate matter (UPM) or diesel exhaust particles (DEP) were intratracheally (i.t.) instilled in C57Bl6/n mice and several endpoints measured at 4, 10 and 24 h. Mice were also repeatedly exposed to 100 μg per animal on three consecutive days with endpoints measured 24 h after the last instillation. Results: Exposure to 200 μg per mouse UPM enhanced arterial thrombosis, but neither UPM nor DEP significantly enhanced venous thrombosis. Both types of PM induced dose-dependent increases in broncho-alveolar lavage fluid (BALF) total cell numbers (mainly neutrophils) and cytokines (IL-6, KC, MCP-1, RANTES, MIP-1α), with peaks at 4 h and overall higher values for UPM than for DEP. Systemic inflammation was limited to increased serum IL-6 levels, 4 h after UPM. Both types of PM induced similar and dose-dependent but modest increases in factor (F)VII, FVIII and fibrinogen. Three repeated instillations did not or only modestly enhance the proinflammatory and procoagulant status. Conclusions: Compared with DEP, UPM induced more pronounced pulmonary inflammation, but both particle types triggered similar and mild short-term systemic effects. Hence, acute exposure to PM triggers activation of primary hemostasis in the mouse, but no substantial secondary hemostasis activation, resulting in arterial but not venous thrombogenicity.

The current authors evaluated whether a system of co-cultures of relevant cells (pneumocytes (A549), macrophages (THP-1), mast cells (HMC-1) and endothelial cells (EAHY926)) would mimic the responses to particles with a 50% cut-off aerodynamic diameter of 10 microm (PM(10)) previously reported in vivo. The role of mast cells was considered of special interest. Single cultures, bicultures (A549 + HMC-1 in a 10:1 ratio; THP-1 + HMC-1 in a 2:1 ratio) and tricultures (A549 + THP-1 + HMC-1 in a 10:2:1 ratio) were exposed to urban PM(10) (24 h at 0, 10, 30 or 100 microg x cm(-2)). Additionally, EAHY926 cells were introduced in inserts above the tricultures. The released cytokines were evaluated with a fluorescence-activated cell sorter array system. THP-1 + HMC-1 bicultures and the tricultures released more granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1beta, interleukin (IL)-1beta, IL-8, IL-6, tumour necrosis factor-alpha and MIP-1alpha in response to PM(10) than the sum of the single cultures. Tricultures with EAHY926 released more G-CSF, MIP-1alpha, IL-8 and MIP-1beta than the EAHY926 single culture. The bicultures, tricultures and tricultures with EAHY926 provide results that are consistent with the local and systemic effects previously described for particulate matter effects, i.e. inflammation, endothelial dysfunction and bone marrow cell mobilisation. Mast cells seem to play a significant role in the co-culture responses.

Several original epidemiologic studies showing associations between specific environmental or occupational exposures and adverse respiratory effects were published in 2007. Such studies in large populations often rely, by necessity, on crude endpoints, such as mortality, respiratory symptoms obtained by questionnaires, or simple pulmonary function measurements. Although such observational studies may point to causal links between (presumed) exposure and disease, they must be completed by further epidemiologic studies that use more refined endpoints and/or more sophisticated designs, and by experimental studies that are needed to provide plausibility as well as mechanistic explanations for the observed findings. Thus, in an editorial, Kaufman (1) pointed to the importance to integrate epidemiologic and toxicology approaches to elucidate the mechanism of the effects of air pollution on health.

In the evaluation of particulate matter (PM) toxicity, one faces the challenge of identifying components that could be addressed as markers of toxicity. This study examines the use of statistical methods to determine which components present in the complex mixtures are related to toxic effects. The work is based on data previously published, where we demonstrated that particles collected in different zones of Mexico City showed different ability of inducing cell death, causing DNA damage, or creating proinflammatory effects. Empirically, we correlated those differences to variations in the concentration of transitional metals or endotoxin. In order to test those correlations, in this study we evaluated the role of the concentration of six transitional metals (Ni, V, Zn, Cu, Fe, and Pb) and endotoxin using the following tools: Pearson's regression analysis, correlation matrix between components, and multiple regression followed by a stepwise analysis. Due to collinearity found among several of the components, only Ni, Zn, and Pb were considered as independent variables. Among these, Ni and Zn show better correlations and help to explain the loss in viability, whereas Pb is the component that betters explains the proinflammatory effects. The statistical identification of composition markers facilitates the generation of a hypothesis relating the role played by the composition of PM and its biological effects.

Exposure to airborne particles has been associated with an increase in cardiopulmonary events. Endothelial cells could be playing an important role in the response to airborne particles due their involvement in proinflammatory events, and there is some evidence of particle translocation from lung into circulation. One of the initiating events of inflammation is endothelial activation. We determined the concentration-response effect of a particulate matter with different aerodynamic sizes (PM2.5 [particulate matter with aerodynamic diameter of 2.5 microm and less] and PM10 [particulate matter with aerodynamic diameter of 10 microm and less]) obtained from Mexico City on human umbilical vein endothelial cells (HUVEC). The adhesion of monocytic U937 cells to HUVEC and the expression of early (E- and P-selectins) and late (ICAM-1, PECAM-1, VCAM-1) adhesion molecules were tested. Adhesion of U937 cells to HUVEC was evaluated by coculture experiments using [3H]thymidine-labeled U937 cells and the expression of adhesion molecules was evaluated by flow cytometry. Tumor necrosis factor (TNF)-alpha was used as a positive control of endothelial activation. Our results showed that both PM2.5 and PM10 induced the adhesion of U937 cells to HUVEC, and their maximal effect was observed at 20 microg/cm2. This adhesion was associated with an increase in the expression of all adhesion molecules evaluated for PM10, and E-selectin, P-selectin, and ICAM-1 for PM2.5. In general, maximum expression of adhesion molecules induced by PM2.5 and PM10 was obtained with 20 microg/cm2; however, PM10-induced expression was observed from 5 microg/cm2. E-selectin and ICAM-1 had the strongest expression in response to particles. In conclusion, PM2.5 and PM10 induce the activation of HUVEC, leading to monocytic adhesion via the expression of adhesion molecules, suggesting that these particles may participate in the development of inflammatory diseases. The role of these events in the development of diseases such as atherosclerosis is likely to be evaluated.

Purpose of review The mechanisms related to adverse respiratory and cardiovascular effects in populations exposed to particulate matter are under debate and different models have been used to further our understanding of the various aspects of those effects. In this review we present some studies that may give new insights into the cellular and systemic mechanisms related to particulate matter toxicity. Recent findings Strong epidemiological evidence is now available regarding exposure markers and health effects. This is evident in the correlation between carbon content in macrophages and decrease in lung function, an increase in the risk of chronic obstructive pulmonary disease, lung cancer and postnatal mortality. The role of outdoor temperature and a missing allele for GSTM1 and the impact of these factors on cardiovascular effects are also reported. At the experimental level, the effects of particulate matter and the interactions between different cell types, the role of toll-like receptor-2 and 4, the translocation of particles through cell monolayers and the activation of endothelial cells by particulate matter are also discussed. The role of composition is under intense debate, and different statistical analyses have been proposed. Summary Experimental studies on the effects of particulate matter are giving plausibility to the epidemiological findings, but the possible mechanisms of action are also becoming a hot topic.