RESUMO
In order to get better knowledge of mechanical properties from microscopic to macroscopic scale of biopolymers, viscoelastic bulk properties of aqueous solutions of sodium alginate were studied at different scales by combining macroscopic shear rheology (Hz), diffusing-wave spectroscopy microrheology (kHz-MHz) and Brillouin spectroscopy (GHz). Structural properties were also directly probed by small-angle X-ray scattering (SAXS). The results demonstrate a change from polyelectrolyte behavior to neutral polymer behavior by increasing polymer concentration with the determination of characteristic sizes (persistence length, correlation length). The viscoelastic properties probed at the phonon wavelength much higher than the ones obtained at low frequency reflect the variation of microscopic viscosity. First experiments obtained by metabolic activity assays with mouse embryonic fibroblasts showed biocompatibility of sodium alginate aqueous solutions in the studied range of concentrations (2.5-10 g L-1) and consequently their potential biomedical applications.
RESUMO
We report on the development of a new model of alveolar air-tissue interface on a chip. The model consists of an array of suspended hexagonal monolayers of gelatin nanofibers supported by microframes and a microfluidic device for the patch integration. The suspended monolayers are deformed to a central displacement of 40-80 µm at the air-liquid interface by application of air pressure in the range of 200-1,000 Pa. With respect to the diameter of the monolayers, that is, 500 µm, this displacement corresponds to a linear strain of 2-10% in agreement with the physiological strain range in the lung alveoli. The culture of A549 cells on the monolayers for an incubation time of 1-3 days showed viability in the model. We exerted a periodic strain of 5% at a frequency of 0.2 Hz for 1 hr to the cells. We found that the cells were strongly coupled to the nanofibers, but the strain reduced the coupling and induced remodeling of the actin cytoskeleton, which led to a better tissue formation. Our model can serve as a versatile tool in lung investigations such as in inhalation toxicology and therapy.
Assuntos
Fenômenos Biomecânicos/fisiologia , Técnicas de Cultura de Células , Dispositivos Lab-On-A-Chip , Alvéolos Pulmonares , Células A549 , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Sobrevivência Celular/fisiologia , Humanos , Nanofibras , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/fisiologiaRESUMO
Regulations on ambient particulate matter (PM) are becoming more stringent because of adverse health effects arising from PM exposure. PM-induced oxidant production is a key mechanism behind the observed health effects and is heavily dependent on PM composition. Measurement of the intrinsic oxidative potential (OP) of PM could provide an integrated indicator of PM bioreactivity and could serve as a better metric of PM hazard exposure than PM mass concentration. The OP of two chemically contrasted PM2.5 samples was compared through four acellular assays, and OP predictive capability was evaluated in different cellular assays on two in vitro lung cell models. PM2.5 collected in Paris at a site close to the traffic exhibited a systematically higher OP in all assays compared to PM2.5 enriched in particles from domestic wood burning. Similar results were obtained for oxidative stress, expression of antioxidant enzymes, and pro-inflammatory chemokine in human bronchial epithelial and endothelial cells. The strongest correlations between OP assays and cellular responses were observed with the antioxidant (ascorbic acid and glutathione) depletion (OPAO) assay. Multivariate regression analysis from OP daily measurements suggested that OPAO was strongly correlated with polycyclic aromatic hydrocarbons at the traffic site while it was correlated with potassium for the domestic wood burning sample.
Assuntos
Poluentes Atmosféricos , Antioxidantes , Células Endoteliais , Humanos , Oxirredução , Estresse Oxidativo , Tamanho da Partícula , Material ParticuladoRESUMO
Nanomaterials are invading our environment due to their increasing use in a very broad range of sectors making human exposure foreseeable during the life cycle of these materials. Inhalation is one of the most frequent routes of exposure in case of unintentional exposure and the small size of nanomaterials allows them to reach the deep lung. Understanding the fate and effects of nanomaterials is a great challenge for scientists as they exhibit a huge physico-chemical diversity that drives their biological reactivity. It is critical to determine the fate of nanomaterials at their route of entry in the organism as this will determine their local and/or systemic effects. In this review we will describe the epithelial barriers and the clearance processes of the respiratory tract. The mechanisms involved in the internalization of nanomaterials by respiratory cells and their ability to cross the epithelial barrier will be presented, highlighting methodologies and the role of the nanomaterial physico-chemical properties.
Assuntos
Pulmão/metabolismo , Nanopartículas , Mucosa Respiratória/metabolismo , Animais , Humanos , Nanopartículas/química , Nanopartículas/metabolismo , Nanopartículas/uso terapêuticoRESUMO
Oxidative stress has increasingly been demonstrated as playing a key role in the biological response induced by nanoparticles (NPs). The acellular cytochrome c oxidation assay has been proposed to determine the intrinsic oxidant-generating capacity of NPs. Yet, there is a need to improve this method to allow a rapid screening to classify NPs in terms of toxicity. We adapted the cytochrome c assay to take into account NP interference, to improve its sensitivity and to develop a high-throughput method. The intrinsic oxidative ability of a panel of NPs (carbon black, Mn2O3, Cu, Ag, BaSO4, CeO2, TiO2 and ZnO) was measured with this enhanced test and compared to other acellular redox assays. To assess whether their oxidative potential correlates with cellular responses, we studied the effect of insoluble NPs on the human bronchial epithelial cell line NCI-H292 by measuring the cytotoxicity (WST-1 assay), pro-inflammatory response (IL-8 cytokine production and expression) and antioxidant defense induction (SOD2 and HO-1 expression). The adapted cytochrome c assay had a greatly increased sensitivity allowing the ranking of NPs in terms of their oxidative potential by using the developed high-throughput technique. Besides, a high oxidative potential revealed to be predictive for toxic effects as Mn2O3 NPs induced a strong oxidation of cytochrome c and a dose-dependent cytotoxicity, pro-inflammatory response and antioxidant enzyme expression. BaSO4, which presented no intrinsic oxidative potential, had no cellular effects. Nevertheless, CeO2 and TiO2 NPs demonstrated no acellular oxidant-generating capacity but induced moderate cellular responses. In conclusion, the novel cytochrome c oxidation assay could be used for high-throughput screening of the intrinsic oxidative potential of NPs. However, acellular redox assays may not be sufficient to discriminate among low-toxicity NPs, and additional tests are thus needed.
Assuntos
Citocromos c/química , Ensaios de Triagem em Larga Escala , Indicadores e Reagentes/química , Nanopartículas Metálicas/toxicidade , Oxidantes/toxicidade , Testes de Toxicidade , Animais , Brônquios/efeitos dos fármacos , Brônquios/imunologia , Brônquios/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Fenômenos Químicos , Cavalos , Humanos , Nanopartículas Metálicas/química , Oxidantes/química , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Tamanho da Partícula , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismo , Reprodutibilidade dos Testes , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Propriedades de SuperfícieRESUMO
Nanoparticles (NP) have a tendency to agglomerate after dispersion in physiological media, which can be prevented by the addition of serum. This may however result in modification of the toxic potential of particles due to the formation of protein corona. Our study aimed to analyze the role of serum that is added to improve the dispersion of 10 nm TiO2 NPs on in vitro and in vivo effects following the exposure via the respiratory route. We characterized NP size, surface charge, sedimentation rate, the presence of protein corona and the oxidant-generating capacity after NP dispersion in the presence/absence of serum. The effect of serum on NP internalization, cytotoxicity and pro-inflammatory responses was assessed in a human pulmonary cell line, NCI-H292. Serum in the dispersion medium led to a slower sedimentation, but an enhanced cellular uptake of TiO2 NPs. Despite this greater uptake, the pro-inflammatory response in NCI-H292 cells was lower after serum supplementation (used either as a dispersant or as a cell culture additive), which may be due to a reduced intrinsic oxidative potential of TiO2 NPs. Interestingly, serum could be added 2 h after the NP treatment without affecting the pro-inflammatory response. We also determined the acute pulmonary and hepatic toxicity in vivo 24 h after intratracheal instillation of TiO2 NPs in C57BL/6N mice. The use of serum resulted in an underestimation of the local acute inflammatory response in the lung, while a systemic response on glutathione reduction remained unaffected. In conclusion, serum as a dispersion agent for TiO2 NPs can lead to an underestimation of the acute pro-inflammatory response in vitro and in vivo. To avoid potential unwanted effects of dispersants and medium components, we recommend that the protocol of NM preparation should be thoroughly tested, and reflect as close as possible realistic exposure conditions.
Assuntos
Fígado/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Oxidantes/toxicidade , Veículos Farmacêuticos/química , Mucosa Respiratória/efeitos dos fármacos , Soro/química , Titânio/toxicidade , Absorção Fisiológica , Administração por Inalação , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fenômenos Químicos , Feminino , Fígado/imunologia , Fígado/metabolismo , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Camundongos Endogâmicos C57BL , Oxidantes/administração & dosagem , Oxidantes/química , Oxidantes/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Tamanho da Partícula , Distribuição Aleatória , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Propriedades de Superfície , Suspensões , Titânio/administração & dosagem , Titânio/química , Titânio/metabolismo , Testes de Toxicidade AgudaRESUMO
BACKGROUND: The lung epithelium constitutes the first barrier against invading pathogens and also a major surface potentially exposed to nanoparticles. In order to ensure and preserve lung epithelial barrier function, the alveolar compartment possesses local defence mechanisms that are able to control bacterial infection. For instance, alveolar macrophages are professional phagocytic cells that engulf bacteria and environmental contaminants (including nanoparticles) and secrete pro-inflammatory cytokines to effectively eliminate the invading bacteria/contaminants. The consequences of nanoparticle exposure in the context of lung infection have not been studied in detail. Previous reports have shown that sequential lung exposure to nanoparticles and bacteria may impair bacterial clearance resulting in increased lung bacterial loads, associated with a reduction in the phagocytic capacity of alveolar macrophages. RESULTS: Here we have studied the consequences of SiO2 nanoparticle exposure on Pseudomonas aeruginosa clearance, Pseudomonas aeruginosa-induced inflammation and lung injury in a mouse model of acute pneumonia. We observed that pre-exposure to SiO2 nanoparticles increased mice susceptibility to lethal pneumonia but did not modify lung clearance of a bioluminescent Pseudomonas aeruginosa strain. Furthermore, internalisation of SiO2 nanoparticles by primary alveolar macrophages did not reduce the capacity of the cells to clear Pseudomonas aeruginosa. In our murine model, SiO2 nanoparticle pre-exposure preferentially enhanced Pseudomonas aeruginosa-induced lung permeability (the latter assessed by the measurement of alveolar albumin and IgM concentrations) rather than contributing to Pseudomonas aeruginosa-induced lung inflammation (as measured by leukocyte recruitment and cytokine concentration in the alveolar compartment). CONCLUSIONS: We show that pre-exposure to SiO2 nanoparticles increases mice susceptibility to lethal pneumonia but independently of macrophage phagocytic function. The deleterious effects of SiO2 nanoparticle exposure during Pseudomonas aeruginosa-induced pneumonia are related to alterations of the alveolar-capillary barrier rather than to modulation of the inflammatory responses.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Nanopartículas/toxicidade , Pneumonia Bacteriana/induzido quimicamente , Infecções por Pseudomonas/induzido quimicamente , Pseudomonas aeruginosa/patogenicidade , Alvéolos Pulmonares/efeitos dos fármacos , Óxidos de Selênio/toxicidade , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/microbiologia , Citocinas/análise , Imunoglobulina M/análise , Exposição por Inalação , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/imunologia , Masculino , Camundongos Endogâmicos C57BL , Nanopartículas/química , Tamanho da Partícula , Fagocitose/efeitos dos fármacos , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/microbiologia , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Alvéolos Pulmonares/irrigação sanguínea , Óxidos de Selênio/química , Propriedades de Superfície , Análise de SobrevidaRESUMO
This study proposes global threat scores to prioritize the harmfulness of anthropogenic fine and ultrafine metallic particles (FMP) emitted into the atmosphere at the global scale. (Eco)toxicity of physicochemically characterized FMP oxides for metals currently observed in the atmosphere (CdO, CuO, PbO, PbSO(4), Sb(2)O(3), and ZnO) was assessed by performing complementary in vitro tests: ecotoxicity, human bioaccessibility, cytotoxicity, and oxidative potential. Using an innovative methodology based on the combination of (eco)toxicity and physicochemical results, the following hazard classification of the particles is proposed: CdCl2~CdO>CuO>PbO>ZnO>PbSO(4)>Sb(2)O(3). Both cadmium compounds exhibited the highest threat score due to their high cytotoxicity and bioaccessible dose, whatever their solubility and speciation, suggesting that cadmium toxicity is due to its chemical form rather than its physical form. In contrast, the Sb(2)O(3) threat score was the lowest due to particles with low specific area and solubility, with no effects except a slight oxidative stress. As FMP physicochemical properties reveal differences in specific area, crystallization systems, dissolution process, and speciation, various mechanisms may influence their biological impact. Finally, this newly developed and global approach could be widely used in various contexts of pollution by complex metal particles and may improve risk management.
Assuntos
Poluentes Ambientais/toxicidade , Nanopartículas Metálicas/toxicidade , Metais/toxicidade , Material Particulado/toxicidade , Oligoelementos/toxicidade , Aliivibrio fischeri , Compostos de Cádmio/toxicidade , Linhagem Celular Tumoral , Cobre/toxicidade , Ecotoxicologia/normas , Humanos , Chumbo/toxicidade , Nanopartículas Metálicas/química , Metais/química , Estresse Oxidativo , Óxidos/toxicidade , Material Particulado/química , Medição de Risco , Óxido de Zinco/toxicidadeRESUMO
A thorough understanding of the interactions of nanomaterials with biological systems and the resulting activation of signal transduction pathways is essential for the development of safe and consumer friendly nanotechnology. Here we present an overview of signaling pathways induced by nanomaterial exposures and describe the possible correlation of their physicochemical characteristics with biological outcomes. In addition to the hierarchical oxidative stress model and a review of the intrinsic and cell-mediated mechanisms of reactive oxygen species (ROS) generating capacities of nanomaterials, we also discuss other oxidative stress dependent and independent cellular signaling pathways. Induction of the inflammasome, calcium signaling, and endoplasmic reticulum stress are reviewed. Furthermore, the uptake mechanisms can be of crucial importance for the cytotoxicity of nanomaterials and membrane-dependent signaling pathways have also been shown to be responsible for cellular effects of nanomaterials. Epigenetic regulation by nanomaterials, effects of nanoparticle-protein interactions on cell signaling pathways, and the induction of various cell death modalities by nanomaterials are described. We describe the common trigger mechanisms shared by various nanomaterials to induce cell death pathways and describe the interplay of different modalities in orchestrating the final outcome after nanomaterial exposures. A better understanding of signal modulations induced by nanomaterials is not only essential for the synthesis and design of safer nanomaterials but will also help to discover potential nanomedical applications of these materials. Several biomedical applications based on the different signaling pathways induced by nanomaterials are already proposed and will certainly gain a great deal of attraction in the near future.
Assuntos
Espaço Intracelular/metabolismo , Nanoestruturas/toxicidade , Transdução de Sinais/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Humanos , Nanoestruturas/química , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Ligação Proteica , Proteínas/metabolismoRESUMO
Epidemiological studies showed a positive association between exposure to PM2.5 and the severity of influenza virus infection. However, the mechanisms by which PM2.5 can disrupt antiviral defence are still unclear. From this perspective, the objective of this study was to evaluate the effects of PM2.5 on antiviral signalling in the respiratory epithelium using the bronchial Calu-3 cell line grown at the air-liquid interface. Pre-exposure to PM2.5 before infection with the influenza virus was investigated, as well as a co-exposure. Although a physical interaction between the virus and the particles seems possible, no effect of PM2.5 on viral replication was observed during co-exposure, although a downregulation of IFN-ß release was associated to PM2.5 exposure. However, pre-exposure slightly increased the viral nucleoprotein production and the pro-inflammatory response. Conversely, the level of the myxovirus resistance protein A (MxA), an interferon-stimulated gene (ISG) induced by IFN-ß, was reduced. Therefore, these results suggest that pre-exposure to PM2.5 could alter the antiviral response of bronchial epithelial cells, increasing their susceptibility to viral infection.
Assuntos
Influenza Humana , Orthomyxoviridae , Viroses , Humanos , Interferons , Influenza Humana/genética , Influenza Humana/metabolismo , Mucosa Respiratória , Antivirais , Epitélio/metabolismo , Material Particulado/toxicidadeRESUMO
BACKGROUND: The involvement of particulate matter (PM) in cardiorespiratory diseases is now established in developed countries whereas in developing areas such as Africa with a high level of specific pollution, PM pollution and its effects are poorly studied. Our objective was to characterize the biological reactivity of urban African aerosols on human bronchial epithelial cells in relation to PM physico-chemical properties to identify toxic sources. METHODS: Size-speciated aerosol chemical composition was analyzed in Bamako (BK, Mali, 2 samples with one having desert dust event BK1) and Dakar (DK; Senegal) for Ultrafine UF, Fine F and Coarse C PM. PM reactivity was studied in human bronchial epithelial cells investigating six biomarkers (oxidative stress responsive genes and pro-inflammatory cytokines). RESULTS: PM mass concentrations were mainly distributed in coarse mode (60%) and were impressive in BK1 due to the desert dust event. BK2 and DK samples showed a high content of total carbon characteristic of urban areas. The DK sample had huge PAH quantities in bulk aerosol compared with BK that had more water soluble organic carbon and metals. Whatever the site, UF and F PM triggered the mRNA expression of the different biomarkers whereas coarse PM had little or no effect. The GM-CSF biomarker was the most discriminating and showed the strongest pro-inflammatory effect of BK2 PM. The analysis of gene expression signature and of their correlation with main PM compounds revealed that PM-induced responses are mainly related to organic compounds. The toxicity of African aerosols is carried by the finest PM as with Parisian aerosols, but when considering PM mass concentrations, the African population is more highly exposed to toxic particulate pollution than French population. Regarding the prevailing sources in each site, aerosol biological impacts are higher for incomplete combustion sources resulting from two-wheel vehicles and domestic fires than from diesel vehicles (Dakar). Desert dust events seem to produce fewer biological impacts than anthropogenic sources. DISCUSSION: Our study shows that combustion sources contribute to the high toxicity of F and UF PM of African urban aerosols, and underlines the importance of emission mitigation and the imperative need to evaluate and to regulate particulate pollution in Africa.
Assuntos
Brônquios/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Pneumopatias/induzido quimicamente , Material Particulado/toxicidade , Saúde da População Urbana , Emissões de Veículos/toxicidade , Aerossóis , Biomarcadores/metabolismo , Brônquios/patologia , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Exposição por Inalação/efeitos adversos , Pneumopatias/genética , Pneumopatias/imunologia , Pneumopatias/metabolismo , Mali , Estresse Oxidativo/efeitos dos fármacos , Tamanho da Partícula , Material Particulado/análise , RNA Mensageiro/metabolismo , Medição de Risco , Senegal , Emissões de Veículos/análiseRESUMO
BACKGROUND: The uptake of nanoparticles (NPs) by cells remains to be better characterized in order to understand the mechanisms of potential NP toxicity as well as for a reliable risk assessment. Real NP uptake is still difficult to evaluate because of the adsorption of NPs on the cellular surface. RESULTS: Here we used two approaches to distinguish adsorbed fluorescently labeled NPs from the internalized ones. The extracellular fluorescence was either quenched by Trypan Blue or the uptake was analyzed using imaging flow cytometry. We used this novel technique to define the inside of the cell to accurately study the uptake of fluorescently labeled (SiO2) and even non fluorescent but light diffracting NPs (TiO2). Time course, dose-dependence as well as the influence of surface charges on the uptake were shown in the pulmonary epithelial cell line NCI-H292. By setting up an integrative approach combining these flow cytometric analyses with confocal microscopy we deciphered the endocytic pathway involved in SiO2 NP uptake. Functional studies using energy depletion, pharmacological inhibitors, siRNA-clathrin heavy chain induced gene silencing and colocalization of NPs with proteins specific for different endocytic vesicles allowed us to determine macropinocytosis as the internalization pathway for SiO2 NPs in NCI-H292 cells. CONCLUSION: The integrative approach we propose here using the innovative imaging flow cytometry combined with confocal microscopy could be used to identify the physico-chemical characteristics of NPs involved in their uptake in view to redesign safe NPs.
Assuntos
Endocitose , Células Epiteliais/efeitos dos fármacos , Citometria de Fluxo/métodos , Microscopia Confocal/métodos , Nanopartículas , Dióxido de Silício , Adsorção , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Endocitose/fisiologia , Células Epiteliais/metabolismo , Corantes Fluorescentes/química , Humanos , Nanopartículas/química , Nanopartículas/toxicidade , Tamanho da Partícula , Dióxido de Silício/química , Dióxido de Silício/toxicidade , Propriedades de Superfície , Azul Tripano/químicaRESUMO
BACKGROUND: It has been shown that nanomaterials (NMs) are able to translocate to secondary tissues one of the important being the kidneys. Oxidative stress has been implicated as a possible mechanism for NM toxicity, hence effects on the human renal proximal tubule epithelial cells (HK-2) treated with a panel of engineered nanomaterials (NMs) consisting of two zinc oxide particles (ZnO - coated - NM 110 and uncoated - NM 111), two multi walled carbon nanotubes (MWCNT) (NM 400 and NM 402), one silver (NM 300) and five TiO2 NMs (NM 101, NRCWE 001, 002, 003 and 004) were evaluated. METHODS: In order to assess the toxicological impact of the engineered NMs on HK-2 cells - WST-1 cytotoxicity assay, FACSArray, HE oxidation and the comet assays were utilised. For statistical analysis, the experimental values were compared to their corresponding controls using an ANOVA with Tukey's multiple comparison. RESULTS: We found the two ZnO NMs (24 hr LC50 - 2.5 µg/cm2) and silver NM (24 hr LC50 - 10 µg/cm2) were highly cytotoxic to the cells. The LC50 was not attained in the presence of any of the other engineered nanomaterials (up to 80 µg/cm2). All nanomaterials significantly increased IL8 and IL6 production. Meanwhile no significant change in TNF-α or MCP-1 was detectable. The most notable increase in ROS was noted following treatment with the Ag and the two ZnO NMs. Finally, genotoxicity was measured at sub-lethal concentrations. We found a small but significant increase in DNA damage following exposure to seven of the ten NMs investigated (NM 111, NRCWE 001 and NRCWE 003 being the exception) with this increase being most visible following exposure to Ag and the positively charged TiO2. CONCLUSIONS: While the NMs could be categorised as low and highly cytotoxic, sub-lethal effects such as cytokine production and genotoxicity were observed with some of the low toxicity materials.
Assuntos
Engenharia Biomédica/métodos , Citotoxinas/toxicidade , Dano ao DNA/efeitos dos fármacos , Mediadores da Inflamação , Nanoestruturas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Animais , Bovinos , Linhagem Celular , Linhagem Celular Transformada , Citotoxinas/química , Dano ao DNA/fisiologia , Humanos , Mediadores da Inflamação/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Nanoestruturas/química , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Long term exposure to particulate air pollution is known to increase respiratory morbidity and mortality. In urban areas with dense traffic most of these particles are generated by vehicles, via engine exhaust or wear processes. Non-exhaust particles come from wear processes such as those concerning brakes and their toxicity is little studied. To improve our understanding of the lung toxicity mechanisms of the nanometric fraction of brake wear nanoparticles (BWNPs), we studied whether these particles affect the barrier properties of the respiratory epithelium considering particle translocation, mucus production and repair efficiency. The Calu-3 cell line grown in two-compartment chambers was used to mimic the bronchial epithelial barrier. BWNPs detected by single-particle ICP-MS were shown to cross the epithelial tissue in small amounts without affecting the barrier integrity properties, because the permeability to Lucifer yellow was not increased and there was no cytotoxicity as assessed by the release of lactate-dehydrogenase. The interaction of BWNPs with the barrier did not induce a pro-inflammatory response, but increased the expression and production of MU5AC, a mucin, by a mechanism involving the epidermal growth factor receptor pathway. During a wound healing assay, BWNP-loaded cells exhibited the same ability to migrate, but those at the edge of the wound showed higher 5-ethynyl-2'-deoxyuridine incorporation, suggesting a higher proliferation rate. Altogether these results showed that BW. NPs do not exert overt cytotoxicity and inflammation but can translocate through the epithelial barrier in small amounts and increase mucus production, a key feature of acute inflammatory and chronic obstructive pulmonary diseases. Their loading in epithelial cells may impair the repair process through increased proliferation.
Assuntos
Poluição do Ar , Nanopartículas , Células Epiteliais/metabolismo , Epitélio , Nanopartículas/toxicidade , PoeiraRESUMO
There is still a lack of in vitro human models to evaluate the chronic toxicity of drugs and environmental pollutants. Here, we used a 3D model of the human bronchial epithelium to assess repeated exposures to xenobiotics. The Calu-3 human bronchial cell line was exposed to silver nanoparticles (AgNP) 5 times during 12 days, at the air-liquid interface, to mimic single and repeated exposure to inhaled particles. Repeated exposures induced a stronger induction of the metal stress response and a steady oxidative stress over time. A sustained translocation of silver was observed after each exposure without any loss of the epithelial barrier integrity. The proteomic analysis of the mucus revealed changes in the secreted protein profiles associated with the epithelial immune response after repeated exposures only. These results demonstrate that advanced in vitro models are efficient to investigate the adaptive response of human cells submitted to repeated xenobiotic exposures.
Assuntos
Nanopartículas Metálicas , Prata , Humanos , Prata/toxicidade , Nanopartículas Metálicas/toxicidade , Proteômica , Xenobióticos/toxicidade , Linhagem Celular , Células EpiteliaisRESUMO
Human exposure to PM(2.5) (particulate matter with an aerodynamic diameter below 2.5 µm) is known to be responsible for airway inflammation and may also induce airway remodelling. In respiratory epithelial cells exposed to PM(2.5), releases of pro-inflammatory cytokines such as granulocyte macrophage-colony stimulating factor (GM-CSF) and growth factor ligands of the epidermal growth factor receptor (EGFR) are increased. The present study aimed at determining the involvement of EGFR ligands by autocrine effects in PM(2.5)-induced GM-CSF release. PM(2.5) exposure triggers GM-CSF release by human bronchial epithelial (HBE) cells. This release is dependent on EGFR activation by ligand binding as it is inhibited by AG1478, an inhibitor of EGFR tyrosine kinase activity as well as by a neutralizing anti-EGFR antibody. The use of conditioned medium from cells previously exposed to PM(2.5) demonstrates that PM(2.5)-exposed cells release soluble EGFR ligands able to induce GM-CSF release by an autocrine manner. It was further demonstrated by inhibiting tumour-necrosis factor-alpha converting enzyme (TACE) that is involved in some EGFR ligand shedding. TAPI-2 and GM-6001, two TACE inhibitors, prevented the PM(2.5)-induced GM-CSF release as well as the silencing of TACE by siRNA. We provide evidence that the pro-inflammatory response induced by PM(2.5) exposure on HBE cells, results from an autocrine effect of EGFR ligands released by TACE activity. This autocrine loop by eliciting and sustaining inflammation could contribute to exacerbation of airway remodelling in respiratory-compromised individuals.
Assuntos
Células Epiteliais/efeitos dos fármacos , Receptores ErbB/efeitos dos fármacos , Inflamação/induzido quimicamente , Material Particulado/toxicidade , Proteínas ADAM/efeitos dos fármacos , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM17 , Remodelação das Vias Aéreas/efeitos dos fármacos , Comunicação Autócrina/efeitos dos fármacos , Brônquios/citologia , Brônquios/efeitos dos fármacos , Linhagem Celular , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Inativação Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Inflamação/patologia , Tamanho da Partícula , Quinazolinas/farmacologia , RNA Interferente Pequeno/metabolismo , Tirfostinas/farmacologiaRESUMO
Particulate pollution is suspected to contribute to obstructive lung diseases characterized by chronic inflammation, mucus hypersecretion and bronchial remodeling. Our aim was to study the effect of real-world particulate matter (PM) on the expression of a mucin, MUC5AC, focusing on the role of the epidermal growth factor receptor (EGFR) pathway. MUC5AC induction was studied in vivo in mice trachea and in vitro in human bronchial epithelial cells (HBEC) exposed to urban fine PM. Fine PM were able to induce MUC5AC mRNA in mice trachea after 48 h of exposure (50 µg PM/mouse), and MUC5AC mRNA and protein in HBEC after 24 h of exposure (from 5 µg PM/cm(2)). It was associated with the increased expression of amphiregulin (AREG), an EGFR ligand. Experiments with conditioned media (media from PM-treated cells) demonstrated the involvement of AREG on MUC5AC induction as MUC5AC induction by media from PM-treated cells was prevented in the presence of either EGFR- or AREG-neutralizing antibodies. The effect of an inhibitor of a metalloprotease involved in the AREG shedding confirmed the autocrine loop made by AREG leading to MUC5AC induction by fine PM. We also demonstrated that IL-8 pro-inflammatory cytokine induction was dependent on the same autocrine mechanisms. We demonstrate for the first time that MUC5AC expression and production is increased by short-term exposure to fine PM through an autocrine effect of AREG. Our study provides mechanistic explanations to the exacerbation of obstructive lung diseases induced by particulate pollution characterized by mucus hypersecretion and chronic inflammation.
Assuntos
Comunicação Autócrina/efeitos dos fármacos , Glicoproteínas/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Mucina-5AC/biossíntese , Material Particulado/toxicidade , Anfirregulina , Animais , Linhagem Celular , Meios de Cultivo Condicionados , Família de Proteínas EGF , Ensaio de Imunoadsorção Enzimática , Receptores ErbB/biossíntese , Receptores ErbB/genética , Citometria de Fluxo , Imunofluorescência , Injeções Espinhais , Interleucina-8/metabolismo , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Tamanho da Partícula , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador alfa/metabolismoRESUMO
Epidemiological studies in urban areas have linked increasing respiratory and cardiovascular pathologies with atmospheric particulate matter (PM) from anthropic activities. However, the biological fate of metal-rich PM industrial emissions in urban areas of developed countries remains understudied. Lead toxicity and bioaccessibility assessments were therefore performed on emissions from a lead recycling plant, using complementary chemical acellular tests and toxicological assays, as a function of PM size (PM(10-2.5), PM(2.5-1) and PM(1)) and origin (furnace, refining and channeled emissions). Process PM displayed differences in metal content, granulometry, and percentage of inhalable fraction as a function of their origin. Lead gastric bioaccessibility was relatively low (maximum 25%) versus previous studies; although, because of high total lead concentrations, significant metal quantities were solubilized in simulated gastrointestinal fluids. Regardless of origin, the finest PM(1) particles induced the most significant pro-inflammatory response in human bronchial epithelial cells. Moreover, this biological response correlated with pro-oxidant potential assay results, suggesting some biological predictive value for acellular tests. Pulmonary effects from lead-rich PM could be driven by thiol complexation with either lead ions or directly on the particulate surface. Finally, health concern of PM was discussed on the basis of pro-inflammatory effects, accellular test results, and PM size distribution.
Assuntos
Poluentes Atmosféricos/toxicidade , Brônquios/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Chumbo/toxicidade , Metalurgia , Material Particulado/toxicidade , Poluentes Atmosféricos/análise , Poluentes Atmosféricos/metabolismo , Animais , Disponibilidade Biológica , Brônquios/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Fontes de Energia Elétrica , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fator Estimulador de Colônias de Granulócitos/metabolismo , Humanos , Técnicas In Vitro , Chumbo/análise , Chumbo/metabolismo , Tamanho da Partícula , Material Particulado/análise , Material Particulado/metabolismo , Reciclagem , SuínosRESUMO
Increasing evidence linking nanoparticles (NPs) with different cellular outcomes necessitate an urgent need for the better understanding of cellular signalling pathways triggered by NPs. Oxidative stress has largely been reported to be implicated in NP-induced toxicity. It could activate a wide variety of cellular events such as cell cycle arrest, apoptosis, inflammation and induction of antioxidant enzymes. These responses occur after the activation of different cellular pathways. In this context, three groups of MAP kinase cascades [ERK (extracellular signal-regulated kinases), p38 mitogen-activated protein kinase and JNK (c-Jun N-terminal kinases)] as well as redox-sensitive transcription factors such as NFκB and Nrf-2 were specially investigated. The ability of NPs to interact with these signalling pathways could partially explain their cytotoxicity. The induction of apoptosis is also closely related to the modulation of signalling pathways induced by NPs. Newly emerged scientific areas of research are the studies on interactions between NPs and biological molecules in body fluids, cellular microenvironment, intracellular components or secreted cellular proteins such as cytokines, growth factors and enzymes and use of engineered NPs to target various signal transduction pathways in cancer therapy. Recently published data present the ability of NPs to interact with membrane receptors leading to a possible aggregation of these receptors. These interactions could lead to a sustained modulation of specific signalling in the target cells or paracrine and even "by-stander" effects of the neighbouring cells or tissues. However, oxidative stress is not sufficient to explain specific mechanisms which could be induced by NPs, and these new findings emphasize the need to revise the paradigm of oxidative stress to explain the effects of NPs.
Assuntos
Nanopartículas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Humanos , Inflamação/induzido quimicamente , Nanopartículas/uso terapêuticoRESUMO
The increased levels of fine particles in the atmosphere are suspected of aggravating cardiopulmonary diseases, but the determinants of particle toxicity are poorly understood. This work aims at studying the role of composition and size in the toxicity of size-segregated particulate matter (PM) collected at different sites on human bronchial epithelial cells. PM were sampled at a traffic urban site (Urb S) and a rural site (Rur S) during the pesticide-spreading period. Ultrafine (UF), fine (F), and coarse (C) PM were characterized by their shape and chemical composition. Whatever the site, the finest PM (UF and F) induced the mRNA expression of CYP1A1, a biomarker of polyaromatic hydrocarbons (PAH) exposure, NQO-1 and heme HO-1, two antioxidant responsive element-driven genes; and two effect biomarkers, GM-CSF, a proinflammatory cytokine and amphiregulin (AR), a growth factor. C PM have a low or no effect. Interestingly, AR is more strongly induced by rural PM at the same mass exposure. These discrepancies suggest involvement of PM chemical composition: rural PM bearing the characteristics of aged aerosols with a high content of water-soluble components, and PM at urban kerbside sites containing mainly water-insoluble components. To conclude, we provide evidence that the finest PM fractions, whatever their origin, are more prone to induce exposure and effect biomarkers. The AR differential expression suggests a source-dependent effect requiring further investigation because of the role of this growth factor in airway remodeling, a characteristic feature of chronic lung respiratory diseases exacerbated by particulate pollution.