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Human exposure to PM (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, 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-induced GM-CSF release. PM 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 demonstrates that PM-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-induced GM-CSF release as well as the silencing of TACE by siRNA. We provide evidence that the pro-inflammatory response induced by PM 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. [ABSTRACT FROM AUTHOR]

Abstract: Particulate matter (PM) is suspected to play a role in environmentally-induced pathologies. Due to its complex composition, the contribution of each PM components to PM-induced biological effects remains unclear. Four samples of Paris PM2.5 having different polyaromatic hydrocarbons and metals contents were compared with each other and with their respective aqueous and organic extracts used alone or in combination. The four PM2.5 samples similarly induced granulocyte macrophage-colony stimulating factor (GM-CSF) release, a pro-inflammatory cytokine, by human bronchial epithelial cells. It results from the activation of upstream signalling pathways and the modulation of the cellular redox state that is different according to PM2.5 samples. The PM-aqueous extracts contained soluble metals involved in hydroxyl radical production in abiotic conditions. However they slightly contributed to the intracellular reactive oxygen species production and GM-CSF release by comparison with organic extracts. Organic compounds transactivated the xenobiotic responsive element (XRE) and antioxidant responsive element (ARE), leading to increased cytochrome P450 1A1 expression and NADPH-quinone oxydoreductase-1 expression respectively but to different extend according to PM samples underlying differences in their bioavailability. Our study underlines that chemical composition of particles per se is insufficient to predict cellular effects and that the interaction and the bioavailability of the various components were critical. [Copyright &y& Elsevier]

Abstract: The ubiquitous presence of nanoparticles (NPs) together with increasing evidence linking them to negative health effects points towards the need to develop the understanding of mechanisms by which they exert toxic effects. This study was designed to investigate the role of surface area and oxidative stress in the cellular effects of two chemically distinct NPs, carbon black (CB) and titanium dioxide (TiO2), on the bronchial epithelial cell line (16HBE14o-). CB and TiO2 NPs were taken up by 16HBE cells in a dose-dependent manner and were localized within the endosomes or free in the cytoplasm. Oxidative stress produced inside the cell by NPs was well correlated to the BET surface area and endocytosis of NPs. Contrary to intracellular conditions only CB NPs produced reactive oxygen species (ROS) under abiotic conditions. Exposure of cells to NPs resulted in an increased granulocyte macrophage colony stimulating factor (GM-CSF) mRNA expression and secretion. Inflammatory effects of NPs were dependent on the surface area and were mediated through oxidative stress as they were inhibited by catalase. It can be concluded that NP induced oxidative stress and pro-inflammatory responses are well correlated not only with the BET (Brunauer, Emmett and Teller) surface of the individual NPs but also with the internalized amount of NPs. Differences of even few nanometers in primary particle size lead to significant changes in inflammatory and oxidative stress responses. [Copyright &y& Elsevier]

BACKGROUND AND OBJECTIVES: Because pulmonary circulation is the primary vascular target of inhaled particulate matter (PM), and nitric oxide is a major vasculoprotective agent, in this study we investigated the effect of various particles on the NO--cyclic guanosine monophosphate (cGMP) pathway in pulmonary arteries. METHODS: We used intrapulmonary arteries and/or endothelial cells, either exposed in vitro to particles or removed from PM-instilled animals for assessment of vasomotricity, cGMP and reactive oxygen species (ROS) levels, and cytokine/chemokine release. RESULTS: Endothelial NO-dependent relaxation and cGMP accumulation induced by acetylcholine (ACh) were both decreased after 24 hr exposure of rat intrapulmonary arteries to standard reference material 1648 (SRM1648; urban PM). Relaxation due to NO donors was also decreased by SRM1648, whereas responsiveness to cGMP analogue remained unaffected. Unlike SRM1648, ultrafine carbon black and ultrafine and fine titanium dioxide (TiO2) manufactured particles did not impair NO-mediated relaxation. SRM1648-induced decrease in relaxation response to ACh was prevented by dexamethasone (an anti-inflammatory agent) but not by antioxidants. Accordingly, SRM1648 increased the release of proinflammatory mediators (tumor necrosis factor-α, interleukin-8) from intrapulmonary arteries or pulmonary artery endothelial cells, but did not elevate ROS levels within intrapulmonary arteries. Decreased relaxation in response to ACh was also evidenced in intrapulmonary arteries removed from rats intratracheally instilled with SRM1648, but not with fine TiO2. CONCLUSION: In contrast to manufactured particles (including nanoparticles), urban PM impairs NO but not cGMP responsiveness in intrapulmonary arteries. We attribute this effect to oxidative-stress--independent inflammatory response, resulting in decreased guanylyl cyclase activation by NO. Such impairment of the NO pathway may contribute to urban-PM--induced cardiovascular dysfunction. [ABSTRACT FROM AUTHOR]

Particulate atmospheric pollutants interact with the human airway epithelium, which releases cytokines, chemokines, and EGF receptor (EGFR) ligands leading to proinflammatory responses. There is little information concerning the short-term effects of EGFR activation by extracellular ligands on ionic regulation of airway surface lining fluids. We identified in the membrane of human epithelial bronchial cells (16HBE14o- line) an endogenous calcium- and voltage-dependent, outwardly rectifying small-conductance chloride channel (CACC), and we examined the effects of EGF on CACC activity. Ion channel currents were recorded with the patch-clamp technique. In cell-attached membrane patches, CACC were activated by exposure of the external surface of the cells to physiological concentrations of EGF without any change in cytosolic Ca2+ concentration ([Ca2+]i) and inhibited by tyrphostin AG-1478 (an inhibitor of EGFR that also blocks EGF-dependent Src family kinase activation). EGF activation of c-Src protein in 16HBE14o- cells was observed, and the signaling pathway elicited by EGFR was blocked by tyrphostin AG-1478. In excised inside-out membrane patches CACC were activated by exposure of the cytoplasmic face of the channels to the human recombinant Src(p60c-crc) kinase with endogenous or exogenous ATP and inhibited by λ-protein phosphatase. Secretion of EGFR ligands by epithelial airway cells exposed to pollutants would then elicit a rapid and direct ionic response of CACC mediated by EGFR activation via a Src kinase family-dependent signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract: Exposure to ambient particulate matter (PM) is responsible for airway inflammation and tissue remodeling. Urban PM2.5 (aerodynamic diameter <2.5μm) is a complex mixture rich in soots and containing hydrosoluble and organic components. We previously showed that the exposure of airway epithelial cells to PM2.5 triggers the release of amphiregulin (AR), ligand of the epidermal growth factor receptor (EGFR) involved in proinflammatory and repair responses. The effect of Paris PM2.5 organic and aqueous fractions in AR expression and secretion was investigated on the bronchial epithelial cell line 16HBE and normal human nasal epithelial (NHNE) cells. Both a macroarray specific for inflammation pathways and RT-PCR showed an AR upregulation in organic extract-treated 16HBE cells. AR release is induced in 16HBE and NHNE cells grown on plastic and exposed to native PM2.5, organic extract and to a lesser extent washed PM2.5 (deprived of its hydrosoluble content) and aqueous extract. Furthermore, as assessed by using NHNE cells grown on Transwell inserts, this secretion is polarized toward the basolateral side where the EGFR is expressed. To conclude, both PM2.5 organic and hydrosoluble components are involved in the expression and secretion of AR; organic compounds exhibiting a strong effect when they are easily bioavailable. [Copyright &y& Elsevier]

Oxygen species may be formed in the air spaces of the respiratory tract in response to environmental pollution such as particulate matter. The mechanisms and target molecules of these oxidants are still mainly unknown but may involve modifications of the ionic homeostasis in epithelial cells. Cytosolic concentrations of Ca2+ (Fura2) and Na+ (SBFI) and short-circuit current (Isc) were followed in primary cultures of human nasal epithelial cells and in the cell line 16HBE14o− after exposure to H2O2 or ·OH (H2O2+Fe2+). Cells were grown on glass coverslips for ionic imaging or on permeable snapwell inserts for Isc studies. Exposure of the apical as well as the basal side of the cultures to H2O2 or ·OH induced a concentration-dependent transient increase in Isc which is due to a transient secretion of Cl−. Cai also increased transiently with approximately the same kinetics. The response was dependent on the release of calcium from intracellular stores. Nai on the contrary increased steadily over more than an hour. When the apical membrane was permeabilized with gramicidin, ·OH inhibited the Na+ current (a measure of Na+-K+-ATPase activity in the baso-lateral membrane). The arrest of the pump was significant after 30 min exposure to oxidant. On the other hand no increase in the apical or baso-lateral sodium conductances could be detected. The progressive arrest of the Na+/K+-pump may contribute to the sustained elevation of Nai. This strong modification in the cellular ionic homeostasis may participate in the stress response of the respiratory epithelium through alterations in signal transduction pathways. [Copyright &y& Elsevier]

Alternative methods to animal testing are used in fundamental and clinical research, for the realization of studies for regulatory purposes, and also screening operations in the development of new molecules. They are based on in vitro (cell models) or in silico (mathematical models) replacement methods. They have been largely promoted by the 3Rs rule (Replace, Reduce, Refine) which aims at regulating animal experimentation. For biomedical research, these different methods are valuable tools for better understanding the physiology of organisms and the mechanisms of the effects of chemicals and physical agents on them., (© Société de Biologie, 2023.)

The outbreak of COVID-19 raised numerous questions on the interactions between the occurrence of new infections, the environment, climate and health. The European Union requested the H2020 HERA project which aims at setting priorities in research on environment, climate and health, to identify relevant research needs regarding Covid-19. The emergence and spread of SARS-CoV-2 appears to be related to urbanization, habitat destruction, live animal trade, intensive livestock farming and global travel. The contribution of climate and air pollution requires additional studies. Importantly, the severity of COVID-19 depends on the interactions between the viral infection, ageing and chronic diseases such as metabolic, respiratory and cardiovascular diseases and obesity which are themselves influenced by environmental stressors. The mechanisms of these interactions deserve additional scrutiny. Both the pandemic and the social response to the disease have elicited an array of behavioural and societal changes that may remain long after the pandemic and that may have long term health effects including on mental health. Recovery plans are currently being discussed or implemented and the environmental and health impacts of those plans are not clearly foreseen. Clearly, COVID-19 will have a long-lasting impact on the environmental health field and will open new research perspectives and policy needs., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Because of an increasing exposure to environmental and occupational nanoparticles (NPs), the potential risk of these materials for human health should be better assessed. Since one of the main routes of entry of NPs is via the lungs, it is of paramount importance to further characterize their impact on the respiratory system. Here, we have studied the uptake of fluorescently labeled SiO₂ NPs (50 and 100 nm) by epithelial cells (NCI-H292) and alveolar macrophages (MHS) in the presence or absence of pulmonary surfactant. The quantification of NP uptake was performed by measuring cell-associated fluorescence using flow cytometry and spectrometric techniques in order to identify the most suitable methodology. Internalization was shown to be time and dose dependent, and differences in terms of uptake were noted between epithelial cells and macrophages. In the light of our observations, we conclude that flow cytometry is a more reliable technique for the study of NP internalization, and importantly, that the hydrophobic fraction of lung surfactant is critical for downregulating NP uptake in both cell types.

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.

Exposure of epithelial respiratory cells to the alkylating agent, mechlorethamine (HN2), induces anoikis initiated by mitochondrial depolarization and caspase-2 activation. The mechanisms of disruption of cell interactions were investigated and expression of integrins, E-cadherin, and actin were therefore studied after HN2 treatment. In the adherent cells, an early disruption of F-actin occurred associated with cell rounding. Inhibitors of caspase-2 resulted in attenuating of the decline of adhesion proteins and microfilaments. HN2-induced down-regulation of beta1 integrin, E-cadherin expression and F-actin pattern occurred in detached cells but were efficiently prevented by inhibitors of mitochondrial permeabilization. Moreover, inhibiting mitochondrial depolarization improved significantly both cell survival and capacity of detached cells to re-adhere. These findings confirm the pro-survival integrins and E-cadherin mediated signalling pathway. The central role of mitochondria in HN2-induced cell detachment is reinforced, suggesting that mitochondria acts as a key executor of reduced cell adherence during anoikis and could be responsible of an inside-out signalling. Present data support the potential of these therapeutics, generated via the inhibition of mitochondrial depolarization, as protectors against the alkylating agent lesions.

Particulate atmospheric pollutants interact with the human airway epithelium, which releases cytokines, chemokines, and EGF receptor (EGFR) ligands leading to proinflammatory responses. There is little information concerning the short-term effects of EGFR activation by extracellular ligands on ionic regulation of airway surface lining fluids. We identified in the membrane of human epithelial bronchial cells (16HBE14o(-) line) an endogenous calcium- and voltage-dependent, outwardly rectifying small-conductance chloride channel (CACC), and we examined the effects of EGF on CACC activity. Ion channel currents were recorded with the patch-clamp technique. In cell-attached membrane patches, CACC were activated by exposure of the external surface of the cells to physiological concentrations of EGF without any change in cytosolic Ca(2+) concentration ([Ca(2+)](i)) and inhibited by tyrphostin AG-1478 (an inhibitor of EGFR that also blocks EGF-dependent Src family kinase activation). EGF activation of c-Src protein in 16HBE14o(-) cells was observed, and the signaling pathway elicited by EGFR was blocked by tyrphostin AG-1478. In excised inside-out membrane patches CACC were activated by exposure of the cytoplasmic face of the channels to the human recombinant Src(p60(c-src)) kinase with endogenous or exogenous ATP and inhibited by lambda-protein phosphatase. Secretion of EGFR ligands by epithelial airway cells exposed to pollutants would then elicit a rapid and direct ionic response of CACC mediated by EGFR activation via a Src kinase family-dependent signaling pathway.

Epidemiological studies have clearly shown a relationship between respiratory diseases and air pollution. Ozone and ambient particles are the main pollutants contributing to the exacerbation of these pathologies. Their toxicity resides in their ability to generate an oxidative stress. The level of oxidative stress and the specificity of the cellular responses result from complex interactions between pro- and anti-oxidants, leading to differentiated cellular strategies. Hierarchical biological responses: adaptation, inflammation, lesions, can be determined according to the oxidative insult and individual anti-oxidant capacities. A better health risk assessment could be achieved by taking into account the oxidative properties of air pollution especially those of ultrafine particles.

Exposure to ambient particulate matter (PM) is responsible for airway inflammation and tissue remodeling. Urban PM(2.5) (aerodynamic diameter <2.5microm) is a complex mixture rich in soots and containing hydrosoluble and organic components. We previously showed that the exposure of airway epithelial cells to PM(2.5) triggers the release of amphiregulin (AR), ligand of the epidermal growth factor receptor (EGFR) involved in proinflammatory and repair responses. The effect of Paris PM(2.5) organic and aqueous fractions in AR expression and secretion was investigated on the bronchial epithelial cell line 16HBE and normal human nasal epithelial (NHNE) cells. Both a macroarray specific for inflammation pathways and RT-PCR showed an AR upregulation in organic extract-treated 16HBE cells. AR release is induced in 16HBE and NHNE cells grown on plastic and exposed to native PM(2.5), organic extract and to a lesser extent washed PM(2.5) (deprived of its hydrosoluble content) and aqueous extract. Furthermore, as assessed by using NHNE cells grown on Transwell inserts, this secretion is polarized toward the basolateral side where the EGFR is expressed. To conclude, both PM(2.5) organic and hydrosoluble components are involved in the expression and secretion of AR; organic compounds exhibiting a strong effect when they are easily bioavailable.

Ambient particulate matter (PM) is known to induce inflammation in the respiratory tract of exposed subjects. The aim of the present study was to detect, in bronchial epithelial cells, candidate inflammatory genes exhibiting transcriptional modifications following urban PM2.5 exposure. Paris urban PM2.5 sampled either at a curbside or a background station in winter and in summer was tested in comparison with diesel exhaust particles (DEP) at 10 microg/cm2 on human bronchial epithelial (16-HBE) cells (18 h of exposure). The gene profiling study performed using a 375 cDNA cytokine expression array highlighted the differential expression of certain genes, three of which were selected as genes of interest: the IL-1 alpha cytokine, the GRO-alpha chemokine, and amphiregulin, a ligand of the EGF receptor. Their increased expression was confirmed by RT-PCR and/or by Northern blotting in bronchial epithelial cells. In the culture medium of particle-treated cultures, increased release of GRO-alpha and amphiregulin was shown. The particle component responsible for protein release varied for the two genes. The organic extract seemed to be mainly involved in amphiregulin expression and secretion, whereas both the aqueous and organic extracts induced GRO-alpha release. In conclusion, in bronchial epithelial cells, Paris PM2.5 increased mRNA and protein expression of GRO-alpha and AR involved in the chemoattraction process and bronchial remodeling, respectively.

Doxycycline (DOX), a synthetic tetracycline, may have potential utility in the management of cancers and in the treatment of chronic inflammatory diseases due to its role in growth, invasion and metastasis of many tumors, on cell proliferation and as inducer of apoptosis. Some studies established its role in the treatment of lesions induced by mustards, warfare agents causing severe damage with blistering and tissue detachment in exposed areas of the body. In the present study, the effect of Dox was investigated in a human bronchial epithelial cell line. Dox induced a time- and concentration-dependent cell proliferation inhibition, associated with a cell cycle arrest in S phase, a decrease in viability due to apoptosis and necrosis, and cell detachment. This latter was partly correlated with early activation of caspase-3 before detachment, and with mitochondrial alteration. Cell transfection with a Bcl-2 encoding vector showed a decrease both in mitochondrial depolarization and cell detachment. Dox-induced apoptosis included decrease in Bcl-2 expression, increase in Bak expression and caspase-3 and -9 activation but appeared to be p53- and Bax-independent. A better comprehension of the Dox-induced apoptotic pathway could allow to abolish its toxic effects, improving the therapeutic efficiency of Dox.

In the present study, the toxicity of yperite, SM, and its structural analogue mechlorethamine, HN2, was investigated in a human bronchial epithelial cell line 16HBE. Cell detachment was initiated by caspase-2 activation, down-regulation of Bcl-2 and loss of mitochondrial membrane potential. Only in detached cells, mustards induced apoptosis associated with increase in p53 expression, Bax activation, decrease in Bcl-2 expression, opening of the mitochondrial permeability transition pore, release of cytochrome c, caspase-2, -3, -8, -9 and -13 activation and DNA fragmentation. Apoptosis, occurring only in detached cells, could be recognized as anoikis and the mitochondrion, involved both in cell detachment and subsequent cell death, appears to be a crucial checkpoint. Based on our understanding of the apoptotic pathway triggered by mustards, we demonstrated that inhibition of the mitochondrial pathway by ebselen, melatonin and cyclosporine A markedly prevented mustard-induced anoikis, pointing to these drugs as interesting candidates for the treatment of mustard-induced airway epithelial lesions.

Respiratory tissues can be damaged by the exposure of airway epithelial cells to reactive oxygen species that generate oxidative stress. We studied the effects of the hydroxyl radical *OH, for which there is no natural intra- or extracellular scavenger, on a Ca(2+)-activated chloride channel (CACC) that participates in Cl(-) secretion in the apical membrane of airway epithelial cells. We identified and characterized CACC in cell-attached and in inside-out excised membrane patches from the apical membrane of cultured nonciliated human nasal epithelial cells. In these cells, the CACC was outwardly rectified, Ca(2+)/calmodulin-kinase II, and voltage dependent. The channel was activated in cell-attached and inside-out patches in a bath solution containing millimolar [Ca(2+)] and ran down quickly. The channel was reversibly or irreversibly activated by exposure of the internal surface of the membrane to *OH, which depended on the concentration and the duration of exposure to H(2)O(2). CACC activity evoked by oxidative stress was inhibited by 1,3-dimethyl-2-thiurea, an antioxidant that scavenges hydroxyl radicals, and by the reduced form of glutathione. The oxidized SH residues could be close to the Ca(2+)/calmodulin kinase site. The reversible or irreversible activation of CACC after a period of oxidative stress without change in [Ca(2+)] is a new observation. CACC play a direct role in mucus production by goblet cells and may thus contribute to the pathogenesis of asthma.