Suitability of human and mammalian cells of different origin for the assessment of genotoxicity of metal and polymeric engineered nanoparticles.

Nanogenotoxicity is a crucial endpoint in safety testing of nanomaterials as it addresses potential mutagenicity, which has implications for risks of both genetic disease and carcinogenesis. Within the NanoTEST project, we investigated the genotoxic potential of well-characterised nanoparticles (NPs): titanium dioxide (TiO2) NPs of nominal size 20 nm, iron oxide (8 nm) both uncoated (U-Fe3O4) and oleic acid coated (OC-Fe3O4), rhodamine-labelled amorphous silica 25 (Fl-25 SiO2) and 50 nm (Fl-50 SiO) and polylactic glycolic acid polyethylene oxide polymeric NPs - as well as Endorem® as a negative control for detection of strand breaks and oxidised DNA lesions with the alkaline comet assay. Using primary cells and cell lines derived from blood (human lymphocytes and lymphoblastoid TK6 cells), vascular/central nervous system (human endothelial human cerebral endothelial cells), liver (rat hepatocytes and Kupffer cells), kidney (monkey Cos-1 and human HEK293 cells), lung (human bronchial 16HBE14o cells) and placenta (human BeWo b30), we were interested in which in vitro cell model is sufficient to detect positive (genotoxic) and negative (non-genotoxic) responses. All in vitro studies were harmonized, i.e. NPs from the same batch, and identical dispersion protocols (for TiO2 NPs, two dispersions were used), exposure time, concentration range, culture conditions and time-courses were used. The results from the statistical evaluation show that OC-Fe3O4 and TiO2 NPs are genotoxic in the experimental conditions used. When all NPs were included in the analysis, no differences were seen among cell lines - demonstrating the usefulness of the assay in all cells to identify genotoxic and non-genotoxic NPs. The TK6 cells, human lymphocytes, BeWo b30 and kidney cells seem to be the most reliable for detecting a dose-response.

Toxicity evaluation of engineered nanoparticles for medical applications using pulmonary epithelial cells.

There are a multitude of nanoparticles (NPs) which have shown great potentials for medical applications. A few of them are already used for lung therapeutic and diagnostic purposes. However, there are few toxicological studies which determine possible adverse pulmonary responses. It is thus important to propose in vitro screening strategies to evaluate the pulmonary toxicity of NPs used in nanomedicine. Our goal was to determine the cellular effects of several biomedical NPs with different physico-chemical characteristics (chemical nature, size and coating) to establish suitable tests and useful benchmark NPs. The effects of poly(lactic-co-glycolic acid) (PLGA), silica, iron oxide and titanium dioxide NPs were studied using human bronchial (16HBE) and alveolar epithelial cells (A549). We evaluated cytotoxicity, reactive oxygen species (ROS) production and pro-inflammatory response in both cell lines. We demonstrated that PLGA NPs are good candidates for negative control NPs and SiO2 NPs were revealed to be the best benchmark NPs. Coating of Fe3O4 with sodium oleate, a known biocompatible compound, led to an unexpected increase in cytotoxicity. Moreover, 16HBE cells are more sensitive than A549 cells and propidium iodide uptake is a more sensitive cytotoxicity test than WST-1. The measurement of oxidative stress does not systematically allow us to predict cellular responses and different other cellular endpoints should also be addressed. We conclude that a battery of assays and cell lines are necessary to accurately evaluate the pulmonary effects of NPs and that PLGA and SiO2 NPs are suitable candidates respectively for negative and positive controls.

Deciphering the mechanisms of cellular uptake of engineered nanoparticles by accurate evaluation of internalization using imaging flow cytometry.

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.

Internalization of SiO2 nanoparticles by alveolar macrophages and lung epithelial cells and its modulation by the lung surfactant substitute Curosurf.

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.

Autocrine effect of EGFR ligands on the pro-inflammatory response induced by PM(2.5) exposure in human bronchial epithelial cells.

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.

Nanoparticles: molecular targets and cell signalling.

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.

Polycyclic aromatic hydrocarbon components contribute to the mitochondria-antiapoptotic effect of fine particulate matter on human bronchial epithelial cells via the aryl hydrocarbon receptor.

BACKGROUND: Nowadays, effects of fine particulate matter (PM2.5) are well-documented and related to oxidative stress and pro-inflammatory response. Nevertheless, epidemiological studies show that PM2.5 exposure is correlated with an increase of pulmonary cancers and the remodeling of the airway epithelium involving the regulation of cell death processes. Here, we investigated the components of Parisian PM2.5 involved in either the induction or the inhibition of cell death quantified by different parameters of apoptosis and delineated the mechanism underlying this effect. RESULTS: In this study, we showed that low levels of Parisian PM2.5 are not cytotoxic for three different cell lines and primary cultures of human bronchial epithelial cells. Conversely, a 4 hour-pretreatment with PM2.5 prevent mitochondria-driven apoptosis triggered by broad spectrum inducers (A23187, staurosporine and oligomycin) by reducing the mitochondrial transmembrane potential loss, the subsequent ROS production, phosphatidylserine externalization, plasma membrane permeabilization and typical morphological outcomes (cell size decrease, massive chromatin and nuclear condensation, formation of apoptotic bodies). The use of recombinant EGF and specific inhibitor led us to rule out the involvement of the classical EGFR signaling pathway as well as the proinflammatory cytokines secretion. Experiments performed with different compounds of PM2.5 suggest that endotoxins as well as carbon black do not participate to the antiapoptotic effect of PM2.5. Instead, the water-soluble fraction, washed particles and organic compounds such as polycyclic aromatic hydrocarbons (PAH) could mimic this antiapoptotic activity. Finally, the activation or silencing of the aryl hydrocarbon receptor (AhR) showed that it is involved into the molecular mechanism of the antiapoptotic effect of PM2.5 at the mitochondrial checkpoint of apoptosis. CONCLUSIONS: The PM2.5-antiapoptotic effect in addition to the well-documented inflammatory response might explain the maintenance of a prolonged inflammation state induced after pollution exposure and might delay repair processes of injured tissues.

Carbon black and titanium dioxide nanoparticles elicit distinct apoptotic pathways in bronchial epithelial cells.

BACKGROUND: Increasing environmental and occupational exposures to nanoparticles (NPs) warrant deeper insight into the toxicological mechanisms induced by these materials. The present study was designed to characterize the cell death induced by carbon black (CB) and titanium dioxide (TiO2) NPs in bronchial epithelial cells (16HBE14o- cell line and primary cells) and to investigate the implicated molecular pathways. RESULTS: Detailed time course studies revealed that both CB (13 nm) and TiO2(15 nm) NP exposed cells exhibit typical morphological (decreased cell size, membrane blebbing, peripheral chromatin condensation, apoptotic body formation) and biochemical (caspase activation and DNA fragmentation) features of apoptotic cell death. A decrease in mitochondrial membrane potential, activation of Bax and release of cytochrome c from mitochondria were only observed in case of CB NPs whereas lipid peroxidation, lysosomal membrane destabilization and cathepsin B release were observed during the apoptotic process induced by TiO2 NPs. Furthermore, ROS production was observed after exposure to CB and TiO2 but hydrogen peroxide (H2O2) production was only involved in apoptosis induction by CB NPs. CONCLUSIONS: Both CB and TiO2 NPs induce apoptotic cell death in bronchial epithelial cells. CB NPs induce apoptosis by a ROS dependent mitochondrial pathway whereas TiO2 NPs induce cell death through lysosomal membrane destabilization and lipid peroxidation. Although the final outcome is similar (apoptosis), the molecular pathways activated by NPs differ depending upon the chemical nature of the NPs.

Carbon black and titanium dioxide nanoparticles induce pro-inflammatory responses in bronchial epithelial cells: need for multiparametric evaluation due to adsorption artifacts.

The initiation of an inflammatory process is the main adverse effect observed following the exposure of the airway epithelium to nanoparticles (NPs). This study was designed to explore the pro-inflammatory potential of two different NPs of similar size but of different compositions (CB 13 nm and TiO(2) 15 nm) on a human bronchial epithelial cell line (16HBE14o-). The expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL-6), and tumor necrosis factor alpha (TNFalpha) was evaluated in terms of mRNA, intracellular proteins, and released cytokines. Exposure to NPs induced a dose-dependent expression of all these cytokines, depending upon the chemical composition of NPs. The released cytokines appeared to be an inaccurate methodology to evaluate the pro-inflammatory response. Indeed, NPs adsorbed cytokines, and the binding was dependent on the nature of both the cytokine and NPs. Furthermore, addition of fetal calf serum or bovine serum albumin improved the detection of cytokines but also reduced cellular responses. Use of different detergents (Tween, Triton, and NP40) demonstrated limited efficiency to desorb cytokines from NPs. Thus, this study demonstrated the pro-inflammatory potential for CB and TiO(2) NP but underlines the methodological artifacts faced during the in vitro evaluation of cytokine release that necessitates a multiparametric evaluation.

Oxidative stress and proinflammatory effects of carbon black and titanium dioxide nanoparticles: role of particle surface area and internalized amount.

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 (TiO(2)), on the bronchial epithelial cell line (16HBE14o-). CB and TiO(2) 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.

Role of Paris PM(2.5) components in the pro-inflammatory response induced in airway epithelial cells.

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 PM(2.5) 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 PM(2.5) 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 PM(2.5) 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.

Arylamine N-acetyltransferase activity in bronchial epithelial cells and its inhibition by cellular oxidants.

Bronchial epithelial cells express xenobiotic-metabolizing enzymes (XMEs) that are involved in the biotransformation of inhaled toxic compounds. The activities of these XMEs in the lung may modulate respiratory toxicity and have been linked to several diseases of the airways. Arylamine N-acetyltransferases (NAT) are conjugating XMEs that play a key role in the biotransformation of aromatic amine pollutants such as the tobacco-smoke carcinogens 4-aminobiphenyl (4-ABP) and beta-naphthylamine (beta-NA). We show here that functional human NAT1 or its murine counterpart Nat2 are present in different lung epithelial cells i.e. Clara cells, type II alveolar cells and bronchial epithelial cells, thus indicating that inhaled aromatic amines may undergo NAT-dependent biotransformation in lung epithelium. Exposure of these cells to pathophysiologically relevant amounts of oxidants known to contribute to lung dysfunction, such as H(2)O(2) or peroxynitrite, was found to impair the NAT1/Nat2-dependent cellular biotransformation of aromatic amines. Genetic and non genetic impairment of intracellular NAT enzyme activities has been suggested to compromise the important detoxification pathway of aromatic amine N-acetylation and subsequently to contribute to an exacerbation of untoward effects of these pollutants on health. Our study suggests that oxidative/nitroxidative stress in lung epithelial cells, due to air pollution and/or inflammation, could contribute to local and/or systemic dysfunctions through the alteration of the functions of pulmonary NAT enzymes.

Impairment of NO-dependent relaxation in intralobar pulmonary arteries: comparison of urban particulate matter and manufactured nanoparticles.

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-alpha, 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.

EGF mediates calcium-activated chloride channel activation in the human bronchial epithelial cell line 16HBE14o-: involvement of tyrosine kinase p60c-src.

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.

Interleukin-13 interferes with CFTR and AQP5 expression and localization during human airway epithelial cell differentiation.

Interleukin-13 (IL-13) is a central regulator of Th2-dominated respiratory disorders such as asthma. Lesions of the airway epithelial barrier frequently observed in chronic respiratory inflammatory diseases are repaired through proliferation, migration and differentiation of epithelial cells. Our work is focused on the effects of IL-13 in human cellular models of airway epithelial cell regeneration. We have previously shown that IL-13 altered epithelial cell polarity during mucociliary differentiation of human nasal epithelial cells. In particular, the cytokine inhibited ezrin expression and interfered with its apical localization during epithelial cell differentiation in vitro. Here we show that CFTR expression is enhanced in the presence of the cytokine, that two additional CFTR protein isoforms are expressed in IL-13-treated cells and that part of the protein is retained within the endoplasmic reticulum. We further show that aquaporin 5 expression, a water channel localized within the apical membrane of epithelial cells, is completely abolished in the presence of the cytokine. These results show that IL-13 interferes with ion and water channel expression and localization during epithelial regeneration and may thereby influence mucus composition and hydration.

Effects of PM2.5 components in the release of amphiregulin by human airway epithelial cells.

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.

Fine urban atmospheric particulate matter modulates inflammatory gene and protein expression in human bronchial epithelial cells.

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.

Inhibition of caspase-dependent mitochondrial permeability transition protects airway epithelial cells against mustard-induced apoptosis.

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.

Responses of well-differentiated nasal epithelial cells exposed to particles: role of the epithelium in airway inflammation.

Numerous epidemiological studies support the contention that ambient air pollution particles can adversely affect human health. To explain the acute inflammatory process in airways exposed to particles, a number of in vitro studies have been performed on cells grown submerged on plastic and poorly differentiated, and on cell lines, the physiology of which is somewhat different from that of well-differentiated cells. In order to obtain results using a model system in which epithelial cells are similar to those of the human airway in vivo, apical membranes of well-differentiated human nasal epithelial (HNE) cells cultured in an air-liquid interface (ALI) were exposed for 24 h to diesel exhaust particles (DEP) and Paris urban air particles (PM(2.5)). DEP and PM(2.5) (10-80 microg/cm(2)) stimulated both IL-8 and amphiregulin (ligand of EGFR) secretion exclusively towards the basal compartment. In contrast, there was no IL-1beta secretion and only weak non-reproducible secretion of TNF-alpha. IL-6 and GM-CSF were consistently stimulated towards the apical compartment and only when cells were exposed to PM(2.5). ICAM-1 protein expression on cell surfaces remained low after particle exposure, although it increased after TNF-alpha treatment. Internalization of particles, which is believed to initiate oxidative stress and proinflammatory cytokine expression, was restricted to small nanoparticles (< or =40 nm). Production of reactive oxygen species (ROS) was detected, and DEP were more efficient than PM(2.5). Collectively, our results suggest that airway epithelial cells exposed to particles augment the local inflammatory response in the lung but cannot alone initiate a systemic inflammatory response.

Mechanisms of doxycycline-induced cytotoxicity on human bronchial epithelial cells.

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.