Pulmonary surfactant inhibition of nanoparticle uptake by alveolar epithelial cells.

Pulmonary surfactant forms a sub-micrometer thick fluid layer that covers the surface of alveolar lumen and inhaled nanoparticles therefore come in to contact with surfactant prior to any interaction with epithelial cells. We investigate the role of the surfactant as a protective physical barrier by modeling the interactions using silica-Curosurf-alveolar epithelial cell system in vitro. Electron microscopy displays that the vesicles are preserved in the presence of nanoparticles while nanoparticle-lipid interaction leads to formation of mixed aggregates. Fluorescence microscopy reveals that the surfactant decreases the uptake of nanoparticles by up to two orders of magnitude in two models of alveolar epithelial cells, A549 and NCI-H441, irrespective of immersed culture on glass or air-liquid interface culture on transwell. Confocal microscopy corroborates the results by showing nanoparticle-lipid colocalization interacting with the cells. Our work thus supports the idea that pulmonary surfactant plays a protective role against inhaled nanoparticles. The effect of surfactant should therefore be considered in predictive assessment of nanoparticle toxicity or drug nanocarrier uptake. Models based on the one presented in this work may be used for preclinical tests with engineered nanoparticles.

Supported pulmonary surfactant bilayers on silica nanoparticles: formulation, stability and impact on lung epithelial cells.

Studies have shown that following exposure to particulate matter, ultrafine fractions (<100 nm) may deposit along the respiratory tract down to the alveolar region. To assess the effects of nanoparticles on the lungs, it is essential to address the question of their biophysicochemical interaction with the different pulmonary environments, including the lung lining fluids and the epithelia. Here we examine one of these interactive scenarios and study the role of supported lipid bilayers (SLB) in the effect of 40 nm fluorescent silica particles on living cells. We first study the particle phase behavior in the presence of Curosurf®, a pulmonary surfactant substitute used in replacement therapies. It is found that Curosurf® vesicles interact strongly with the nanoparticles, but do not spontaneously form SLBs. To achieve this goal, we use sonication to reshape the vesicular membranes and induce lipid fusion around the particles. Centrifugal sedimentation and electron microscopy are carried out to determine the optimum coating conditions and layer thickness. We then explore the impact of surfactant SLBs on the cytotoxic potential and interactions towards a malignant epithelial cell line. All in vitro assays indicate that SLBs mitigate the particle toxicity and internalization rates. In the cytoplasm, the particle localization is also strongly coating dependent. It is concluded that SLBs profoundly affect cellular interactions and functions in vitro and could represent an alternative strategy for particle coating. The current data also shed some light on the potential mechanisms pertaining to the particle or pathogen transport through the air-blood barrier.

Involvement of oxidative stress and calcium signaling in airborne particulate matter - induced damages in human pulmonary artery endothelial cells.

Recent studies have revealed that particulate matter (PM) exert deleterious effects on vascular function. Pulmonary artery endothelial cells (HPAEC), which are involved in the vasomotricity regulation, can be a direct target of inhaled particles. Modifications in calcium homeostasis and oxidative stress are critical events involved in the physiopathology of vascular diseases. The objectives of this study were to assess the effects of PM2.5 on oxidative stress and calcium signaling in HPAEC. Different endpoints were studied, (i) intrinsic and intracellular production of reactive oxygen species (ROS) by the H2DCF-DA probe, (ii) intrinsic, intracellular and mitochondrial production of superoxide anion (O2-) by electronic paramagnetic resonance spectroscopy and MitoSOX probe, (iii) reactive nitrosative species (RNS) production by Griess reaction, and (vi) calcium signaling by the Fluo-4 probe. In acellular conditions, PM2.5 leads to an intrinsic free radical production (ROS, O2-) and a 4h-exposure to PM2.5 (5-15µg/cm2), induced, in HPAEC, an increase of RNS, of global ROS and of cytoplasmic and mitochondrial O2- levels. The basal intracellular calcium ion level [Ca2+]i was also increased after 4h-exposure to PM2.5 and a pre-treatment with superoxide dismutase and catalase significantly reduced this response. This study provides evidence that the alteration of intracellular calcium homeostasis induced by PM2.5 is closely correlated to an increase of oxidative stress.

Proinflammatory effect of fine and ultrafine particulate matter using size-resolved urban aerosols from Paris.

Epidemiological and experimental studies have underlined that exposure to particulate matter (PM) leads mainly to airway inflammation, but the roles of particle size and chemical composition associated to such adverse health outcomes need to be better investigated. This study was performed to validate novel strategies of particle sampling, recovery and cell exposure in order to evaluate the pro-inflammatory potential of fine and ultrafine particles from a fractionated aerosol. Samplings of Paris background aerosols using 13-stage low pressure impactors (0.03-10 microm) gave bimodal mass distributions with an accumulation mode centered on a median diameter of 0.42 microm and a coarse one on 3.25 microm. PM 1 accounted for 70% and PM 0.1 for 12% of PM 10. The latter mainly comprised carbon-chained aggregates. The development of an efficient and reproducible method to recover fine (PM 1-0.1) and ultrafine (PM 0.1-0.03) particulate matter has permitted experimental comparison of the impact of such particles on human bronchial epithelial cells (HBECs). In this study we have compared the relative effects of fine and ultrafine particles at non-cytotoxic concentrations over 24h on the production of the pro-inflammatory cytokine GM-CSF by HBECs. Combining two cell exposure strategies to the size-fraction particles according to either their proportion (isovolume exposure) or their quantity in the aerosol (isomass exposure), we showed that both ultrafine and fine particles induced a concentration-dependent GM-CSF release by HBECs which is significant from 1 microg cm(-2). In conclusion, short duration samplings using 13-stage impactors enable to obtain size-resolved PM in sufficient quantities to carry out toxicological investigations. These findings are promising in view to conduct a more intensive study joining chemical and toxicological assays.

Expression and role of EGFR ligands induced in airway cells by PM2.5 and its components.

The aim of the current study was to establish the epidermal growth factor receptor (EGFR) ligand expression profile in human airway epithelial cells exposed to either particulate matter (PM) with an aerodynamic diameter <2.5 microm (PM(2.5)) or its components and the involvement of EGFR ligands in PM(2.5)-provoked airway inflammation. EGFR ligand mRNA and protein expression were studied in a human bronchial epithelial cell line and normal nasal cells exposed to noncytotoxic concentrations of PM(2.5) or its components. The autocrine role of EGFR ligands in airway epithelial cell pro-inflammation was determined by adding conditioned media from PM(2.5)-treated cells to fresh cells and measuring the secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF), a pro-inflammatory biomarker. PM(2.5)increased amphiregulin, transforming growth factor-alpha and heparin-binding EGF-like growth factor mRNA expression and protein secretion, with a slight contribution of aqueous metallic compounds and a strong participation of organic components putatively attributed to PM polyaromatic hydrocarbon content. PM(2.5)-induced EGFR ligands were involved in cellular GM-CSF release. The current study revealed upregulation of several epidermal growth factor receptor ligands by airway epithelial cells exposed to particulate matter with an aerodynamic diameter <2.5 microm and their contribution to bronchial epithelial cell granulocyte-macrophage colony-stimulating factor secretion by an autocrine action, suggesting that these ligands could elicit and sustain the particulate matter-induced airway pro-inflammatory response and contribute to bronchial remodelling.

Human airway epithelial cells in culture for studying the molecular mechanisms of the inflammatory response triggered by diesel exhaust particles.

Epidemiological studies have shown that particulate air pollution is linked to the increase of morbidity and mortality due to respiratory diseases. Diesel exhaust particles (DEPs), which are the most important part of PM2.5 in Western European and Japanese urban areas, have been suspected. The mechanisms of proinflammatory response induced by DEPS were elucidated using a human epithelial cell line (16-HBE). It has been shown that DEPs can be phagocytosed by HBE cells, inducing the release of cytokines. MAP kinase pathways (i.e., ERK1/2 and P38) were triggered as well as the activation of the nuclear factor NF-kappaB. Reactive oxygen species (ROS) were strongly incriminated in this response because DEPs induce the increase of intracellular hydroperoxides and antioxidants inhibit the release of DEP-induced cytokines, the activation of MAP kinases and NF-kappaB. Organic compounds adsorbed on DEPs seemed to be involved in the response and the production of ROS. Moreover, we have demonstrated that DEPs can activate CYP1A1 in HBE cells. These experimental results give biological plausibility to the epidemiological findings.

Organic compounds from diesel exhaust particles elicit a proinflammatory response in human airway epithelial cells and induce cytochrome p450 1A1 expression.

Diesel exhaust particles (DEP) are known to enhance inflammatory responses in human volunteers. In cultured human bronchial epithelial (16HBE) cells, they induce the release of proinflammatory cytokines after triggering transduction pathways, including nuclear factor (NF)-kappaB activation and mitogen-activated protein kinase (MAPK) phosphorylation. This study compares the effects of native DEP (nDEP), organic extracts of DEP (OE-DEP), and carbonaceous particles, represented by stripped DEP (sDEP) and carbon black particles (CB), in order to clarify their respective roles. OE-DEP and nDEP induce granulocyte macrophage colony-stimulating factor (GM-CSF) release, NF-kappaB activation, and MAPK phosphorylation. The carbonaceous core generally induces less intense effects. Reactive oxygen species are produced in 16HBE cells and are involved in GM-CSF release and in the stimulation of NF-kappaB DNA binding by nDEP and OE-DEP. We demonstrate, for the first time, in airway epithelial cells in vitro that nDEP induce the expression of the CYP1A1, a cytochrome P450 specifically involved in polycyclic aromatic hydrocarbons metabolism, thereby demonstrating the critical role of organic compounds in the DEP-induced proinflammatory response. Understanding the respective contributions of DEP components in these effects is important for vehicle manufacturers in order to improve their exhaust gas post-treatment technologies. In conclusion, the DEP-induced inflammatory response in airway epithelial cells mainly involves organic compounds such as PAH, which induce CYP1A1 gene expression.

Similar cellular effects induced by diesel exhaust particles from a representative diesel vehicle recovered from filters and Standard Reference Material 1650.

Standard reference diesel exhaust particles (DEP) SRM 1650 are often used to evaluate the toxicity of DEP. However, these particles did not necessarily reflect the effects of DEP representative of present diesel automobiles. This study was designed to compare the effects of SRM 1650 to DEP from representative cars (RC-DEP) on airway epithelial cells. Therefore we established a method to recover RC-DEP impacted on filters after emission from diesel automobiles on test beds. Electron microscopy and flow cytometry showed that these two types of particles were phagocytosed to the same extent by epithelial cells. This phagocytosis is not dependent on the adsorbed organic compounds in contrast to the cytotoxic effect evaluated by measurements of LDH release. This is emphasized by the fact that RC-DEP equipped with an oxidation catalyst are less cytotoxic than particles from a non-equipped vehicle or SRM 1650. This type of catalyst also reduces significantly the release of GM-CSF by bronchial epithelial cells. We have shown in the present paper that SRM 1650 may be used as a surrogate of DEP. However, exhaust gas post-treatment devices of current diesel automobiles reduce the cytotoxicity as well as the inflammatory response of these particles.

Efficient protection of human bronchial epithelial cells against sulfur and nitrogen mustard cytotoxicity using drug combinations.

The aim of this study was to test the efficacy of several candidate molecules against sulfur mustard (SM) and nitrogen mustard (HN2) using a human bronchial-epithelial cell line (16HBE14o-). Candidate molecules were chosen on the basis of the known cytotoxicity mechanisms of mustards or their efficacy previously observed on other cellular models. It included the sulfhydryl-containing molecules N-acetyl-cysteine (NAC) and WR-1065, the nucleophile hexamethylenetetramine (HMT), the energy-level stabilizer niacinamide (NC), the antioxidant dimethylthiourea (DMTU), L-arginine analogues such as L-thiocitrulline (L-TC) and L-nitroarginine methyl ester (L-NAME), and the anti-gelatinase doxycycline (DOX). Their efficacy was determined using 2-(4-[3-iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2Htetrazolium (WST-1) reduction by viable cells 24 h after initial exposure to 100 microM HN2 or SM. On individual immediate cotreatment, some molecules exhibited selective protection against only one mustard, such as DMTU and WR-1065 against HN2 and DOX against SM, whereas NAC and L-TC were effective against both SM and HN2 cytotoxicity. However, as the level of protection against SM was always weak compared to HN2, several combinations were investigated against SM to improve the protection. The effective combinations (L-TC + DOX, NAC + DOX, NAC + DMTU, NAC + HMT, NC + DOX) combined agents, reducing the bioavailability of the mustard with compounds possibly acting on the consequences of alkylation. One of these combinations, NAC + DOX, appeared to be the most interesting, as these agents are already used in human therapy. It exhibited good efficacy in delayed cotreatment (up to 90 min) against SM.

Responses of the rabbit tracheal epithelium in vitro to H(2)O(2)-induced oxidative stress.

A model of rabbit tracheal epithelial (RTE) cells in primary culture was used to characterize specific and repair responses of airway epithelial cells to oxidative stress. Two well-known reactive oxygen species (ROS) generating systems were used: H(2)O(2) alone or in combination with Fe(2+) to produce the hydroxyl radical. RTE cells exhibited lipid peroxidation when exposed to H(2)O(2) + Fe(2+). Moreover, catalase (CAT) activity decreased after a 1-hour treatment in 3-day-old cultures but increased in 7-day-old cultures which have higher antioxidant enzyme activities. Superoxide dismutase (SOD) activity was never affected. In addition, RTE cells displayed a repair response leading to squamous metaplasia. H(2)O(2) + Fe(2+) treatment resulted in a time-dependent increase in the steady-state level of c-myc mRNA while c-jun and c-fos were not activated. Moreover, a chronic exposure induced the expression of the squamous phenotype characterized by the expression of the cytokeratin 13 confirmed both at the message and protein levels. RTE cells in primary culture react early to H(2)O(2) + Fe(2+) exposure by an increase in c-myc expression and by modifications in CAT activity. Further, a lipid peroxidation occurs and the tracheal epithelium evolves to squamous metaplasia.

Differential effects of several retinoid receptor-selective ligands on squamous differentiation and apoptosis in airway epithelial cells.

The roles of the different retinoid receptors on the differentiation of rabbit tracheal epithelial (RbTE) cells in primary culture were analysed using selective agonists for the retinoid acid receptor subtypes RARalpha (CD336), RARbeta (CD2019), RARgamma (CD437), an RAR panagonist (CD367), a retinoid X receptor RXR panagonist (CD2624) and an antagonist for RARbeta/gamma (CD2665). Squamous differentiation was assessed via expression of cytokeratins CK13/CK4 and transglutaminase I (TGI), specific markers of metaplasia. Treatment with RARalpha and beta agonists or RAR panagonist, but not the RARgamma agonist or RXR agonist, is required for the inhibition of squamous metaplasia, evidenced by inhibition of CK13/CK4 and TGI expression. The expression of CK10 cytokeratin of keratinizing epithelia, CK14/CK5 basal cell cytokeratins, and CK6 marker of cell proliferation decreases upon exposure of the RARaalpha/beta and RXR agonists. The RARgamma agonist CD437, inactive in the decrease in CK13/CK4, CK10 and CK14, reduces CK5/CK6 amounts. CD437 is responsible for a dose-dependent apoptotic response. Nuclear labelling with propidium iodide (PI) and electron microscopy revealed chromatin condensation and nuclear fragmentation. DNA cleavage and cell fragmentation were confirmed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The RARbetagamma antagonist was also slightly active. The results indicate that CD437 causes growth arrest in the early S-phase of the cell cycle and prevents the transition G1-S-phase. CD437 was demonstrated to induce apoptosis in the S-phase cells identified by bromodeoxyuridine (BrdU) incorporation. In conclusion, RARalpha/beta ligands are effective inhibitors of squamous differentiation. On the contrary, RARgamma ligand appears to be inefficient in metaplasia inhibition, but the selective RARgamma agonist CD437 induces growth arrest and apoptosis of basal proliferative cells.

Protection from cytotoxic effects induced by the nitrogen mustard mechlorethamine on human bronchial epithelial cells in vitro.

The present study was undertaken to find potent molecules against the toxicity of nitrogen mustard mechlorethamine (HN2) on respiratory epithelial cells, using a human bronchial epithelial cell line (16HBE14o-) as an in vitro model. The compounds examined included inhibitors of poly(ADP-ribose) polymerase (PARP), sulfhydryl-group donors as nucleophiles, and iron chelators and inhibitors of lipid peroxidation as antioxidants. Their effectiveness was determined upon observance of metabolic dysfunction induced by HN2 following a 4-h exposure, using (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and ATP-level assays as indicators. Moreover, the fluorescent probe, monobromobimane (mBBr), and 2',7'-dichlorofluorescin-diacetate (H2DCF-DA) were used to assess intracellular sulfhydryl and peroxide level modifications by flow cytometry, respectively, following a 3-h exposure. At last, cell death was assessed by flow cytometry using the propidium iodide (PI)-dye-exclusion assay following 24-h exposure. PARP inhibitors (niacinamide, 3-aminobenzamide, 6(5H)-phenanthridinone), and two sulfhydryl-group donors (N-acetylcysteine, WR-1065) were found to be effective in preventing HN2-induced metabolic dysfunction when added in immediate or delayed treatment with HN2. Only N-acetylcysteine, however, was found to prevent cell death induced by HN2, though it must be present at the time of the HN2 challenge. Flow cytometric measurements of intracellular sulfhydryl levels strongly suggested that N-acetylcysteine and WR-1065 are preventive in alkylation of cellular compounds, mainly by direct extracellular interaction with HN2. PARP inhibitors prevent secondary deleterious effects induced by HN2, considering metabolism dysfunction as the endpoint. Elsewhere, the oxidative stress appears to be a side effect in HN2 toxicity only upon considering the inefficiency of several antioxidants.

Mechanisms of GM-CSF increase by diesel exhaust particles in human airway epithelial cells.

We have previously shown that exposure to diesel exhaust particles (DEPs) stimulates human airway epithelial cells to secrete the inflammatory cytokines interleukin-8, interleukin-1beta, and granulocyte-macrophage colony-stimulating factor (GM-CSF) involved in allergic diseases. In the present paper, we studied the mechanisms underlying the increase in GM-CSF release elicited by DEPs using the human bronchial epithelial cell line 16HBE14o-. RT-PCR analysis has shown an increase in GM-CSF mRNA levels after DEP treatments. Comparison of the effects of DEPs, extracted DEPs, or extracts of DEPs has shown that the increase in GM-CSF release is mainly due to the adsorbed organic compounds and not to the metals present on the DEP surface because the metal chelator desferrioxamine had no inhibitory effect. Furthermore, radical scavengers inhibited the DEP-induced GM-CSF release, showing involvement of reactive oxygen species in this response. Moreover genistein, a tyrosine kinase inhibitor, abrogated the effects of DEPs on GM-CSF release, whereas protein kinase (PK) C, PKA, cyclooxygenase, or lipoxygenase inhibitors had no effect. PD-98059, an inhibitor of mitogen-activated protein kinase, diminished the effects of DEPs, whereas SB-203580, an inhibitor of p38 mitogen-activated protein kinase, had a lower effect, and DEPs did actually increase the active, phosphorylated form of the extracellular signal-regulated kinase as shown by Western blotting. In addition, cytochalasin D, which inhibits the phagocytosis of DEPs, reduced the increase in GM-CSF release after DEP treatment. Together, these data suggest that the increase in GM-CSF release is mainly due to the adsorbed organic compounds and that the effect of native DEPs requires endocytosis of the particles. Reactive oxygen species and tyrosine kinase(s) may be involved in the DEP-triggered signaling of the GM-CSF response.

Activation of Transcription Factors by Diesel Exhaust Particles in Human Bronchial Epithelial Cells in Vitro.

Diesel exhaust particles (DEP) are suspected to be involved in the aggravation of inflammatory respiratory diseases. We have shown previously, in human bronchial epithelial cell line 16HBE 14o-, that DEP induced the release of the proinflammatory cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-8 (IL-8) after 24 h of exposure. Gene expression of these cytokines is regulated by transcription factors including NF-κB and AP-1, which are known to be sensitive to oxidative stress. Their activation by DEP was investigated in comparison with a pure oxidant, H2O2 A 4-h exposure to DEP (10 µg/cm(2)) or to H2O2 (100 µM) increased NF-κB DNA binding in 16HBE cells as assessed by electrophoretic mobility shift assay. AP-1 was only activated by H202 in the same conditions. Organic extracts of DEP increased NF-κB DNA binding as did native DEP, suggesting the role of the polycyclic aromatic hydrocarbons (PAH) in this NF-κB increased DNA binding. Dimethylthiourea (DMTU), an antioxidant, inhibited the NF-κB DNA binding induced by DEP, suggesting an involvement of reactive oxygen species (ROS) in the transduction pathways leading to NF-κB activation. Moreover, the MEK pathway inhibitor PD98059 inhibited DEP-induced NF-κB DNA binding. The role of Erk 1/2 was likely implicated, since DEP induced an increase of Erk phosphorylation.

Diesel exhaust particles are taken up by human airway epithelial cells in vitro and alter cytokine production.

The involvement of diesel exhaust particles (DEPs) in respiratory diseases was evaluated by studying their effects on two in vitro models of human airway epithelial cells. The cytotoxicity of DEPs, their phagocytosis, and the resulting immune response were investigated in a human bronchial epithelial cell line (16HBE14o-) as well as in human nasal epithelial cells in primary culture. DEP exposure induced a time- and dose-dependent membrane damage. Transmission electron microscopy showed that DEPs underwent endocytosis by epithelial cells and translocated through the epithelial cell sheet. Flow cytometric measurements allowed establishment of the time and dose dependency of this phagocytosis and its nonspecificity with different particles (DEPs, carbon black, and latex particles). DEPs also induced a time-dependent increase in interleukin-8, granulocyte-macrophage colony-stimulating factor, and interleukin-1beta release. This inflammatory response occurred later than phagocytosis, and its extent seems to depend on the content of adsorbed organic compounds because carbon black had no effect on cytokine release. Furthermore, exhaust gas posttreatments, which diminished the adsorbed organic compounds, reduced the DEP-induced increase in granulocyte-macrophage colony-stimulating factor release. These results suggest that DEPs could 1) be phagocytosed by airway epithelial cells and 2) induce a specific inflammatory response.

Airborne particles evoke an inflammatory response in human airway epithelium. Activation of transcription factors.

PM10, the commonly used indicator of respirable environmental suspended particulate matter with a mean aerodynamic diameter of less than 10 microm, is composed of organic or elemental carbon aggregates containing various metals, acid salts, organic pollutants (polyaromatic hydrocarbons, quinones, nitroaromatic hydrocarbons, etc.), and biological contaminants. In urban and industrial areas, fossil fuel combustion products (e.g., diesel exhaust particles and residual oil fly ash) are the main contributors to PM10. Epidemiological data show that air pollution particulates cause adverse pulmonary health effects, especially in individuals with preexisting lung diseases. A critical cell type that encounters particles after inhalation and that is affected in a number of respiratory diseases is the epithelial cell of the airway and alveoli. In vitro studies have shown that PM10 is responsible for the production and the release of inflammatory cytokines by the respiratory tract epithelium as well as for the activation of the transcription factor NFkappaB. As many of the adsorbed materials on the particle surface are direct oxidants (metals, quinones) and indirectly produce reactive oxygen species, it is hypothesized that oxidative stress may be a component of the mechanisms by which particles activate cytokine production and NFkappaB in epithelial cells.

Use of fluorescent probes to assess the early sulfhydryl depletion and oxidative stress induced by mechlorethamine in human bronchial epithelial cells.

In this study, we report in vitro methods using fluorescent probes to assess thiol depletion and the oxidative stress induced by mechlorethamine (HN2), a nitrogen mustard, on a human bronchial epithelial cell line (16HBE14o-). Monobromobimane (mBBr) and 2',7'-dichlorofluorescin-diacetate (H(2)DCF-DA) were respectively used to monitor the intracellular thiol and peroxide levels. Fluorescent measurements were realized on gated live cells with a flow cytometer and a microspectrofluorimeter. Results clearly show that HN2 induced an early reduction of free sulfhydryl groups inside the cell correlated with an increase in the intracellular peroxides content. HN2 effects were time and dose dependent. The use of these fluorescent probes provide a useful and rapid methods for future screening of protective molecules against the early sulfydryl depletion and oxidative stress induced by HN2 on human airway epithelium.

Diesel Exhaust Particles Increase NF-kappaB DNA Binding Activity and c-FOS Proto-oncogene Expression in Human Bronchial Epithelial Cells.

There is increasing evidence that diesel exhaust particles (DEP) could be incriminated in respiratory diseases. They have been shown to induce an inflammatory response in the lung and are suspected to be carcinogenic because of the presence of polyaromatic hydrocarbons (PAH) on their surface. DEP were tested on a human bronchial epithelial cell line (16HBE) in comparison with carbon black particles (CB) devoid of PAH. DEP and CB at 10mug/cm(2) induced the release of the lactate dehydrogenase (LDH) by 16HBE cells from 48hr of exposure. DEP at 5mug/cm(2) but not CB activated the binding of the nuclear factor kappaB (NF-kappaB) to DNA from 2hr of exposure up to 15hr. NF-kappaB is a transcription factor involved in the expression of some cytokines such as IL-8 and GM-CSF which have been shown to be released by 16HBE cells after DEP exposure. In addition, DEP as well as CB induced the expression of the c-fos proto-oncogene. Taken together, these new data suggest that the activation of NF-kappaB and the expression of c-fos could contribute to the proliferation and chronic inflammation processes induced in lungs after DEP exposure.

Defense and repair mechanisms in the airway epithelium exposed to oxidative stress: effects of analogues of retinoic acid.

A model of rabbit tracheal epithelial (RTE) cells in vitro was developed in order to investigate the effects, on the airway epithelium, of reactive oxygen species (ROS) generated by H2O2 in association or not in association with Fe2+. The immediate consequence of a 24 h exposure of RTE cells to an oxidative stress was an increase in catalase activity whereas superoxide dismutase activity was not modified. A latter consequence of a chronic ROS insult was the induction of a repair mechanism which led to squamous metaplasia (SM). SM is characterized by a stratification of the epithelium, the expression of the cytokeratin 13 and the appearance of cells with cross-linked envelopes. Reversible modifications between mucociliary and squamous phenotype are modulated by retinoids. The action of retinoids is selective: metaplasia is inhibited by agonists of RAR alpha and beta receptors and not by an agonist of RAR gamma receptors.

Early cytotoxic effects of mechlorethamine, a nitrogen mustard, on mammalian airway epithelium.

The aim of this in vitro study was to characterize the early effects of short-term exposure to low concentrations of mechlorethamine (HN2), a nitrogen mustard, on rabbit tracheal primary cultures. Marked inhibition of cell growth was observed without recovery until 14 days after the treatment with sublethal doses of HN2. Cell detachment associated with rearrangement of the cytoskeletal proteins was also noted as early as 1 hr after treatment. Moreover, cells treated with HN2 at the sublethal dose of 50 mum (LC(10)) for 1 hr showed early lipid peroxidation and cellular membrane damage. This was correlated with a significant increase in the activities of antioxidant enzymes associated with an increase in glutathione content. These early events appeared several hours before arrest of the cell cycle progression. On the other hand, long-term treatment with HN2 at a sublethal dose led to modification of cytokeratin expression and appeared to induce squamous metaplasia.