Diesel exhaust particulate emissions and in vitro toxicity from Euro 3 and Euro 6 vehicles.

Incomplete combustion processes in diesel engines produce particulate matter (PM) that significantly contributes to air pollution. Currently, there remains a knowledge gap in relation to the physical and chemical characteristics and also the biological reactivity of the PM emitted from old- and new-generation diesel vehicles. In this study, the emissions from a Euro 3 diesel vehicle were compared to those from a Euro 6 car during the regeneration of a diesel particulate filter (DPF). Different driving cycles were used to collect two types of diesel exhaust particles (DEPs). The particle size distribution was monitored using an engine exhaust particle sizer spectrometer and an electrical low-pressure impactor. Although the Euro 6 vehicle emitted particulates only during DPF regeneration that primarily occurs for a few minutes at high speeds, such emissions are characterized by a higher number of ultrafine particles (<0.1 µm) compared to those from the Euro 3 diesel vehicle. The emitted particles possess different characteristics. For example, Euro 6 DEPs exhibit a lower PAH content than do Euro 3 samples; however, they are enriched in metals that were poorly detected or undetected in Euro 3 emissions. The biological effects of the two DEPs were investigated in human bronchial BEAS-2B cells exposed to 50 µg/mL of PM (corresponding to 5.2 µg/cm2), and the results revealed that Euro 3 DEPs activated the typical inflammatory and pro-carcinogenic pathways induced by combustion-derived particles, while Euro 6 DEPs were less effective in regard to activating such biological responses. Although further investigations are required, it is evident that the different in vitro effects elicited by Euro 3 and Euro 6 DEPs can be correlated with the variable chemical compositions (metals and PAHs) of the emitted particles that play a pivotal role in the inflammatory and carcinogenic potential of airborne PM.

Signaling Control of Mucociliary Epithelia: Stem Cells, Cell Fates, and the Plasticity of Cell Identity in Development and Disease.

Mucociliary epithelia are composed of multiciliated, secretory, and stem cells and line various organs in vertebrates such as the respiratory tract. By means of mucociliary clearance, those epithelia provide a first line of defense against inhaled particles and pathogens. Mucociliary clearance relies on the correct composition of cell types, that is, the proper balance of ciliated and secretory cells. A failure to generate and to maintain correct cell type composition and function results in impaired clearance and high risk to infections, such as in congenital diseases (e.g., ciliopathies) as well as in acquired diseases, including asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF). While it remains incompletely resolved how precisely cell types are specified and maintained in development and disease, many studies have revealed important mechanisms regarding the signaling control in mucociliary cell types in various species. Those studies not only provided insights into the signaling contribution to organ development and regeneration but also highlighted the remarkable plasticity of cell identity encountered in mucociliary maintenance, including frequent trans-differentiation events during homeostasis and specifically in disease. This review will summarize major findings and provide perspectives regarding the future of mucociliary research and the treatment of chronic airway diseases associated with tissue remodeling.

Comparative Genotoxicity and Mutagenicity of Cigarette, Cigarillo, and Shisha Tobacco Products in Epithelial and Cardiac Cells.

Epidemiology studies link cigarillos and shisha tobacco (delivered through a hookah waterpipe) to increased risk for cardiopulmonary diseases. Here we performed a comparative chemical constituent analysis between 3 cigarettes, 3 cigarillos, and 8 shisha tobacco products. The potency for genotoxicity and oxidative stress of each product's generated total particulate matter (TPM) was also assessed using immortalized oral, lung, and cardiac cell lines to represent target tissues. Levels of the carcinogenic carbonyl formaldehyde were 32- to 95-fold greater, while acrolein was similar across the shisha aerosols generated by charcoal heating compared to cigarettes and cigarillos. Electric-mediated aerosol generation dramatically increased acrolein to levels exceeding those in cigarettes and cigarillos by up to 43-fold. Equivalent cytotoxic-mediated cell death and dose response for genotoxicity through induction of mutagenicity and DNA strand breaks was seen between cigarettes and cigarillos, while minimal to no effect was observed with shisha tobacco products. In contrast, increased potency of TPM from cigarillos compared to cigarettes for inducing oxidative stress via reactive oxygen radicals and lipid peroxidation across cell lines was evident, while positivity was seen for shisha tobacco products albeit at much lower levels. Together, these studies provide new insight into the potential harmful effects of cigarillos for causing tobacco-associated diseases. The high level of carbonyls in shisha products, that in turn is impacted by the heating mechanism, reside largely in the gas phase which will distribute throughout the respiratory tract and systemic circulation to likely increase genotoxic stress.

Partial Rescue of F508del-CFTR Stability and Trafficking Defects by Double Corrector Treatment.

Deletion of phenylalanine at position 508 (F508del) in the CFTR chloride channel is the most frequent mutation in cystic fibrosis (CF) patients. F508del impairs the stability and folding of the CFTR protein, thus resulting in mistrafficking and premature degradation. F508del-CFTR defects can be overcome with small molecules termed correctors. We investigated the efficacy and properties of VX-445, a newly developed corrector, which is one of the three active principles present in a drug (Trikafta®/Kaftrio®) recently approved for the treatment of CF patients with F508del mutation. We found that VX-445, particularly in combination with type I (VX-809, VX-661) and type II (corr-4a) correctors, elicits a large rescue of F508del-CFTR function. In particular, in primary bronchial epithelial cells of CF patients, the maximal rescue obtained with corrector combinations including VX-445 was close to 60-70% of CFTR function in non-CF cells. Despite this high efficacy, analysis of ubiquitylation, resistance to thermoaggregation, protein half-life, and subcellular localization revealed that corrector combinations did not fully normalize F508del-CFTR behavior. Our study indicates that it is still possible to further improve mutant CFTR rescue with the development of corrector combinations having maximal effects on mutant CFTR structural and functional properties.

Porphyromonas gingivalis gingipains potentially affect MUC5AC gene expression and protein levels in respiratory epithelial cells.

Porphyromonas gingivalis (Pg) is a periodontopathic pathogen that may affect MUC5AC-related mucus hypersecretion along airway epithelial cells. Here, we attempted to establish whether Pg virulence factors (lipopolysaccharide, FimA fimbriae, gingipains) affect MUC5AC in immortalized and primary bronchial cells. We report that MUC5AC gene expression and protein levels are affected by Pg culture supernatant, but not by lipopolysaccharide or FimA fimbriae. Cells treated with either Pg single (Kgp or Rgp) or double (Kgp/Rgp) mutants had altered levels of MUC5AC gene expression and protein levels, and MUC5AC staining of double mutant-treated mouse lung cells showed that MUC5AC protein levels were unaffected. Taken together, we propose that Pg gingipains may be the primary virulence factor that influences both MUC5AC gene expression and protein levels.

Effects of deltamethrin and thiacloprid on cell viability, colony formation and DNA double-strand breaks in human bronchial epithelial cells.

Deltamethrin (DEL) and thiacloprid (THIA) are commonly used insecticides applied either separately or as a mixture. We aimed to investigate the effects of DEL and THIA on cell viability, proliferation and DNA damage in human bronchial epithelial cells (BEAS-2B) because their effects in lung cells are not known. Our results indicate that all concentrations of DEL and THIA statistically decreased colony formation, plating efficiency and survival fraction in a concentration-dependent manner in BEAS-2B cells expect the lowest concentration for 24 h. MTT assay showed that treatment of DEL + THIA increased the cytotoxicity at higher concentrations. DEL + THIA significantly induced the foci formation of phosphorylated H2AX protein and p53 binding protein 1 at the highest concentration (44 µM DEL+666 µM THIA) for 120 h. Because gH2AX foci number was still higher in the recovery group given an additional 24 h after 120 h, the recovery period was not sufficient for DNA double-strand breaks repair.

Sphingolipids and plasma membrane hydrolases in human primary bronchial cells during differentiation and their altered patterns in cystic fibrosis.

Human primary bronchial epithelial cells differentiated in vitro represent a valuable tool to study lung diseases such as cystic fibrosis (CF), an inherited disorder caused by mutations in the gene coding for the Cystic Fibrosis Transmembrane Conductance Regulator. In CF, sphingolipids, a ubiquitous class of bioactive lipids mainly associated with the outer layer of the plasma membrane, seem to play a crucial role in the establishment of the severe lung complications. Nevertheless, no information on the involvement of sphingolipids and their metabolism in the differentiation of primary bronchial epithelial cells are available so far. Here we show that ceramide and globotriaosylceramide increased during cell differentiation, whereas glucosylceramide and gangliosides content decreased. In addition, we found that apical plasma membrane of differentiated bronchial cells is characterized by a higher content of sphingolipids in comparison to the other cell membranes and that activity of sphingolipids catabolic enzymes associated with this membrane results altered with respect to the total cell activities. In particular, the apical membrane of CF cells was characterized by high levels of ceramide and glucosylceramide, known to have proinflammatory activity. On this basis, our data further support the role of sphingolipids in the onset of CF lung pathology.

Induction of multidrug resistance-associated protein 3 expression by diesel exhaust particle extract in human bronchial epithelial BEAS-2B cells.

Diesel exhaust particles (DEPs) are common environmental air pollutants known to impair expression and activity of drug detoxifying proteins, including hepatic ATP-binding cassette (ABC) drug transporters. The present study was designed to determine whether organic DEP extract (DEPe) may also target ABC drug transporters in bronchial cells. DEPe (10 µg/mL) was demonstrated to induce mRNA and protein expression of the multidrug resistance-associated protein (MRP) 3 in cultured bronchial epithelial BEAS-2B cells, whereas mRNA levels of other MRPs, multidrug resistance gene 1 or breast cancer resistance protein were unchanged, reduced or not detected. DEPe also increased MRP3 mRNA expression in normal human bronchial epithelial cells. Inhibition of the aryl hydrocarbon receptor (AhR) pathway by AhR antagonist or AhR silencing, as well as the silencing of nuclear-factor-E2-related factor 2 (Nrf2) repressed DEPe-mediated MRP3 induction. This underlines the implication of the AhR and Nrf2 signaling cascades in DEPe-mediated MRP3 regulation. DEPe was additionally demonstrated to directly inhibit MRP activity in BEAS-2B cells, in a concentration-dependent manner. Taken together, these data indicate that DEPs may impair expression and activity of MRPs, notably MRP3, in human bronchial cells, which may have consequences in terms of lung barrier and toxicity for humans exposed to diesel pollution.

Molecular characterization of gene regulatory networks in primary human tracheal and bronchial epithelial cells.

BACKGROUND: Robust methods to culture primary airway epithelial cells were developed several decades ago and these cells provide the model of choice to investigate many diseases of the human lung. However, the molecular signature of cells from different regions of the airway epithelium has not been well characterized. METHODS: We utilize DNase-seq and RNA-seq to examine the molecular signatures of primary cells derived from human tracheal and bronchial tissues, as well as healthy and diseased (cystic fibrosis (CF)) donor lung tissue. RESULTS: Our data reveal an airway cell signature that is divergent from other epithelial cell types and from common airway epithelial cell lines. The differences between tracheal and bronchial cells are clearly evident as are common regulatory features. Only minor variation is seen between bronchial cells from healthy or CF donors. CONCLUSIONS: These data are a valuable resource for functional genomics analysis of airway epithelial tissues in human disease.

DUOX expression in human keratinocytes and bronchial epithelial cells: Influence of vanadate.

Dual oxygenases (DUOX) 1 and 2, expressed in many animal tissues, participate in host defense at mucosal surfaces and may have important signaling roles through generation of reactive oxygen. Present work addresses their expression in cultured human epidermal keratinocytes and effects of cytokines and metal/metalloid compounds. Both DUOX1 and 2 were expressed at much higher levels after confluence than in the preconfluent state. Maximal DUOX1 mRNA levels were 50 fold those of DUOX2. DUOX1 and 2 were induced ≈3 fold by interleukin 4, but only DUOX1 was induced by interferon gamma (IFNγ). In human bronchial HBE1 cells, by contrast, interleukin 4 induced only DUOX 1, and IFNγ induced only DUOX2. A survey in the keratinocytes of metal/metalloid compounds showed that arsenite, antimonite, chromate, cadmium, copper, lead and vanadate suppressed DUOX1 levels but did not prevent interleukin 4 stimulation. Effects on DUOX2 were less dramatic, except that vanadate potentiated the stimulation by IFNγ up to 7 fold. The results indicate that epithelial cell types of different tissue origins can differ in their cytokine regulation and that epidermal cells can exhibit striking alterations in response due to certain metal/metalloid exposures.

Linking the generation of DNA adducts to lung cancer.

Worldwide, lung cancer is the leading cause of cancer death. DNA adducts are considered a reliable biomarker that reflects carcinogen exposure to tobacco smoke, but the central question is what is the relationship of DNA adducts and cancer? Therefore, we investigated this relationship by a meta-analysis of twenty-two studies with bronchial adducts for a total of 1091 subjects, 887 lung cancer cases and 204 apparently healthy individuals with no evidence of lung cancer. Our study shows that these adducts are significantly associated to increase lung cancer risk. The value of Mean Ratiolung-cancer (MR) of bronchial adducts resulting from the random effects model was 2.64, 95% C.I. 2.00-3.50, in overall lung cancer cases as compared to controls. The significant difference, with lung cancer patients having significant higher levels of bronchial adducts than controls, persisted after stratification for smoking habits. The MRlung-cancer value between lung cancer patients and controls for smokers was 2.03, 95% C.I. 1.42-2.91, for ex-smokers 3.27, 95% C.I. 1.49-7.18, and for non-smokers was 3.81, 95% C.I. 1.85-7.85. Next, we found that the generation of bronchial adducts is significantly related to inhalation exposure to tobacco smoke carcinogens confirming its association with volatile carcinogens. The MRsmoking estimate of bronchial adducts resulting from meta-regression was 2.28, 95% Confidence Interval (C.I.) 1.10-4.73, in overall smokers in respect to non-smokers. The present work provides strengthening of the hypothesis that bronchial adducts are not simply relate to exposure, but are a cause of chemical-induced lung cancer.

MiRNA Expression in Cystic Fibrosis Bronchial Epithelial Cells.

Bronchial epithelial cells represent an invaluable tool to elucidate molecular signaling regulation in cystic fibrosis (CF). CF is a lethal genetic condition characterized by chronic inflammation in which bronchial epithelial cells play a pivotal role. Here we describe their use in analysis of microRNA (miRNA) and their target genes following a two-step RT-PCR miRNA profiling method in bronchial cell specimens from CF and control individuals where 667 human miRNA were examined. We also describe an approach to experimental modulation of these miRNA in vitro.

Cytotoxic effects of composite dust on human bronchial epithelial cells.

INTRODUCTION: Previous research revealed that during routine abrasive procedures like polishing, shaping or removing of composites, high amounts of respirable dust particles (<5µm) including nano-sized particles (<100nm) may be released. OBJECTIVE: To determine the cytotoxic potential of composite dust particles on bronchial epithelium cells. METHODS: Composite dust of five commercial composites (one nano-composite, two nano-hybrid and two hybrid composites) was generated following a clinically relevant protocol. Polymerized composite samples were cut with a rough diamond bur (grain size 100µm, speed 200,000rpm) and all composite dust was collected in a sterile chamber. Human bronchial epithelial cells (16HBE14o-) were exposed to serially diluted suspensions of composite dust in cell culture medium at concentrations between 1.1 and 3.3mg/ml. After 24h-exposure, cell viability and membrane integrity were assessed by the WST-1 and the LDH leakage assay, respectively. The release of IL-1ß and IL-6 was evaluated. The composite dust particles were characterized by transmission electron microscopy and by dynamic and electrophoretic light scattering. RESULTS: Neither membrane damage nor release of IL-1ß was detected over the complete concentration range. However, metabolic activity gradually declined for concentrations higher than 660µg/ml and the release of IL-6 was reduced when cells were exposed to the highest concentrations of dust. SIGNIFICANCE: Composite dust prepared by conventional dental abrasion methods only affected human bronchial epithelial cells in very high concentrations.

CDK14 expression is down-regulated by cigarette smoke in vivo and in vitro.

In this study, DNA arrays have been employed to monitor gene expression patterns in testis of mice exposed to tobacco smoke for 24 weeks and compared to control animals. The results of the analysis revealed significant changes in expression of several genes that may have a role in spermatogenesis. Cdk14 was chosen for further characterization because of a suggested role in the testis and in regulation of Wnt signaling. RT-PCR analysis confirmed down regulation of Cdk14 in mice exposed to cigarette smoke (CS). Cdk14 is expressed in all testicular cells; spermatogonia- and Sertoli-derived cell lines treated with cigarette smoke extract (CSE) in vitro showed down-regulation of CDK14 mRNA and protein levels as well as down-regulation of ß-catenin levels. CS-induced down-regulation of CDK14 mRNA and protein levels was also observed in several lung epithelium-derived cell lines including primary normal human bronchial epithelial cells (NHBE), suggesting that the effect is not restricted to the testis. Similar to testicular cells, CS-induced down-regulation of CDK14 in lung cells correlated with decreased levels of ß-catenin, a finding suggesting impaired Wnt signaling. In the lungs, CDK14 was localized to the alveolar and bronchial epithelium.

In vitro cadmium effects on ECM gene expression in human bronchial epithelial cells.

Occupational and environmental exposure to the heavy metal cadmium (Cd) and its inhalation from cigarette smoke are associated with emphysema. Many growth factors and extracellular matrix (ECM) cell signaling molecules are directly involved in the epithelial bronchial cell pathway. This study investigated the direct effects of Cd on the production of several ECM components in human bronchial epithelial cells (BEAS-2B) that were exposed in vitro for 48 h to sub-toxic and toxic concentrations of Cd. Gene expression of collagens, metalloproteases (MMPs), integrins, tenascin and vitronectin were quantified by RT-PCR. To study apoptosis cascade, annexin assay and cellular cytotoxicity by MTT assay were performed. We also investigated whether an imbalance in the TGFß/TGFß receptor (TGFßR) expression mediated Cd effects. The results showed the sub-toxic Cd dose significantly increased tenascin, vitronectin, ß1 and ß5 integrin gene expression. The toxic Cd dose decreased type IV and V collagen, α1, α2 and ß3 integrins. Both Cd doses down-regulated type I collagen and up-regulated metalloproteases. Each Cd dose caused a different imbalance in the complex pattern of TGFß and its receptors. No alteration in classic apoptotic marker protein expression was observed in presence of the sub-toxic dose of Cd, suggesting this metal alters ECM production without apoptotic activation. In conclusion, all these data show even sub-toxic Cd dose exposure alters the specific gene expression of several ECM components that are crucially implicated in the mechanical properties of lung parenchyma supporting the hypothesis that the mechanism underlying Cd-induced lung disease may involve downstream changes in TGFß/TGFßR signaling.

Efectos de la contaminación del aire en células humanas de pulmón / Effects of air pollution in human lung cells

Introducción. Las PM2.5 son componentes de la atmósfera de la Ciudad de México. Contienen, entre otros compuestos, los hidrocarburos aromáticos policíclicos, que tienen efectos tóxicos conocidos. Debido a las diferencias en la composición de las PM2.5 en las diferentes zonas de la Ciudad de México, y la falta de información sobre sus efectos, el objetivo del estudio fue evaluar la cito y genotoxicidad de la fracción orgánica soluble que contienen los hidrocarburos aromáticos policíclicos, de las PM2.5 provenientes de las estaciones de monitoreo Noreste (NE), Centro (C) y Suroeste (SO) de la Ciudad de México, en cultivo de células NL-20 humanas durante 24 horas. Métodos. Se extrajo la fracción orgánica soluble de los filtros con las PM2.5 de las diferentes estaciones de monitoreo. Se cultivaron las células bronquiales humanas y, posteriormente, se realizaron los ensayos de exposición a la fracción orgánica soluble para evaluar el efecto en la viabilidad y en la inducción de genotoxicidad. Resultados. Los resultados mostraron que 0.1 μg/μl de fracción orgánica soluble de la estación Centro fue la más citotóxica, reduciendo a 52.4% y 54.2% la viabilidad celular, en las temporadas tanto de sequía como de lluvia, respectivamente. Esta fracción orgánica soluble indujo anormalidades celulares, como multinucleación y atipia nuclear. Los porcentajes contrastaron con los obtenidos de la estación NE que fueron 91.2% y 85% a la misma concentración, respectivamente (p <0.05). La concentración de 0.1 μg/μl, tanto de la estación NE como de la del C, fue genotóxica. Conclusiones. La fracción orgánica soluble de la zona Centro fue la más citotóxica, ya que es la zona con mayor concentración de automóviles que son la fuente principal de hidrocarburos aromáticos policíclicos.

Background. PM2.5 are components of the atmosphere of Mexico City and contain polycyclic aromatic hydrocarbons (PAH), which induce toxic effects. Due to different compositions of the PM2.5 in all zones of Mexico City and the lack of information about their effects, the main purpose of this study was to evaluate the cytotoxicity and genotoxicity due to soluble organic fractions (SOFs), which contains PAH isolated from the PM2.5 collected from several monitoring stations in Mexico City (northeast, downtown, and southwest) in a cell culture of human line NL-20 during a 24-h period. Methods. We extracted the soluble organic fraction of PM2.5 filters from the different monitoring stations. Human bronchial cells were cultured and subsequently assays were performed on the exposure of SOFs to evaluate the effect on the viability and induction of genotoxicity. Results. Results show that 0.1 μg/μl of SOF from the downtown station was more cytotoxic, reducing cell viability to 52.4% and 54.2% in both dry and rainy periods, respectively. Also, cellular anomalies were induced such as multinucleation and nuclear atypia. These percentages of cytotoxicity contrasted against those obtained from SOFs from the northeast area that were 91.2% and 85% at the same concentration during both dry and rainy periods, respectively (p <0.05). Only at 0.1 μg/μl SOF were the results genotoxic from the northeast and downtown (p <0.05). Conclusions. SOFs from the downtown zone were the most cytotoxic due to the high concentration of automobiles as the main sources of PAH.