Lung Epithelial Cell Model Research Articles

Occurrence of molecular abnormalities of cell cycle in L132 cells after in vitro short-term exposure to air pollution PM 2.5

To improve the knowledge of the underlying mechanisms implying in air pollution Particulate Matter (PM)-induced lung toxicity in humans, we were interested in the sequential occurrence of molecular abnormalities from TP53- RB gene signaling pathway activation in the L132 target human lung epithelial cell model. The most toxicologically relevant physical and chemical characteristics of air pollution PM 2.5 collected in Dunkerque, a French highly-industrialized sea-side city, were determined. L132 cells were exposed during 24, 48 and 72 h to Dunkerque City's PM 2.5 (i.e. Lethal Concentration (LC) 10 = 18.84 μg PM/mL or 5.02 μg PM/cm 2; LC 50 = 75.36 μg PM/mL or 20.10 μg PM/cm 2), TiO 2 and desorbed PM (i.e. dPM; EqLC 10 = 15.42 μg/mL or 4.11 μg PM/cm 2; EqLC 50 = 61.71 μg/mL or 16.46 μg PM/cm 2), benzene (7 μM) or Benzo[a]Pyrene (B[a]P; 1 μM). Dunkerque City's PM 2.5 altered the gene expression and/or the protein concentration of several key cell cycle controllers from TP53- RB gene signaling pathway (i.e. P53; BCL2; P21; cyclin D1, cyclin-dependent kinase 1; retinoblastoma protein) in L132 cells, thereby leading to the occurrence of cell proliferation and apoptosis together. The activation of the critical cell cycle controllers under study might be related to PM-induced oxidative stress, through the possible involvement of covalent metals in redox systems, the metabolic activation of organic chemicals by enzyme-catalyzed reactions, and phagocytosis. Taken together, these results might ask the critical question whether there is a balance or, in contrast, rather an imbalance between the cell proliferation and the apoptosis occurring in PM-exposed L132 cells, with possible consequences in term of PM-induced lung tumorgenesis.

RT-PCR analysis of ABC, SLC and SLCO drug transporters in human lung epithelial cell models

AbstractObjectivesCarrier-mediated transport mechanisms play crucial roles in drug absorption and elimination processes, as well as in the transport of endogenous molecules affecting cellular regulation and function. In this study we used RT-PCR analysis to characterise the mRNA transcript expression of a wide range of membrane carrier transporters in several in-vitro lung epithelial cell models. Transporters studied included: 11 ATP-binding cassette (ABC) transporters, 11 solute carrier (SLC) transporters and 9 solute carrier organic anion (SLCO) transporters.MethodsThe cell culture models included both established cell lines (A549, Calu-3, 16HBE14o-, BEAS-2B) and freshly isolated lung epithelial cells in primary culture (human bronchial and alveolar epithelial cells).Key findingsThe expression profiles of several clinically relevant drug transporters were characterised using RT-PCR analysis. Our results showed differential transporter expression in cell culture models from different regions of the lung and also highlighted disparities when comparing lung cell lines with primary cell culture models. Differences in transporter expression between cell models of pulmonary and gastrointestinal origin were also noted.ConclusionsThe information will guide and validate the use of in-vitro lung epithelial cell lines in the study of pulmonary administered drugs and candidate molecules.

Relative Contributions of Active Mediated Transport and Passive Diffusion of Fluoroquinolones with Various Lipophilicities in a Calu-3 Lung Epithelial Cell Model

The transport characteristics of six fluoroquinolones (FQs) with various lipophilicities were compared in a Calu-3 cell model. For each FQ, an active polarized transport was observed in the direction of the apical side. However, the apparent permeability of FQs resulted from active transport and passive diffusion that were highly variable between compounds and mainly governed by lipophilicity. Therefore, active transport was predominant for compounds with relatively low lipophilicity but minor for FQs with higher lipophilicity.

RT-PCR analysis of ABC, SLC and SLCO drug transporters in human lung epithelial cell models

Carrier-mediated transport mechanisms play crucial roles in drug absorption and elimination processes, as well as in the transport of endogenous molecules affecting cellular regulation and function. In this study we used RT-PCR analysis to characterise the mRNA transcript expression of a wide range of membrane carrier transporters in several in-vitro lung epithelial cell models. Transporters studied included: 11 ATP-binding cassette (ABC) transporters, 11 solute carrier (SLC) transporters and 9 solute carrier organic anion (SLCO) transporters. The cell culture models included both established cell lines (A549, Calu-3, 16HBE14o-, BEAS-2B) and freshly isolated lung epithelial cells in primary culture (human bronchial and alveolar epithelial cells). The expression profiles of several clinically relevant drug transporters were characterised using RT-PCR analysis. Our results showed differential transporter expression in cell culture models from different regions of the lung and also highlighted disparities when comparing lung cell lines with primary cell culture models. Differences in transporter expression between cell models of pulmonary and gastrointestinal origin were also noted. The information will guide and validate the use of in-vitro lung epithelial cell lines in the study of pulmonary administered drugs and candidate molecules.

P-Glycoprotein-Mediated Transport of Moxifloxacin in a Calu-3 Lung Epithelial Cell Model

Moxifloxacin (MXF) is a fluoroquinolone antibiotic that is effective against respiratory infections. However, the mechanisms of MXF lung diffusion are unknown. Active transport in other tissues has been suggested for several members of the fluoroquinolone family. In this study, transport of MXF was systematically investigated across a Calu-3 lung epithelial cell model. MXF showed polarized transport, with the secretory permeability being twice as high as the absorptive permeability. The secretory permeability was concentration dependent (apparent P(max) = 13.6 x 10(-6) cm x s(-1); apparent K(m) = 147 microM), suggesting saturated transport at concentrations higher than 350 microg/ml. The P-glycoprotein inhibitor PSC-833 inhibited MXF transport in both directions, whereas probenecid, a multidrug resistance-related protein inhibitor, appeared to have no effect in the Calu-3 model. Moreover, rifampin, a known inducer of efflux transport proteins, upregulated the expression of P-glycoprotein in Calu-3 cells and enhanced MXF active transport. In conclusion, this study clearly indicates that MXF is subject to P-glycoprotein-mediated active transport in the Calu-3 model. This P-glycoprotein-dependent secretion may lead to higher MXF epithelial lining fluid concentrations than those in plasma. Furthermore, drug-drug interactions may be expected when MXF is combined with other P-glycoprotein substrates or modulators.

Genotoxic potential of Polycyclic Aromatic Hydrocarbons-coated onto airborne Particulate Matter (PM 2.5) in human lung epithelial A549 cells

To improve the knowledge of the underlying mechanisms of action involved in air pollution Particulate Matter (PM)-induced toxicity in human lungs, with a particular interest of the crucial role played by coated-organic chemicals, we were interested in the metabolic activation of Polycyclic Aromatic Hydrocarbons (PAH)-coated onto air pollution PM, and, thereafter, the formation of PAH–DNA adducts in a human lung epithelial cell model (A549 cell line). Cells were exposed to Dunkerque city’s PM 2.5 at its Lethal Concentrations at 10% and 50% (i.e. LC 10 = 23.72 μg/mL or 6.33 μg/cm 2, and LC 50 = 118.60 μg/mL or 31.63 μg/cm 2), and the study of Cytochrome P450 (CYP) 1A1 gene expression (i.e. RT-PCR) and protein activity (i.e. EROD activity), and the formation of PAH–DNA adducts (i.e. 32P-postlabeling), were investigated after 24, 48, and/or 72 h. PAH, PolyChlorinated Dibenzo- p-Dioxins and -Furans (PCDD/F), Dioxin-Like PolyChlorinated Biphenyls (DLPCB), and PolyChlorinated Biphenyls (PCB)-coated onto collected PM were determined (i.e. GC/MS and HRGC/HRMS, respectively), Negative (i.e. TiO 2 or desorbed PM, dPM; EqLC 10 = 19.42 μg/mL or 5.18 μg/cm 2, and EqLC 50 = 97.13 μg/mL or 25.90 μg/cm 2), and positive (i.e. benzo( a)pyrene; 1 μM) controls were included in the experimental design. Statistically significant increases of CYP1A1 gene expression and protein activity were observed in A549 cells, 24, 48 and 72 h after their exposure to dPM, suggesting thereby that the employed outgassing method was not efficient enough to remove total PAH. Both the CYP1A1 gene expression and EROD activity were highly induced 24, 48 and 72 h after cell exposure to PM. However, only very low levels of PAH–DNA adducts, also not reliably quantifiable, were reported 72 h after cell exposure to dPM, and, particularly, PM. The relatively low levels of PAH together with the presence of PCDD/F, DLPCB, and PCB-coated onto Dunkerque City’s PM 2.5 could notably contribute to explain the borderline detection of PAH–DNA adducts in dPM and/or PM-exposed A549 cells. Hence, remaining very low doses of PAH in dPM or relatively low doses of PAH-coated onto PM were involved in enzymatic induction, a key feature in PAH-toxicity, but failed to show a clear genotoxicity in this in vitro study. We also concluded that, in the human lung epithelial cell model we used, and in the experimental conditions we chose, bulky-DNA adduct formation was apparently not a major factor involved in the Dunkerque City’s PM 2.5-induced toxicity.

Analysis of mRNA for ABC‐, SLC‐ and SLCO‐transporter in human respiratory epithelial cells

The role of transporter proteins in drug disposition in the lung remains mostly unknown to date. Here, RT‐PCR is used to determine the presence of mRNA transcripts for drug transporters in human lung epithelial cell models. In the cell lines, A549, Calu‐3 and 16HBE14o‐, expression of mRNA transcripts for all ABC‐transporters tested, except for MRP8, was found, while in hBEpC and hAEpC, mRNA transcripts for all ABC‐transporters were present. When the cell lines were examined for expression of SLC‐transporters, all tested cells were found positive for OCT1, OCTN2 and PEPT2. PEPT1, OCTN1 and OAT4 expression was noted in all cells, except hBEpC. OCT3 was positively identified in A549, Calu‐3 and hBEpC. None of the cells showed positive transcripts for OCT2, OAT1, ‐2 and ‐3. Levels of SLCO transcripts varied between all cell lines tested, although none were found in 16HBE14o‐. Transcripts for OATP3A1 and OATP4A1 were found in all other cell lines. Additionally to A549 and Calu‐3, mRNA for OATP5A1 and OATP1B3 was found in BEAS‐2B and for OATP1A2 in BEAS‐2B and hBEpC, whereas mRNA for OATP1B1 and OATP1C1 was only detected in A549 and Calu‐3. This study shows that there are significant differences in the expression pattern of ABC‐, SLC‐ and SLCO‐transporters between cell types of the respiratory epithelium. These data are essential for the appropriate use and interpretation of in vitro data obtained with the described cell cultures.

SARS-CoV replication and pathogenesis in an in vitro model of the human conducting airway epithelium

SARS coronavirus (SARS-CoV) emerged in 2002 as an important cause of severe lower respiratory tract infection in humans and in vitro models of the lung are needed to elucidate cellular targets and the consequences of viral infection. The severe and sudden onset of symptoms, resulting in an atypical pneumonia with dry cough and persistent high fever in cases of severe acute respiratory virus brought to light the importance of coronaviruses as potentially lethal human pathogens and the identification of several zoonotic reservoirs has made the reemergence of new strains and future epidemics all the more possible. In this chapter, we describe the pathology of SARS-CoV infection in humans and explore the use of two models of the human conducting airway to develop a better understanding of the replication and pathogenesis of SARS-CoV in relevant in vitro systems. The first culture model is a human bronchial epithelial cell line Calu-3 that can be inoculated by viruses either as a non-polarized monolayer of cells or polarized cells with tight junctions and microvilli. The second model system, derived from primary cells isolated from human airway epithelium and grown on Transwells, form a pseudostratified mucociliary epithelium that recapitulates the morphological and physiological features of the human conducting airway in vivo. Experimental results using these lung epithelial cell models demonstrate that in contrast to the pathology reported in late stage cases SARS-CoV replicates to high titers in epithelial cells of the conducting airway. The SARS-CoV receptor, human angiotensin 1 converting enzyme 2 (hACE2), was detected exclusively on the apical surface of cells in polarized Calu-3 cells and human airway epithelial cultures (HAE), indicating that hACE2 was accessible by SARS-CoV after lumenal airway delivery. Furthermore, in HAE, hACE2 was exclusively localized to ciliated airway epithelial cells. In support of the hACE2 localization data, the most productive route of inoculation and progeny virion egress in both polarized Calu-3 and ciliated cells of HAE was the apical surface suggesting mechanisms to release large quantities of virus into the lumen of the human lung. Preincubation of the apical surface of cultures with antisera directed against hACE2 reduced viral titers by two logs while antisera against DC-SIGN/DC-SIGNR did not reduce viral replication levels suggesting that hACE2 is the primary receptor for entry of SARS-CoV into the ciliated cells of HAE cultures. To assess infectivity in ciliated airway cultures derived from susceptible animal species we generated a recombinant SARS-CoV by deletion of open reading frame 7a/7b (ORF 7a/7b) and insertion of the green fluorescent protein (GFP) resulting in SARS-CoV GFP. SARS-CoV GFP replicated to similar titers as wild type viruses in Vero E6, MA104, and CaCo2 cells. In addition, SARS-CoV replication in airway epithelial cultures generated from Golden Syrian hamster tracheas reached similar titers to the human cultures by 72 h post-infection. Efficient SARS-CoV infection of ciliated cell-types in HAE provides a useful in vitro model of human lung origin to study characteristics of SARS-CoV replication and pathogenesis.

A TGFβ-induced Smad complex directly activates PKA in pancreatic acinar cells and regulates pancreatic acinar cell growth

Background: TGFβ is an important regulator of growth and differentiation in pancreas. We have recently demonstrated that TGFβ activates PKA in a mink lung epithelial cell model through complex formation of Smads with PKA independent of increases in cAMP (Mol Cell Biol 2004; 24: 2169-2180). In this study, we sought to evaluate whether TGFβ can activate PKA in pancreatic acinar cells, the mechanism by which PKA is activated, and PKA’s role in TGFβ-mediated pancreatic growth regulation. Methods: Isolated mouse pancreatic acini were treated with TGFβ and in vitro kinase assays for PKA activity were performed using a biotinylated PKA peptide substrate.

Opposed arsenite-induced signaling pathways promote cell proliferation or apoptosis in cultured lung cells.

Arsenic is a well-known carcinogen that possibly promotes tumors and the development of various types of cancer in individuals chronically exposed to arsenic in their work or living environment. Many studies have demonstrated the activation of mitogen-activated protein kinase (MAPK) in several cell types by using lethal concentrations of arsenic in the range of 50-500 micro M. Since the exposure of humans to arsenic is normally at a much lower level in the workplace or in daily life, it is more relevant to study the effects of arsenic at this lower exposure level. In the present study we aimed at redefining the role of signal transduction pathways in arsenic-induced malignant transformation as well as apoptosis using our established in vitro rat lung epithelial cell model system. Our results indicate a molecular mechanism by which MAPK pathways might differentially contribute to cell growth regulation and cell death in response to different dosages of arsenite. A low level (2 micro M) of arsenite stimulated extracellular signal-regulated kinase (ERK) signaling pathway and enhanced cell proliferation, and this arsenite-induced ERK activity was blocked by MEK inhibitor, PD98059. In contrast, a high level (40 micro M) of arsenite stimulated the c-Jun N-terminal kinase (JNK) signaling pathway and induced cell apoptosis, and this arsenite-induced JNK activity was blocked by JNK inhibitor II, SP600125. The implications of these findings are that a high concentration of arsenic exposure causes apoptosis, whereas a low concentration of arsenic exposure is carcinogenic and may result in aberrant cell accumulation.

Jacobs receives Astrazeneca contract

To improve the knowledge of the underlying mechanisms implying in air pollution Particulate Matter (PM)-induced lung toxicity in humans, we were interested in the sequential occurrence of molecular abnormalities from TP53-RB gene signaling pathway activation in the L132 target human lung epithelial cell model. The most toxicologically relevant physical and chemical characteristics of air pollution PM2.5 collected in Dunkerque, a French highly-industrialized sea-side city, were determined. L132 cells were exposed during 24, 48 and 72 h to Dunkerque City's PM2.5 (i.e. Lethal Concentration (LC)10 = 18.84 μg PM/mL or 5.02 μg PM/cm2; LC50 = 75.36 μg PM/mL or 20.10 μg PM/cm2), TiO2 and desorbed PM (i.e. dPM; EqLC10 = 15.42 μg/mL or 4.11 μg PM/cm2; EqLC50 = 61.71 μg/mL or 16.46 μg PM/cm2), benzene (7 μM) or Benzo[a]Pyrene (B[a]P; 1 μM). Dunkerque City's PM2.5 altered the gene expression and/or the protein concentration of several key cell cycle controllers from TP53-RB gene signaling pathway (i.e. P53; BCL2; P21; cyclin D1, cyclin-dependent kinase 1; retinoblastoma protein) in L132 cells, thereby leading to the occurrence of cell proliferation and apoptosis together. The activation of the critical cell cycle controllers under study might be related to PM-induced oxidative stress, through the possible involvement of covalent metals in redox systems, the metabolic activation of organic chemicals by enzyme-catalyzed reactions, and phagocytosis. Taken together, these results might ask the critical question whether there is a balance or, in contrast, rather an imbalance between the cell proliferation and the apoptosis occurring in PM-exposed L132 cells, with possible consequences in term of PM-induced lung tumorgenesis.