Rat Lung Epithelial Cells Research Articles

Induction of COX-2 by acrolein in rat lung epithelial cells

Acrolein is a highly reactive alpha, beta-unsaturated aldehyde, and a product of lipid peroxidation reactions. Acrolein is also an environmental pollutant and a key component of cigarette smoke, and has been implicated in multiple respiratory diseases. Lung tissue is a primary target for acrolein toxicity in smokers and may lead to chronic lung inflammation and lung cancer. Chronic inflammation, associated with expression of cyclooxygenase-2 (COX-2) and prostaglandins, are predisposing factors for malignancy. In this study, we investigated the induction of COX-2 by acrolein in rat lung epithelial cells and its related signaling cascade. Induction of COX-2 by acrolein was significant at 6 h post-treatment and was dependent upon NFkappaB activation. The activation of NFkappaB by acrolein was induced as a result of degradation of IkappaBalpha over the time of treatment. In addition, the upstream signaling cascade involved Raf-1/ERK activation by acrolein in the COX-2 induction and was inhibited by GW5074 (a Ras/Raf-1/ERK inhibitor), thereby providing evidence for the role of this cascade in this process. The results of these studies offer an explanation for the mechanism of COX-2 induction by acrolein in rat lung epithelial cells.

Uranium induces oxidative stress in lung epithelial cells

Uranium compounds are widely used in the nuclear fuel cycle, antitank weapons, tank armor, and also as a pigment to color ceramics and glass. Effective management of waste uranium compounds is necessary to prevent exposure to avoid adverse health effects on the population. Health risks associated with uranium exposure includes kidney disease and respiratory disorders. In addition, several published results have shown uranium or depleted uranium causes DNA damage, mutagenicity, cancer and neurological defects. In the current study, uranium toxicity was evaluated in rat lung epithelial cells. The study shows uranium induces significant oxidative stress in rat lung epithelial cells followed by concomitant decrease in the antioxidant potential of the cells. Treatment with uranium to rat lung epithelial cells also decreased cell proliferation after 72 h in culture. The decrease in cell proliferation was attributed to loss of total glutathione and superoxide dismutase in the presence of uranium. Thus the results indicate the ineffectiveness of antioxidant system's response to the oxidative stress induced by uranium in the cells.

Ultrafine carbon particles induce apoptosis and proliferation in rat lung epithelial cells via specific signaling pathways both using EGF-R

Apoptosis and proliferation are important causes of adverse health effects induced by inhaled ultrafine particles. The molecular mechanisms of particle cell interactions mediating these end points are therefore a major topic of current particle toxicology and molecular preventive medicine. Initial studies revealed that ultrafine particles induce apoptosis and proliferation in parallel in rat lung epithelial cells, dependent on time and dosage. With these end points, two antagonistic reactions seem to be induced by the same extracellular stimulus. It was therefore investigated whether proliferation is induced directly by the particles or as a compensation of particle-caused cell death. Experimental conditions excluding compensatory proliferation demonstrated that both end points are induced independently by specific signaling pathways. Events eliciting signaling cascades leading to apoptosis and proliferation were studied with specific inhibitors of membrane receptors. Epidermal growth factor receptor (EGF-R) kinase activity was identified as essential for apoptosis as well as for proliferation. As ultrafine particle-induced proliferation alone was dependent on the activation of beta1-integrins, these membrane receptors are suggested to mediate the specificity of EGF-R signaling concerning the decision as to whether apoptosis or proliferation is triggered. Accordingly, MAP kinase signaling downstream of EGF-R showed comparable specificity with regard to receptor-dependent induction of apoptosis and proliferation. As key mediators of signaling cascades, the activation of extracellular signal-regulated kinases 1 and 2 proved to be specific for proliferation in a beta1-integrin-dependent manner, whereas phosphorylation of c-Jun NH2-terminal kinases 1 and 2 was correlated with the induction of apoptosis.

Induction of CYP1A1 in rat lung cells following in vivo and in vitro exposure to quartz

Respirable quartz has been classified as a human lung carcinogen, but the mechanism by which quartz exposure leads to lung cancer has not been clarified. Consistently higher risks of lung cancer are reported in smokers with quartz exposure and we therefore hypothesised that quartz exposure may alter the expression of enzyme systems involved in activation/detoxification of pre-carcinogens in cigarette smoke. More specifically we studied cytochrome P4501A1 (CYP1A1) expression using reverse transcriptase polymerase chain reaction and immunohistochemistry (IHC) upon in vitro and in vivo quartz exposure. In vitro incubation of rat lung epithelial cells with DQ12 quartz for 24 h showed a dose-dependent induction of CYP1A1-mRNA. On the other hand, CYP1A1 message was not increased in lung epithelial cells isolated from rats at 3, 28 or 90 days after intratracheal instillation of 2 mg DQ12. Following IHC for CYP1A1 protein in rat lung sections from later time-points (180 and 360 days), we observed an increase in the number of CYP1A1 positive cells. After in vivo quartz exposure, protein expression of the Aryl hydrocarbon receptor (AhR) was increased and nuclear translocation of AhR was observed at the same time-points. In conclusion, our findings demonstrate an effect of quartz exposure on chronic CYP1A1 expression in vivo, whereas the in vitro models show an immediate upregulation. We suggest that this upregulation of CYP1A1 may act as a co-carcinogenic pathway in quartz exposed workers by activation of pre-carcinogens such as those present in cigarette smoke.

Acquired tolerance in cadmium-adapted lung epithelial cells: Roles of the c-Jun N-terminal kinase signaling pathway and basal level of metallothionein

Cadmium-resistant cells were developed in our laboratory with rat lung epithelial cells (LECs) by stepwise exposure of LECs to cadmium chloride from 1 microM to 20 microM after 20 passages. To investigate the Cd-resistant phenotype in a long-term perspective, cadmium-resistant cells adapted to 20 microM cadmium (Cd(R)) were then cultured in the absence of cadmium for various passages [Cd(R)(-n)]. All these adapted cells were significantly protected from cadmium toxicity as compared to parental cadmium-sensitive LECs (Cd(S)). The cadmium-resistant phenotype of adapted cells was relatively stable in the absence of cadmium for as long as 40 passages. Basal mRNA level of metallothionein-1 (MT-1) was dramatically higher in Cd(R) than in Cd(R)(-), which may account for the higher Cd-resistance of Cd(R) than Cd(R)(-). MT-1 mRNA level decreased drastically in Cd(R) after cadmium removal, suggesting that the high basal level of MT-1 in Cd(R) may be only partially responsible for cadmium-resistance. Treatment of cells with high levels of cadmium resulted in decreased phosphorylation of c-Jun N-terminal kinase (JNK1/2) in adapted cells than in sensitive cells and this cadmium-induced JNK activity was blocked by JNK inhibitor II, SP600125. Ro318220, a strong activator of JNK, reverted cadmium-sensitive phenotype in adapted cells. Taken together, our results suggest that during cadmium adaptation, cells develop tolerance to cell death, generally due to perturbation of the JNK signaling pathway and the nonresponsiveness of JNK phosphorylation is critical for the Cd-tolerance in these cells.

Proteomic and biochemical analyses of in vitro carcinogen‐induced lung cell transformation: Synergism between arsenic and benzo[a]pyrene

Chronic coexposures to carcinogens inorganic arsenic and benzo[a]pyrene (B[a]P) are common in the living environment. However, little is known about their effects exerted at the proteome level. Our previous study in rat lung epithelial cells showed that cell transformation frequency increased by more than 100-fold when arsenic was given in combination with B[a]P than cells either exposed to arsenic or B[a]P alone. This demonstrated a synergism between them. Here, we reported that alterations to the proteome varied and were more pronounced in the transformed cells that were exposed to a combination of arsenic and B[a]P than to B[a]P and much less to arsenic alone when compared to passage-matched control cells. In general, three proteins belonging to intermediate filaments were found to be significantly down-regulated and six proteins belonging to antioxidative stress-, chaperone-, and glycolytic proteins were up-regulated in these transformed cells. These transformed cells were also associated with an increase of proliferation and de-differentiation. Taken together, our findings suggest that although arsenic or B[a]P alone is sufficient to induce cell transformation and alter the proteome to a similar extent, the effects of coexposure are much more pronounced. This further substantiates the notion that these carcinogens act in concert during cocarcinogenesis.

Effect of diesel exhaust particles on mRNA expression of viral and bacterial receptors in rat lung epithelial L2 cells

Epidemiological studies have shown that particulate matter (PM) is associated with adverse respiratory health effects. Although infection in the respiratory organ is one of the most important health risks the association of infection with PM is not fully understood. As we had hypothesized that diesel exhaust particles (DEP), one of the major component of PM, may induce the expression of receptors for viruses and bacteria at invasion sites, we studied the effect of DEP on the mRNA expression of intercellular adhesion molecule-1 (ICAM-1), low-density lipoprotein (LDL) and platelet-activating factor (PAF) receptors, which are invasion sites of virus and bacteria, on rat lung epithelial cells. The real-time quantitative polymerase chain reaction (PCR) method was used for the evaluation. All of these mRNAs were up-regulated by 3, 10, and 30 μg/ml of DEP in a concentration-dependent manner. The up-regulation of each was associated with the mRNA expression of heme oxygenase-1 (HO-1), a marker of oxidative stress. Our present results show that DEP up-regulated the mRNA expression of viral and bacterial receptors. This up-regulation might be associated with DEP-induced oxidative stress. These results thus suggest that DEP may enhance the risk of pneumonia by increasing the density of bacterial and viral invasion sites in the lungs.

Identification of an E-box motif as a transcriptional repressor element in the proximal promoter region of the GCLC gene in rat lung epithelial L2 cells

Glutathione (GSH) is a critical antioxidant for protecting the airway epithelium from oxidant injury and its levels are mainly controlled by glutamate-cysteine ligase (GCL), which is the rate-limiting enzyme in GSH synthesis. A full understanding of the gene regulation mechanism of this important enzyme may disclose the role it plays in respiratory diseases. GCL is made up of two differentially regulated subunits, a catalytic or heavy subunit (GCLC) and a modifier or light subunit (GCLM). Many studies in this field led to the findings of important positive regulatory regions of the GCLC promoter. For a detailed analysis of this gene regulation in the respiratory system, we cloned a 1.76-kb 5′-flanking region of the rat GCLC gene, inserted into a luciferase reporter vector. Exonuclease III was used to cut the 5′-flanking region of the rat GCLC gene unidirectionally into deletion mutants of different lengths. Sequential deletion analysis revealed that regions from −403 to −111 and from −705 to −613 are involved in positive regulation and the region from −745 to −705 is involved in negative regulation of the GCL gene in rat lung epithelial L2 cells. Specific proteins binding to these regions were confirmed by electrophoretic mobility-shift assays (EMSAs) and antibody supershift assays. An E-box motif was found in the negative regulatory region −745 to −705. Site-directed mutagenesis proved that the functional element in this negative regulatory region was a putative E-box element. EMSA and supershift assays showed that USF1 and USF2 can specifically bind to the E-box element. Overexpression of USFs in L2 cells led to a decreased activity of the GCLC promoter. Western blotting demonstrated that the expression of GCLC protein was decreased in the retroviral USFs-expressing cells than in nontransfected (no DNA added) cells, suggesting that USF binding to the E-box at −729/−724 serves to trans-repress GCLC gene expression. These findings indicate that the E-box is an important transcriptional suppressor element in the GCLC promoter in rat lung epithelial L2 cells. Inhibition of interaction between the E-box and the USF may provide an effective means of ameliorating oxidant injury of the lung.

Persistent Nicotine Treatment Potentiates Amplification of the Dihydrofolate Reductase Gene in Rat Lung Epithelial Cells as a Consequence of Ras Activation

Although nicotine has been suggested to promote lung carcinogenesis, the mechanism of its action in this process remains unknown. The present investigation demonstrates that the treatment of rat lung epithelial cells with nicotine for various periods differentially mobilizes multiple intracellular pathways. Protein kinase C and phosphoinositide 3-OH-kinase are transiently activated after the treatment. Also, Ras and its downstream effector ERK1/2 are activated after long term exposure to nicotine. The activation of Ras by nicotine treatment is responsible for the subsequent perturbation of the methotrexate (MTX)-mediated G1 cell cycle restriction as well as an increase in production of reactive oxygen species. When p53 expression is suppressed by introducing E6, persistent exposure to nicotine enables dihydrofolate reductase gene amplification in the presence of methotrexate (MTX) and the formation of the MTX-resistant colonies. Altering the activity of phosphoinositide 3-OH-kinase has no effect on dihydrofolate reductase amplification. However, the suppression of protein kinase C dramatically affects the colony formation in soft agar. Thus, our data suggest that persistent exposure to nicotine perturbs the G1 checkpoint and causes DNA damage through the increase of the production of reactive oxygen species. However, a third element rendered by loss of p53 is required for the initiation of the process of gene amplification. Under p53-deficient conditions, the establishment of a full oncogenic transformation, in response to long term nicotine exposure, is achieved through the cooperation of multiple signaling pathways.

Lung Fluid Balance During Development

After completing this article, readers should be able to: 1. Describe the major driving forces for liquid formation in and liquid removal from the lumen of the fetal lung. 2. Delineate the role of a normal volume of liquid within the lumen of the fetal lung for normal lung growth during development. 3. Describe the effects of labor on Na,K-ATPase activity in lung epithelial cells and clearance of lung liquid after birth. 4. Explain the route by which most residual liquid drains from the lung. 5. List the conditions associated with preterm birth that may contribute to delayed removal of fetal lung liquid. Curious students of mammalian development recognized more than a century ago that the lungs are filled with liquid during fetal life, (1) but the origin of that liquid remained obscure until 1948, when an unexpected discovery by two French scientists dispelled the prevailing view that fetal lung liquid derived from intrauterine inhalation of amniotic fluid. In studies designed to explore the ontogeny of the pituitary-adrenal axis, tracheal ligation of fetal rabbits for 9 days resulted in fluid distention of the lungs. (2) This serendipitous finding, subsequently confirmed and embellished by others, (3)(4)(5) established that the fetal lung itself, rather than the amniotic sac, is the source of the liquid that fills the lung during development. This liquid forms a slowly expanding structural template that prevents collapse and promotes growth of the fetal lung. Subsequent studies, conducted mostly with sheep, showed that liquid in the lumen of the fetal lung forms as a result of chloride secretion in the respiratory epithelium, (6)(7) a process that can be inhibited by diuretics that block Na,K-2Cl cotransport. (8)(9) In vitro experiments using cultured explants of lung tissue and monolayers of epithelial cells harvested from human fetal lung have …

Cells in fluidic environments are sensitive to flow frequency

Virtually all cells accommodate to their mechanical environment. In particular, cells subject to flow respond to rapid changes in fluid shear stress (SS), cyclic stretch (CS), and pressure. Recent studies have focused on the effect of pulsatility on cellular behavior. Since cells of many different tissue beds are constantly exposed to fluid flows over a narrow range of frequencies, we hypothesized that an intrinsic flow frequency that is optimal for determining cell phenotype exists. We report here that cells from various tissue beds (bovine aortic endothelial cells (BAEC), rat small intestine epithelial cells (RSIEC), and rat lung epithelial cells (RLEC)) proliferate maximally when cultured in a perfusion bioreactor under pulsatile conditions at a specific frequency, independent of the applied SS. Vascular endothelial and pulmonary epithelial cell proliferation peaked under 1 Hz pulsatile flow. In contrast, proliferation of gastrointestinal cells, which in their physiological context are subject to no flow or higher wavelength signal, was maximum at 0.125 Hz or under no flow. Moreover, exposure of BAEC to pulsatile flow of varying frequency influenced their nitric oxide synthase activity and prostacyclin production, which reached maximum values at 1 Hz. Notably, the "optimal" frequencies for the cell types examined correspond to the physiologic operating range of the organs from where they were initially derived. These findings suggest that frequency, independent of shear, is an essential determinant of cell response in pulsatile environments.

Induction of nuclear factor-κB activation through TAK1 and NIK by diesel exhaust particles in L2 cell lines

Diesel exhaust particles (DEPs) are known to induce allergic responses in airway epithelial cells, such as the production of various cytokines via nuclear factor-kappa B (NF-κB). However, the intracellular signal transduction pathways underlying this phenomenon have not been fully examined. This study showed that DEP induced NF-κB activity via transforming growth factor-β activated kinase 1 (TAK1) and NF-κB-inducing kinase (NIK) in L2 rat lung epithelial cells. DEP induced the NF-kB dependent reporter activity approximately two- to three-fold in L2 cells. However, this effect was abolished by the expression of the dominant negative forms of TAK1 or NIK. Furthermore, it was shown that DEP induced TAK1 phosphorylation in the L2 cells. These results suggest that TAK1 and NIK are important mediators of DEP-induced NF-κB activation.

A proteome analysis of the arsenite response in cultured lung cells: evidence for in vitro oxidative stress-induced apoptosis.

Arsenite is well documented as a chemotherapeutic agent capable of inducing cell death. However, the cellular response at the molecular level has not been studied extensively. In the present study, we provide for the first time a proteomic analysis of rat LECs (lung epithelial cells) treated with arsenite, with the aim of identifying defence proteins, probably expressed to protect the cells during the course of arsenic-induced apoptosis. Comparative proteome analysis was conducted on LECs and LECs treated with 40 microM arsenite to identify global changes in their protein expression profiles. Over 1000 protein spots were separated by two-dimensional electrophoresis and visualized by silver staining. Seven proteins changed expression levels significantly and were identified by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry and database searching. The proteins up-regulated were mostly HSPs (heat-shock proteins) and antioxidative stress proteins, including HSP70, aldose reductase, haem oxygenase-1, HSP27, ferritin light chain and alphaB-crystallin. The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase was down-regulated. Pretreatment with the thiol antioxidants glutathione or N-acetylcysteine before arsenite insult effectively abrogated the induction of these defence proteins and sustained cell viability, whereas antioxidants were protective only at earlier time points if they were added to cells after arsenite. Taken together, our results demonstrate that high levels of arsenite cause oxidative stress-induced apoptosis.

Inflammatory Time Course after Quartz Instillation

Inflammation has been suggested as the key factor in the development of quartz-induced fibrosis and carcinogenesis, and particle surface properties are argued as an important characteristic responsible for these pathologic alterations. To evaluate the effect of surface modification on acute and subchronic inflammation, female Wistar rats were intratracheally instilled with 2 mg native quartz, or quartz coated either with polyvinyl-pyridine-N-oxide or with aluminium lactate. Various markers of lung toxicity, inflammation, and oxidative stress were found to be enhanced at 3, 7, 21, and 90 d after instillation of native quartz. Quartz-treated animals also showed enhanced immunostaining of nuclear factor-kappaB (NF-kappaB) in alveolar macrophages and lung epithelium, as well as reduced IkappaBalpha levels in whole lung homogenate. Both surface modifications were found to inhibit most of the effects as observed with native quartz. NF-kappaB activation was also observed in vitro in rat lung epithelial cells following treatment with lavage fluid from quartz-treated animals, as well as with conditioned medium of quartz-treated macrophages, and these effects appeared to be at least partly tumor necrosis factor-alpha-independent. In conclusion, the persistent subchronic inflammatory lung response after quartz exposure appears to be particle surface-driven and is associated with NF-kappaB activation in both alveolar macrophages and the lung epithelium.

Mitogen-activated protein kinases modulate H(2)O(2)-induced apoptosis in primary rat alveolar epithelial cells.

Increasing evidence suggests a role for apoptosis in the maintenance of the alveolar epithelium under normal and pathological conditions. However, the signaling pathways modulating alveolar type II (ATII) cell apoptosis remain poorly defined. Here we investigated the role of MAPKs as modulators of oxidant-mediated ATII cell apoptosis using in vitro models of H(2)O(2)-stress. H(2)O(2), delivered either as a bolus or as a flux, lead to time- and concentration-dependent increases in ATII cells apoptosis. Increased apoptosis in primary rat ATII cells was detected at H(2)O(2) concentrations and production rates in the physiological range (1 microM) and peaked at 100 microM H(2)O(2). Immortalized rat lung epithelial cells (RLE), in contrast, required millimolar concentration of H(2)O(2) for maximal responses. H(2)O(2)-induced apoptosis was preceded by rapid activation of all three classes of mitogen-activated protein kinases (MAPKs): ERK, JNK, and p38. Specific inhibition of JNK using antisense oligonucleotides and ERK and p38 using PD98059 or SB202190, respectively, indicated a pro-apoptotic role for JNK pathway and an anti-apoptotic role for ERK- and p38-initiated signaling events. Our data show that the balance between the activation of JNK, ERK, and p38 is a critical determinant of cell fate, suggesting that pharmacological interventions on the MAPK pathways may be useful in the treatment of oxidant-related lung injury.

Comparative study of PM2.5 - and PM10 - induced oxidative stress in rat lung epithelial cells

Accurate estimation of the exposure-response relationship between ambient urban particulate matters (PM) and public health is important for regulatory perspective of ambient urban particulate matters (PM). Ambient PM contains various transition metals and organic compounds. PM10 (aerodynamic diameter less than 10 micro m) is known to induce diverse diseases such as chronic cough, bronchitis, chest illness, etc. However, recent evaluation of PM2.5 (aerodynamic diameter less than 2.5 micro m) against health outcomes has suggested that the fine particles may be more closely associated with adverse respiratory health effects than particles of larger size. This study was performed to evaluate PM2.5-induced oxidative stress in rat lung epithelial cell in order to provide basic data for the risk assessment of PM2.5. PM2.5 showed higher cytotoxicity than PM10. Also, PM 2.5 induced more malondialdehyde (MDA) formation than PM10. In Hoechst 33258 dye staining and DNA fragmentation assay, apopotic changes were clearly detected in PM2.5 treated cells in compared to PM10. Expression of catalase mRNA was increased by PM2.5 rather than PM10. PM2.5 induced higher Mth1 mRNA than PM10. In pBR322 DNA treated with PM2.5, production of single strand breakage of DNA was higher than that of PM10. In Western blot analysis, PM2.5 induced more Nrf-2 protein, associated with diverse transcriptional and anti-oxidative stress enzymes, compared to PM10. Our data suggest that PM2.5 rather than PM10 may be responsible for PM-induced toxicity. Additional efforts are needed to establish the environmental standard of PM2.5.

Foetal rat lung epithelial (FRLE) cells: alterations in cellular homeostasis and gene expression in response to etoposide, hydrogen peroxide and sodium butyrate.

Genomics technology offers a way of detecting the effects of a toxin on the expression of many genes in a single experiment. We have previously partially characterised a foetal rat lung epithelial (FRLE) cell line and shown that it is suitable for use in a pneumocytotoxicity screen. In this study, we wanted to ascertain whether we could use alterations in FRLE cell gene expression as a sensitive marker of cell stress. Sodium butyrate and etoposide were shown to arrest FRLE cell cycle at G0/G1 and G2/M phase of the cell cycle, respectively and this was associated with a decrease in the number of cells in culture. Following 24 h of culture both compounds caused a statistically significant increase in the mRNA levels of the cell cycle inhibitory protein, gadd153, whereas p21 was statistically altered by etoposide only. Hydrogen peroxide induced growth arrest at low concentrations (< or =250 microM) following 24 h of culture. We could not detect an increase in apoptosis or in the mRNA levels of the pro-apoptotic protein bax in FRLE cells following culture with hydrogen peroxide or etoposide. Thus, it was possible to correlate cellular perturbations in FRLE cells with alterations in gene expression, demonstrating that these cells are suitable for use in a toxicity screen.

Dexamethasone responsive element in the rat Na, K-ATPase β 1 gene coding region

The Na, K-ATPase plays an essential role in active alveolar epithelial fluid resorption. In fetal and adult alveolar epithelial cells, glucocorticoids (GC) increase Na, K-ATPase activity, mRNA levels, and transcription rate of the β 1 subunit. In this study, we describe a glucocorticoid responsive element (GRE) in the coding region of the rat Na, K-ATPase β 1 gene in a rat lung epithelial cell line. Transient transfection experiments with the β 1 subunit coding region with or without the 5′ and 3′ untranslated regions demonstrated responsiveness to dexamethasone induction and also identified a GRE at +434 in exon IV. The +434 GRE conferred dexamethasone responsiveness in a heterologous thymidine kinase promoter irrespective of its orientation to the β 1 promoter. Transcriptional upregulation by dexamethasone was abolished in +434 mutants. Electrophoretic mobility shift assays (EMSA) demonstrated specific binding of nuclear proteins to the +434 GRE and the presence of the GC receptor. This specific binding was inhibited by a GRE previously described in the rat Na, K-ATPase β 1 gene at −631. In conclusion, we identified a GRE at +434 in the exon IV of the rat Na, K-ATPase β 1 gene.

Foetal rat lung epithelial (FRLE) cells: partial characterisation and response to pneumotoxins

Cultured cell lines are routinely used for in vitro toxicity screens, reducing the requirement for animal studies during the development of new pharmaceutical, agrochemical and cosmetic products. The foetal rat lung epithelial (FRLE) cell line was originally derived from alveolar type II cells (ATII) of the lung. The aims of this study were to further characterise FRLE cells and investigate their potential for screening for pneumotoxins. The cells were found to have retained some of the features of their progenitor cells, namely the expression of cytokeratin proteins, specifically cytokeratin 18, and the ability to actively accumulate the non-selective contact herbicide paraquat. However, the cells have lost the ability to synthesise surfactant protein mRNA and no longer contain multiple lamellar bodies. Toxins that damage ATII cells in vivo (cadmium chloride, cobalt chloride and paraquat) were found to induce cytotoxicity in FRLE cells, as did the non-specific pneumotoxin nitrofurantoin, and hydrogen peroxide. However, the cells were less sensitive to the effects of compounds that require metabolic activation (1-nitronaphthalene, coumarin and butylated hydroxytoluene) and the hepatotoxin bromobenzene. Thus, FRLE cells appear to be a good in vitro model for monitoring the potential toxicity to ATII cells and could be used as an initial screen for pneumotoxicity.

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.