Human Bronchoepithelial Cells Research Articles

JNK1, but not JNK2, is required for COX-2 induction by nickel compounds

Activation of the signaling pathways leading to gene expression regulation is critical in the carcinogenic effects of nickel exposure. In the present study, we found nickel exposure could induce cyclooxygenase-2 (COX-2) expression at transcriptional and protein levels in both human bronchoepithelial cells (Beas-2B) and murine embryonic fibroblasts (MEFs). We further provided direct evidence for the specific involvement of the JNK1 signaling pathway in the COX-2 induction using specific gene knockout approaches. Our results demonstrated that COX-2 induction by nickel was impaired in JNK1(-/-) MEFs, but not in JNK2(-/-) MEFs. Moreover, re-constitutional expression of JNK1 restored COX-2 induction, confirming the specific requirement of JNK1 in COX-2 induction. Further investigation revealed that JNK1 mediated the nickel-induced COX-2 expression in a c-Jun/AP-1-dependent manner. Ectopic expression of TAM67, a c-Jun dominant negative mutant, also suppressed the COX-2 induction. Our results demonstrate that the JNK1/c-Jun/AP-1 pathway, but not the JNK2 pathway, plays a critical role in nickel-induced COX-2 expression.

Crystalline nickel sulfide-induced genomic instability in transformed human broncho-epithelial cells

To detect the genomic instability in the 16 human broncho-epithelial (16HBE) cells induced by crystalline nickel sulfide so as to provide the scientific basis for further study of nickel-induced cancer molecular mechanism. To analyse the genomic instability in transformed 16HBE cells induced by crystalline nickel sulfide by random amplified polymorphic DNA (RAPD). All the 7 random primers selected could amplify 1 - 6 clear PCR bands. There were no significant differences between transformed 16HBE cells and negative control cells in the 4th, 5th, and 7th primers, but in the rest 4 primers there were significant differences, with special PCR bands for the same primer, indicating that genomic instability in transformed 16 HBE cells was induced by crystalline nickel sulfide. Crystalline nickel sulfide could induce genomic instability in 16HBE cells.

62 Tumour necrosis factor-alpha cell signalling events in human bronchoepithelial cells

62 Tumour necrosis factor-alpha cell signalling events in human bronchoepithelial cells

O-138 Tumour necrosis factor-alpha cell signalling events in human bronchoepithelial cells

O-138 Tumour necrosis factor-alpha cell signalling events in human bronchoepithelial cells

Increased Gene Expression of Novel Cytosolic and Secretory Phospholipase A2Types in Human Airway Epithelial Cells Induced by Tumor Necrosis Factor-αand IFN-γ

Phospholipase A(2) (PLA(2)) is a growing family of enzymes that may play a major role in inflammation. We investigated the effect of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on the gene expression of 19 different PLA(2) types (IB, IIA, IID, IIE, IIF, III, IVA, IVB, IVC, V, VIA, VIB, VIIA, VIIB, VIIIA, VIIIB, X, XII, and XIII) in human bronchoepithelial (BEAS-2B) and nasal epithelial (RPMI 2650) cells. The cells were stimulated with TNF-alpha or IFN-gamma for different lengths of time (1, 4, 18, and 48 h), and the mRNA levels of the different PLA(2) types were determined by reverse transcriptase-PCR (RT-PCR) and normalized to those of the housekeeping gene, GAPDH. In both cell lines, TNF-alpha increased the expression of PLA(2) IVA and IVC, and IFN-gamma increased the expression of PLA(2) IIA and IID. No influence on the gene expression of PLA(2)-activating protein (PLAP) was noted on cytokine stimulation. These findings indicate that TNF-alpha and IFN-gamma induce gene expression of two novel cytosolic and secretory PLA(2) types (IVC and IID, respectively) in human airway epithelial cells. The possibility that these PLA(2) types are involved in cytokine-mediated inflammation in the respiratory tract is inferred.

Rapid reduction of intracellular glutathione in human bronchial epithelial cells exposed to occupational levels of toluene diisocyanate.

Toluene diisocyanate (TDI) is a recognized chemical asthmogen, yet the mechanism of this toxicity and the molecular reactions involved have not been elucidated. We have previously shown that TDI vapor forms adducts with the apical surface of the respiratory epithelium, and that it colocalizes with ciliary tubulin. In vitro, we have shown rapid reaction of TDI with glutathione (GSH) and transfer of the bisGS-TDI adduct to a sulfhydryl-containing major histocompatibility complex peptide. This study sought to determine if intracellular GSH is altered following exposure to TDI. We used the dye CellTracker Green (chloromethylfluorescein, CMFDA) for detection of glutathione. One-day and 6-day air-liquid cultures of human bronchoepithelial cells (HBE) were exposed to 20-100 ppb TDI vapor for 5, 15, or 30 min. Cells were subsequently imaged using a confocal microscope. Both 1- and 6-day cultures showed a decrease in intensity of the thiol staining as a function of the TDI exposure dose. Doses as low as 20 ppb, the current permissible exposure limit (PEL) to TDI, resulted in rapid (within 5 min) decreases in fluorescence. The decreased fluorescence was not due to cytotoxicity or decrease in either esterase or glutathione-S-transferase activity, enzymes necessary for activation of the fluorescence of CMFDA. The decrease in glutathione levels was verified using another fluorescent label, ThioGlo(TM) 1, and cell extracts. In addition, the mucus produced by 6-day air-liquid interface HBE cells in response to TDI exposure appeared to be protective, as HBE cells underlying mucus retained more fluorescence than did cells in the same cultures that were not covered with mucus. These results, along with previous data, strongly suggest that TDI enters pulmonary cells and reacts rapidly with intracellular GSH, and that this can occur at the current PEL of 20 ppb. This rapid reaction suggests the importance of cellular thiols in TDI-induced pulmonary disease.

Intracellular S-glutathionyl adducts in murine lung and human bronchoepithelial cells after exposure to diisocyanatotoluene.

Diisocyanatotoluene (toluene diisocyanate, TDI), a 4:1 mixture of 2, 4- and 2,6-isomers used in the preparation of polyurethanes, causes occupational asthma by an as yet unknown mechanism. We previously showed that it forms adducts with the apical surface of the bronchoepithelium in vivo, and with ciliary microtubules in cultured human bronchoepithelial (HBE) cells. These results suggested that TDI may not enter HBE cells. In vitro studies, however, showed that TDI avidly forms bis adducts with glutathione (GSH) and that these adducts transfer monoisocyanato-monoglutathionyl-TDI to a sulfhydryl-containing peptide. This study sought to elucidate intracellular reactions of TDI. Using an electron paramagnetic resonance spectrometric (EPR) method, we established that the level of thiol-dependent quenching of phenoxyl radicals of etoposide was decreased >40% in pulmonary tissue of mice that received TDI intrabronchially. Similarly, HBE cells exposed to 100 ppb TDI vapor experienced a >30% reduction in thiol levels as determined with a thiol-specific fluorescent probe (ThioGlo 1). HPLC/UV analysis of lysates from HBE cells exposed to 200 and 500 ppb TDI vapor suggested a dose-related formation of S-glutathionyl adducts. Data from the 500 ppb TDI-treated HBE cells verified the identity of the 2-monoglutathionyl-4-monoisocyanato adduct. The results provide firm evidence that TDI enters pulmonary cells and reacts with GSH. This rapid reaction leading to formation of S-glutathionyl adducts of TDI suggests the importance of cellular thiols in TDI-induced pulmonary disease.

Interactions between human bronchoepithelial cells and lung fibroblasts after ozone exposure in vitro

Long-term exposure to ozone has been shown to cause lung fibrosis and increased collagen synthesis by fibroblasts in experimental animals. As the bronchial epithelium appears to play a major regulatory role in inflammatory processes, we investigated whether ozone induces bronchoepithelial cells in vitro to increase gene expression of procollagens and other fibrogenic mediators in human lung fibroblasts. Membrane cultures of human airway epithelial cells (BEAS-2B) in the presence or absence of lung fibroblast (HFL-1) cultures were exposed to air or 500 ppb ozone for 1 h, followed by (co-)incubation periods of 11 and 23 h. After ozone exposure of the co-cultures, there were substantial increases of steady-state mRNA levels of both α 1 procollagens type I and III as well as TGF β 1 in the fibroblasts above the corresponding air control levels. In the absence of ozone, the presence of epithelial cells always caused significant decreases in the basal steady-state mRNA levels of both procollagens as compared to their absence. There were no significant effects of ozone on the secretion or gene expression of TGF β 2, PDGF or IL-8 in any cell type. In contrast, co-culture condition induced altered patterns of IL-8 gene expression or of PDGF production in fibroblasts and bronchoepithelial cells, respectively, both in the absence or presence of ozone. In summary, our data demonstrate that the effect of ozone on fibroblasts was mediated by epithelial cells and that mutual regulatory interactions between the different cell types occur. Thus, our co-cultivation system in vitro appears to be able to mimic the in vivo situation providing insight into the nature of cellular interactions and modulation by ozone, which may occur in the whole organism after long-term exposure.

Induction of sister chromatid exchanges (SCE) in human tracheal epithelial cells by the fractions PM-10 and PM-2.5 of airborne particulates

Recent epidemiological studies in the United States and in Europe indicate, that the coarse fraction (PM-10) of airborne particulates, smaller than 10 μm and particularly the fine fraction (PM-2.5) smaller than 2.5 μm are responsible for adverse health effects, causing an increasing morbidity and mortality. Furthermore, an association was reported between air pollution, especially the levels of the fine respirable particles and death from lung cancer. The epithelium of the respiratory tract is the major target of airborne particulates and the location of the most common cancer in man, bronchogenic carcinoma. The genotoxic activity of the coarse (PM-10) and the fine fraction (PM-2.5) of airborne particulates leading to mutation and cancer can be analyzed using in vitro models of human bronchoepithelial cells. In our study collection of the coarse (PM-10) and the fine fraction (PM-2.5) of airborne particulates was conducted in the winter of 1996 in the highly industrialized Rhine-Ruhr region (Germany). For collection we selected an urban area (Düsseldorf), an industrialized area Duisburg and a rural area (Borken). Airborne particulates were collected with a Low Volume M-10 dichotomous sampler (Graseby-Andersen) equipped with glass fiber filters. Chemical substances were extracted from filters with di-chloromethane and quantitatively transferred to dimethylsulfoxide (DMSO). As target cells for testing the genotoxic activity we used cultures of the human bronchioepithelial cell line (BEAS-2B). As a sensitive cytogenetic endpoint for evaluation of the genotoxic activity of extracts of airborne particulates we utilized the induction of ‘sister chromatid exchanges’ (SCE). The coarse fraction PM-10 and especially the fine fraction PM-2.5 of airborne particulates from all three locations caused a strong dose-related induction of ‘sister chromatid exchanges’. The fine fractions PM-2.5 from the three locations exerted a stronger genotoxic activity than the corresponding coarse fractions PM-10. While airborne particulates from Düsseldorf and Duisburg revealed a comparable genotoxic activity, the samples from Borken disclosed a lower genotoxicity. It is important that especially the fine fraction PM-2.5, exerted a strong genotoxicity equivalent to substances of airborne particulates from less than 0.5 m 3 of air. Results of this study and earlier reports demonstrate that the human tracheobronchial epithelial cell line (BEAS-2B) in vitro offer a reliable and sensitive in vitro model for genotoxicity testing of airborne particulates, especially of the coarse (PM-10) and fine fraction (PM-2.5).

Induction of manganese superoxide dismutase gene expression in bronchoepithelial cells after rockwool exposure.

Superoxide dismutases play an important protective role in the lung defense against the pro-oxidative effect of fibrous dusts (e.g. crocidolite fibers). Particularly crocidolite, but also other asbestos fibers, are known to induce cellular antioxidant defense. Although rockwool, a man-made fiber made from rock, is used widely for insulation purposes, its effects on the superoxide dismutases in bronchoepithelial cells have not been investigated. Thus, the purpose of this study was to determine whether human bronchoepithelial cells (BEAS 2B) respond to rockwool fibers (115-4 experimental rockwool fiber) by induction of MnSOD mRNA and an increase of MnSOD activity levels. The results were compared with BEAS 2B cells exposed to silica (alpha-quartz: DQ12; SiO2) and UICC (Union Internationale Contre le Cancer) crocidolite (concentrations of all dusts: 0, 2, 5, 10, 25, 50 micrograms/cm2 = 0, 2.4, 6, 12, 30, 60 micrograms/ml; 24-h exposure) as control fibers. Scanning electron microscopy confirmed close dust cell contact under all experimental settings. Very low MnSOD mRNA baseline levels rose significantly (p < 0.001) in BEAS 2B cells exposed to all three dusts at 2 micrograms/cm2. However, at > 25 micrograms/cm2 MnSOD mRNA levels in silica- and crocidolite- but not in rockwool-exposed cells decreased. Slight (no significance) increases of MnSOD activity were observed which decreased at higher dust (> 5 micrograms/cm2) concentrations. These results suggest that: (1) like crocidolite and silica, rockwool accelerates MnSOD gene expression in bronchoepithelial cells; (2) an increase of MnSOD mRNA levels is not accompanied by MnSOD activity elevation; (3) in contrast to rockwool, high concentrations (> or = 25 micrograms/cm2) of crocidolite and silica reduced MnSOD activity and MnSOD mRNA levels. Because oxidants (H2O2) and crocidolite fibers were shown to reduce SOD activity, lack of active MnSOD protein may be caused by inactivation on a post-translational level. Furthermore, the decline of MnSOD mRNA and MnSOD activity levels coincides with increasing cytotoxicity. In conclusion, rockwool was demonstrated to induce MnSOD gene expression, perhaps because of its pro-oxidative effect in bronchoepithelial cells. In contrast to crocidolite and silica, rockwool fibers are not cytotoxic in this experimental setting.

Human bronchoepithelial cells in vitro as a tool for detection of genotoxic activity of airborne particulates

Human bronchoepithelial cells in vitro as a tool for detection of genotoxic activity of airborne particulates

Bioassays of benzo(a)pyrene and lung cancer

Bioassays of benzo(a)pyrene and long cancer Wu Zhong-hang’, Chen Jia-kun’, Zhan De-jin’, Jin Bo’, He Ling’, Du Ying-xiu’ and Joseph M. Wu D 0 Guangz,hou institute for Chemical Carcinogenesis, Gaangzhou Medical College, Guangzhou. China, ’ Depanment of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York, USA Benzo(a)pyrene (B(a)P) is an ubiquitous contaminant generated by combustion of tobacco, coal. organic compounds, etc. Human exposure primarily occurs through inhalation of polluted air and cigarette smoke. Epidemiological studies have shown a correlation between lung cancer and exposure to B(a)P. The mutagenic and carcinogenic effects of the procarcinogen B(a)P require its biotransformation in a speciesand tissue-specific manner. Thus, it is difficult to extrapolate the results from animals and cells to humans because of interand intraspecies variability. In this study. immunochemically-confirmed human fetal broncho-epithelial cells (HFBE) were prepared from an aborted fetus and cultured in vitro to investigate the genotoxicity of the metabolites of B(a)P. l Anti-7,8dihydrodiol-9.10.epoxybenzo(a)pyrene (anti-BPDE), syn-7,8-dihydrodiol-9,lO-epoxybenzo(a)pyrene(syn-BPDE), 9-hydroxybenzo(a)pyrene(9-OH-B(a)P), 3-hydroxybenzo(a)pyrene (3-OH-B(a)P) and 7,8dihydrodiolbenzo(a)pyrene (7,8diol-B(a)P) were used. l Microsomal fractions were prepared from human fetal liver and lung tissues. The genotoxicity potential of the metabolites was evaluated using unscheduled DNA synthesis (UDS) and the micronucleus test. l