DNA damage can impair normal cellular functions and result in various pathophysiological processes including cardiovascular diseases and cancer. We compared the genotoxic potential of diverse DNA damaging agents, and focused on their effects on the DNA damage response (DDR) and cell fate in human lung cells BEAS-2B. Polycyclic aromatic hydrocarbons [PAHs; benzo[a]pyrene (B[a]P), 1-nitropyrene (1-NP)] induced DNA strand breaks and oxidative damage to DNA; anticancer drugs doxorubicin (DOX) and 5-bromo-2'-deoxyuridine (BrdU) were less effective. DOX triggered the most robust p53 signaling indicating activation of DDR, followed by cell cycle arrest in the G2/M phase, induction of apoptosis and senescence, possibly due to the severe and irreparable DNA lesions. BrdU not only activated p53, but also increased the percentage of G1-phased cells and caused a massive accumulation of senescent cells. In contrast, regardless the activation of p53, both PAHs did not substantially affect the cell cycle distribution or senescence. Finally, a small fraction of cells accumulated only in the G2/M phase and exhibited increased cell death after the prolonged incubation with B[a]P. Overall, we characterized differential responses to diverse DNA damaging agents resulting in specific cell fate and highlighted the key role of DNA lesion type and the p53 signaling persistence.

• MeSH
• apoptóza MeSH
• bromodeoxyuridin farmakologie   MeSH
• DNA MeSH
• lidé MeSH
• nádorový supresorový protein p53 * metabolismus   MeSH
• plíce metabolismus   MeSH
• polycyklické aromatické uhlovodíky * toxicita   MeSH
• poškození DNA MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Components of cyanobacterial water blooms were quantified in aerosols above agitated water surfaces of five freshwater bodies. The thoracic and respirable aerosol fraction (0.1-10 μm) was sampled using a high-volume sampler. Cyanotoxins microcystins were detected by LC-MS/MS at levels 0.3-13.5 ng/mL (water) and < 35-415 fg/m3 (aerosol). Lipopolysaccharides (endotoxins) were quantified by Pyrogene rFC assay at levels < 10-119 EU/mL (water) and 0.13-0.64 EU/m3 (aerosol). Cyanobacterial DNA was detected by qPCR at concentrations corresponding to 104-105 cells eq./mL (water) and 101-103 cells eq./m3 (aerosol). Lipopolysaccharides isolated from bloom samples induced IL-6 and IL-8 cytokine release in human bronchial epithelial cells Beas-2B, while extracted cyanobacterial metabolites induced both pro-inflammatory and cytotoxic effects. Bloom components detected in aerosols and their bioactivities observed in upper respiratory airway epithelial cells together indicate that aerosols formed during cyanobacterial water blooms could induce respiratory irritation and inflammatory injuries, and thus present an inhalation health risk.

• MeSH
• aerosoly MeSH
• chromatografie kapalinová MeSH
• lidé MeSH
• lipopolysacharidy analýza   MeSH
• mikrocystiny toxicita   MeSH
• sinice * metabolismus   MeSH
• sladká voda analýza   MeSH
• tandemová hmotnostní spektrometrie MeSH
• toxiny kmene Cyanobacteria * MeSH
• voda MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Non-small cell lung cancer (NSCLC) results in high mortality and has gained increasing attention. C-Phycocyanin (C-PC) has been identified as a potential therapeutic inhibitor for NSCLC, but its underlying mechanism remains obscure. The gene expression of the long noncoding RNA neighbour of BRCAI RNA 2 (NBR2) in NSCLC cells was evaluated by quantitative reverse transcription-PCR. The cell capacity for proliferation and migration was examined by EdU and wound-healing assays. Furthermore, the viability and apoptosis of cells was measured with CCK-8 and annexin V/PI, respectively. Next, the protein level of activation of adenosine monophosphate- activated protein kinase and the rapamycin kinase (mTOR) signalling pathway-associated molecules was evaluated by western blotting. H292 cells were pre-treated with C-PC or transfected with plasmids encoding NBR2 or the shNBR2 plasmid, to over-express or knock down NBR2 expression, respectively. NBR2 expression was robustly down-regulated in NSCLC cell lines compared with a normal cell line (BEAS-2B). NBR2 over-expression inhibited migration and promoted apoptosis of H292 cells. Treatment of H292 cells with C-PC enhanced NBR2 levels in a dose- and time-dependent manner. Downregulation of NBR2 in H292 cells inhibited the activity of C-PC on cell proliferation, viability and clone formation. Further mechanistic investigation showed that the down-regulation of NBR2 abolished the modulatory effects of C-PC on the AMPK/mTOR signalling pathway. In conclusion, C-PC inhibits H292 cell growth by enhancing the NBR2/AMPK signalling pathway.

• MeSH
• adenosinmonofosfát farmakologie   terapeutické užití   MeSH
• annexin A5 farmakologie   terapeutické užití   MeSH
• apoptóza MeSH
• fykocyanin metabolismus   farmakologie   terapeutické užití   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory plic * farmakoterapie   genetika   metabolismus   MeSH
• nemalobuněčný karcinom plic * farmakoterapie   genetika   metabolismus   MeSH
• pohyb buněk MeSH
• proliferace buněk MeSH
• proteinkinasy aktivované AMP genetika   metabolismus   farmakologie   MeSH
• RNA dlouhá nekódující * genetika   MeSH
• sinkalid metabolismus   farmakologie   terapeutické užití   MeSH
• sirolimus farmakologie   terapeutické užití   MeSH
• TOR serin-threoninkinasy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

In this work, levofloxacin (LVX), a third-generation fluoroquinolone antibiotic, is encapsulated within amphiphilic polymeric nanoparticles of a chitosan-g-poly(methyl methacrylate) produced by self-assembly and physically stabilized by ionotropic crosslinking with sodium tripolyphosphate. Non-crosslinked nanoparticles display a size of 29 nm and a zeta-potential of +36 mV, while the crosslinked counterparts display 45 nm and +24 mV, respectively. The cell compatibility, uptake, and intracellular trafficking are characterized in the murine alveolar macrophage cell line MH-S and the human bronchial epithelial cell line BEAS-2B in vitro. Internalization events are detected after 10 min and the uptake is inhibited by several endocytosis inhibitors, indicating the involvement of complex endocytic pathways. In addition, the nanoparticles are detected in the lysosomal compartment. Then, the antibacterial efficacy of LVX-loaded nanoformulations (50% w/w drug content) is assessed in MH-S and BEAS-2B cells infected with Staphylococcus aureus and the bacterial burden is decreased by 49% and 46%, respectively. In contrast, free LVX leads to a decrease of 8% and 5%, respectively, in the same infected cell lines. Finally, intravenous injection to a zebrafish larval model shows that the nanoparticles accumulate in macrophages and endothelium and demonstrate the promise of these amphiphilic nanoparticles to target intracellular infections.

• MeSH
• antibakteriální látky farmakologie   MeSH
• chitosan * MeSH
• dánio pruhované MeSH
• lidé MeSH
• makrofágy metabolismus   MeSH
• myši MeSH
• nanočástice * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Acute lung injury (ALI) caused by lipopolysaccharide (LPS) is a common, severe clinical syndrome. Injury caused by inflammation and oxidative stress in vascular endothelial and alveolar epithelial cells is a vital process in the pathogenesis of ALI. Toll-like receptor 9 (TLR9) is highly expressed in LPS-induced ALI rats. In this study, Beas-2B human pulmonary epithelial cells and A549 alveolar epithelial cells were stimulated by LPS, resulting in the upregulation of TLR9 in a concentrationdependent manner. Furthermore, TLR9 overexpression and interference vectors were transfected before LPS administration to explore the role of TLR9 in LPS-induced ALI in vitro. The findings revealed that inhibition of TLR9 reduced inflammation and oxidative stress while suppressing apoptosis of LPS-induced Beas-2B and A549 cells, whereas TLR9 overexpression aggravated these conditions. Moreover, TLR9 inhibition resulted in downregulated protein expression of myeloid differentiation protein 88 (MyD88) and activator activator protein 1 (AP-1), as well as phosphorylation of nuclear factor-?B (NF-kappaB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). The phosphorylation of extracellular-regulated protein kinases 1/2 was upregulated compared to that of cells subjected to only LPS administration, and this was reversed by TLR9 overexpression. These results indicate that inhibition of TLR9 plays a protective role against LPS-induced inflammation and oxidative stress in Beas-2B and A549 cells, possibly via the MyD88/NF-kappaB and MyD88/MAPKs/AP-1 pathways.

• MeSH
• akutní poškození plic * chemicky indukované   metabolismus   prevence a kontrola   MeSH
• epitelové buňky patologie   MeSH
• krysa rodu rattus MeSH
• lipopolysacharidy * metabolismus   toxicita   MeSH
• myeloidní diferenciační faktor 88 metabolismus   MeSH
• NF-kappa B metabolismus   MeSH
• oxidační stres MeSH
• signální transdukce MeSH
• toll-like receptor 4 metabolismus   MeSH
• toll-like receptor 9 genetika   metabolismus   MeSH
• transkripční faktor AP-1 metabolismus   MeSH
• zánět chemicky indukované   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Air pollution caused by road traffic has an unfavorable impact on the environment and also on human health. It has previously been shown, that complete gasoline emissions lead to toxic effects in cell models originating from human airways. Here we focused on extractable organic matter (EOM) from particulate matter, collected from gasoline emissions from fuels with different ethanol content. We performed cytotoxicity evaluation, quantification of mucin and extracellular reactive oxygen species (ROS) production, DNA breaks detection, and selected gene deregulation analysis, after one and five days of exposure of human bronchial epithelial model (BEAS-2B) and a 3D model of the human airway (MucilAirTM). Our data suggest that the longer exposure had more pronounced effects on the parameters of cytotoxicity and mucin production, while the impacts on ROS generation and DNA integrity were limited. In both cell models the expression of CYP1A1 was induced, regardless of the exposure period or EOM tested. Several other genes, including FMO2, IL1A, or TNF, were deregulated depending on the exposure time. In conclusion, ethanol content in the fuels did not significantly impact the toxicity of EOM. Biological effects were mostly linked to xenobiotics metabolism and inflammatory response. BEAS-2B cells were more sensitive to the treatment.

• MeSH
• benzin * MeSH
• bronchy cytologie   MeSH
• buněčné linie MeSH
• cytochrom P-450 CYP1A1 genetika   MeSH
• epitelové buňky účinky léků   metabolismus   MeSH
• histony metabolismus   MeSH
• interleukin-1alfa genetika   MeSH
• látky znečišťující vzduch toxicita   MeSH
• lidé MeSH
• oxygenasy genetika   MeSH
• pevné částice toxicita   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• regulace genové exprese účinky léků   MeSH
• TNF-alfa genetika   MeSH
• výfukové emise vozidel toxicita   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The evaluation of the frequency of micronuclei (MN) is a broadly utilised approach in in vitro toxicity testing. Nevertheless, the specific properties of nanomaterials (NMs) give rise to concerns regarding the optimal methodological variants of the MN assay. In bronchial epithelial cells (BEAS-2B), we tested the genotoxicity of five types of NMs (TiO2: NM101, NM103; SiO2: NM200; Ag: NM300K, NM302) using four variants of MN protocols, differing in the time of exposure and the application of cytochalasin-B combined with the simultaneous and delayed co-treatment with NMs. Using transmission electron microscopy, we evaluated the impact of cytochalasin-B on the transport of NMs into the cells. To assess the behaviour of NMs in a culture media for individual testing conditions, we used dynamic light scattering measurement. The presence of NMs in the cells, their intracellular aggregation and dispersion properties were comparable when tests with or without cytochalasin-B were performed. The genotoxic potential of various TiO2 and Ag particles differed (NM101 < NM103 and NM302 < NM300K, respectively). The application of cytochalasin-B tended to increase the percentage of aberrant cells. In conclusion, the comparison of the testing strategies revealed that the level of DNA damage induced by NMs is affected by the selected methodological approach. This fact should be considered in the interpretation of the results of genotoxicity tests.

• Publikační typ
• časopisecké články MeSH

Gasoline engine emissions have been classified as possibly carcinogenic to humans and represent a significant health risk. In this study, we used MucilAir™, a three-dimensional (3D) model of the human airway, and BEAS-2B, cells originating from the human bronchial epithelium, grown at the air-liquid interface to assess the toxicity of ordinary gasoline exhaust produced by a direct injection spark ignition engine. The transepithelial electrical resistance (TEER), production of mucin, and lactate dehydrogenase (LDH) and adenylate kinase (AK) activities were analyzed after one day and five days of exposure. The induction of double-stranded DNA breaks was measured by the detection of histone H2AX phosphorylation. Next-generation sequencing was used to analyze the modulation of expression of the relevant 370 genes. The exposure to gasoline emissions affected the integrity, as well as LDH and AK leakage in the 3D model, particularly after longer exposure periods. Mucin production was mostly decreased with the exception of longer BEAS-2B treatment, for which a significant increase was detected. DNA damage was detected after five days of exposure in the 3D model, but not in BEAS-2B cells. The expression of CYP1A1 and GSTA3 was modulated in MucilAir™ tissues after 5 days of treatment. In BEAS-2B cells, the expression of 39 mRNAs was affected after short exposure, most of them were upregulated. The five days of exposure modulated the expression of 11 genes in this cell line. In conclusion, the ordinary gasoline emissions induced a toxic response in MucilAir™. In BEAS-2B cells, the biological response was less pronounced, mostly limited to gene expression changes.

• MeSH
• adenylátkinasa metabolismus   MeSH
• bronchy cytologie   MeSH
• dvouřetězcové zlomy DNA MeSH
• elektrická impedance MeSH
• epitelové buňky účinky léků   metabolismus   MeSH
• kultivované buňky MeSH
• L-laktátdehydrogenasa metabolismus   MeSH
• lidé MeSH
• muciny metabolismus   MeSH
• testy toxicity metody   MeSH
• transkriptom MeSH
• výfukové emise vozidel toxicita   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Lower respiratory tract infection due to Pseudomonas aeruginosa has become increasingly challenging, resulting in a worse morbidity and mortality. Airway remodeling is a common phenomenon in this process, to which epithelial-mesenchymal transition (EMT) may contribute as an important promoter. Previous studies showed that epithelium-specific integrin αvβ6-mediated EMT was involved in pulmonary fibrosis via transforming growth factor-β1 (TGF-β1) signaling, but whether integrin αvβ6 plays a role in the P. aeruginosa-associated airway remodeling remains unknown. BEAS-2B cells were incubated with lipopolysaccharide (LPS) from P. aeruginosa in the presence or the absence of integrin αvβ6-blocking antibodies. Morphologic changes were observed by an inverted microscopy. The EMT markers were detected using Western blotting and immunofluorescence. The activation of TGF-β1-Smad2/3 signaling pathway was assessed. Furthermore, matrix metalloproteinase (MMP)-2 and -9 in the medium were measured using ELISA. P. aeruginosa's LPS decreased the expression of the epithelial marker E-cadherin and promoted the mesenchymal markers, vimentin and α-smooth muscle actin in BEAS-2B cells. The expression of integrin αvβ6 was significantly increased during EMT process. Blocking integrin αvβ6 could attenuate P. aeruginosa's LPS-induced EMT markers' expression via TGF-β1-Smad2/3 signaling pathway. Furthermore, blocking integrin αvβ6 could prevent morphologic changes and oversecretion of MMP-2 and -9. Integrin αvβ6 mediates epithelial-mesenchymal transition in human bronchial epithelial cells induced by lipopolysaccharides of P. aeruginosa via TGF-β1-Smad2/3 signaling pathway and might be a promising therapeutic target for P. aeruginosa-associated airway remodeling.

• MeSH
• antigeny nádorové genetika   metabolismus   MeSH
• epitelo-mezenchymální tranzice * MeSH
• epitelové buňky cytologie   účinky léků   metabolismus   MeSH
• integriny genetika   metabolismus   MeSH
• lidé MeSH
• lipopolysacharidy metabolismus   MeSH
• matrixové metaloproteinasy genetika   metabolismus   MeSH
• protein Smad2 genetika   metabolismus   MeSH
• protein Smad3 genetika   metabolismus   MeSH
• pseudomonádové infekce genetika   metabolismus   mikrobiologie   patofyziologie   MeSH
• Pseudomonas aeruginosa metabolismus   MeSH
• signální transdukce MeSH
• transformující růstový faktor beta1 genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

MiR-222 and miR-126 are associated with asbestos exposure and the ensuing malignancy, but the mechanism(s) of their regulation remain unclear. We evaluated the mechanism by which asbestos regulates miR-222 and miR-126 expression in the context of cancer etiology. An 'in vitro' model of carcinogen-induced cell transformation was used based on exposing bronchial epithelium BEAS-2B cells to three different carcinogens including asbestos. Involvement of the EGFR pathway and the role of epigenetics have been investigated in carcinogen-transformed cells and in malignant mesothelioma, a neoplastic disease associated with asbestos exposure. Increased expression of miR-222 and miR-126 were found in asbestos-transformed cells, but not in cells exposed to arsenic and chrome. Asbestos-mediated activation of the EGFR pathway and macrophages-induced inflammation resulted in miR-222 upregulation, which was reversed by EGFR inhibition. Conversely, asbestos-induced miR-126 expression was affected neither by EGFR modulation nor inflammation. Rather than methylation of the miR-126 host gene EGFL7, epigenetic mechanism involving DNMT1- and PARP1-mediated chromatin remodeling was found to upregulate of miR-126 in asbestos-exposed cells, while miR-126 was downregulated in malignant cells. Analysis of MM tissue supported the role of PARP1 in miR-126 regulation. Therefore, activation of the EGFR pathway and the PARP1-mediated epigenetic regulation both play a role in asbestos-induced miRNA expression, associated with in asbestos-induced carcinogenesis and tumor progression.

• MeSH
• azbest škodlivé účinky   MeSH
• karcinogeny chemie   MeSH
• lidé MeSH
• maligní mezoteliom MeSH
• mezoteliom genetika   patologie   MeSH
• mikro RNA metabolismus   MeSH
• nádory plic genetika   patologie   MeSH
• senioři MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH