Oxidative stress and activation of H2A.X in lung alveolar epithelial cells (A549) by nanoparticulate carbon black.

Fine airborne particulate matter enter the respiratory system, induce oxidative stress and initiate DNA damage. The aim of our study was the estimation of cell viability, oxidative stress, DNA damage, cell cycle alterations and activation of histone H2A.X. Experiments were done on lung alveolar epithelial (A549) cells grown for 24 h with 200 µg mL-1 coarse carbon black (CB), or nanoparticulate CB (NPCB). Neither CB nor glutathione depletion altered cell viability, growth rates, and H2A.X expression while NPCB decreased cell viability, increased oxidative stress and DNA damage. The cell cycle was blocked at G0/G1. NPCB but not CB increased expression and activation of H2A.X at mRNA and protein levels. Co-expression data point to γH2A.X as a major NPCB target, and show the interdependence of γH2A.X and oxidative stress. We conclude, that NPCB increases γ-H2A.X expression in A549 cells at mRNA and protein levels and stimulates H2A.X (Ser139), phosphorylation, associated with oxidative stress, the DNA damage response and G1 cell cycle arrest.

Co-expression of Hsp70 Protein and Autophagy Marker Protein LC3 in A549 Cells and THP1 Cells Exposed to Nanoparticles of Air Pollution.

The ability of air particulate matter (PM) to cause reactive oxygen species-driven protein damage is associated with both COPD and lung cancer, but the mechanisms are unsettled. In this study, we investigated the co-expression of Hsp70 and the autophagy marker protein LC3 in A549 cells (alveolar epithelial cell line) and THP-1 cells (monocyte/macrophage cells) grown in media supplemented with 100 µg/mL of four types of PM: carbon black (CB), urban dust (UD), nanoparticulate CB (NPCB), and nanoparticulate CB coated with benzo(a)pyrene (NPCB-BaP). Fluorescent monoclonal antibodies and flow cytometry were used to assess the expression and co-expression of HSP70 and LC3 proteins. Hsp70 expression was significantly increased by all PM, while LC3 was decreased by CB in A549 cells, unchanged by CB and UD in THP-1 cells and increased by NPCB and NPCB-BaP in both cell types. All PMs increased the Hsp70/LC3 ratio in binary scatterplots; the relationship was positive and linear, which may reflect chaperone-dependent autophagy. The UD was the only PM type that affected the slopes of the spatial trend lines and altered binary patterns of Hsp70/LC3 distribution in THP1 cells. These findings provide an insight into the molecular mechanisms regulating proteostasis in PM-exposed cells through the chaperone-autophagy system in the cytoplasm.

Sialidase Attenuates Epidermal Growth Factor Response and Abolishes Antiproliferative Effects of Erlotinib in A549Alveolar Epithelial Cells.

Erlotinib is a widely used, reversible tyrosine kinase inhibitor (TKI), targeting pro-proliferative signaling of epidermal growth factor receptor (EGFR). The drug is approved for the first-line treatment of patients with metastatic non-small cell lung cancer with EGFR mutations. Extracellular glycans can affect EGFR expression, dimerization, phosphorylation, and EGF binding. In this study we investigated the effects of EGF and erlotinib on the cell cycle of naive and sialidase (alpha-neuraminidase)-pretreated human A549 alveolar epithelial cells. A549 cells were labeled with propidium iodide, and fractions of cells in different phases of cycle were quantified by flow cytometry. We found that neither did desialilation nor EGF, as well as erlotinib treatment, increase the number of damaged cells (subG0/G1 cell fraction), while erlotinib did significantly increase the number of G0/G1 cells and decrease S + G2/M cell fractions. In naive cells, EGF increased proliferating cell numbers by more than 40%, and this effect was blocked by erlotinib. In desialylated cells, however, proliferation was significantly decreased by about 29%, and EGF and erlotinib did not exert significant effects. We conclude that changes in alveolar epithelial cell membrane glycosylation may affect function of growth-promoting receptors and erlotinib effectiveness.

Phagocytosis and Autophagy in THP-1 Cells Exposed to Urban Dust: Possible Role of LC3-Associated Phagocytosis and Canonical Autophagy.

Exposure to ambient particulate matter (PM) increases mortality and morbidity due to respiratory and cardiovascular diseases. The aim of this study was to assess the effect of standardized urban dust (UD) on phagocytosis and autophagy in a monocyte-macrophage cell line (THP-1 cells). The cells were grown for 24 h in the medium supplemented with 200 µg·mL-1 coarse carbon black (CB) or UD. In some experiments glutathione (GSH) was depleted in THP-1 cells by buthionine sulfoximine. The cells were double stained with green latex beads (phagocytosis) and with red autophagy marker (LC3) and were evaluated in a flow cytometer. In naïve THP-1 cells, about 61% of them were classified as "negative", while 39% were classified as "double-positive". Both GSH depletion and UD treatment produced three distinct subpopulations of cells on bivariate scatterplots. A new subpopulation of cells (about 24% of the total number) appeared, with a lower autophagy and phagocytosis, but with a higher autophagy/phagocytosis ratio, when compared to highly positive cells. CB affected, to some extent, phagocytosis without a substantial effect on autophagy. In conclusion, the research on distinct pathways of immune cell activation may be relevant to the diagnostics and therapy of PM-induced pneumotoxicity, inflammation, and tumorigenesis.