Effect of PM_2.5 on AMPK and eEF2 in BEAS-2B cells.

<p>Cells were untreated or exposed to 100 μg/ml PM_2.5 for 5–7 weeks before removal of particles for 24 h (lane 2) or re-exposure to 100 μg/ml PM_2.5 for 1–24 h (lanes 3–5). (<b>A)</b> Immunoblotting of the activating phosphorylation of AMPK at Thr172 (n = 3) and (<b>B</b>, <b>C</b>) the inhibitory phosphorylation of eEF2 at Thr56. The AMPK inhibitor dorsomorphin (10 μM) was added 1 h prior to re-exposure to PM_2.5. (n = 2) Detection of GAP-DH or total eEF2 was used as loading controls.</p

PM_2.5-induced morphological changes in shape and paracellular gap formation and activation of p38 MAPK.

<p>Photomicrographs at 200x magnification from (<b>A</b>) untreated BEAS-2B cells or (<b>B</b>) cells exposed to 100 μg/ml of PM_2.5 for 5 weeks. (<b>C</b>) Phosphorylation of p38 MAPK at Thr180/Tyr182 upon exposure to PM_2.5. BEAS-2B cells were left untreated (lane 1), exposed to 100 μg/ml of PM_2.5 for 5 weeks and then particles were removed for 24 h (lane 2), or cells were re-exposed to PM_2.5 for 1 to 24 h (lanes 3–5). The immunoblots were normalized using an antibody, detecting total p38 MAPK. Results are shown from 5 (<b>A</b>, <b>B</b>) and 3 (<b>C</b>) different experiments.</p

Effect of PM_2.5 on lysosomal integrity, inflammasome activation, and apoptosis in BEAS-2B cells.

<p>Cells were left untreated or exposed to 100 μg/ml PM_2.5 one week before complete cell death. (<b>A</b>) Acridine orange staining and flow cytometry to determine lysosomal destabilization 72 h after re-exposure to PM_2.5. Fluorescence intensity, displayed as the geometric mean of PM exposed BEAS-2B cells vs. untreated control cells (student’s t-test, *, <i>p</i> < 0.05; n = 3). (<b>B</b>) qRT-PCR for IL1β gene expression and (<b>C</b>) IL1β ELISA to quantify extracellular release of mature IL-1β 48 h after re-exposure to PM_2.5 (student’s t-test, n = 3). (<b>D</b>) Immunodetection of procaspase-1 and its cleaved, activated fragment (p20) in whole cell lysates 0–24 h after re-exposure to PM_2.5. (<b>E</b>) MTT cellular viability assay using the caspase-1 inhibitor z-WEHD-FMK (10 μM), added 1 h prior to re-exposure to PM_2.5 for 72 h. Results show percentage of MTT conversion compared to untreated control cells (2-way ANOVA followed by the Bonferroni’s post-hoc test, ^###, <i>p</i> < 0.001; n = 3). (<b>F</b>) Immunodetection of procaspase-3 processing to its active, cleaved form and (<b>G</b>) cleavage of the cellular caspase-3 substrate PARP in whole cellular lysates, prepared 24 h after re-exposure to PM_2.5 (n = 3). Detection of α-tubulin served as a loading control. (<b>H</b>) MTT cellular viability assay using the p38 inhibitor SB203580 (10 μM) or the caspase-3/7 inhibitor Q-VD-OPh (10 μM), added 1 h prior to re-exposure to PM_2.5 for 72 h. Statistically significant differences within groups are shown for BEAS-2B cells, left untreated or exposed to 100 μg/ml PM_2.5 (2-way ANOVA followed by the Bonferroni’s post-hoc test, ^###, <i>p</i> < 0.001) and PM_2.5-exposed cells vs. PM_2.5-exposed cells in the presence of SB203580 (2-way ANOVA followed by the Bonferroni’s post-hoc test, ***, <i>p</i> < 0.05) or Q-VD-OPh (2-way ANOVA followed by the Bonferroni’s post-hoc test, *, <i>p</i> < 0.001); n = 3.</p

Representative TEM images of BEAS-2B cells after short-term exposure (D–I) or long-term exposure (J–O) to PM_2.5 (100 μg/ml).

<p>(<b>A</b>, <b>B</b>) PM_2.5 and (<b>C</b>) BEAS-2B cells without PM_2.5 as controls. (<b>D–F</b>) Internalization mechanism of PM_2.5. (<b>G</b>) Fusion of a PM_2.5 containing membrane-bound vesicle with an autophagosome (double membrane vesicle). (<b>H</b>) Vesicle (amphisome) after fusion of a membrane-bound vesicle with PM_2.5 and an autophagosome. (<b>I</b>) Complex fusion product with PM_2.5. (<b>J</b>) Large fusion products containing PM_2.5. The white arrow indicates a ruptured membrane. (<b>K</b>) Amphisome/fusion product with PM_2.5, released into the cytosol. (<b>L</b>) White arrow indicates released PM_2.5 from the fusion products in the cytosol. (<b>M</b>) Swollen mitochondria. (<b>N</b>, <b>O</b>) Necrotic BEAS-2B cells. Scale bars are indicated in nm.</p

Effect of PM_2.5 on the actin cytoskeleton, p38 MAPK and HSP27.

<p>Microscopy of BEAS-2B cells after staining with 0.2 U/ml TRITC phalloidin for 40 min (n = 2). Cells not treated with PM_2.5 (<b>A</b>, <b>C</b>), cells re-exposed to 100 μg/ml of PM_2.5 for 8 h after long-term culture with PM_2.5 for 5 weeks (<b>B</b>, <b>D</b>). The p38 MAPK inhibitor SB203580 (10 μM) was added 1 h prior to re-exposure to PM_2.5 (<b>C</b>, <b>D</b>). Effect of PM_2.5 on RhoA activity and HSP27 phosphorylation (<b>E–G</b>). BEAS-2B cells were left untreated or were exposed to 100 μg/ml PM_2.5 for 5 weeks before removal of particles for 24 h or re-exposure to 100 μg/ml of PM_2.5 for 1–24 h. Immunoblots show precipitation of active RhoA with GST-Rothekin from total cellular lysates (upper panel) and total amount of RhoA in cellular lysates (lower panel), (<b>E</b>; n = 3); phosphorylation of HSP27 at Ser82, (<b>F</b>; n = 3), addition of 10 μM of the p38 MAPK inhibitor SB203580 1 h prior to re-exposure to PM_2.5 (<b>G</b>; n = 2). GAP-DH was used as a loading control.</p

Effect of PM_2.5 on autophagy and cell viability.

<p>Autophagy was studied by immunodetection of, truncated LC3B (<b>A</b>, <b>B</b>), beclin-1 up-regulation (<b>D</b>), or ULK-1 phosphorylation at Ser555 (<b>E</b>) in whole cell lysates of BEAS-2B cells, left untreated or exposed to 100 μg/ml PM_2.5 for 5–7 weeks before removal of particles for 24 h or re-exposure to 100 μg/ml PM_2.5 for 1–24 h. Autophagy was prevented by 10 μM E-64d plus 5 μM pepstatin, added 1 h before re-exposure to PM_2.5 (<b>B</b>). The AMPK inhibitor dorsomorphin (10 μM) was added 1 h prior to re-exposure to PM_2.5 (<b>E</b>). Detection of GAP-DH served as a loading control. (<b>A</b>, <b>B</b>, <b>D</b>, <b>E</b>) MTT cell viability assay was performed one week before complete cell death of BEAS-2B cells after persistent treatment with 100 μg/ml PM_2.5 and re-exposure for 72 h. SB203580 (10 μM), E-64d (10 μM) plus pepstatin (5 μM) (<b>C</b>) or dorsomorphin (1 μM) (<b>F</b>) were added 1 h prior to addition of PM_2.5. Results, displayed as percentage of MTT conversion of PM_2.5-exposed cells compared to untreated control cells. Statistically significant differences within groups are shown for BEAS-2B cells, left untreated or exposed to 100 μg/ml PM_2.5 (^###, <i>p</i> < 0.001) and PM_2.5-exposed cells vs. PM_2.5-exposed cells in the presence of 10 μM SB203580 (*, <i>p</i> < 0.05), 10 μM E-64d plus 5 μM pepstatin (**, <i>p</i> < 0.01) or 1 μM dorsomorphin (***, <i>p</i> < 0.001). <b>A</b>–<b>D</b>: n = 3, <b>E</b>: n = 2, <b>F</b>: n = 3; statistical analysis by 2-way ANOVA followed by the Bonferroni’s post-hoc test, respectively.</p

Effect of PM_2.5 on cell numbers and cell cycle.

<p>(<b>A</b>) BEAS-2B cells counted by a trypan blue exclusion assay using a Neubauer counting chamber every three days after passage and exposure to PM_2.5. Statistically significant differences represent the mean ± SD, n = 5 (1-way ANOVA, **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001) (<b>B</b>) Cell cycle distribution analyzed by flow cytometry after 3–4 weeks of exposure to PM_2.5 and 72 h re-exposure to PM_2.5. Results are displayed as percentage of PM_2.5-exposed cells compared to untreated control cells. Statistically significant differences represent the mean ± SD, n = 4 (2-way ANOVA followed by the Bonferroni’s post-hoc test, *, <i>p</i> < 0.05; ***, <i>p</i> < 0.001). (<b>C</b>) Accumulation of p21CIP1/WAF1, analyzed by immunoblotting. Untreated cells (lane 1), exposed to 100 μg/ml PM_2.5 for 3–4 weeks before removal of particles for 24 h (lane 2), or re-exposed to 100 μg/ml of PM_2.5 for 1–24 h (lanes 3–5). GAP-DH served as a loading control (n = 3).</p

Fluorescence Microscopy Analysis of Particulate Matter from Biomass Burning: Polyaromatic Hydrocarbons as Main Contributors

<div><p>New efficient approaches to the characterization of fly ash and particulate matter (PM) have to be developed in order to better understand their impacts on environment and health. Polycyclic aromatic hydrocarbons (PAH) contained in PM from biomass burning have been identified as genotoxic and cytotoxic, and some tools already exist to quantify their contribution to PM. Optical fluorescence microscopy is proposed as a rapid and relatively economical method to allow the quantification of PAH in different particles emitted from biomass combustion. In this study samples were collected in the flue gas of biomass-combustion facilities with nominal output ranging from 40 kW to 17.3 MW. The fly ash samples were collected with various flue gas treatment devices (multicyclone, baghouse filter, electrostatic precipitator); PM samples were fractionated from the flue gas with a DEKATI® DGI impactor. A method using fluorescence observations (at 470 nm), white-light observations and image processing has been developed with the aim of quantifying fluorescence per sample. Organic components of PM and fly ash, such as PAH, humic-like substances (HULIS) and water-soluble organic carbon (WSOC) were also quantified. Fluorescence microscopy analysis method assessment was first realized with fly ash that was artificially coated with PAH and HULIS. Total amounts of PAH in the three size fractions of actual PM from biomass burning strongly correlated with the intensities of fluorescence. These encouraging results contribute to the development of a faster and cheaper method of quantifying particle-bound PAH.</p><p>Copyright 2015 American Association for Aerosol Research</p></div