Urban Aerosol Particulate Matter Promotes Necrosis and Autophagy via Reactive Oxygen Species-Mediated Cellular Disorders that are Accompanied by Cell Cycle Arrest in Retinal Pigment Epithelial Cells

Abstract

Urban particulate matter (UPM) is recognized as a grave public health problem worldwide. Although a few studies have linked UPM to ocular surface diseases, few studies have reported on retinal dysfunction. Thus, the aim of the present study was to evaluate the influence of UPM on the retina and identify the main mechanism of UPM toxicity. In this study, we found that UPM significantly induced cytotoxicity with morphological changes in ARPE-19 human retinal pigment epithelial (RPE) cells and increased necrosis and autophagy but not apoptosis. Furthermore, UPM significantly increased G2/M arrest and simultaneously induced alterations in cell cycle regulators. In addition, DNA damage and mitochondrial dysfunction were remarkably enhanced by UPM. However, the pretreatment with the potent reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) effectively suppressed UPM-mediated cytotoxicity, necrosis, autophagy, and cell cycle arrest. Moreover, NAC markedly restored UPM-induced DNA damage and mitochondrial dysfunction. Meanwhile, UPM increased the expression of mitophagy-regulated proteins, but NAC had no effect on mitophagy. Taken together, although further studies are needed to identify the role of mitophagy in UPM-induced RPE injury, the present study provides the first evidence that ROS-mediated cellular damage through necrosis and autophagy is one of the mechanisms of UPM-induced retinal disorders.

Keywords: mitophagy; necrosis; reactive oxygen species (ROS); retinal pigment epithelial (RPE) cells; urban aerosol particulate matter (UPM).

Figure 1

Urban particulate matter (UPM) induces slight cytotoxicity in ARPE-19 cells. ARPE-19 cells were incubated with UPM for 24 h. (A) Cytotoxicity was measured by a CCK-8 assay. Data are expressed as the mean ± SD (n = 6). * p < 0.05, ** p < 0.01, and *** p < 0.001 when compared to untreated cells. (B) Morphological cellular changes were observed under an inverted microscope. (C) DAPI staining was performed to visualize the morphological changes in the nucleus and was pictured under a fluorescence microscope. Scale bar: 75 μm.

Figure 2

UPM induces necrosis and autophagy without apoptosis in ARPE-19 cells. (A–E) The cells were treated with the indicated concentration of UPM for 24 h. (A) The representative histograms of the cells stained with annexin V/propidium iodine (PI) and analyzed using a flow cytometer with necrotic cells identified as annexin V⁻/PI⁺ cells (upper left quadrant) and apoptotic cells defined as annexin V-positive cells (lower left quadrant). (B) The percentages of necrotic cells. (C) The expression of apoptosis-regulatory proteins. (D) The expression of Bad in mitochondrial fraction. (E) Relative activities of caspase-3, -8, and -9. (F) The representative histograms of ARPE-19 cells pretreated with 200 μM necrostatin-1 (Nec-1) for 1 h and incubated with 200 μg/mL UPM for 24 h and were stained with annexin V/PI. (G) The percentages of necrotic cells. Data are expressed as the mean ± SD (n = 5). ** p < 0.01 and *** p < 0.001 when compared to untreated cells. ^### p < 0.001 compared to 200 μg/mL UPM-treated cells. (H) Cyto-ID-stained cells (autophagic cells, green) and DAPI-stained cells (nuclei, blue) were captured under fluorescence microscopy. Scale bar: 75 μm.

Figure 3

UPM mediates G2/M cell cycle arrest in ARPE-19 cells. (A) The representative histograms of ARPE-19 cells incubated with PI for flow cytometry analysis. (B) The average percentages of ARPE-19 cells incubated with PI in each phase of the cell, except for the cells at the sub-G1 phase. (C) The expression of cell cycle-regulatory proteins. (D) The representative histograms of ARPE-19 cells pretreated with 200 μM necrostatin-1 for 1 h and then treated with 200 μg/mL UPM for 24 h. The cells were stained with PI. (E) The average percentages of ARPE-19 cells pretreated with 200 μM necrostatin-1 for 1 h and treated with 200 μg/mL UPM for 24 h in each phase of the cell cycle, except for the cells at the sub-G1 phase.

Figure 4

UPM triggers DNA and mitochondrial disorders in ARPE-19 cells. (A) Fluorescence images of ARPE-19 cells stained with 10 μM DCF-DA. Intracellular reactive oxygen species (ROS) generation was identified as a DCF-DA intensity that was observed under a fluorescence microscope. Scale bar: 200 μm. (B) Fluorescence images of the cells immunostained with γH2AX antibody (green) and visualized using a fluorescence microscope. DAPI was used to counterstain the nuclei (blue). Scale bar: 75 μm. (C) Fluorescence images of the cells stained with 10 μM JC-1 with the monomer showing green fluorescence defining low MMP (∆Ψm) and aggregates showing red fluorescence characterizing high MMP (∆Ψm). Scale bar: 75 μm. (D) Quantification of JC-1 red and green intensity. Data are expressed as the mean ± SD (n = 5). *** p < 0.001 when compared to untreated cells. (E) The expression of mitophagy-regulated proteins.

Figure 5

Suppression of ROS attenuates UPM-induced necrosis, auophagy, and cell cycle arrest in AREP-19 cells. The cells were pretreated with 5 mM NAC for 1 h and then treated with 200 μg/mL UPM for 24 h. (A) Cell viability measured by a CCK-8 assay. Data are expressed as the mean ± SD (n = 4). * p < 0.05 and *** p < 0.001 when compared to untreated cells. ^# p < 0.05 and ^### p < 0.001 compared to 200 μg/mL UPM-treated cells. (B) The shape of the cell and the morphological changes in the nucleus. Scale bar: 75 μm. (C) Representative histograms of cell death mode. (F) Cell cycle profiles derived from flow cytometry analysis. (D) The percentages of necrotic cells. Data are expressed as the mean ± SD (n = 5). *** p < 0.001 when compared to untreated cells. *** p < 0.001 compared to 200 μg/mL UPM-treated cells. (E) Cyto-ID-stained cells (autophagic cells, green) and DAPI-stained cells (nuclei, blue) captured under fluorescence microscopy. Scale bar: 75 μm. (G) The average percentages of cells in each phase of the cell cycle.

Figure 6

Inhibition of ROS suppresses UPM-induced DNA and mitochondrial damage in AREP-19 cells. (A) Representative fluorescence images of DCF-DA-stained cells. Scale bar: 200 μm. (B) The fluorescence expression of γH2AX (green). DAPI was used to counterstain the nuclei (blue). Scale bar: 75 μm. (C) Representative images of JC-1 fluorescence staining. Monomers showing green fluorescence define low MMP, and aggregates showing red fluorescence characterize high MMP. Scale bar: 25 μm. (D) Quantification of JC-1 red and green intensities. Data are expressed as the mean ± SD (n = 4). * p < 0.05 and *** p < 0.001 when compared to untreated cells. ^### p < 0.001 compared to 200 μg/mL UPM-treated cells. (E) The expression of mitophagy-regulated proteins.

Figure 7

UPM promotes necrosis and autophagy via ROS-mediated cellular disorders that are accompanied by cell cycle arrest and mitophagy in ARPE-19 cells. UPM induces cytotoxicity, which is due to autophagic and necrotic cell death with cell cycle arrest at the G2/M phase. Furthermore, UPM markedly enhances DNA damage with the overexpression of γH2AX. In addition, UPM notably increases mitochondrial dysfunction with MMP (∆Ψm) loss and induces mitophagy. This UPM-mediated cellular damage is attributed to intracellular ROS production but not mitophagy. In conclusion, ROS-mediated cellular damage with autophagy and necrosis may be the critical mechanisms underlying UPM-induced retinal disorders.