Autophagic activity in response to nanoparticle exposure of A549 cells

We have shown previously that lung adenocarcinoma (A549) cells internalize polystyrene nanoparticles (PNP). Once intracellular, PNP stimulate autophagy in A549 cells and over time are delivered to lysosomes, with subsequent egress from the cells by lysosomal exocytosis. We also found evidence that A549 cell exposure to either PNP or ambient air pollution ultrafine particles (UFP) leads to lysosomal dysfunction. In the current study, we assessed if there is a potential limit to autophagic capacity in A549 cells. In the presence of chloroquine (40 μM, 1 hr), A549 cells were exposed to a fluorescent marker, DAPRed (Dojindo; Rockville, MD), that labels autophagosomes and autolysosomes. Autophagic activity was assessed based on the fluorescence intensity of DAPRed, which was measured in serial z sections over the entire cell volume in live, single cells using confocal laser scanning microscopy. Single exposure (50 nM Rapamycin as positive control, 80 μg/mL of 20 nm carboxylated PNP, or 1 μg/mL of UFP (<180 nm, collected in Los Angeles)) and double exposure (Rapamycin + PNP or Rapamycin + UFP) models were utilized to determine the kinetics of autophagic activity over 24 hr. Data on autophagic activity were also obtained by measurement of fluorescence intensities using LC3-GFP-RFP in response to PNP/UFP exposures. LC3-GFP-RFP was transduced (700:1 virus particle per cell ratio; Thermo Fisher Scientific; Waltham, MA) into A549 cells and expression of LC3-GFP-RFP was assessed using confocal microscopy. We found that autophagic activity remained relatively constant in unexposed A549 cells (negative control) as opposed to a rapid increase (>3 times negative control) with a peak at ~3-5 hr post exposure in Rapamycin-exposed cells. Both PNP and UFP exposure resulted in gradual elevations in autophagic activity, reaching a steady state (at ~2.5 times negative control) at ~8-10 hr post exposure with no appreciable change from ~10 to 24 hr. Combining Rapamycin with PNP or UFP caused a rapid rise in autophagic activity (peaking <5 hr post exposure); however, no difference in steady state autophagic capacity (~2.5 times negative control) was observed in comparison to either PNP or UFP exposure over 24 hr. Similar time courses of elevated autophagic activity were observed by increased fluorescence of LC3-GFP-RFP in response to 24 hr exposure to Rapamycin, PNP or UFP. In addition, higher expression of LC3-RFP indicated higher levels of autophagosome fusion with autolysosomes (increased autophagic flux). These data suggest that environmental stressors might exert their harmful effects, at least in part, by limiting available autophagic capacity, thereby making nanoparticle-exposed cells more susceptible to secondary injury. © FASEB.