Increased lung cell susceptibility to injury due to autophagic overload after exposure to nanoparticles

Autophagy is an intracellular process that helps maintain cellular homeostasis. In this study, we measured autophagic flux and cellular injury in alveolar epithelial cells (AEC) and lung adenocarcinoma (A549) cells exposed to polystyrene nanoparticles (PNP: 20 nm, carboxylated, near-infrared dye-labeled) and a secondary insult to explore the effects of limited autophagic capacity. Rat AEC monolayers (RAECM) and A549 cells were exposed for 24 hrs to i) PNP, ii) rotenone (RT: inhibits mitochondrial respiratory chain complex I) or tunicamycin (TN: disrupts protein synthesis and induces the unfolded protein response), or iii) RT or TN for an additional 24 hrs after 12 hrs of PNP preincubation. Release of lactate dehydrogenase (LDH) served as an index of cellular damage. Autophagic flux was assessed using confocal imaging to quantify the rate of autophagosome production. PNP taken up into AEC and A549 cells induced autophagy, eliciting increased autophagic flux and reaching steady-state at ~10-24 hrs. When RAECM or A549 cells were exposed to RT or TN alone for up to 24 hrs, autophagy was also activated, reaching steady-state autophagic flux similar to PNP exposure alone. In the presence of both PNP and RT (or TN), steady-state autophagic flux was similar to that with PNP, RT or TN alone. Cellular damage due to RT or TN, in both AEC and A549 cells, became more severe after PNP preexposure, indicating that induction of autophagy by PNP makes AEC and A549 cells more susceptible to injury by each of two secondary insults due to finite autophagic capacity.