Kinetics of macroautophagy in lung adenocarcinoma (A549) cells exposed to nanoparticles
Authors: Sipos Arnold , Kim Kwang-Jin , Alvarez Juan , Crandall Edward 
Published: May 2023

Macroautophagy is an intracellular process helping to maintain normal cellular function. Dysregulation of autophagy is recognized in certain diseases, but it is unknown how maintenance of cellular homeostasis might be affected by the kinetics of autophagy in response to various stimuli. In this study, we measured autophagy kinetics in A549 cells exposed to nanoparticles (NP) (including ambient ultrafine particles (UFP)) and/or Rapamycin. A549 cells were apically exposed (t=0-24hrs) in four experimental settings: (1) control exposure only to culture fluid; (2) exposure to polystyrene NP (PNP), UFP or Rapamycin; (3) concurrent exposure to PNP and Rapamycin or UFP and Rapamycin; and, (4) sequential (in both orders) exposure to a first agent (PNP, UFP or Rapamycin) at t=0-5hrs, followed by exposure to an additional agent (Rapamycin after PNP (or UFP) and PNP (or UFP) after Rapamycin) at t=5-24hrs. Autophagic activity was assessed using fluorescence intensity of DAPRed (a marker for autophagosomes and autolysosomes; 0.1 μM; Dojindo; Rockville, MD) or BacMam RFP-GFP-LC3B (Thermo Fisher; Waltham, MA) in the presence or absence of 40 μM chloroquine measured over the entire cell volume of live single cells using confocal laser scanning microscopy. Autophagic flux was calculated as the difference of observed fluorescence intensity of DAPRed or RFP-GFP-LC3B in the presence and absence of chloroquine. Autophagic flux increased in all experimental modalities, including both single agent and double agent exposures, and reached steady state in all cases ~2 times control from ~8 to 24 hrs, suggesting the presence of an upper limit to autophagic activity. Rapamycin exposure alone induced a rapid rise in autophagic flux, peaking at ~3hrs post exposure, followed by a lowered steady state autophagic flux for up to 24hrs. Exposure to PNP or UFP alone also increased autophagic flux, gradually reaching steady state at ~10-24hrs. During concurrent double exposure to Rapamycin and PNP or UFP at t=0, the steady state level of autophagic flux remained comparable to that observed with single exposures to Rapamycin, PNP or UFP for up to 24 hrs. Under sequential exposure conditions, the steady state level of autophagic flux did not exceed the steady state level observed in single or concurrent exposures. In summary, exposure to nanoparticles activates macroautophagy in a time-dependent manner in A549 cells. Autophagic activity demonstrated comparable steady state levels after exposure to single or dual stimuli, indicating an effective upper limit of autophagic flux in A549 cells under our experimental conditions. These findings suggest that environmental stressors might exert their harmful effects, at least in part, by limiting available autophagic capacity, thereby making NP-exposed cells more susceptible to secondary injury due to autophagic overload. Funding: NIH; WRMPPF This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.