Apoptotic volume decrease (AVD) in A₅₄₉ cells exposed to water-soluble fraction of particulate matter (PM₁₀)

Exposure to atmospheric particulate matter (PM) is recognized as a human health risk factor of great concern. The present work aimed to study the cellular mechanisms underlying cytotoxic effects of airborne particulate matter <10 µm in size (PM₁₀), sampled in an urban background site from January to May 2020, on A549 cells. In particular, the study addressed if PM₁₀ exposure can be a main factor in the induction of the Apoptotic Volume Decrease (AVD), which is one of the first events of apoptosis, and if the generation of intracellular oxidative stress can be involved in the PM₁₀ induction of apoptosis in A549 cells. The cytotoxicity of PM₁₀ samples was measured by MTT test on cells exposed for 24 h to the PM₁₀ aqueous extracts, cell volume changes were monitored by morphometric analysis of the cells, apoptosis appearance was detected by annexin V and the induction of intracellular oxidative stress was evaluated by the ROS sensitive CM-H₂DCFDA fluorescent probe. The results showed cytotoxic effects ascribable to apoptotic death in A549 cells exposed for 24 h to aqueous extracts of airborne winter PM₁₀ samples characterized by high PM₁₀ value and organic carbon content. The detected reduced cell viability in winter samples ranged from 55% to 100%. Normotonic cell volume reduction (ranging from about 60% to 30% cell volume decrease) after PM₁₀ exposure was already detectable after the first 30 min clearly indicating the ability of PM₁₀, mainly arising from biomass burning, to induce Apoptotic Volume Decrease (AVD) in A549 cells. AVD was prevented by the pre-treatment with 0.5 mM SITS indicating the activation of Cl^- efflux presumably through the activation of VRAC channels. The exposure of A549 cells to PM₁₀ aqueous extracts was able to induce intracellular oxidative stress detected by using the ROS-sensitive probe CM-H₂DCFDA. The PM₁₀-induced oxidative stress was statistically significantly correlated with cell viability inhibition and with apoptotic cell shrinkage. It was already evident after 15 min exposure representing one of the first cellular effects caused by PM exposure. This result suggests the role of oxidative stress in the PM₁₀ induction of AVD as one of the first steps in cytotoxicity.