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

Abstract

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.

Keywords: A549 cells; AVD; air pollution; apoptosis; oxidative stress; particulate matter.

FIGURE 1

Cytotoxicity of PM₁₀ expressed as % cell viability inhibition assessed by the MTT test on A549 cells exposed for 24 h to PM₁₀ aqueous extracts.

FIGURE 2

(A, B) Cell volume change in A549 cells exposed to the aqueous extracts of the 8 PM10 samples expressed as percentage cell size compared to not exposed cells (control). Representative brightfield image of not exposed cells; representative images of PM₁₀ exposed cells (sample of 14-1-2020). brightfield and annexin V stained. (B) Correlation analysis between cell viability inhibition and % of cell shrinkage.

FIGURE 3

Time-course of cell volume change in A549 cells during the first 2 h of exposure to the aqueous extracts of 4 PM10 samples. Cell volume changes were expressed as percentage cell size compared to the size of the cells at time 0. *p < 0.05; **p < 0.01.

FIGURE 4

(A–D) Effect of SITS (0.5 mM) on the time-course of cell volume change in A549 cells during the first 2 h of exposure to the aqueous extracts each of the 4 PM10 samples. Cell volume changes were expressed as percentage cell size compared to the size of the cells at time 0. *p < 0.05; **p < 0.01.

FIGURE 5

(A) Effect of the exposure (24 h) to increasing concentrations of the same PM₁₀ extract (representative experiment on the sample of 21-1-2020) on the intracellular fluorescence intensity of A549 cells charged with the ROS-sensitive probe CM-H2DCFDA and the corresponding confocal images (B); (C) Intracellular fluorescence of A549 cells exposed to the eight PM₁₀ aqueous extracts for 24 h and charged with the ROS sensitive probe CM-H2DCFDA (extract dilution used for all the samples: 50%).

FIGURE 6

(A) Correlation analysis between cell viability inhibition and intracellular oxidative stress expressed as fold increase of the fluorescence intensity of the CM-H2DCFDA probe compared to control. (B) Correlation analysis between apoptotic cell shrinkage evaluated after 24 h and intracellular oxidative stress (see above) ***p < 0.001; **p < 0.01.

FIGURE 7

Intracellular fluorescence of A549 cells exposed to the four PM₁₀ aqueous extracts for 15 min and charged with the ROS sensitive probe CM-H₂DCFDA (extract dilution used for all the samples: 50%). The statistical analysis of data was performed by one way ANOVA and Dunnett’s multiple comparison test.