The study was performed on eight PM₁₀ samples chosen from the whole sampling campaign carried out from December to May 2020 in a site located in southeastern Italy (Aradeo, Puglia, Italy) already described in a previous work (Guascito et al., 2023). The sampling site was located in the center of the municipality of Aradeo (Lecce, Italy) (40◦07′47″ N; 18◦07′56″ E) with a population of about 10,000 inhabitants. The site is an urban background site potentially influenced by the local urban activities, biomass burning and agricultural activities, and the nearby industrial activities including a cement production plant located at about 7.2 km in the northeast direction. Daily PM₁₀ samples (starting from midnight) were collected by an automatic low-volume sampler at 38.3 L·min⁻¹ (Zambelli Explorer Plus) on PFTE filters (Whatman, 47 mm in diameter) located on the roof of the City Hall at about 14 m above the ground.

As widely described by Guascito et al. (2023), the larger contribution of PM₁₀ in the study site originated from biomass burning.

Gravimetric determination of PM₁₀ samples was done according to UNI EN 12341 (2014) by weighing the filters (three replicates before and after sampling), following stabilization for 48 h in a conditioned room (for details see Guascito et al., 2023). The weighing was performed using a microbalance Sartorious Cubis (model MSx6.6S, ±1 μg resolution). Quality control of gravimetric results was done using field blanks and periodic (once per week) control of the inlet flow rate of the samplers with external flow meters. Organic (OC) and elemental carbon (EC) were determined by a Sunset laboratory carbon analyser (Sunset Laboratory Inc., Tigard OR, United States) using thermo-optical transmittance (TOT) with the EUSAAR2 protocol (Cesari et al., 2018). The analyser was calibrated using a sucrose solution as an external standard (2.198 g/L in water, CPAchem Ltd., Bulgaria). Linear calibration had a slope of 0.97 (±0.01), a negligible intercept, (0.1 ± 0.2), and a determination coefficient R ² = 0.99.

The chemical composition of PM₁₀ sampled was determined via ICP-MS (PerkinElmer NexION 1000 and NexION 300x) for the main metals and ion chromatography (ICS1100, Thermo Scientific) for the water-soluble ions according to Guascito et al. (2023).

Cell viability was evaluated by MTT test on A549 cells exposed for 24 h to the water-soluble fractions of PM₁₀ extracted from the whole PFTE filter for each of the eight samples according to Lionetto et al. (2021). Extraction was carried out in 10 mL ultrapure water (Milli-Q) using an ultrasonic bath. Four cycles of sonication for a total of 80 min were performed and each cycle was followed by 1 min vortex agitation (according to Lionetto et al., 2021). Then, the extracts were filtered using PTFE (polytetrafluoroethylene) 0.45 μm pore syringe filters. The assay measures the metabolic activity of the cells as an indicator of cell viability, assessing the mitochondrial NAD(P)H-dependent oxidoreductase enzyme activity which reduces a yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide to formazan that accumulates as crystals within healthy cells. These are dissolved with DMSO and the absorbance of the resulting colored solution is spectrophotometrically analyzed at 570 nm (Cytation 5, BioTek Instruments, Winooski, VT, United States). Six replicates per sample were carried out. The relative viability of the cells was calculated as follows: R e l a t i v e   v i a b i l i t y   o f   c e l l s   % = [ t r e a t e d   c e l l s   O D / u n t r e a t e d   c e l l s   O D ]   x   100

A549 cells adherent to the bottom of a 96 multiwell were exposed to the PM₁₀ aqueous extracts for 24 h and visualized by Cytation 5 cell imaging multimode reader (Agilent, Santa Clara, CA, United States) (observation objective: ×40). Cell volume changes were monitored by morphometric analysis of the cells and were expressed as a percentage of the cell area of 2-D cell images after PM₁₀ exposure vs. the cell area of control cells (cells not exposed to the PM₁₀ extracts) according to Giordano et al. (2020) and Lionetto et al. (2010). At least a minimum of 100 cells/field and 5 fields per well were analyzed.

One of the earliest events of apoptosis is the translocation of the membrane phospholipid from the inner to the outer leaflet of the plasma membrane. Once exposed to the extracellular environment, binding sites on phosphatidylserine become available for Annexin V, a 36 kDa Ca²⁺-dependent phospholipid-binding protein that has a high affinity for the anionic phospholipid phosphatidylserine (Leventis and Grinstein, 2010).

A549 cells exposed to PM₁₀ aqueous extracts for 24 h were incubated with 1 μg/mL annexin V (Alexa Fluor^® 488) for 15 min and viewed by Cytation 5 cell imaging multimode reader according to Gelles et al. (2019).

The intracellular oxidative stress was assessed using the ROS-sensitive cell-permeant probe 5-(6)-Chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CMH₂DCFDA) (Ex/Em: 492–495/517–527 nm) (Thermo Fisher Scientific, Waltham, MA, United States) according to Lionetto et al. (2021); Giordano et al. (2020). The probe, once in the intracellular compartment, loses its acetate group, which is cleaved by cellular esterases, and undergoes hydrolysis. The resulting DCFH carboxylate anion is trapped inside the cell and once oxidated by intracellular ROS produces the fluorescent product DCF (Ameziane-El-Hassani and Dupuy, 2013). Fluorescence intensity was measured using the Cytation 5 cell imaging multimode reader. The results were expressed as a fold increase in the fluorescence intensity compared to the negative control (untreated cells). More details of the methodology are reported in Giordano et al. (2020).

Cells charged with CM-H₂DCFDA were also visualized by confical microscopy. Briefly, A549 cells were plated at a density of 1 × 10⁵ cells per mL into a chambered coverslip (IBIDI, Gräfelfing, Germany), incubated for 24 h for the adhesion of the cells to the bottom of the plate, then exposed for 24 h with the aqueous PM₁₀ extracts and finally charged with CM-H₂DCFDA as reported above. The cells were viewed using a ×100 NA plan apochromatic objective mounted on a NIKON TE300 inverted microscope coupled to a NIKON C1 confocal laser scanning unit (Nikon, Tokyo, Japan). The Argon 488-nm laser line was used. Unlabeled cells did not exhibit any detectable fluorescence under the conditions used. Images were acquired and analyzed by EZ-C1 NIKON software.

Data are given as the mean ± S.E.M. The statistical significance was analyzed by one-way ANOVA, and Dunnett’s multiple comparison test.

The average values of PM₁₀ concentration and carbon content of the 8 samples used in the present study are reported in Table 1. The sample set included six winter samples characterized by higher PM₁₀ concentrations and two spring samples characterized by lower PM₁₀ values in agreement with the typical seasonal PM₁₀ concentration of the area (Cesari et a., 2018). On average the same trend was also observed for carbon content. Moreover, it must be considered that the sampling campaign was carried out in 2020, when Italy was subjected to a national lockdown and limitation to the movement of people because of the COVID-19 pandemic. The first six samples were collected before the lockdown period, while the other two May samples were collected just after the lockdown when the restart of activities was slow. Some previous evidence indicated that the lockdown has slightly reduced the PM₁₀ concentration in the air at least in this region of Italy (Dinoi et al., 2021). The mean chemical composition of the PM₁₀ in the study site is reported in Supplementary Table S1.

Cell viability after 24 h exposure to water-soluble extract of the airborne PM₁₀ samples was assessed by MTT assay on A549 cells. In Figure 1 the MTT results obtained on the eight PM₁₀ samples chosen for the objective of the present study are reported. Inhibition of the mitochondrial NAD(P)H-dependent cellular oxidoreductase enzymatic activity after exposure of A549 cells for 24 h to the aqueous extracts of PM₁₀ compared to control cells was observed in the sample data set in agreement with a previous study on the whole sampling campaign (Guascito et al., 2023). The entity of the inhibition varied from about 100% (detected in January samples) to values of about 9% recorded in May samples. The dose-dependence response of the assay for each aqueous extract was preliminarily checked by exposure of A549 cells to increasing dilutions of the same extract, as reported in our previous studies (Lionetto et al., 2019; Lionetto et al., 2021). On average, winter samples showed a higher cytotoxic potential as indicated by the high percentage reduction of cell viability, particularly in January and February, compared to the two May samples according to Guascito et al. (2023) in parallel to PM₁₀ concentration.

To deepen the analysis of the mechanisms involved in PM₁₀-induced cytotoxicity on A549 cells, cell morphology was analyzed on PM₁₀ exposed cells in parallel to MTT assay using Cytation 5 cell imaging multimode reader on the same samples.

After 24 h exposure to PM₁₀ aqueous extracts, A549 cells showed typical cell shrinkage detected as a percentage decrease of the cell area of 2-D images compared to not exposed (control) cells (Figure 2A). Cell shrinkage levels were in agreement with the NAD(P)H-dependent cellular oxidoreductase enzymatic activity reduction, as indicated by the correlation analysis (Figure 2B). Particularly high cell volume reduction, above 50%, was observed in the samples which showed the highest inhibition of mitochondrial NAD(P)H-dependent oxidoreductase activity. Cell shrinkage was also accompanied by other typical morphological signs of apoptosis such as cell rounding, surface roughness, blebs formation, and annexin V positivity, visualized by the delectable green contour of the cell plasma membrane of cells treated with 1 μg/mL annexin V (Alexa Fluor^® 488) following exposure to PM₁₀ extract as shown in Figure 2A. This figure reports representative images obtained from the sample of 14-1-2020. Similar apoptotic positive evidence (cell rounding, surface roughness, blebs formation, and annexin V positivity) were obtained also for the other 5 winter samples but was not detectable for the two May samples (not shown).

After detecting reduced vitality and apoptosis appearance in A549 cells following 24 h exposure to PM₁₀ aqueous extracts, we tested the hypothesis of the possible involvement of PM₁₀ exposure in the induction of the Apoptotic Volume Decrease (AVD), which is one of the first events of apoptosis, occurring early in the first 1–2 h (Chang et al., 2000; Maeno et al., 2000). Therefore, the time course of the volume changes was monitored in A549 cells during the first 2 h of exposure to PM₁₀ aqueous extracts by time-lapse imaging of the cells every 30 min. The AVD analysis was focused on 4 representative samples, two January samples, characterized by the highest values of vitality reduction and a marked cell shrinkage after 24 h exposure, and the two samples of May, characterized by a reduction of the NAD(P)H-dependent cellular oxidoreductase activity below 15% and without apparent apoptotic signs. For each of the four filter extracts, three independent experiments were performed.

The cells exposed to the aqueous extracts of January 18 and 21 showed a significant reduction of cell volume compared to control cells already after 30 min exposure and the decrease continued in the 2 h observation reaching 16% and 27% respectively (Figure 3). If these results are compared with the overall apoptotic cell volume reduction observed after 24 h in the same extracts, the AVD response observed in the first 2 h corresponds to one-third and one-half respectively of the overall cell shrinkage produced.

On the other hand, the cells exposed to the other two spring extracts (9-5-2022 and 28-5-2022) did not show any significant cell size reduction compared to the control cells.

AVD is known to be caused by the loss of K⁺ and Cl⁻ from the cell (Yu and Choi, 2000). Therefore, in order to investigate the nature of the PM₁₀-induced early isotonic cell shrinkage, A549 cells were preincubated with 0.5 mM disulfonic stilbene derivative (4-Acetamido-40-isothiocyanato-stilbene-2,20-disulfonic acid), a known inhibitor of Cl⁻ channels (Kokubun et al., 1991) including the ubiquitously expressed volume-regulated anion channels (VRACs) (Maeno et al., 2000; Hoffman et al., 2015; Okada et al., 2021), which have been demonstrated to be involved in AVD in other cell types (Maeno et al., 2000). The cells were viewed by time-lapse microscopy. After (4-Acetamido-40-isothiocyanato-stilbene-2,20-disulfonic acid) preincubation, the cells were exposed to the PM₁₀ aqueous extracts of 18-1-2020 and 21-1-2020 for 2 h and in this case, the PM-induced isotonic shrinkage was completely prevented (Figures 4A, B). On the other hand, (4-Acetamido-40-isothiocyanato-stilbene-2,20-disulfonic acid) alone was not able to produce any significant alteration in cell size. The other two extracts, not showing AVD, were also tested and no significant effect of (4-Acetamido-40-isothiocyanato-stilbene-2,20-disulfonic acid) treatment was observed (Figures 4C, D).

It is known that reactive oxygen species and oxidative stress play a pivotal role in apoptosis induction in several cell types (Kannan and Jain, 2000; Redza-Dutordoir and Averill-Bates, 2016).

In order to test whether oxidative stress was involved in PM₁₀-induced apoptosis in A549 cells, intracellular oxidative stress levels were measured in A549 exposed for 24 h to all the eight PM₁₀ aqueous extracts of the study using the cell-permeable probe CM-H₂DCFDA. Preliminarily, the cells were exposed to different dilutions of the same extract. Figure 5A shows the representative dose response for one of the tested samples (21-1-2021) revealing that PM₁₀ was able to induce the generation of intracellular oxidative stress in a dose-dependent way. The confocal representative images of cells exposed to different dilutions of the PM₁₀ extracts and then charged with the fluorescent probe are reported in Figure 5B. The intracellular fluorescence intensity of the exposed cells increases with the concentration of the PM₁₀ aqueous extract. At the highest dilution tested the appearance of apoptotic blebs is clearly evident.

The analysis of the PM₁₀-induced intracellular oxidative stress was applied to the eight extracts revealing a highly significant fluorescence increase compared to control in the six winter samples after 24 h, while no significant effect was observed in the two spring samples (Figure 5C). The oxidative stress data were statistically correlated with vitality inhibition data and cell shrinkage results (Figures 6A, B) suggesting that the generation of intracellular oxidative stress can be one of the main underlying mechanisms of the PM₁₀ induction of apoptosis in A549 cells.

The investigation of the effect of PM₁₀ exposure on intracellular oxidative stress induction was deepened with a short-term analysis of the intracellular fluorescence of A549 cells charged with CM-H₂DCFDA and exposed for 15 min to the 4 aqueous PM₁₀ extracts used for AVD analysis. As reported in Figure 7, the cells exposed to the aqueous extracts of 18-1-2020 and 21-1-2020 expressed a significantly increased fluorescence compared to control cells already after 15 min exposure, suggesting that the induction of oxidative stress by PM₁₀ was an early event, timely correspondent with the AVD induction sustained by VRAC channels.