).Int. J. Mol. Sci. 2021, 22,7 ofFigure five. UV-Vis absorption spectra (A) and action
).Int. J. Mol. Sci. 2021, 22,7 ofFigure five. UV-Vis absorption spectra (A) and action spectra of singlet oxygen photogeneration (B) by 0.two mg/mL of ambient particles: winter (blue circles), spring (green diamonds), summer (red squares), autumn (brown hexagons). Information points are connected using a B-spline for eye guidance. (C) The impact of sodium azide (red lines) on singlet oxygen phosphorescence signals induced by excitation with 360 nm light (black lines). The experiments have been repeated 3 times yielding related benefits and representative spectra are demonstrated.2.five. Light-Induced Lipid Peroxidation by PM In each liposomes and HaCaT cells, the Macrolide Inhibitor Accession examined particles increased the observed levels of lipid hydroperoxides (LOOH), which have been additional elevated by light (Figure six). Inside the case of liposomes (Figure 6A), the photooxidizing impact was highest for autumn particles, exactly where the level of LOOH following 3 h irradiation was 11.2-fold higher than for MDM2 Inhibitor MedChemExpress irradiated control samples with out particles, followed by spring, winter and summer time particles, exactly where the levels have been respectively 9.4-, eight.5- and 7.3-fold higher than for irradiated controls. In cells, the photooxidizing effect from the particles was also most pronounced for autumn particles, displaying a 9-fold greater degree of LOOH just after three h irradiation compared with irradiated manage. The observed photooxidation of unsaturated lipids was weaker for winter, spring, and summer season samples resulting within a 5.six, 3.6- and 2.8-fold enhance ofInt. J. Mol. Sci. 2021, 22,eight ofLOOH, compared to handle, respectively. Changes inside the levels of LOOH observed for control samples had been statistically insignificant. The two analyzed systems demonstrated each season- and light-dependent lipid peroxidation. Some differences inside the information located for the two systems may possibly be attributed to different penetration of ambient particles. In addition, inside the HaCaT model, photogenerated reactive species might interact with various targets besides lipids, e.g., proteins resulting in comparatively decrease LOOH levels when compared with liposomes.Figure 6. Lipid peroxidation induced by light-excited particulate matter (100 /mL) in (A) Liposomes and (B) HaCaT cells. Information are presented as implies and corresponding SD. Asterisks indicate significant differences obtained working with ANOVA with post-hoc Tukey test ( p 0.05 p 0.01 p 0.001). The iodometric assays have been repeated three occasions for statistics.2.six. The Connection amongst Photoactivated PM and Apoptosis The phototoxic impact of PM demonstrated in HaCaT cells raised the query regarding the mechanism of cell death. To examine the situation, flow cytometry with Annexin V/Propidium Iodide was employed to ascertain whether the dead cells had been apoptotic or necrotic (Figure 7A,B). The strongest impact was located for cells exposed to winter and autumn particles, exactly where the percentage of early apoptotic cells reached 60.six and 22.1 , respectively. The price of necrotic cells did not exceed three.4 and did not differ substantially between irradiated and non-irradiated cells. We then analyzed the apoptotic pathway by measuring the activity of caspase 3/7 (Figure 7C). Though cells kept within the dark exhibited related activity of caspase 3/7, regardless of the particle presence, cells exposed to light for two h, showed elevated activity of caspase 3/7. The highest activity of caspase 3/7 (30 larger than in non-irradiated cells), was detected in cells treated with ambient particles collected within the autumn. Cells with particles collected.