).Int. J. Mol. Sci. 2021, 22,7 ofFigure five. UV-Vis absorption spectra (A) and action
).Int. J. Mol. Sci. 2021, 22,7 ofFigure 5. UV-Vis absorption spectra (A) and action spectra of singlet oxygen photogeneration (B) by 0.2 mg/mL of ambient particles: winter (blue circles), spring (green diamonds), summer time (red squares), autumn (brown hexagons). Information points are connected with a B-spline for eye guidance. (C) The effect of sodium azide (red lines) on singlet oxygen phosphorescence signals induced by excitation with 360 nm light (black lines). The experiments were repeated three times yielding similar outcomes and representative spectra are demonstrated.2.5. Light-Induced Lipid Nav1.8 Inhibitor supplier peroxidation by PM In both liposomes and HaCaT cells, the examined particles enhanced the observed levels of lipid hydroperoxides (LOOH), which had been additional elevated by light (Figure six). Within the case of liposomes (Figure 6A), the photooxidizing effect was highest for autumn particles, exactly where the degree of LOOH right after three h irradiation was 11.2-fold larger than for irradiated manage samples without having particles, followed by spring, winter and summer particles, exactly where the levels had been respectively 9.4-, eight.5- and 7.3-fold higher than for irradiated controls. In cells, the photooxidizing effect of the particles was also most pronounced for autumn particles, showing a 9-fold higher level of LOOH immediately after three h irradiation compared with irradiated manage. The observed photooxidation of unsaturated lipids was weaker for winter, spring, and summer time samples resulting within a five.6, 3.6- and two.8-fold increase ofInt. J. Mol. Sci. 2021, 22,8 ofLOOH, compared to control, respectively. Adjustments inside the levels of LOOH observed for control samples were statistically insignificant. The two analyzed systems demonstrated both season- and light-dependent lipid peroxidation. Some differences inside the data identified for the two systems could possibly be attributed to various penetration of ambient particles. Moreover, inside the HaCaT model, photogenerated reactive species may possibly interact with many targets apart from lipids, e.g., proteins resulting in reasonably decrease LOOH levels in comparison with liposomes.Figure 6. Lipid peroxidation induced by light-excited particulate matter (one hundred /mL) in (A) Liposomes and (B) HaCaT cells. Data are presented as indicates and corresponding SD. Asterisks indicate substantial variations obtained working with ANOVA with post-hoc Tukey test ( p 0.05 p 0.01 p 0.001). The iodometric assays have been repeated 3 times for statistics.2.6. The Connection amongst Photoactivated PM and Apoptosis The phototoxic impact of PM demonstrated in HaCaT cells raised the question regarding the mechanism of cell death. To examine the situation, flow cytometry with Annexin V/Propidium Iodide was employed to identify whether the dead cells had been apoptotic or necrotic (Figure 7A,B). The strongest impact was identified for cells exposed to winter and autumn particles, where the percentage of early apoptotic cells reached 60.six and 22.1 , respectively. The rate of necrotic cells did not exceed three.4 and didn’t vary substantially between irradiated and non-irradiated cells. We then analyzed the apoptotic pathway by measuring the activity of caspase 3/7 (Figure 7C). Even though cells kept inside the dark exhibited equivalent 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 MEK Inhibitor MedChemExpress greater than in non-irradiated cells), was detected in cells treated with ambient particles collected inside the autumn. Cells with particles collected.