Exploring the Cellular Impact of Size-Segregated Cigarette Aerosols: Insights into Indoor Particulate Matter Toxicity and Potential Therapeutic Interventions

Abstract

Exposure to anthropogenic aerosols has been associated with a variety of adverse health effects, increased morbidity, and premature death. Although cigarette smoke poses one of the most significant public health threats, the cellular toxicity of particulate matter contained in cigarette smoke has not been systematically interrogated in a size-segregated manner. In this study, we employed a refined particle size classification to collect cigarette aerosols, enabling a comprehensive assessment and comparison of the impacts exerted by cigarette aerosol extract (CAE) on SH-SY5Y, HEK293T, and A549 cells. Exposure to CAE reduced cell viability in a dose-dependent manner, with organic components having a greater impact and SH-SY5Y cells displaying lower tolerance compared to HEK293T and A549 cells. Moreover, CAE was found to cause increased oxidative stress, mitochondrial dysfunction, and increased levels of apoptosis, pyroptosis, and autophagy, leading to increased cell death. Furthermore, we found that rutin, a phytocompound with antioxidant potential, could reduce intracellular reactive oxygen species and protect against CAE-triggered cell death. These findings underscore the therapeutic potential of antioxidant drugs in mitigating the adverse effects of cigarette aerosol exposure for better public health outcomes.

Figure 1

(A) Cigarette aerosol collection procedure and the classification table. (B) Extraction preparation procedure. (C) Mass distribution of CAE. (D) Relative mass distribution of 16 PAHs in cigarette aerosol collected. (E) Relative mass distribution of heavy metal components in cigarette aerosol collected.

Figure 2

Distribution of IC₅₀ values from three repeated tests of CAE effects on three cell lines, SH-SY5Y, HEK293T, and A549. The IC₅₀ values of (A) OP extract and (B) WP extract. I–VI indicate the size of cigarette aerosol obtained with MOUDI listed in Figure 1.

Figure 3

CAE promoted intracellular H₂O₂ generation in living cells, (A) SH-SY5Y, (B) HEK293T, and (C) A549. I–VI indicate the size of cigarette aerosol obtained with MOUDI listed in Figure 1. Data was calculated from three independent experiments (error bar, mean ± s.d.). *, **, and *** represent significant differences (* = p < 0.05), (** = p < 0.01), and (*** = p < 0.005) based on paired t-test in comparison to the control condition. All investigated conditions exhibit p < 0.005 for all three cell lines based on the analysis of variance (ANOVA) test as well.

Figure 4

Influences of CAE on mitochondrial ROS generation, MMP polarization, and ATP generation in living cells, (A) SH-SY5Y (B) HEK293T, and (C) A549. I–VI indicate the size of cigarette aerosol obtained with MOUDI listed in Figure 1. Data was calculated from three independent experiments (error bar, mean ± s.d.). *, **, and *** represent significant differences (* = p < 0.05), (** = p < 0.01), and (*** = p < 0.005) based on paired t-test in comparison to the control condition. All investigated conditions exhibit p < 0.005 for all three cell lines based on the ANOVA test as well.

Figure 5

Influences of CAE on (A) caspase-3 and (B) caspase-1 activities, (C) representative immunofluorescence images of LC3-II, (D) quantified analysis of LC3-II images, and (E) autophagy activities in SH-SY5Y, HEK293T, and A549 cell lines. Data was calculated from three independent experiments (error bar, mean ± s.d.). *, **, and *** represent significant differences (* = p < 0.05), (** = p < 0.01), and (*** = p < 0.005) based on paired t-test in comparison to the control condition without CAE treatment.

Figure 6

Rescue effects of rutin. (A). Cell viability of SH-SY5Y cells, HEK293T cells, and A549 cells after treatment with various concentrations of rutin for 24 h at 37 °C verified by the MTT assay. (B). Cell viability of SH-SY5Y cells, HEK293T cells, and A549 cells after treatment of OP CAE in the presence of 200 μM rutin for 24 h at 37 °C verified by the MTT assay. (C). Cell viability of SH-SY5Y cells, HEK293T cells, and A549 cells after treatment with OP CAE in the presence of various concentrations of rutin for 24 h at 37 °C verified by the MTT assay. (D) Intracellular H₂O₂ generation in SH-SY5Y, HEK293T, and A549 cells after treatment of OP CAE in the presence of various concentrations of rutin for 80 min at 37 °C. *, **, and *** represent significant differences (* = p < 0.05), (** = p < 0.01), and (*** = p < 0.005). N (repeat of experiment) is 3 for each condition based on paired t-test in comparison to the control condition. All investigated conditions exhibit p < 0.005 for all three cell lines based on the ANOVA test as well.

Figure 7

(A). Summary mechanism of CAE-induced cell death and (B) rescue effect of rutin treatment in living cells.