Polystyrene nanoplastics mediate oxidative stress, senescence, and apoptosis in a human alveolar epithelial cell line

Abstract

Background: Nanoplastics, an emerging form of pollution, are easily consumed by organisms and pose a significant threat to biological functions due to their size, expansive surface area, and potent ability to penetrate biological systems. Recent findings indicate an increasing presence of airborne nanoplastics in atmospheric samples, such as polystyrene (PS), raising concerns about potential risks to the human respiratory system.

Methods: This study investigates the impact of 800 nm diameter-PS nanoparticles (PS-NPs) on A549, a human lung adenocarcinoma cell line, examining cell viability, redox balance, senescence, apoptosis, and internalization. We also analyzed the expression of hallmark genes of these processes.

Results: We demonstrated that PS-NPs of 800 nm in diameter significantly affected cell viability, inducing oxidative stress, cellular senescence, and apoptosis. PS-NPs also penetrated the cytoplasm of A549 cells. These nanoparticles triggered the transcription of genes comprised in the antioxidant network [SOD1 (protein name: superoxide dismutase 1, soluble), SOD2 (protein name: superoxide dismutase 2, mitochondrial), CAT (protein name: catalase), Gpx1 (protein name: glutathione peroxidase 1), and HMOX1 (protein name: heme oxygenase 1)], senescence-associated secretory phenotype [Cdkn1a (protein name: cyclin-dependent kinase inhibitor 1A), IL1A (protein name: interleukin 1 alpha), IL1B (protein name: interleukin 1 beta), IL6 (protein name: interleukin 6), and CXCL8 (protein name: C-X-C motif chemokine ligand 8)], and others involved in the apoptosis modulation [BAX (protein name: Bcl2 associated X, apoptosis regulator), CASP3 (protein name: caspase 3), and BCL2 (protein name: Bcl2, apoptosis regulator)].

Conclusion: Collectively, this investigation underscores the importance of concentration (dose-dependent effect) and exposure duration as pivotal factors in assessing the toxic effects of PS-NPs on alveolar epithelial cells. Greater attention needs to be directed toward comprehending the risks of cancer development associated with air pollution and the ensuing environmental toxicological impacts on humans and other terrestrial mammals.

Keywords: alveolar epithelial cells; apoptosis; microplastics; nanoplastics; oxidative stress; polystyrene; senescence; toxicity.

Figure 1

Effects of PS-NPs on A549 cell viability. Increasing concentrations of PS-NPs (10–500 μg/mL) were added to A549 cells (3,000 cells/well). MTS assay was performed after 24, 48, and 96 h of incubation. Each value represents the mean ± SD of six independent experiments. p-values are expressed as ^****p ≤ 0.0001, ^***p ≤ 0.001, ^**p ≤ 0.01, and ^*p ≤ 0.05 vs. untreated cells (CTR).

Figure 2

Effects of PS-NPs on the intracellular generation of reactive oxygen species in A549 cells. Increasing concentrations of PS-NPs (10–500 μg/mL) were added to A549 cells (3,000 cells/well). ROS-GloTM H₂O₂ assay was performed after 24, 48, and 96 h of incubation. Each value represents the mean ± SD of six independent experiments. p-values are expressed as ^**p ≤ 0.01 and ^*p ≤ 0.05 vs. untreated cells (CTR).

Figure 3

Effects of PS-NPs on the expression of oxidative stress-related genes in A549 cells. Selected concentrations of PS-NPs (10, 100, and 500 μg/mL) were added to A549 cells (500,000/well). After 2 (A,C,E,G,I) and 48 h (B,D,F,H,J) of incubation, the mRNA levels of SOD1 (protein name: superoxide dismutase 1) (A,B), SOD2 (protein name: superoxide dismutase 2) (C,D), CAT (protein name: catalase) (E,F), GPX1 (protein name: Glutathione Peroxidase 1) (G,H), HMOX1 (protein name: Heme Oxygenase 1) (I,J), and Gapdh were assessed by qPCR. Relative fold change in gene expression was calculated using untreated A549 cells as control (CTR, fold change = 1) and normalized to GAPDH as a housekeeping gene. Data are reported as mean ± SD of at least three independent experiments; p-values are expressed as ^****p ≤ 0.0001, ^***p ≤ 0.001, ^**p ≤ 0.01, and ^*p ≤ 0.05 vs. Control (CTR).

Figure 4

Effects of PS-NPs on cellular senescence induction. Selected concentrations of PS-NPs (10, 100, and 500 μg/mL) were added to A549 cells (500,000/well) to evaluate effects of PS-NPs on SA-β-Gal activity (A) and the mRNA levels of CDKN1A (B), IL1A (C), IL1B (D), IL6 (E), and IL-8/CXCL8 (F) in A549 cells. The senescence-associated SA-β-Gal activity was evaluated after 96 h of incubation. Data are reported as mean ± SD of three independent experiments (A). The mRNA levels of CDKN1A (protein name: Cyclin-Dependent Kinase Inhibitor 1A, also called p21), IL1A (protein name: Interleukin 1 Alpha), IL1B (protein name: Interleukin 1 beta), IL6 (protein name: Interleukin 6), IL-8/CXCL8 (protein name: Interleukin 8), and GAPDH were assessed by qPCR after 48 h of incubation. Relative fold change in gene expression was calculated using untreated A549 cells as control (CTR, fold change = 1) and normalized to GAPDH as a housekeeping gene. Data are reported as mean ± SD of at least three independent experiments; p-values are expressed as ^****p ≤ 0.0001, ^***p ≤ 0.001, ^**p ≤ 0.01, and ^*p ≤ 0.05 vs. control (CTR).

Figure 5

Effect of PS-NPs on apoptosis in A549 cells. Selected concentrations of PS-NPs (10, 100, and 500 μg/mL) were added to A549 cells (500,000/well). Apoptosis was measured by Annexin-FITC/PI assay by flow cytometry after 96 h of incubation. Apoptotic cell population (Annexin-Vpos/PIneg and Annexin-Vpos/PIpos) can be discriminated from vital (Annexin-Vneg/PIneg) or necrotic cells (AnnexinVneg/PIpos), according to their fluorescence emission. (A) Dot plots are the most representative of the four experiments. (B) The graph represents the mean percentage ± SD of four experiments. Control (CTR) corresponds to untreated A549 cells. p-values are expressed as ^*p ≤ 0.05 vs. control (CTR). The mRNA levels of BAX (C), CASP3 (D), BCL2 (E), and GAPDH were assessed by qPCR after 2 h of incubation. Relative fold change in gene expression was calculated using untreated A549 cells as control (CTR, fold change = 1) and normalized to GAPDH as a housekeeping gene. Data are reported as mean ± SD of at least three independent experiments; p-values are expressed as ^***p ≤ 0.001, ^**p ≤ 0.01, and ^*p ≤ 0.05 vs. control (CTR).

Figure 6

Cellular intake of PS-NPs in A549 cells after 100 μg/mL or 500 μg/mL treatment. Transmission electron microscopy (TEM) analysis revealed the movement of nano-sized plastics into membrane structures (A,B,D,F,G–K). Active transport through the cell membrane (insets in G–K) may represent a significant gateway into the cell after treatment with different PS-NP concentrations (100 and 500 μg). Panels (C,E) reported images acquired from optical microscopy. Bars: (A,B,D,F) 2 μm; (C,E) 50 μm; (G–J) 0.5 μm; (K) 1 μm.