Ozone Aggravated the Toxicity of Fine Particulate Matter by Impairing Membrane Stability and Facilitating Particle Internalization

Abstract

The combined pollution of fine particulate matter (PM_2.5) and ozone (O₃) is increasing synergistically on a global scale, posing a serious threat to human health. However, the joint toxicity and the underlying mechanisms associated with co-exposure to PM_2.5 and O₃ remain poorly understood. Through complementary in vivo animal models and in vitro cellular assays, the results demonstrate that although there was no synergistic cytotoxicity effect between PM_2.5 and O₃, the presence of O₃ significantly enhanced the genotoxicity of PM_2.5 by inducing severe DNA double-strand breaks. Furthermore, O₃ exposure significantly exacerbated the bioaccumulation of PM_2.5 by disturbing the cellular membrane integrity, thus leading to synergistic toxicity in bronchial cells and mouse lungs. Astaxanthin (AST) effectively antagonized the adverse effects of PM_2.5 and O₃ co-exposure by maintaining cell membrane integrity. These findings enhance our understanding of the pathophysiological mechanisms induced by co-exposure to PM_2.5 and O₃, and provide a promising therapeutic strategy for treating respiratory diseases caused by unavoidable exposure to these pollutants.

Keywords: cell membrane damage; detoxification; fine particulate matter; joint toxicity; ozone.

Figure 1

Characterization of PM_2.5 at varying concentrations and in different media. (A) TEM images of PM_2.5. Scale bar = 200 nm. (B) The average hydrated particle sizes and (C) zeta potentials for 25, 50, and 100 μg/mL PM_2.5 in different media. (D) EPR spectra of EPFRs in PM_2.5 with or without O₃ pre-treatment. Blue line: EPR spectra of 50 μg/mL PM_2.5. Red line: EPR spectra of 50 μg/mL PM_2.5 after 1 ppm O₃ pre-treatment (1 h). Grey line: EPR spectra of PBS used as a control solution.

Figure 2

Cytotoxicity and genotoxicity of PM_2.5 and O₃. (A) Experimental flowchart. (B) The dose-dependent changes in cellular viability induced by O₃. Purple column: BEAS-2B cells were exposed to 0.2, 0.4, 0.6, 0.8, and 1 ppm O₃ for 1 h. Pink column: cells were maintained in culture conditions for another 24 h after O₃ exposure (1 h). (C) The survival fraction of BEAS-2B cells exposed to different concentrations of PM_2.5 (25, 50, and 100 μg/mL) for 24 h. The combined effects of O₃ and PM_2.5 on (D) survival fraction and (E) γ-H2AX protein levels of BEAS-2B cells. Cells were treated with PM_2.5 (24 h) or O₃ (1 h in O₃ + 24 h in culture condition) or combined-treated with 1 ppm O₃ for 1 h, and this was immediately followed by 50 μg/mL PM_2.5 exposure for another 24 h. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared to control group; ns, no significance.

Figure 3

Membrane damage caused by PM_2.5 and O₃, respectively. The dose-dependent changes in (A) LDH release and (C) membrane potential induced by O₃. Purple column: BEAS-2B cells were exposed to 0.2, 0.4, 0.6, 0.8, and 1 ppm O₃ for 1 h. Pink column: cells were maintained in culture conditions for another 24 h after O₃ exposure (1 h). The (B) LDH release and (D) membrane potential of BEAS-2B cells to 0, 25, 50, and 100 μg/mL of PM_2.5 for 24 h. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared to control group; ns, no significance.

Figure 4

Combined effects of O₃ and PM_2.5 on membrane damage and PM_2.5 bioaccumulation. (A) LDH release, (B) membrane potential, and (C) Ca²⁺ flux. (D) Direct observation of cell membrane rupture (green arrows) and PM_2.5 bioaccumulation (red arrows) in BEAS-2B cells. Scale bar = 500 nm. Cells were pre-treated with O₃ (1 h in O₃ + 24 h in culture conditions) or PM_2.5 (24 h), or combined-treated with 1 ppm O₃ for 1 h, followed by 50 μg/mL PM_2.5 treatment for another 24 h. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared to the control group.

Figure 5

Suppression of PM_2.5- and O₃-induced toxicity by AST in vitro and in vivo. (A) γ-H2AX protein levels, (B) membrane potential, and (C) Ca²⁺ flux in cells treated with AST (10 μM) and 50 μg/mL PM_2.5 for 24 h after pre-treatment with 1 ppm O₃ for 1 h. (D) Direct observation of the protective role of AST on cell membrane breakage (green arrow) and PM_2.5 bioaccumulation (red arrows) induced by PM_2.5 and O₃ co-exposure. Scale bar = 500 nm. (E) H&E of lung tissues collected from PM_2.5, O_3, and AST-treated mice. Scale bar = 50 µm. Red arrows indicate PM_2.5 particles. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared to control group.