The composition of PM2.5 is complex and is related closely to respiratory diseases such as lung injury, fibrosis, and lung cancer(Guo et al.2022;Wang et al.2023). At present, research on the mechanism of in vitro PM2.5-induced cytotoxicity still focuses on whole PM2.5 particles or a single component, with the combination of solid dust and non-solid substances studied only rarely. In the current experiments, BaP-loaded gypsum dust was the compound used to supplement the solutions that were then used to study the toxic mechanism of specific components in PM2.5. Gypsum is a monoclinic crystalline mineral, with its main chemical composition being calcium sulfate. As a consequence of its microporous structure and heating and dewatering characteristics, gypsum has excellent sound insulation, heat insulation, and fire performance and is therefore used widely in industrial materials and building materials. BaP is a class of PAHs with a pentacyclic structure and has a very strong carcinogenic effect(Baek et al.1991;Lu et al.2020). Elnaz et al.(2021)showed that nano-selenium chemically modified by CaSO4 increased the toxicity in breast cancer cells compared with that observed for nano-selenium alone, and that CaSO4 and nano-selenium had a synergistic effect on cytotoxicity, which may have been related to activation of apoptosis signals and/or pH regulation properties. These effects were also confirmed by the results of our experiment. Starting at 0.25 mg/mL, the same dose of BaP-loaded gypsum dust significantly reduced the cell survival rate of 16 HBE cells compared with that caused by gypsum dust and BaP alone (Fig. 1). This indicated that the adsorption of organic matter in solid particles significantly increased the toxicity of the individual components on cells.
Studies have shown that PAHs accumulate in tissues, and even exposure to low concentrations of PM2.5 results in high concentrations of PAHs being detected in local tissues, with biological persistence and high carcinogenic teratogen mutagenicity(Kanae et al.2013). Lv et al.(2023)analyzed the PAHs characteristics of atmospheric PM2.5 in Sichuan Basin, China and showed that the spatial distribution of PAHs varied from region to region, with the total concentration ranging from 1 µg/g to 1 mg/g, of which BaP accounted for about 18% of PAHs. The mechanism of cellular genotoxicity caused by PAHs is due mainly to them forming stable DNA adducts after binding with genetic material DNA, resulting in distortion of the molecular structure of DNA (Qin et al.2020), DNA methylation(Liu et al.2021), and alteration of gene expression (Sørhus et al.2021). Jeremy et al.(2021)infected HT-29 colon cells with different concentrations of BaP and by detecting the level of DNA adducts, showed that their formation was dependent on certain exposure concentrations and time. Therefore, in the current study we mixed 1 µg of BaP with 1 g of gypsum dust to investigate the effect on cytotoxicity of adsorption of organic matter in solid dust. The results of Wright-Giemsa staining showed that at low concentrations of exposure, a large number of spherical lipid droplets appeared in the cytoplasm and nucleus, with intracellular lipid droplets compressing the nucleus and causing swelling and deformation of the cells, with a small number of highly refractive and long strip crystals being visible outside the cell (Figs. 2a and 2b). With an increase in concentration, the crystals gradually changed from a long strip with strong refraction to a grayish brown sheet. When the concentration was 1.25 mg/mL, apoptotic bodies appeared in the cells (Fig. 2f). The experiment also showed that the flake crystals were easily deposited on the top of the cell clusters. Therefore, we speculate that after infection of 16HBE cells with high concentrations of BaP and gypsum dust, chemisorption between BaP and gypsum dust occurs over a certain period of time, resulting in an increase in cytotropism of flake crystals. To further verify that the apoptosis of 16HBE cells can be induced by BaP-loaded gypsum, we investigated apoptosis in these cells using flow cytometry. The results showed that 1.25 mg/mL of BaP-loaded gypsum dust caused a significant increase in the number of early apoptotic cells (Fig. 3b), with the number of apoptotic cells reaching 57.92 ± 1.92% (Fig. 3c).
Studies have shown that extractable organic matter in PM2.5 activates AHR and produces a large number of reactive oxygen species, leading to DNA damage and apoptosis(Fei et al.2020). AHR is a ligand-activated transcription factor, which mediates the toxic reaction of polycyclic aromatic hydrocarbons, dioxins, and other compounds, in addition to participating in some important biological processes, such as signal transduction, cell differentiation, and apoptosis(Ge et al.2012; Jan et al.2020). Under normal circumstances, AHR forms a complex with HSP90 and chaperone p23 in the cytoplasm and exists in a dormant state. When stimulated by external organic pollutants, activated AHR and the aryl hydrocarbon receptor nuclear translocator (ARNT), form a heterodimer that is transported into the nucleus and binds to a xenobiotic responsive element (XRE) that alters the expression of genes controlled by XRE(Jan et al.2020; Wang et al.2020). Han et al.(2020)reported that after BaP binds to Ahr, the aromatic hydrocarbon receptor signaling pathway is activated, which was shown to induce the invasion and migration of BEAS-2B in bronchial epithelial cells. In the current experiment, the 16HBE cells were exposed to 1.25 mg/mL BaP-loaded gypsum dust for 24 h to detect the mRNA expression levels of Ahr, Nrf2, Bax, and Bcl-2. The results showed that the mRNA expression levels of Ahr, Bax, and Bcl-2 were increased significantly in the infected group compared with those measured in the negative control group. Next, we used geldanamycin, an inhibitor of HSP90, to indirectly inhibit Ahr function. After pretreatment with geldanamycin, the mRNA expression levels of Ahr, Nrf2, Bax, and Bcl-2 were reduced significantly compared with those in the infected group (Figs. 4b, c, d, and e). For further verification, the expression levels of Ahr, Bax, and Bcl-2 proteins were measured, with the results showing that the expression levels of these proteins were reduced significantly compared with those observed in the infected group (Fig. 4f and g). Taken together, these results indicate that apoptosis of 16HBE cells induced by BaP-loaded gypsum dust may be caused by activation of the AHR signaling pathway.
In conclusion, short-term exposure to BaP-loaded gypsum significantly enhanced the toxic effects of gypsum or BaP on 16HBE cells in a concentration-dependent manner. Wright-Giemsa staining and flow cytometry were used to show that BaP-loaded gypsum dust induced apoptosis of 16HBE cells. In addition, the expression levels of AHR, NRF2, Bax, and Bcl-2 were detected with geldanamycin, an Indirect inhibitor of the AHR receptor. The results suggested that apoptosis of 16HBE cells induced by BaP-loaded gypsum dust may be due to activation of the AHR signaling pathway. In this experiment, we also found that 16HBE cells contaminated with BaP-loaded gypsum dust were not easily digested by 0.25% pancreatic enzymes, due possibly to the increased adhesion of BaP-loaded gypsum dust. Our next steps are to investigate how the chemistry of BaP-loaded gypsum dust changes in cell culture, determine its impact on cell adhesion, and further investigate its molecular mechanism of inducing 16HBE apoptosis using AHR reception-specific inhibitors.