Differential Pattern of Cell Death and ROS Production in Human Airway Epithelial Cells Exposed to Quinones Combined with Heated-PM2.5 and/or Asian Sand Dust

The combined toxicological effects of airborne particulate matter (PM), such as PM2.5, and Asian sand dust (ASD), with surrounding chemicals, particularly quinones, on human airway epithelial cells remain underexplored. In this study, we established an in vitro combination exposure model using 1,2-naphthoquinones (NQ) and 9,10-phenanthroquinones (PQ) along with heated PM (h-PM2.5 and h-ASD) to investigate their potential synergistic effects. The impacts of quinones and heated PM on tetrazolium dye (WST-1) reduction, cell death, and cytokine and reactive oxygen species (ROS) production were examined. Results revealed that exposure to 9,10-PQ with h-PM2.5 and/or h-ASD dose-dependently increased WST-1 reduction at 1 μM compared to the corresponding control while markedly decreasing it at 10 μM. Higher early apoptotic, late apoptotic, or necrotic cell numbers were detected in 9,10-PQ + h-PM2.5 exposure than in 9,10-PQ + h-ASD or 9,10-PQ + h-PM2.5 + h-ASD. Additionally, 1,2-NQ + h-PM2.5 exposure also resulted in an increase in cell death compared to 1,2-NQ + h-ASD and 1,2-NQ + h-PM2.5 + h-ASD. Quinones with or without h-PM2.5, h-ASD, or h-PM2.5 + h-ASD significantly increased ROS production, especially with h-PM2.5. Our findings suggest that quinones, at relatively low concentrations, induce cell death synergistically in the presence of h-PM2.5 rather than h-ASD and h-PM2.5 + h-ASD, partially through the induction of apoptosis with increased ROS generation.


Introduction
Particulate matter (PM) induces adverse health effects, including respiratory toxicity. PM is a complex mixture of carbon particles in which various organic compounds, including polycyclic aromatic hydrocarbons (PAHs), are adsorbed. PM2.5 is a complex mixture of solid and liquid airborne particles, and different PM2.5 components have different effects on cell inflammatory responses [1]. In China, Xi'an is one of the most heavily polluted cities and has experienced poor air quality in recent years [2]. Several studies in Xi'an have examined the negative effects of ambient air pollutants on various outcomes, including mortality and respiratory diseases [3][4][5]. However, few studies have focused on specific hazards associated with certain PM2.5 components and their influence on inflammatory processes and oxidative stress [6]. In the atmosphere, PAHs are converted to nitro-PAHs, which are more mutagenic and carcinogenic than PAHs because they contain nitrogen molecules. PAHs in the air can also be converted to their respective quinone forms through photooxidation. Additionally, PAHs are metabolized in vivo via transdihydrodiols by

and/or h-ASD on Cytokine Production
We evaluated IL-6 production from the cells after exposure to 0.1, 1, and 10 µM of 9,10-PQ + h-PM2.5 and/or h-ASD. We observed no significant difference after exposure to 0.1 and 1 µM of all compounds compared to the corresponding control. At 10 µM, IL-6 production decreased after exposure to all compounds compared to the corresponding control (p < 0.01). A relative comparison between compounds showed that at 0.1, 1, and 10 µM, the production was comparable between 9,10-PQ and combined exposure at every dose ( Figure 3A).

Discussion
This study found that exposure to 9,10-PQ + h-PM2.5 and/or h-ASD dose-dependently increased WST-1 reduction at 1 μM compared to the corresponding control but markedly decreased it at 10 μM. At 1 μM, exposure to 9,10-PQ + h-PM2.5 increased the number of late apoptotic or necrotic cells compared among all groups. Exposure to 1,2-NQ + h-PM2.5 and/or h-ASD dose-dependently increased WST-1 reduction compared to the corresponding control. 1,2-NQ at 10 μM + h-PM2.5 increased the number of late apoptotic or necrotic cells compared among all groups. Quinones with or without h-PM2.5, h-ASD, or h-PM2.5 + h-ASD dose-dependently increased ROS production compared to the corresponding control. In the cell-free condition, quinones with or without h-PM2.5, h-ASD, or h-PM2.5 + h-ASD also dose-dependently increased ROS production compared to the corresponding control, with quinones + h-PM2. 5 showing a significantly greater increase than quinones + h-ASD or h-PM2.5 + h-ASD.
Although PMs, including PM2.5 and ASD, have respiratory toxicity, relatively few studies have investigated the responsible components, such as chemicals and heavy metals, in or around PMs contributing to this toxicity [15,16]. Other groups, including ours, have shown that the chemical components of PM2.5 have toxic potential and synergistically exacerbate allergic models in vivo [17][18][19][20]. However, most of these studies have examined the individual effects of these components. In the environment, particularly the atmosphere, these components commonly exist with suspended PMs such as PM2.5 and ASD and enter the body through the intestinal and respiratory routes, making it difficult to investigate their health effects in the real world. Therefore, studying the combined effects of these compounds and PMs can provide valuable and precise information about the mechanism of PM toxicity. To stimulate realistic effects, we preferentially selected two potential quinones and examined the combined effects of these quinones with heated PM2.5 and/or ASD in vitro. We used heated PMs in order to exclude some bioeffects from

Discussion
This study found that exposure to 9,10-PQ + h-PM2.5 and/or h-ASD dose-dependently increased WST-1 reduction at 1 µM compared to the corresponding control but markedly decreased it at 10 µM. At 1 µM, exposure to 9,10-PQ + h-PM2.5 increased the number of late apoptotic or necrotic cells compared among all groups. Exposure to 1,2-NQ + h-PM2.5 and/or h-ASD dose-dependently increased WST-1 reduction compared to the corresponding control. 1,2-NQ at 10 µM + h-PM2.5 increased the number of late apoptotic or necrotic cells compared among all groups. Quinones with or without h-PM2.5, h-ASD, or h-PM2.5 + h-ASD dose-dependently increased ROS production compared to the corresponding control. In the cell-free condition, quinones with or without h-PM2.5, h-ASD, or h-PM2.5 + h-ASD also dose-dependently increased ROS production compared to the corresponding control, with quinones + h-PM2.5 showing a significantly greater increase than quinones + h-ASD or h-PM2.5 + h-ASD.
Although PMs, including PM2.5 and ASD, have respiratory toxicity, relatively few studies have investigated the responsible components, such as chemicals and heavy metals, in or around PMs contributing to this toxicity [15,16]. Other groups, including ours, have shown that the chemical components of PM2.5 have toxic potential and synergistically exacerbate allergic models in vivo [17][18][19][20]. However, most of these studies have examined the individual effects of these components. In the environment, particularly the atmosphere, these components commonly exist with suspended PMs such as PM2.5 and ASD and enter the body through the intestinal and respiratory routes, making it difficult to investigate their health effects in the real world. Therefore, studying the combined effects of these compounds and PMs can provide valuable and precise information about the mechanism of PM toxicity. To stimulate realistic effects, we preferentially selected two potential quinones and examined the combined effects of these quinones with heated PM2.5 and/or ASD in vitro. We used heated PMs in order to exclude some bioeffects from other materials/microorganisms attached to these PMs. In fact, we observed that the heating procedure of PM at 360 • C eliminated almost all PAHs, endotoxins, and fungus and also reduced toxicity [21]. As a result, this in vitro exposure model may be applied to other chemicals and/or metals, such as PAHs. In a study, IL-6 and IL-8 release from airway epithelial cells caused by organic extracts from PM2.5 were reduced by a metal chelator [22]. This finding indicates that a combination of organic components and metals in PM2.5 may lead to stronger proinflammatory responses. Indeed, we have previously shown that coexposure to cadmium (Cd) and 9,10-PQ additively/synergistically increased proinflammatory responses in airway epithelial cells, whereas coexposure to Cd and phenanthrene resulted in no acceleration applying to the same in vitro system as the current one [23]. Accordingly, the metal may be selected for the current model in the future. Furthermore, the combined exposure model can examine the effects of these compounds even at relatively low concentrations. Although our in vitro studies have shown that 1-10 µM of 9,10-PQ or 1,2-NQ induces a significant increase in the number of apoptotic or necrotic cells, these concentrations are lower than those reported by other groups [24,25].
In this study, exposure to 9,10-PQ and 1,2-NQ dose-dependently increased WST-1 reduction. However, exposure to both h-PM2.5 and h-ASD did not affect this increase. Tan and Berridge [26] reported that quinone can increase WST-1 reduction via NAD(P)H quinone oxidoreductase-1 (NQO-1) at optimum concentrations. Above these concentrations, inhibitory effects are observed. The present study also indicated that decreased WST-1 reduction correlates with increased cell death. It is possible that at lower concentrations, quinones disrupt the redox cycle while exhibiting strong toxicities at high concentrations. Furthermore, the results of IL-6 and IL-8 production or release from bronchial epithelial cells were similar to those of WST-1 reduction, showing an overall trend. This dose (50 µg/mL) of both particles may not influence the WST-1 reduction increased by quinones. Dosedependent studies are necessary to determine the synergistic effects of these components. Additionally, in vivo studies using a pulmonary route of exposure may yield different results because the respiratory system has various resident cells, such as macrophages, leukocytes, lymphocytes, and endothelial cells.
Exposure to h-ASD or h-PM2.5 + h-ASD increased the number of late apoptotic or necrotic cells compared to vehicles. Conversely, in the cell-free condition, h-ASD increased ROS production compared to vehicle, although the value was considerably lower than that of h-PM2.5 at 50 µg/mL. Previous studies have demonstrated that h-ASD exposure induces apoptosis of epithelial cells, concomitant with increased ROS production [27,28]. However, Piao et al. [29] showed that exposure of human keratinocytes to PM2.5 increased intracellular ROS production, leading to endoplasmic reticulum stress, mitochondrial damage, autophagy, and cell apoptosis. In the present study, 50 µg/mL of h-ASD, but not h-PM2.5, increased apoptotic changes in epithelial cells. Although h-ASD at this dose can induce apoptosis or necrosis of epithelial cells, its effects on the cells may not be mediated by ROS hyperproduction. These results indicate that PMs can induce different types of cell deaths and injuries; thus, further studies are needed.
Interestingly, co-exposure to quinones and PM2.5 markedly induced late apoptosis or necrosis of cells. Previous studies have shown that 1,4-NQ induced apoptosis at 0.1 µM in leukemic cells [30], and 9,10-PQ significantly increased DNA fragmentation at a dose of 20 µM following 12 h of exposure in A549 human pulmonary epithelial cells in vitro [31]. This study is the first, to the best of our knowledge, to demonstrate that relatively low concentrations of these quinones combined with h-PM2.5 dramatically induce cell death by apoptosis or necrosis. Furthermore, the coexistence of quinones and h-PM2.5 was observed to increase ROS generation, although the exact mechanism remains unclear.
In this study, the levels of Pb (780 ng/mg), As (150 ng/mg), Cu (180 ng/mg), Cr (91 ng/mg), Ni (98 ng/mg), and Cd (9.5 ng/mg) in h-PM2.5 were 3.12-fold, 9.4-fold, 4.4-fold, 1.5-fold, 1.8-fold, and 24.4-fold higher than those in h-ASD, respectively. It has been reported that metals with H 2 O 2 have the ability to generate hydroxyl radicals in aqueous buffer solutions at pH 7.4 in the order Fe (II) > V (IV) > Cr (III) > Cu (I) > Co (II)> Ni (II) > Pb (II) > Cd (II) [32]. Therefore, these components in h-PM2.5 may play an important role in ROS generation due to their combination with quinones. ROSinduced oxidative stress is an important part of apoptosis [33]. Higher ROS levels can induce oxidative DNA damage and apoptosis, which contribute to allergic asthma and other respiratory diseases [34]. Enhanced formation of ROS activates cellular signaling mechanisms, inducing the inflammatory response observed in asthma and many other pulmonary conditions, such as chronic obstructive pulmonary disease, cystic fibrosis, idiopathic pulmonary fibrosis, and respiratory distress syndrome [35][36][37][38]. In addition, high ROS levels can damage the DNA, and given their roles as signaling molecules and inflammatory mediators, they can impede apoptosis and activate protooncogenes [39]. These results strongly indicate that these quinones at relatively low concentrations exert toxicity in the presence of h-PM2.5, at least in part, through increased ROS production generated by their mixture (a ROS-dependent pathway). In other words, these quinone components may be, at least in part, responsible for the toxicity of PM2.5 to respiratory cells. Further investigation is required to determine the factors responsible for the phenomenon (e.g., attaching style of quinones + PM2.5, responsible effects on Fas, tumor necrosis factor, proapoptotic proteins, etc.).
In conclusion, this study demonstrated that exposure to two types of quinones, quinones + h-PM2.5 and/or h-ASD, dose-dependently increased WST-1 reduction at lower concentrations compared to the corresponding control, whereas it markedly decreased at higher concentrations. At a lower concentration of 9,10-PQ, exposure to h-ASD or h-PM2.5 + h-ASD increased the number of late apoptotic or necrotic cells, whereas exposure to 9,10-PQ + h-PM2.5 markedly increased the number. At a higher concentration of 1,2-NQ, exposure to h-ASD or h-PM2.5 + h-ASD also increased the number of late apoptotic or necrotic cells, whereas exposure to 1,2-NQ + h-PM2.5 especially increased their number. Quinones with or without h-PM2.5, h-ASD, or h-PM2.5 + h-ASD dose-dependently increased ROS production compared to the corresponding control. In the cell-free condition, quinones with or without h-PM2.5, h-ASD, or h-PM2.5 + h-ASD also dose-dependently increased ROS production compared to the corresponding control. Specifically, quinones + h-PM2.5 significantly increased the value.
These results suggest that exposure to quinone chemicals at low concentrations combined with h-PM2.5 induces apoptosis or necrosis of bronchial epithelial cells, which is concomitant with intracellular or extracellular ROS overproduction. In contrast, h-ASD alone or combined with quinones induces apoptosis or necrosis. The different cell death patterns resulting from the coexistence of quinone chemicals may lead to different lung inflammations and injuries.

Cell Culture
The BEAS-2B cell line, derived from human bronchial epithelial cells transformed by an adenovirus 12-SV40 hybrid virus, was purchased from the European Collection of Cell Cultures (Salisbury, Wiltshire, UK). Airway epithelial cells were seeded in collagen-Icoated 96-or 12-well plates and incubated for 72 h to reach semiconfluence. The cells were incubated in serum-free medium LHC-9 (Life Technologies, Carlsbad, CA, USA) at 37 • C in a humidified atmosphere containing 5% CO 2 .
ASD was purchased as a reference material (CRM No. 30, Gobi Kosa dust) from the National Institute for Environmental Studies, Japan, and was purified. To exclude substances that are sensitive to heat, such as PAH, the ASD was treated by heating at 360 • C for 30 min, resulting in heated-ASD (h-ASD) [21].

WST-1 Reduction Assay
We measured the change of tetrazolium dye (WST-1) as an external electron acceptor for reduction using the Premix WST-1 Cell Proliferation Assay System (TaKaRa Bio, Shiga, Japan). Briefly, after treatment for 21 h, the WST-1 reagent was added to each well of a 96-well plate and mixed by gentle shaking. After incubating BEAS-2B cells with the WST-1 reagent at 37 • C for 3 h, absorbance was measured at a wavelength and a reference wavelength of 450 nm and 630 nm, respectively, using an iMarkMicroplate Absorbance Reader (Bio-Rad Laboratories, Hercules, CA, USA). The results were expressed as the percentage of exposed cells relative to untreated cells (control, 0.1% DMSO).

Quantitation of Inflammatory Proteins in the Culture Supernatant
After cell treatment for 24 h, the cell culture medium was collected and centrifuged at 300× g for 5 min to remove floating cells. The resulting supernatant was stored at −80 • C until further use. The levels of interleukin (IL)-6 and IL-8 in the culture medium were quantified using an ELISA kit (Thermo Scientific, Waltham, MA, USA), following the manufacturer's instructions. The absorbance was measured on the iMark microplate absorbance reader at 450 nm with a reference wavelength of 550 nm.

Statistical Analysis
The data were presented as the mean ± standard deviation for each experimental group (n = 4). The significance of variation among different groups was determined by a one-way analysis of variance. Differences among groups were analyzed using Tukey's multiple comparison tests (Excel Statistics, Social Survey Research Information Co., Ltd., Tokyo, Japan). A p-value < 0.05 was considered statistically significant. For the detection of cell death via apoptosis and necrosis, the cells were pooled together after treatment for 24 h (n = 4).

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author.